Methods for rejuvenating aged tissues by inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH)

Abstract

To provide a method to strengthen the function of aged skeletal muscle of a subject.SOLUTION: The disclosure provides compositions and methods based on the identification of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) as a therapeutic target in aging muscle or dystrophic muscle to improve muscle atrophy, and to increase muscle mass, function and strength. Further provided herein are compositions and methods for the rejuvenation of aged tissue. In particular, 15-PGDH inhibitors, such as SW033291, are used to elevate the levels of prostaglandin E2 (PGE2) in the muscle or tissue.SELECTED DRAWING: Figure 1B

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 860,180, filed June 11, 2019, U.S. Provisional Patent Application No. 62 / 875,915, filed July 18, 2019, U.S. Provisional Patent Application No. 62 / 882,981, filed August 5, 2019, and U.S. Provisional Patent Application No. 62 / 883,025, filed August 5, 2019, each of which is incorporated herein by reference in its entirety.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under contract AG020961 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Muscle wasting diseases cause rapid loss of muscle mass and strength, primarily due to excessive protein degradation, often accompanied by decreased protein synthesis. This loss of muscle function reduces quality of life and increases morbidity and mortality. Although much is known about how muscle atrophy occurs, current therapeutic strategies to effectively prevent or delay atrophy are limited to exercise. Reasonable strategies for increasing muscle mass and strength include altering protein balance, for example, through modulation of the TGF-β family or insulin receptor signaling pathways.

[0004] Prostaglandin E2 (PGE2), also known as dinoprostone, is used in various clinical settings, including inducing labor in women and enhancing hematopoietic stem cell transplantation. PGE2 can be used as an anticoagulant and antithrombotic agent. The role of PGE2 as a lipid mediator capable of resolving inflammation is also well known. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are inhibitors of COX-1 and / or COX-2, suppress inflammation by inhibiting prostanoids, primarily via PGE2 biosynthesis. PGE2 is synthesized from arachidonic acid by the enzymes cyclooxygenase (COX) and prostaglandin E synthase. PGE2 levels are physiologically regulated by the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 15-PGDH catalyzes the inactivation of PGE2 15-OH to a 15-keto group.

[0005] There remains a need in the art for effective treatments to prevent or reverse the loss of proteins in aging and / or atrophied muscles of subjects in need thereof, as well as the resulting loss of muscle fiber and / or myotube size, and the resulting loss of muscle strength, muscle endurance, or muscle mass of atrophied muscles.There also remains a need in the art for effective treatments to prevent or reverse the loss of function in tissues, such as non-skeletal muscle tissues, of subjects with age-related diseases and disorders.The present disclosure meets these needs and provides other advantages. Summary of the Invention

[0006] overview In one aspect, a method for enhancing the function of aged skeletal muscle in a subject is provided, the method comprising administering to the aged skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in one or more senescent cells within the aged skeletal muscle, thereby enhancing the function of the aged skeletal muscle.

[0007] In another aspect, a method for increasing muscle mass, strength, and / or endurance of aging skeletal muscle of a subject is provided, the method comprising administering to the aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in one or more senescent cells within the aging skeletal muscle, thereby increasing muscle mass, strength, and / or endurance of the aging skeletal muscle.

[0008] In another aspect, a method for increasing the level of PGE2 in aged skeletal muscle of a subject is provided, the method comprising administering to the aged skeletal muscle an amount of a 15-PGDH inhibitor effective to increase the level of PGE2 in the aged skeletal muscle, thereby increasing the level of PGE2 in the aged skeletal muscle.

[0009] In any one of the preceding methods, the subject has one or more biomarkers of aging.

[0010] In yet another aspect, a method for rejuvenating aging skeletal muscle in a subject having one or more biomarkers of aging is provided, the method comprising administering to a subject having one or more biomarkers of aging a 15-PGDH inhibitor in an amount effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the subject, thereby rejuvenating the aging skeletal muscle.

[0011] In any one of the aforementioned methods, the one or more biomarkers of aging are selected from the group consisting of increased 15-PGDH levels compared to those present in young skeletal muscle, decreased PGE2 levels compared to those present in young skeletal muscle, increased PGE2 metabolites compared to those present in young skeletal muscle, increased or greater accumulation of senescent cells compared to those present in young skeletal muscle, increased expression of one or more muscle atrophy-related genes (atrogenes) compared to those present in young skeletal muscle, decreased mitochondrial biogenesis and / or mitochondrial function compared to those present in young skeletal muscle, and increased transforming growth factor pathway signaling compared to those present in young skeletal muscle. In some cases, the one or more muscle atrophy-related genes are selected from the group consisting of Atrogin 1 (MAFbx1), MuSA (Fbxo30), and Trim63 (MuRF1). In some cases, the increased transforming growth factor pathway signaling comprises increased expression of one or more genes selected from the group consisting of activin receptors, myostatin, SMAD proteins, and bone morphogenetic proteins. In any one of the aforementioned methods, aged skeletal muscle has an increased accumulation of senescent cells compared to young skeletal muscle. Optionally, the senescent cells express one or more senescence markers. Optionally, the senescent cells have increased levels of one or more senescence markers compared to non-senescent cells. Optionally, the one or more senescence markers are selected from the group consisting of p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6. Optionally, the senescent cells are macrophages. In any one of the aforementioned methods, the aged skeletal muscle has not been injured and / or has not undergone exercise and / or has not undergone regeneration. In any one of the aforementioned methods, the method further comprises administering a senolytic agent to the aged skeletal muscle.In some cases, the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a combination therapy of dasatinib and quercetin, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof. In any one of the aforementioned methods, the 15-PGDH inhibitor is selected from the group consisting of a small molecule compound, a blocking antibody, a nanobody, and a peptide. In any one of the aforementioned methods, the 15-PGDH inhibitor is SW033291. In any one of the aforementioned methods, the 15-PGDH inhibitor is selected from the group consisting of an antisense oligonucleotide, a microRNA, an siRNA, and an shRNA. In any one of the aforementioned methods, the subject is a human. In any one of the aforementioned methods, the subject is at least 30 years old. In any one of the aforementioned methods, the administering step includes systemic administration or local administration. In any one of the aforementioned methods, the level of PGE2 is increased in aged skeletal muscle compared to the level of PGE2 present in aged skeletal muscle before administration of the 15-PGDH inhibitor. In any one of the aforementioned methods, the level of PGE2 is increased by at least 10% compared to the level of PGE2 present in aged skeletal muscle before administration of the 15-PGDH inhibitor. In any one of the aforementioned methods, the level of PGE2 is increased to a level substantially equivalent to that present in young skeletal muscle. In any one of the aforementioned methods, the level of PGE2 is increased to a level within about 50% or less of that present in young skeletal muscle. In any one of the aforementioned methods, the method results in an increase in the cross-sectional area and / or diameter of muscle fibers and / or myotubes. In any one of the aforementioned methods, the method results in an increase in the cross-sectional area and / or diameter of oxidative (type IIa) fibers and / or glycolytic (type IIb) fibers. In any one of the aforementioned methods, the 15-PGDH inhibitor reduces or blocks 15-PGDH expression. In any one of the foregoing methods, the 15-PGDH inhibitor reduces or blocks the enzymatic activity of 15-PGDH.In any one of the aforementioned methods, the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, in aged skeletal muscle. In any one of the aforementioned methods, the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, in aged skeletal muscle compared to the aged skeletal muscle before administration of the 15-PGDH inhibitor. In any one of the aforementioned methods, the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, in aged skeletal muscle to a level substantially equivalent to that present in young skeletal muscle. In any one of the aforementioned methods, the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, in aged skeletal muscle to a level within about 50% or less of the level present in young skeletal muscle. In any one of the aforementioned methods, the method results in enhanced function of aged skeletal muscle. In any one of the aforementioned methods, the method results in enhanced function of aged skeletal muscle compared to the aged skeletal muscle before administration of the 15-PGDH inhibitor. In any one of the aforementioned methods, the method results in an enhancement of aging skeletal muscle function to a level substantially equivalent to that present in young skeletal muscle. In any one of the aforementioned methods, the method results in an enhancement of aging skeletal muscle function to a level within about 50% or less of that present in young skeletal muscle. In any one of the aforementioned methods, the function is increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof. In any one of the aforementioned methods, the method results in a reduction in the level of PGE2 metabolites in aging skeletal muscle compared to that of aging skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. In some cases, the PGE2 metabolites are selected from the group consisting of 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2. In any one of the aforementioned methods, the subject has sarcopenia due to aging. In any one of the foregoing methods, the expression level of one or more muscle atrophy-associated genes is reduced compared to aged skeletal muscle prior to administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle.In any one of the foregoing methods, the expression level of one or more components of a mitochondrial complex is increased compared to aged skeletal muscle prior to administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. In some cases, one or more components of the mitochondrial complex are selected from the group consisting of Ndufal 1, Ndufal 2, Ndufal 3, Ndufa2, Ndufa3, Ndufa4, Ndufa5, Ndufal O, Ndufb5, Ndufcl, Ndufs4, Ndufs8, Ndufvl, Ndufv2, Uqcrb, Uqcrcl, Uqcrh, Uqcrq, Ucqr10, Cox8b, Cox7a1, Cox7a2, Cox7b, Cox6c, Cox5a, Cox5b, Atp5f1, Atp5g1, Atp5h, Atp5j2, Atp5o, Atp5e, and Atp5k. In any one of the aforementioned methods, the expression level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α) is increased compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. In any one of the aforementioned methods, the expression level of one or more genes selected from the group consisting of Tnfaip1, Klhdc8a, Fbxw11, Tnfaip3, Herc3, Herc2, Hdac4, Traf6, Ankib1, Mib1, Pja2, Ubr3, Thbs1, Smad3, Acvr2a, Rgmb, Tgfb2, and Mstn is decreased compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. In any one of the aforementioned methods, the method is unrelated to increasing proliferation of muscle stem cells (MuSCs) in the subject. In any one of the preceding methods, the administering step comprises administering once daily, twice daily, once weekly, or once monthly.

[0012] In yet another aspect, a method for rejuvenating aged non-skeletal muscle tissue of a subject is provided, the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH level, thereby rejuvenating the aged non-skeletal muscle tissue. Optionally, administration increases the level of PGE2 in the aged non-skeletal muscle tissue of the subject. Optionally, the level of PGE2 in the aged non-skeletal muscle tissue increases compared to the aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor. Optionally, the level of PGE2 in the aged non-skeletal muscle tissue increases by at least 10% compared to the aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor. Optionally, the level of PGE2 in the aged non-skeletal muscle tissue increases to a level substantially equivalent to that present in young non-skeletal muscle tissue. Optionally, the level of PGE2 in the aged non-skeletal muscle tissue increases to a level within about 50% or less of that present in young non-skeletal muscle tissue. In some cases, the aging non-skeletal muscle tissue is selected from the group consisting of epidermal tissue, epithelial tissue, vascular tissue, cardiac muscle, brain, bone, cartilage, sensory organs, kidney, thyroid gland, lung, smooth muscle, brown fat, spleen, liver, heart, small intestine, colon, skin, ovary and other reproductive tissue, hair, dental tissue, blood, cochlea, and any combination thereof.In some cases, the subject has one or more biomarkers of aging.In some cases, the one or more biomarkers of aging are selected from the group consisting of: increased 15-PGDH levels compared to young non-skeletal muscle tissue, decreased PGE2 levels compared to young non-skeletal muscle tissue, increased PGE2 metabolites compared to young non-skeletal muscle tissue, increased or more accumulated senescent cells compared to young non-skeletal muscle tissue, increased expression of one or more muscle atrophy-related genes compared to young non-skeletal muscle tissue, decreased mitochondrial biogenesis and / or mitochondrial function compared to young non-skeletal muscle tissue, and increased transforming growth factor pathway signaling compared to young non-skeletal muscle tissue. In some cases, aged non-skeletal muscle tissue has an increased accumulation of senescent cells compared to young non-skeletal muscle tissue. In some cases, the senescent cells express one or more markers of senescence.In some cases, senescent cells have increased levels of one or more senescence markers compared to non-senescent cells. In some cases, the one or more senescence markers are selected from the group consisting of p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6. In some cases, the senescent cells are macrophages. In some cases, the method further comprises administering a senolytic agent to aging non-skeletal muscle tissue. In some cases, the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a combination therapy of dasatinib and quercetin, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof. In some cases, the 15-PGDH inhibitor is selected from the group consisting of a small molecule compound, a blocking antibody, a nanobody, and a peptide. In some cases, the 15-PGDH inhibitor is SW033291. In some cases, the 15-PGDH inhibitor is selected from the group consisting of antisense oligonucleotides, microRNA, siRNA, and shRNA. In some cases, the subject is a human. In some cases, the subject is at least 30 years old. In some cases, the 15-PGDH inhibitor reduces or inhibits 15-PGDH expression. In some cases, the 15-PGDH inhibitor reduces or inhibits the enzymatic activity of 15-PGDH. In some cases, the function of aged non-skeletal muscle is enhanced compared to the function of aged non-skeletal muscle before administration of the 15-PGDH inhibitor. In some cases, the function of aged non-skeletal muscle tissue is enhanced by at least 10% compared to the function of aged non-skeletal muscle before administration of the 15-PGDH inhibitor. In some cases, the function of aged non-skeletal muscle tissue is enhanced to a level substantially equivalent to that present in young non-skeletal muscle tissue. In some cases, the function of aged non-skeletal muscle tissue is enhanced to a level within about 50% or less of that present in young non-skeletal muscle tissue. In some cases, the function includes increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof.In some cases, the method results in a reduction in the level of a PGE2 metabolite in aged non-skeletal muscle tissue compared to the aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young non-skeletal muscle. In some cases, the PGE2 metabolite is selected from the group consisting of 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2.

[0013] In yet another aspect, a method for enhancing the function of a subject's skeletal muscle is provided, the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the skeletal muscle, thereby enhancing the function of the subject's skeletal muscle, wherein the skeletal muscle is healthy and the method is unrelated to increasing the proliferation of the subject's muscle stem cells (MuSCs). Optionally, the skeletal muscle is undamaged. Optionally, the skeletal muscle has not undergone regeneration. Optionally, the skeletal muscle has not undergone significant or substantial exercise. Optionally, the function is enhanced compared to the skeletal muscle before administration of the 15-PGDH inhibitor. Optionally, the function is increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof. Optionally, the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, compared to the skeletal muscle before administration of the 15-PGDH inhibitor. Optionally, the skeletal muscle is young skeletal muscle. Optionally, the subject is under 30 years old. In some cases, the skeletal muscle is aged skeletal muscle. In some cases, the subject is over 30 years old.

[0014] In another aspect, the present disclosure provides a method for increasing muscle mass, strength, and / or endurance in aged and / or atrophied muscles of a subject, the method comprising administering to the subject a therapeutically effective amount of a 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor, wherein administration of the 15-PGDH inhibitor increases muscle fiber and / or myotube size in the aged and / or atrophied muscles of the subject.

[0015] In some embodiments, the subject has a condition or disease associated with muscle wasting selected from the group consisting of sarcopenia, diabetes, muscular dystrophy, sarcopenic obesity, neuropathy, cancer cachexia, HIV cachexia, muscle immobility, muscle disuse, frailty, and combinations thereof. In some embodiments, the subject is a human. In some embodiments, the human is over 30 years old (e.g., an adult with age-related sarcopenia). In some embodiments, the human is a child (e.g., a child with muscular dystrophy, such as Duchenne muscular dystrophy). In some embodiments, the method further comprises selecting the human for treatment with a 15-PGDH inhibitor based on its age.

[0016] In some embodiments, the method further comprises selecting a human for treatment with a 15-PGDH inhibitor based on a diagnosis of diabetes, frailty, muscular dystrophy, sarcopenic obesity, neuropathy, cancer cachexia or HIV cachexia, or muscle wasting due to immobility or disuse. In some embodiments, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0017] In some embodiments, the 15-PGDH inhibitor inactivates 15-PGDH or inhibits 15-PGDH activity (e.g., enzymatic activity). In some embodiments, the 15-PGDH inhibitor reduces the stability of 15-PGDH. In some embodiments, the 15-PGDH inhibitor is a small molecule compound, a blocking antibody, a nanobody, or a peptide. In some embodiments, the small molecule compound is SW033291. In some embodiments, the 15-PGDH inhibitor reduces or inhibits 15-PGDH expression. In some embodiments, the 15-PGDH inhibitor is an antisense oligonucleotide, microRNA, siRNA, or shRNA. In some embodiments, the 15-PGDH inhibitor is a modified RNA, such as a modified mRNA (mmRNA).

[0018] In some embodiments, the muscle is skeletal muscle. In some embodiments, the muscle has not been injured and / or has not undergone exercise and / or regeneration. In some embodiments, the inhibitor increases the size of myofibers and / or myotubes in the subject's aged and / or atrophied muscles, regardless of muscle injury, exercise, or regeneration. In some embodiments, a therapeutically effective amount of a 15-PGDH inhibitor increases the muscle mass or the cross-sectional area or diameter of myofibers and / or myotubes in the subject's aged and / or atrophied muscles. In some embodiments, a therapeutically effective amount of a 15-PGDH inhibitor increases muscle strength, muscle function, muscle mass, and / or muscle endurance, regardless of or without increasing the proliferation of the subject's muscle stem cells (MuSCs). In some embodiments, a therapeutically effective amount of a 15-PGDH inhibitor increases, elevates, or restores prostaglandin E2 (PGE2) levels in the subject's aged and / or atrophied muscles. In some embodiments, a therapeutically effective amount of a 15-PGDH inhibitor reduces PGE2 metabolite levels in aging and / or atrophying muscles of a subject.

[0019] In some embodiments, the PGE2 metabolite is 15-keto-PGE2 or 13,14-dihydro-15-keto-PGE2 (PGEM). In some embodiments, the administration of the 15-PGDH inhibitor comprises systemic administration or local administration. In some embodiments, aged and / or atrophied muscle has increased accumulation of senescent cells (e.g., compared to young muscle).

[0020] In some embodiments, the method further comprises administering to the subject a senolytic agent. In some embodiments, the senolytic agent is selected from the group consisting of a Bcl2 inhibitor (e.g., navitoclax (ABT-263), ABT-737), a pan-tyrosine kinase inhibitor (e.g., dasatinib), a flavonoid (e.g., quercetin), a peptide that interferes with FOXO4-p53 interaction (e.g., FOXO4-DRI), a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor (e.g., 17-DMAG), and combinations thereof.

[0021] In some embodiments, administration of a 15-PGDH inhibitor results in a decrease in the level or activity of atrogin 1 in aged and / or atrophied muscles of a subject. In some embodiments, administration of a 15-PGDH inhibitor results in an increase in EP4 activity in aged and / or atrophied muscles of a subject. In some embodiments, administration of a 15-PGDH inhibitor results in protection against muscle cell death, particularly protection of mature muscle cells.

[0022] The present disclosure provides, inter alia, compositions and methods for improving the health, function, and / or performance of non-skeletal muscle tissue in a subject with an age-related condition or disease by inhibiting 15-PGDH in the subject.

[0023] In one aspect, the present disclosure provides a method for increasing the function of non-skeletal muscle tissue of a subject having an age-related disorder, the method comprising administering to the subject a therapeutically effective amount of a 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitor, wherein administration of the 15-PGDH inhibitor increases or restores levels of PGE2 and / or PGD2 in the non-skeletal muscle tissue of the subject.

[0024] In some embodiments of the method, the age-related disorder is selected from the group consisting of cardiovascular disease, chronic respiratory disease, nutritional disease, renal disease, gastrointestinal or digestive disease, neurological disorder, sensory disorder, hearing disorder, skin or subcutaneous disease, cerebrovascular disease, osteoporosis, osteoarthritis, premature aging disease, and combinations thereof. In some embodiments, the cardiovascular disease is atrial fibrillation, stroke, ischemic heart disease, cardiomyopathy, endocarditis, intracerebral hemorrhage, hypertension, or a combination thereof. In some embodiments, the chronic respiratory disease is chronic obstructive pulmonary disease, asbestosis, silicosis, or a combination thereof. In some embodiments, the nutritional disease is trachoma, diarrheal disease, encephalitis, or a combination thereof. In some embodiments, the renal disease is chronic kidney disease. In some embodiments, the gastrointestinal or digestive disease is NASH, pancreatitis, ulcer, intestinal obstruction, or a combination thereof. In some embodiments, the neurological disorder is Alzheimer's disease, dementia, Parkinson's disease, or a combination thereof. In some embodiments, the sensory disorder is hearing loss, vision loss, loss of smell or taste, macular degeneration, retinitis pigmentosa, glaucoma, or a combination thereof. In some embodiments, the skin or subcutaneous disease is cellulitis, ulcers, fungal skin disease, pyoderma, or a combination thereof. In some embodiments, the premature aging disease is osteogenesis imperfecta, Bloom's syndrome, Cockayne's syndrome, Hutchinson-Gilford progeria syndrome, mandibuloacral dysplasia, progeria, progeria-like syndrome, Rothmund-Thomson syndrome, Seip syndrome, Werner syndrome, Down's syndrome, acroprogeria, Rothmund-Thomson syndrome, an immunodeficiency causing a premature aging syndrome, e.g., ataxia-telangiectasia, or an infectious disease causing premature aging, e.g., HIV.

[0025] In some embodiments of the method, the subject is a human. In some embodiments, the method further comprises selecting the human for treatment with a 15-PGDH inhibitor based on a diagnosis of age-related disorders. In some embodiments, the non-skeletal muscle tissue is selected from the group consisting of epidermis, epithelium, blood vessels, cardiac muscle, brain, bone, cartilage, sensory organs, kidney, thyroid, lung, smooth muscle, brown fat, spleen, liver, heart, brain, small intestine, colon, skin, ovary and other reproductive tissues, hair, dental tissue, cochlea, oligodendrocyte, and combinations thereof.

[0026] In some embodiments of the method, the 15-PGDH inhibitor inactivates 15-PGDH or blocks 15-PGDH activity. In some embodiments, the 15-PGDH inhibitor reduces or blocks the enzymatic activity of 15-PGDH. In some embodiments, the 15-PGDH inhibitor is a small molecule compound, a blocking antibody, a nanobody, or a peptide. In some embodiments, the small molecule compound is SW033291. In some embodiments, the 15-PGDH inhibitor reduces or blocks 15-PGDH expression. In some embodiments, the 15-PGDH inhibitor is an antisense oligonucleotide, microRNA, siRNA, or shRNA.

[0027] In some embodiments of the method, administration of a 15-PGDH inhibitor increases or restores PGE2 levels in the subject's non-skeletal muscle tissue. In some embodiments, a therapeutically effective amount of a 15-PGDH inhibitor reduces PGE2 metabolite levels and / or PGD2 metabolite levels in the subject's non-skeletal muscle tissue. In some embodiments, the PGE2 metabolite is 15-keto-PGE2 or 13,14-dihydro-15-keto-PGE2 (PGEM). In some embodiments, the PGD2 metabolite is 15-keto-PGD2 or 13,14-dihydro-15-keto-PGD2. In some embodiments, a therapeutically effective amount of a 15-PGDH inhibitor increases protein synthesis, increases cell proliferation, increases cell survival, lengthens telomeres, and / or reduces protein degradation in the subject's non-skeletal muscle tissue. In some embodiments, administration of a 15-PGDH inhibitor includes systemic administration. In some embodiments, administering the 15-PGDH inhibitor comprises local administration. In some embodiments, non-skeletal muscle tissue has increased accumulation of senescent cells (e.g., compared to young non-skeletal muscle tissue). In some embodiments, the method further comprises administering to the subject a senolytic agent. In some embodiments, the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof.

[0028] [The present invention 1001] A method for enhancing the function of aging skeletal muscle in a subject, comprising administering to the aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in one or more senescent cells within the aging skeletal muscle, thereby enhancing the function of the aging skeletal muscle. [The present invention 1002] A method for increasing muscle mass, strength, and / or endurance of aging skeletal muscle in a subject, comprising administering to the aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in one or more senescent cells within the aging skeletal muscle, thereby increasing muscle mass, strength, and / or endurance of the aging skeletal muscle. [The present invention 1003] A method for increasing the level of PGE2 in aged skeletal muscle of a subject, the method comprising the step of administering to the aged skeletal muscle an amount of a 15-PGDH inhibitor effective to increase the level of PGE2 in the aged skeletal muscle, thereby increasing the level of PGE2 in the aged skeletal muscle. [The present invention 1004] The method of any of claims 1001 to 1003, wherein said subject has one or more biomarkers of aging. [The present invention 1005] A method for rejuvenating aging skeletal muscle in a subject having one or more biomarkers of aging, comprising administering to the subject having one or more biomarkers of aging an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the subject, thereby rejuvenating the aging skeletal muscle. [The present invention 1006] The method of any one of claims 1004 to 1005, wherein the one or more biomarkers of aging are selected from the group consisting of an increased level of 15-PGDH compared to the level present in young skeletal muscle, a decreased level of PGE2 compared to the level present in young skeletal muscle, an increased level of PGE2 metabolites compared to the level present in young skeletal muscle, an increased or greater accumulation of senescent cells compared to the level present in young skeletal muscle, an increased expression of one or more muscle atrophy-related genes (atrogenes) compared to the level present in young skeletal muscle, a decreased mitochondrial biogenesis and / or mitochondrial function compared to the level present in young skeletal muscle, and an increased transforming growth factor pathway signaling compared to the level present in young skeletal muscle. [The present invention 1007] 1006. The method of claim 10, wherein said one or more muscle atrophy-related genes are selected from the group consisting of Atrogin 1 (MAFbx1), MuSA (Fbxo30), and Trim63 (MuRF1). [The present invention 1008] The method of claim 1006, wherein the increased transforming growth factor pathway signaling comprises increased expression of one or more genes selected from the group consisting of activin receptors, myostatin, SMAD proteins, and bone morphogenetic proteins. [The present invention 1009] The method of any one of claims 1001 to 1008, wherein the accumulation of senescent cells is increased in the aged skeletal muscle compared to young skeletal muscle. [The present invention 1010] 1009. The method of any of claims 1001, 1002, or 1009, wherein said senescent cells express one or more senescence markers. [The present invention 1011] 1010. The method of any of claims 1001, 1002, 1009, or 1010, wherein said senescent cells have increased levels of one or more markers of senescence compared to non-senescent cells. [The present invention 1012] 1012. The method of any one of claims 1010 to 1011, wherein said one or more senescence markers are selected from the group consisting of p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6. [The present invention 1013] The method of any one of claims 1001, 1002, or 1019 to 1012, wherein the senescent cells are macrophages. [The present invention 1014] The method of any of claims 1001 to 1013, wherein said aged skeletal muscle has not been damaged and / or has not undergone exercise and / or has not undergone regeneration. [The present invention 1015] The method of any one of claims 1001 to 1014, further comprising the step of administering a senescent cell removing agent to the aged skeletal muscle. [The present invention 1016] The method of claim 1015, wherein the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a combination therapy of dasatinib and quercetin, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof. [The present invention 1017] 1017. The method of any one of claims 1001 to 1016, wherein said 15-PGDH inhibitor is selected from the group consisting of a small molecule compound, a blocking antibody, a nanobody, and a peptide. [The present invention 1018] The method of any one of claims 1001 to 1017, wherein the 15-PGDH inhibitor is SW033291. [The present invention 1019] The method of any one of claims 1001 to 1016, wherein said 15-PGDH inhibitor is selected from the group consisting of antisense oligonucleotides, microRNA, siRNA, and shRNA. [The present invention 1020] The method of any one of claims 1001 to 1019, wherein the subject is a human. [The present invention 1021] 1021. The method of any one of claims 1001 to 1020, wherein the subject is at least 30 years old. [The present invention 1022] 1022. The method of any one of claims 1001 to 1021, wherein said administering comprises systemic administration or local administration. [The present invention 1023] The method of any of claims 1001 to 1022, wherein the level of PGE2 is increased in aged skeletal muscle compared to the level of PGE2 present in the aged skeletal muscle before administration of said 15-PGDH inhibitor. [The present invention 1024] The method of any of claims 1001 to 1023, wherein the level of PGE2 is increased by at least 10% compared to the level of PGE2 present in aged skeletal muscle before administration of said 15-PGDH inhibitor. [The present invention 1025] The method of any of claims 1001 to 1024, wherein the level of PGE2 is increased to a level substantially equivalent to that present in young skeletal muscle. [The present invention 1026] The method of any of claims 1001 to 1025, wherein the level of PGE2 is increased to a level that is within about 50% or less of the level present in young skeletal muscle. [The present invention 1027] The method of any of claims 1001 to 1026, which results in an increase in the cross-sectional area and / or diameter of muscle fibers and / or myotubes. [The present invention 1028] The method of any of claims 1001 to 1027, which results in an increase in the cross-sectional area and / or diameter of oxidative (type IIa) fibers and / or glycolytic (type IIb) fibers. [The present invention 1029] The method of any of claims 1001 to 1028, wherein said 15-PGDH inhibitor reduces or blocks 15-PGDH expression. [The present invention 1030] The method of any of claims 1001 to 1029, wherein said 15-PGDH inhibitor reduces or inhibits the enzymatic activity of 15-PGDH. [The present invention 1031] The method of any one of inventions 1001 to 1030, which results in an increase in muscle mass, muscle strength, muscle endurance, or any combination thereof, of said aged skeletal muscle. [The present invention 1032] Any of the methods of present inventions 1001 to 1031, which results in an increase in muscle mass, muscle strength, muscle endurance, or any combination thereof, in aged skeletal muscle compared to the aged skeletal muscle before administration of the 15-PGDH inhibitor. [The present invention 1033] Any of the methods of inventions 1001 to 1032, which results in an increase in muscle mass, muscle strength, muscle endurance, or any combination thereof, in said aged skeletal muscle to a level substantially equivalent to that present in young skeletal muscle. [The present invention 1034] Any of the methods of inventions 1001 to 1033, which results in an increase in muscle mass, muscle strength, muscle endurance, or any combination thereof, in said aged skeletal muscle to a level that is within about 50% or less of the level present in young skeletal muscle. [This invention 1035] The method of any one of claims 1001 to 1034, which results in the enhancement of the function of aged skeletal muscle. [The present invention 1036] The method of any of claims 1001 to 1035, which results in an enhancement of the function of aged skeletal muscle compared to the aged skeletal muscle before administration of said 15-PGDH inhibitor. [This invention 1037] 1037. The method of any of claims 1001 to 1036, which results in an enhancement of the function of said aged skeletal muscle to a level substantially equivalent to that present in young skeletal muscle. [The present invention 1038] 1038. The method of any of claims 1001 to 1037, which results in an enhancement of the function of said aged skeletal muscle to a level that is within about 50% or less of the level present in young skeletal muscle. [This invention 1039] 9. The method of any of claims 1035 to 1038, wherein said function is increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof. [The present invention 1040] Any of the methods of inventions 1001 to 1039, which result in a reduction in the level of PGE2 metabolites in aged skeletal muscle compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to the level present in young skeletal muscle. [This invention 1041] 1040. The method of claim 1040, wherein said PGE2 metabolite is selected from the group consisting of 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2. [The present invention 1042] The method of any one of claims 1001 to 1041, wherein the subject has sarcopenia caused by aging. [This invention 1043] Any of the methods of inventions 1001 to 1042, wherein the expression level of one or more muscle atrophy-related genes is reduced compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. [This invention 1044] Any of the methods of inventions 1001 to 1043, wherein the expression level of one or more components of a mitochondrial complex is increased compared to aged skeletal muscle before administration of said 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. [This invention 1045] 104. The method of claim 1044, wherein said one or more components of a mitochondrial complex are selected from the group consisting of Ndufal 1, Ndufal 2, Ndufal 3, Ndufa2, Ndufa3, Ndufa4, Ndufa5, Ndufal 10, Ndufb5, Ndufc1, Ndufs4, Ndufs8, Ndufv1, Ndufv2, Uqcrb, Uqcrc1, Uqcrh, Uqcrq, Ucqr10, Cox8b, Cox7a1, Cox7a2, Cox7b, Cox6c, Cox5a, Cox5b, Atp5f1, Atp5g1, Atp5h, Atp5j2, Atp5o, Atp5e, and Atp5k. [The present invention 1046] Any of the methods of claims 1001 to 1045, wherein the expression level of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (Pgc1α) is increased compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. [This invention 1047] Any of the methods of inventions 1001 to 1046, wherein the expression level of one or more genes selected from the group consisting of Tnfaip1, Klhdc8a, Fbxw11, Tnfaip3, Herc3, Herc2, Hdac4, Traf6, Ankib1, Mib1, Pja2, Ubr3, Thbs1, Smad3, Acvr2a, Rgmb, Tgfb2, and Mstn is reduced compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle. [This invention 1048] The method of any of claims 1001 to 1047, wherein the method is unrelated to increasing proliferation of muscle stem cells (MuSCs) in said subject. [This invention 1049] The method of any of claims 1001 to 1048, wherein said administering comprises once-daily, twice-daily, once-weekly, or once-monthly administration. [The present invention 1050] A method for rejuvenating aged non-skeletal muscle tissue in a subject, the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the subject, thereby rejuvenating the aged non-skeletal muscle tissue. [This invention 1051] 1050. The method of claim 1050, wherein said administering increases the level of PGE2 in aged non-skeletal muscle tissue of said subject. [This invention 1052] 1052. The method of any one of claims 1050 to 1051, wherein the level of PGE2 in aged non-skeletal muscle tissue is increased compared to aged non-skeletal muscle tissue before administration of said 15-PGDH inhibitor. [This invention 1053] 1053. The method of any of claims 1050 to 1052, wherein the level of PGE2 in aged non-skeletal muscle tissue is increased by at least 10% compared to the aged non-skeletal muscle tissue before administration of said 15-PGDH inhibitor. [This invention 1054] The method of any of claims 1050 to 1053, wherein the level of PGE2 in said aged non-skeletal muscle tissue is increased to a level substantially equivalent to that present in young non-skeletal muscle tissue. [This invention 1055] The method of any of claims 1050 to 1054, wherein the level of PGE2 in said aged non-skeletal muscle tissue is increased to a level that is within about 50% or less of the level present in young non-skeletal muscle tissue. [This invention 1056] 1056. The method of any of claims 1050 to 1055, wherein said aged non-skeletal muscle tissue is selected from the group consisting of epidermal tissue, epithelial tissue, vascular tissue, cardiac muscle, brain, bone, cartilage, sensory organs, kidney, thyroid, lung, smooth muscle, brown fat, spleen, liver, heart, small intestine, colon, skin, ovaries and other reproductive tissues, hair, dental tissue, blood, cochlea, and any combination thereof. [This invention 1057] The method of any of claims 1050 to 1056, wherein said subject has one or more biomarkers of aging. [This invention 1058] 1057. The method of claim 1057, wherein said one or more biomarkers of aging are selected from the group consisting of increased 15-PGDH levels compared to young non-skeletal muscle tissue, decreased PGE2 levels compared to young non-skeletal muscle tissue, increased PGE2 metabolites compared to young non-skeletal muscle tissue, increased or greater accumulation of senescent cells compared to young non-skeletal muscle tissue, increased expression of one or more muscle atrophy-associated genes compared to young non-skeletal muscle tissue, decreased mitochondrial biogenesis and / or mitochondrial function compared to young non-skeletal muscle tissue, and increased transforming growth factor pathway signaling compared to young non-skeletal muscle tissue. [This invention 1059] The method of any one of claims 1050 to 1058, wherein the accumulation of senescent cells is increased in said aged non-skeletal muscle tissue compared to young non-skeletal muscle tissue. [The present invention 1060] 1059. The method of any one of claims 1058 to 1059, wherein said senescent cells express one or more senescence markers. [This invention 1061] 1061. The method of any of claims 1058 to 1060, wherein the senescent cells have increased levels of one or more senescence markers compared to non-senescent cells. [This invention 1062] 1062. The method of any one of claims 1060 to 1061, wherein said one or more senescence markers are selected from the group consisting of p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6. [This invention 1063] The method of any one of claims 1060 to 1062, wherein the senescent cells are macrophages. [This invention 1064] The method of any of claims 1050 to 1063, further comprising the step of administering a senolytic agent to the aged non-skeletal muscle tissue. [This invention 1065] 1064. The method of claim 1064, wherein the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a combination therapy of dasatinib and quercetin, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof. [The present invention 1066] 1066. The method of any one of claims 1050 to 1065, wherein the 15-PGDH inhibitor is selected from the group consisting of a small molecule compound, a blocking antibody, a nanobody, and a peptide. [This invention 1067] The method of any one of claims 1050 to 1066, wherein the 15-PGDH inhibitor is SW033291. [The present invention 1068] 1066. The method of any one of claims 1050 to 1065, wherein the 15-PGDH inhibitor is selected from the group consisting of antisense oligonucleotides, microRNA, siRNA, and shRNA. [The present invention 1069] The method of any one of claims 1050 to 1068, wherein the subject is a human. [The present invention 1070] 1069. The method of any of claims 1050 to 1069, wherein the subject is at least 30 years old. [This invention 1071] The method of any one of claims 1050 to 1070, wherein the 15-PGDH inhibitor reduces or blocks 15-PGDH expression. [This invention 1072] The method of any one of claims 1050 to 1071, wherein the 15-PGDH inhibitor reduces or inhibits the enzymatic activity of 15-PGDH. [This invention 1073] The method of any one of claims 1050 to 1072, wherein the function of aged non-skeletal muscle is enhanced compared to the function of aged non-skeletal muscle before administration of the 15-PGDH inhibitor. [This invention 1074] The method of any of claims 1050 to 1073, wherein the function of aged non-skeletal muscle tissue is enhanced by at least 10% compared to the function of aged non-skeletal muscle before administration of said 15-PGDH inhibitor. [This invention 1075] 1075. The method of any of claims 1050 to 1074, wherein the function of said aged non-skeletal muscle tissue is enhanced to a level substantially equivalent to that present in young non-skeletal muscle tissue. [This invention 1076] 1076. The method of any of claims 1050 to 1075, wherein the function of said aged non-skeletal muscle tissue is enhanced to a level that is within about 50% or less of the level present in young non-skeletal muscle tissue. [This invention 1077] 1077. The method of any of claims 1073 to 1076, wherein said function comprises increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof. [This invention 1078] Any of the methods of claims 1050 to 1077, which result in a reduction in the level of PGE2 metabolites in aged non-skeletal muscle tissue compared to aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to the level present in young non-skeletal muscle. [This invention 1079] 1078. The method of claim 1078, wherein said PGE2 metabolite is selected from the group consisting of 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2. [The present invention 1080] 1. A method for enhancing skeletal muscle function in a subject, comprising: the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the skeletal muscle, thereby enhancing skeletal muscle function in the subject; wherein the skeletal muscle is healthy, and the method is independent of increasing proliferation of muscle stem cells (MuSCs) in the subject; The method. [This invention 1081] The method of claim 1080, wherein the skeletal muscle is undamaged. [This invention 1082] 1082. The method of any one of claims 1080 to 1081, wherein said skeletal muscle has not undergone regeneration. [This invention 1083] 1083. The method of any one of claims 1080 to 1082, wherein the skeletal muscle has not experienced significant or substantial exercise. [This invention 1084] The method of any one of claims 1080 to 1083, wherein the function is enhanced as compared with that of the skeletal muscle before administration of the 15-PGDH inhibitor. [This invention 1085] 108. The method of any of claims 1080 to 1084, wherein said function is increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof. [The present invention 1086] Any of the methods of inventions 1080 to 1085, which results in an increase in muscle mass, an increase in muscle strength, an increase in muscle endurance, or any combination thereof, compared to skeletal muscle before administration of the 15-PGDH inhibitor. [This invention 1087] The method of any one of claims 1080 to 1086, wherein the skeletal muscle is a young skeletal muscle. [This invention 1088] The method of claim 1087, wherein the subject is under 30 years of age. [This invention 1089] The method of any one of claims 1080 to 1086, wherein the skeletal muscle is an aged skeletal muscle. [The present invention 1090] The method of claim 1089, wherein the subject is over 30 years of age. Other objects, features and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description and figures. [Brief explanation of the drawings]

[0029] [Figure 1A] Figures 1A-1D. Decrease in muscle force and PGE2 levels in aging muscles. (Figure 1A) Plantar flexor tetanus torque in young (2 months, n = 9), middle-aged (18 months, n = 5), and aged (25 months, n = 5) male mice. (Figure 1B) PGE2 catabolic scheme. 13,14-dihydro-15-ketoPGE2 (PGEM). (Figure 1C) 15-PGDH specific enzyme activity assayed in muscle tissue from young (2 months) and aged (25 months) mice (n = 4 per age group). (Figure 1D) PGE2 and PGEM levels in muscle tissue lysates quantified by mass spectrometry (n = 14 mice for young and n = 8 for aged). *P < 0.05, **P < 0.001, ****P < 0.0001. ANOVA test with Bonferroni correction for multiple comparisons (Figure 1A and Figure 1D); Mann-Whitney test (Figure 1C). Mean ± s.e.m. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2] 15-PGDH, a component of senescent cells in aging tissue. Expression of 15-PGDH (Hpgd) in muscle tissue of 20-month-old C57Bl / 6 wild-type mice treated with vehicle (veh) or ABT-263 (ABT) over a 4-week alternating regimen and analyzed 2 months later (n=3 / condition for 2-month-old mice, n=4 / condition for 23-month-old mice). *P<0.05. ANOVA test with Bonferroni correction for multiple comparisons; mean+sem. Abbreviations: mo, months. [Figure 3A]Figures 3A-E. 15-PGDH inhibition improves muscle function in aging mice by increasing endogenous PGE2 levels. (Figure 3A) Aged mice were treated daily with the 15-PGDH inhibitor SW033291 (SW) or vehicle, and muscle function was measured at 1 month. Experimental scheme (top). Left to right: Muscle mass assessed as the weight of dissected gastrocnemius (GA) and tibialis anterior (TA) muscles. Muscle strength assessed as plantar flexion tetanic force (absolute values). Plantar flexion tetanic force (values ​​normalized to baseline). Muscle endurance assessed as time and distance to exhaustion. (Figure 3B) Representative TA cross-sections from aged muscles treated with vehicle or SW for 1 month. DAPI, blue; laminin, green. Bar = 50 μm. (Figure 3C) Myofiber cross-sectional area (CSA) in vehicle-treated and SW-treated aged GA mice (n = 4 / group). (Figure 3D) Mean CSA (n = 4 / group). (Figure 3E) PGE2 and PGEM levels in muscle tissue lysates quantified by mass spectrometry (n = 3 / group). *P < 0.05, **P < 0.001, ****P < 0.0001. Mann-Whitney test (Figures 3A and 3D). ANOVA test with Bonferroni correction for multiple comparisons (Figures 3C and 3E); mean ± s.e.m. Abbreviations: mo, months; i.p., intraperitoneal. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A]Figures 4A-4D. Knockdown of 15-PGDH by AAV9-delivered shRNA improves muscle function in aging mice. Intramuscular (im) injection of AAV9 carrying an shRNA construct against 15-PGDH (sh15PGDH) or a scrambled (scr) control into the GA. (Figure 4A) Experimental scheme. (Figure 4B) 15-PGDH expression levels in scr-infected and sh15PGDH-infected muscles and young controls (n = 5 / group). (Figure 4C) Weight of dissected gastrocnemius (GA) muscles. (Figure 4D) Plantar flexion tetanic force (absolute value). *P < 0.05. ANOVA test with Bonferroni correction for multiple comparisons (Figure 4B); Mann-Whitney test (Figures 4C and 4D). Mean ± s.e.m. Abbreviations: mo, months; im, intramuscular. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A] Figures 5A and 5B. 15-PGDH inhibition improves muscle function in a Duchenne muscular dystrophy mouse model. (Figure 5A) Expression of aging markers and 15-PGDH (Hpgd) in the GA muscle of Duchenne muscular dystrophy (DMD) mice (mdx4cv / mTRKO(G2)) and control (mTRKO(G2)) mice (n = 4 per genotype). (Figure 5B) DMD and control mice were treated daily with the 15-PGDH inhibitor SW033291 (SW) or vehicle, and muscle function was measured at 1 month. Experimental scheme (top). Plantar flexion tetanus force (values ​​normalized to vehicle treatment for each genotype, bottom). *P < 0.05, ****P < 0.0001. Mann-Whitney test (Figures 5A and 5B). Mean ± s.e.m. Abbreviations: mo, months; i.p., intraperitoneal. [Figure 5B] See legend to Figure 5A. [Figure 6A]Figures 6A-6F. PGE2 treatment of cultured myotubes results in the inhibition of muscle atrophy pathways. (Figure 6A) (Left) Expression levels of atrogin 1 in vehicle- and SW-treated aged muscles (n = 3 / condition); (Right) Expression levels of shscr- and sh15PGDH-treated aged muscles (n = 5 / condition). (Figure 6B) Expression levels of PGE2 receptors, EP1-4 (Ptger1-4), during the differentiation time course. (Figure 6C) Expression levels of Pax7 and Myh during the differentiation time course. (Figure 6D) Expression levels of the atrophy marker, atrogin 1 (left), and myotube diameter (center) in differentiated myotubes starved for 24 hours and simultaneously treated with vehicle, PGE2, or SW in the presence of the EP4 antagonist ONO-AE3-208. Representative images of myotubes exposed to PGE2 or vehicle after differentiation (right). Bar = 50 μm. (Figure 6E) Diameter and MYH-staining-positive area of ​​EP4fl / fl or EP4Δ / Δ myotubes. (Figure 6F) Schematic illustration of 15-PGDH regulation in aging and dystrophic mice. Rescue of muscle mass and strength loss in aging or DMD muscles can be achieved by using 15-PGDH inhibitors or senolytic agents to restore PGE2 levels, resulting in reduced levels of the downstream atrophy mediator atrogin-1, muscle hypertrophy, and increased muscle strength in treated DMD or aging mice. *P<0.05, **P<0.001, ***P<0.0005, ****P<0.0001. Mann-Whitney test (Figure 6A, Figure 6D left, and Figure 6E); ANOVA test with Bonferroni correction for multiple comparisons (Figure 6D right); mean ± s.e.m. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 7A]Figures 7A-7C. Mass spectrometry analysis of young and aged muscle for the detection of prostaglandins and PGE2 metabolites. (Figure 7A) Chemical structures, formulas, accurate masses, and molecular weights of the analyzed prostaglandins (PGE2, PGF2α, and PGD2) and PGE2 metabolites (15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2). Internal standards PGF2α-D9 and PGE2-D9 were added to both composite standards. (Figure 7B) Calibration curves for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS) analysis were generated by diluting the stock solutions to final concentrations ranging from 0.1 ng / ml to 500 ng / ml. The standard curve equations and correlation coefficients are shown for each standard. (Figure 7C) Representative chromatograms. Distinct peaks demonstrate excellent chromatographic resolution of the analyzed prostaglandins and their metabolites. cps: counts per second. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8A]Figures 8A-8P. Analysis of eicosanoid levels during aging revealed an increase in the PGE2-degrading enzyme 15-PGDH. (Figure 8A) Catabolic scheme of PGE2 and PGD2. (Figure 8B) PGE2, PGD2, PGF2a, and 13,14-dihydro-15-ketoPGE2 (PGEM) levels in muscle tissue lysates quantified by mass spectrometry (n = 12 mice for young mice, n = 8 for aged mice). (Figure 8C) Representative chromatograms of PGE2 and PGD2 levels analyzed by mass spectrometry in young (2-month-old, left) and aged (25-month-old, right) muscle tissues. (Figure 8D) 15-PGDH specific enzyme activity assayed in tissues from young (2-month-old) and aged (25-month-old) mice. Activity is expressed as the percentage change compared to young mice. (Figure 8E) 15-PGDH (Hpgd) RNAseq expression data from young (3 months) and aged (>24 months) mice (n = 4 and 6, respectively). TPM, transcripts per million. (Figure 8F) 15-PGDH immunoblots from muscle lysates from young (3 months) and aged (25 months) mice (n = 4 each). (Figures 8G-8P) Intramuscular (i.m.) injection of AAV9 carrying an shRNA construct against 15-PGDH (sh15PGDH) or a scrambled (scr) control into the GA of young (3 months) and aged (24 months) C57BL / 6 mice. (Figure 8G) Experimental scheme. (Figure 8H) 15-PGDH expression levels in scr- and sh15PGDH-infected muscles and young controls (n = 5 / group). (Figure 8I) 15-PGDH specific enzyme activity assayed in muscle tissue from scr-infected and sh15PGDH-infected aged muscles normalized to scr treatment (n = 5 mice / age group). (Figure 8J) PGE2, PGD2, and PGF2a levels in muscle tissue lysates quantified by mass spectrometry (n = 4 / group). (Figure 8K) Representative TA cross-sections from scr-infected and sh15PGDH-infected aged muscles. DAPI, blue; laminin, green. Bar = 50 μm. (Figure 8L) Myofiber cross-sectional area (CSA) in scr-infected and sh15PGDH-infected aged GA (n = 7 / group). (Figure 8M) Mean CSA. (Figure 8N) Weight of dissected TA. (Figure 8O) Weight of dissected GA. (Figure 8P) Plantar flexion tetanic force (absolute value).*P<0.05, **P<0.01, ****P<0.0001. ANOVA test with Bonferroni correction for multiple comparisons (Figures 8H and 8L-8P); multiple t-test (Figures 8B, 8D, and 8J); Mann-Whitney test (Figures 8E, 8F, and 8I). Mean ± s.e.m. Abbreviations: Spl. spleen; Mus. muscle; mo. months; im. intramuscular. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 8G] See legend to Figure 8A. [Figure 8H] See legend to Figure 8A. [Figure 8I] See legend to Figure 8A. [Figure 8J] See legend to Figure 8A. [Figure 8K] See legend to Figure 8A. [Figure 8L] See legend to Figure 8A. [Figure 8M] See legend to Figure 8A. [Figure 8N] See legend to Figure 8A. [Figure 8O] See legend to Figure 8A. [Figure 8P] See legend to Figure 8A. [Figure 9A]Figures 9A-9C. Mass spectrometry of young and aged muscle detects prostaglandins and PGE2 metabolites. (Figure 9A) Chemical structures, formulas, accurate masses, and molecular weights of the analyzed prostaglandins (PGE2, PGF2α, and PGD2), PGE2 metabolites (15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2), PGA2 and its metabolite, 13,14-dihydro-15-ketoPGA2, and the internal standards PGF2α-D9, PGE2-D4, and PGD2-D4. (Figure 9B) The PGE2 calibration curve was linear in the range of 0.05 to 500 ng / mL. The standard curve equation and correlation coefficient are shown. (Figure 9C) Representative chromatogram of the standard mixture showing the chromatographic separation of the analyzed prostaglandins and their metabolites. Analyte peak intensities are expressed as cps, counts per second. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10] Mass spectrometry of young and aged muscle. Representative chromatograms show the transition state of the metabolite PGE2 levels analyzed by mass spectrometry from young (2 month, left) and aged (25 month, right) muscle tissue. [Figure 11] 15-PGDH specific activity assay in young and aged tissues. Kinetic measurement of 15-PGDH specific activity in lysates prepared from young (gray) and aged (black) tissues. [Figure 12-1]Figures 12A-D. Transcriptome analysis of quadriceps muscle from young versus aged C57BL / 6 mice. (Figures 12A-D) RNA sequencing was performed on young (3 months) and aged (>24 months) mice (n=4, 6, respectively). (Figure 12A) Heatmap of Euclidean sample distances for young and aged samples after rlog transformation. (Figure 12B) Volcano plot of differentially expressed genes for young versus aged samples. (Figure 12C) Boxplot of TPM values ​​for prostaglandin E2 receptors (Ptger1-4). (Figure 12D) GO term and KEGG analysis of differentially up- and down-regulated genes from (Figure 12B). Abbreviations: mo., months; ns, not significant; TPM Transcripts Per Million. [Figure 12-2] See description of Figure 12-1. [Figure 12-3] See description of Figure 12-1. [Figure 12-4] See description of Figure 12-1. [Figure 13] 15-PGDH levels are elevated in aging muscle. 15-PGDH (Hpgd) microarray expression data obtained from aged human (78 ± 6 years) compared to young (25 ± 3 years) biopsies from vastus lateralis muscle (n = 21, 15, respectively) analyzed from publicly available data GSE25941 (Raue et al. 2012). *P < 0.0001. Mann-Whitney test. [Figure 14A] Figures 14A-C. AAV9-mediated knockdown of 15-PGDH. (Figure 14A) Mass spectrometry quantification of PGE2, PGD2, and PGF2a levels in muscle tissue of young sh15PGDH compared to shscr (n=4 / group). (Figure 14B) Representative images of TA cross sections from scr-infected and sh15PGDH-infected aged muscles. DAPI, blue; GFP, green; laminin, white. (Figure 14C) Plantar flexion tetanic force (compared to baseline). *P<0.05. Multiple t-test (Figure 14A), ANOVA test with Bonferroni correction for multiple comparisons (Figure 14C). Mean + s.e.m. Abbreviations: TA: tibialis anterior; scr: scrambled; ns, not significant. [Figure 14B]See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15A] Figures 15A-15M. Small molecule 15-PGDH inhibition improves muscle function in aging mice by increasing endogenous PGE2 levels. (Figure 15A) Experimental scheme. Young (3-month-old) and aged (>24-month-old) mice were treated daily with the 15-PGDH inhibitor SW033291 (SW) or vehicle, and muscle function was measured at 1 month. (Figure 15B) 15-PGDH specific enzyme activity assayed in muscle tissue from vehicle-treated and SW-treated aged muscles normalized to vehicle treatment (n = 4 mice / age group). (Figure 15C) Eicosanoid levels in muscle tissue lysates quantified by mass spectrometry (n = 10 for young, n = 5 for aged veh, n = 7 for aged SW). (Figure 15D) Representative TA cross-sections from vehicle-treated or SW-treated aged muscles for 1 month. DAPI, blue; laminin, green. Bar = 50 μm. (Fig. 15E) Myofiber cross-sectional area (CSA) in vehicle-treated and SW-treated aged GA (n = 4 / group). (Fig. 15F) Mean CSA. (Fig. 15G) Representative TA cross-sections from 1-month vehicle-treated or SW-treated aged muscles stained for oxidative (MHC2a) and glycolytic (MHC2b) fibers. Laminin, blue; MHC2a, green, and MHC2b, red. Bar = 50 μm. (Fig. 15H) Mean CSA. (Fig. 15I) Cross-sectional area of ​​MHC2a. n = 4 / group. (Fig. 15J) Cross-sectional area of ​​MHC2b. n = 4 / group. (Fig. 15K) Weights of dissected gastrocnemius (GA), tibialis anterior (TA), and soleus muscles. (Fig. 15L) Plantar flexion tetanic force (absolute). (Fig. 15M) Time to exhaustion. *P<0.05, **P<0.01, ****P<0.0001. Mann-Whitney test (Figures 15B and 15H); ANOVA test with Bonferroni correction for multiple comparisons (Figures 15C, 15E, 15F, and 15J-15M). Mean ± s.e.m. Abbreviations: mo, months; i.p., intraperitoneal. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 15G] See legend to Figure 15A. [Figure 15H] See legend to Figure 15A. [Figure 15I] See legend to Figure 15A. [Figure 15J] See legend to Figure 15A. [Figure 15K] See legend to Figure 15A. [Figure 15L] See legend to Figure 15A. [Figure 15M] See legend to Figure 15A. [Figure 16A] Figures 16A-16C. Analysis of aged vehicle-treated and SW-treated muscles. (Figure 16A) Representative chromatograms show the transition state of metabolite PGE2 levels analyzed by mass spectrometry from aged vehicle-treated (left) and SW-treated (right) muscle tissue. (Figure 16B) Mass spectrometry quantification of PGE2, PGD2, and PGF2a levels in SW-treated muscle tissue compared to vehicle treatment (n=4 / group). (Figure 16C) Plantar flexion tetanic force (relative to baseline). **P<0.01. Multiple t-test (Figure 16B), ANOVA test with Bonferroni correction for multiple comparisons (Figure 16C). Abbreviations: ns, not significant. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 17A]Figures 17A-G. 15-PGDH is expressed by cells within the aging muscle microenvironment. (Figure 17A) Expression of 15-PGDH (Hpgd) in sorted macrophages (Cd11b+ / Cd11c- / F4 / 80+ / Cd31-), endothelial cells (Cd31+ / Cd11b- / Cd11c- / F4 / 80-), and myoblasts and stem cells (α7+ / Cd11b- / Cd45- / Cd31- / Sca1-) from young (2-month-old) and aged (25-month-old) hindlimb muscles. (Figure 17B) Expression of p16Ink4a and p21 in FACS-isolated young (2-month-old) and aged (25-month-old) macrophages (n = 3 and 5, respectively). (Figures 17C-17G) 12-month-old INK-ATTAC mice were treated with vehicle or AP20187 (AP) twice weekly for 16 months to remove senescent cells, and skeletal muscle tissue was analyzed at 28 months. (Figure 17C) Experimental scheme. (Figure 17D) Expression of 15-PGDH enzyme (Hpgd) in the quadriceps muscle of young (2-month-old) mice and aged (28-month-old) INK-ATTAC mice treated with vehicle (veh) or AP. n = 5 at 2 months, n = 6 at 28 months treated with veh or AP. (Figure 17E) Eicosanoid levels in muscle tissue lysates quantified by mass spectrometry (n = 10 for young, n = 3 for vehicle-treated, n = 3 for AP-treated). (Fig. 17F) Expression of 15-PGDH (Hpgd) in sorted macrophages and endothelial cells from adult (12 months) and aged INK-ATTAC (28 months) treated with vehicle (veh) or AP, obtained from hindlimb muscles. (Fig. 17G) Weight, grip strength, and treadmill endurance (right) of dissected gastrocnemius (GA) and tibialis anterior (TA) muscles (left) from adult (12 months) and aged INK-ATTAC (28 months) treated with vehicle (veh) or AP. n = 6, 7, and 15, respectively. *P < 0.05, ***P < 0.001, ****P < 0.0001. Multiple t-test (A), ANOVA test with Bonferroni correction for multiple comparisons (Fig. 17E-G). Mean ± sem. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 17E] See legend to Figure 17A. [Figure 17F] See legend to Figure 17A. [Figure 17G] See legend to Figure 17A. [Figure 18A] Figures 18A and 18B. Expression of senescence markers in sorted cells from young and aged mice. (Figure 18A) Sorting of macrophages (Cd11b+ / Cd11c- / F4 / 80+ / Cd31-) from young (3 months) and aged (24 months) mice. (Figure 18B) Expression of p16 and p21 in sorted endothelium (Cd31+ / Cd11b- / Cd11c- / F4 / 80-) from young (2 months) and aged (24 months) mice. (n = 5 mice per condition). *P < 0.05, ****P < 0.0001. Mann-Whitney test (Figure 18B). Mean + sem. Abbreviations: mo., months. [Figure 18B] See legend to Figure 18A. [Figure 19A]Figures 19A-19G. Characterization of INK-ATTAC mice and senescent-ablated aged mice. (Figure 19A) Expression of the indicated senescence markers in the quadriceps muscle of young (2-month-old) mice and aged (28-month-old) INK-ATTAC mice treated with vehicle (veh) or AP. n = 5 for young mice, n = 6 for aged mice treated with vehicle (veh) or AP. (Figure 19B) Representative chromatograms show the transient state of metabolite PGE2 levels analyzed by mass spectrometry from aged INK-ATTAC vehicle-treated (left) muscle tissue and aged INK-ATTAC AP-treated (right) muscle tissue. (Figure 19C) Expression of p21 in sorted macrophages and endothelial cells from adult (12-month-old) INK-ATTAC mice and aged (28-month-old) INK-ATTAC mice treated with vehicle (veh) or AP. (n = 4 / condition). (Figures 19D-19G) 20-month-old C57Bl / 6 wild-type mice were treated with vehicle (veh) or ABT-263 (ABT) for 4 weeks in an alternating regimen and analyzed 2 months later. (Figure 19D) Schematic (top). Expression of aging markers in young or aged C57Bl / 6 wild-type (wt) mice treated with vehicle or ABT-263 (ABT) for 4 weeks in an alternating regimen (n = 3 / condition for young mice, n = 4 / condition for aged mice) (bottom). (Figure 19E) Representative TA cross-sections of young (2-month-old), aged ABT-treated, and aged vehicle-treated muscles (23-month-old). DAPI, blue; 15-PGDH, green; WGA, red. (Bar = 20 μm). (Figure 19F) Quantification of 15-PGDH+ immunostained cells in muscle tissue sections. Muscle cross sections (approximately 5,000-8,000 DAPI-positive cells / section) from n = 4 aged mice treated with ABT and n = 4 mice treated with vehicle control (Figure 19G). Expression of 15-PGDH (Hpgd) (n = 3 for young 2-month-old mice and n = 4 for aged 23-month-old mice / condition). *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA test with Bonferroni correction for multiple comparisons (Figure 19A, Figure 19C, Figure 19D left and Figure 19G). Mann-Whitney test (Figure 19F and Figure 19D right). Mean + s.e.m. Abbreviations: mo., months. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] See legend to Figure 19A. [Figure 19F] See legend to Figure 19A. [Figure 19G] See legend to Figure 19A. [Figure 20A]Figures 20A-20K. Overexpression of 15-PGDH induces muscle atrophy and is rescued by treatment with SW033291. (Figures 20A-20H) Intramuscular (im) injection of AAV9 carrying a CMV construct driving 15-PGDH expression or a control into the tibialis anterior (TA) muscle of young C57BL / 6 (4-month-old) mice. (Figure 20A) Experimental scheme. (Figure 20B) Expression of 15-PGDH (Hpgd) in scr-infected and 15-PGDH OE-infected young muscles (n=5 / group). (Figure 20C) PGE2, PGD2, PGF2a, and PGEM levels in muscle tissue lysates quantified by mass spectrometry (n=4 / group). (Figure 20D) Representative TA cross-sections 1 month after im injection. DAPI, blue; laminin, green. Bar = 50 μm. (Figure 20E) Myofiber cross-sectional area of ​​muscles injected with 15-PGDH overexpression vector and control (n = 3 / group). (Figure 20F) Weight of dissected tibialis anterior (TA) muscles. (Figure 20G) Plantar flexion tetanus force (absolute value). (Figure 20H) Expression levels of MuRF1 (Trim63), atrogin-1 (Fbxo32), p62, Lc3b, Atg4, and Atg6 measured by qPCR (n = 3). (Figures 20I-K) Intramuscular (im) injection of AAV9 carrying a CMV construct driving 15-PGDH expression or control into the TA of young C57BL / 6 (3-month-old) mice, combined with daily intraperitoneal (ip) treatment with the 15-PGDH inhibitor SW033291 (SW) or vehicle (n = 4 mice / group). (Figure 20I) Experimental scheme. (Figure 20J) Weight of dissected TA muscle. (Figure 20K) Plantar flexion tetanic force (absolute value). *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001. ANOVA test with Bonferroni correction for multiple comparisons (Figures 20J and 20K); multiple t-test (Figure 20C); Mann-Whitney test (Figures 20B and 20E-H). Mean ± sem. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 20E] See legend to Figure 20A. [Figure 20F] See legend to Figure 20A. [Figure 20G] See legend to Figure 20A. [Figure 20H] See legend to Figure 20A. [Figure 20I] See legend to Figure 20A. [Figure 20J] See legend to Figure 20A. [Figure 20K] See legend to Figure 20A. [Figure 21A] Figures 21A-21K. PGE2 mediates the beneficial effects of 15-PGDH inhibition. (Figures 21A-21G) Intramuscular (i.m.) injection of AAV9 carrying an shRNA construct against prostaglandin D2 synthase PTGDS (shPTGDS) or a scrambled (scr) control into the gastrocnemius (GA) of aged (>24 months) C57BL / 6 mice. (Figure 21A) Experimental scheme. (Figure 21B) Expression of Ptgds measured by qPCR (n=4 / group). (Figure 21C) PGD2 levels in muscle tissue lysates quantified by mass spectrometry (n=4 / group). (Figure 21D) Weight of dissected GA. (Figure 21E) Plantar flexion tetanic force (normalized to baseline). (Figure 21F) Plantar flexion tetanic force (absolute value). (Figure 21G) Distance to exhaustion on a treadmill. (Figures 21H-21K) EP4f / f mice or littermate controls (EP4+ / +) were intramuscularly (i.m.) injected with AAV9 carrying a construct for the MCK promoter driving Cre expression into the GA. Mice were then treated daily with the 15-PGDH inhibitor SW033291 (SW) or vehicle, and muscle function was measured at 1 month. (Figure 21H) Experimental scheme. (Figure 21I) Dissected GA weight. (Figure 21J) Plantar flexion tetanic force (normalized to baseline). (Figure 21K) Plantar flexion tetanic force (absolute value). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). ANOVA test with Bonferroni correction for multiple comparisons (Figures 21B, 21D-G, and 21I-K); Mann-Whitney test (Figure 21C). Mean ± sem. Abbreviations: mo. months; ip. intraperitoneal; im. intramuscular. [Figure 21B]See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 21D] See legend to Figure 21A. [Figure 21E] See legend to Figure 21A. [Figure 21F] See legend to Figure 21A. [Figure 21G] See legend to Figure 21A. [Figure 21H] See legend to Figure 21A. [Figure 21I] See legend to Figure 21A. [Figure 21J] See legend to Figure 21A. [Figure 21K] See legend to Figure 21A. [Figure 22] Expression of prostaglandin receptors in myotubes. Expression levels of PGE2 receptors, EP1-4 (Ptger1-4), PGD2 receptors (Ptgdr1-2), and PGF2a receptors (Ptgfr) in myotubes (differentiated myotubes at day 4). [Figure 23A] Figures 23A and 23B. PGE2 treatment leads to activation of CREB in muscle. (A) Immunoblot of muscle lysates from young (3 month old) C57BL / 6 mice injected im with PGE2 after 0, 30, or 60 minutes. (B) Quantification of immunoblots in (A). **P<0.01. ANOVA test with Bonferroni correction for multiple comparisons (B). Mean + sem. [Figure 23B] See legend to Figure 23A. [Figure 24A]Figures 24A-24I. 15-PGDH inhibition affects multiple pathways to improve muscle function. (Figures 24A-24C) RNA sequencing analysis of aged muscle mice treated daily with the 15-PGDH inhibitor SW033291 (SW) or vehicle, and muscle function was measured at 1 month (n=3 each). (Figure 24A) KEGG and GO term analysis of up-regulated (left) and down-regulated (right) genes. (Figure 24B) Heat map of mitochondrial genes identified in (Figure 24A). (Figure 24C) Expression levels of Pgc1a by qPCR (n=4 / group). (Figure 24D) Relative quantification of mitochondrial DNA to nuclear DNA (n=4 / group). (Figure 24E) Heat map of protein ubiquitin-related genes (top) and TGF-β signaling pathway (bottom) identified in (Figure 24A). (Figure 24F) Immunoblot of myotubes (MTs) differentiated from myogenic precursors derived from human muscle biopsies treated with PGE2 (10 ng / ml) for 0, 15, or 30 minutes. (Figure 24G) Immunoblot (top) and quantification (bottom) of muscle lysates from aged vehicle- and SW-treated mice (n = 4 each). (Figure 24H) Expression levels of MuRF1 (Trim63), atrogin-1 (Fbxo32), and myostatin (Mstn) in vehicle- and SW-treated mice measured by qPCR (n = 12 for aged veh, n = 8 for aged SW). (Figure 24I) Expression levels of MuRF1 (Trim63), atrogin-1 (Fbxo32), and myostatin (Mstn) in scr- and sh15PGDH-treated mice measured by qPCR (n = 5 for aged shscr, n = 4 for aged sh15PGDH). *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001. ANOVA test with Bonferroni correction for multiple comparisons (Figure 24C); Mann-Whitney test (Figure 24D and Figures 24G-I). Mean ± s.e.m. Abbreviations: KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology; BP: Biological Process; MF: Molecular Function; CC: Cellular Component. [Figure 24B] See legend to Figure 24A. [Figure 24C]See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 24E] See legend to Figure 24A. [Figure 24F] See legend to Figure 24A. [Figure 24G] See legend to Figure 24A. [Figure 24H] See legend to Figure 24A. [Figure 24I] See legend to Figure 24A. [Figure 25A] Figures 25A-25D. PGE2 treatment leads to increased protein synthesis in myotubes. (Figure 25A) Diameter of differentiated myotubes starved for 24 hours and simultaneously treated with vehicle, PGE2 (10 ng / ml), or SW (1 μM) in the presence of the EP4 antagonist ONO-AE3-208 (1 μM). (n=4 / condition) (Figure 25B) Representative images of starved myotubes treated as in (Figure 25A). DAPI, blue; MYH, red. Bar = 50 μm. (Figure 25C) Left: Diameter of differentiated myotubes treated for 4 days with vehicle or PGE2. Right: Representative images of differentiated myotubes treated for 4 days with vehicle or PGE2. DAPI, blue; myosin heavy chain (MYH), red. Bar = 50 μm. DM, differentiation medium. (Figure 25D) Left: Immunoblot of puromycin uptake into differentiated mouse myotubes treated daily (4 days) with PGE2 (10 ng / ml) or vehicle. Cycloheximide was added as a control during puromycin addition. Right: Loading control is shown as Ponceau S staining. ANOVA test (Figure 25A), Mann-Whitney test (Figure 25B) with Bonferroni correction for multiple comparisons. ***P<0.001, ****P<0.0001. Mean + sem. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 25D] See legend to Figure 25A. [Figure 26A]Figures 26A-D. Characterization of 15-PGDH inhibition or knockdown in aging muscle. (Figure 26A) Expression levels of atrophy markers in vehicle-treated and SW-treated aged muscles (n=8 and 5, respectively). (Figure 26B) Expression levels of autophagy markers in young (3-month-old) vehicle- and SW-treated aged muscles (n=4 for young, n=12 for aged veh, n=8 for aged SW). (Figure 26C) Expression levels of inflammatory and senescence markers in vehicle- and SW-treated aged muscles (n=3 / condition). (Figure 26D) Expression levels of inflammatory and senescence markers in shscr- and sh15PGDH AAV9-treated aged muscles (n=5 / condition). Mann-Whitney test (Figures 26A, 26C, and 26D), ANOVA test with Bonferroni correction for multiple comparisons (Figure 26B), *P<0.05, **P<0.01. Mean + sem. Abbreviations: ns, not significant. [Figure 26B] See legend to Figure 26A. [Figure 26C] See legend to Figure 26A. [Figure 26D] See legend to Figure 26A. [Figure 27A] Figures 27A and 27B. The PGE2-degrading enzyme 15-PGDH increases in aged tissues. (Figure 27A) Catabolic scheme of PGE2 and PGD2. (Figure 27B) 15-PGDH specific enzyme activity assayed in tissues from young (2-month-old) and aged (25-month-old) mice. Activity is expressed as percent change relative to young. *P<0.05, **P<0.001, ***P<0.0005. Multiple t-test (Figure 27B). Mean ± sem. Abbreviations: Spl. Spleen; Mus. Muscle. [Figure 27B] See legend to Figure 27A. [Figure 28] 15-PGDH specific activity assay of young and aged tissues. Kinetic measurement of 15-PGDH specific activity in lysates prepared from young (gray) and aged (black) tissues. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description 1. Introduction The present disclosure is based in part on the discovery that loss of PGE2 signaling contributes to skeletal muscle wasting associated with muscle atrophy during aging and muscular dystrophy, and that PGE2 catabolism is dysregulated and has a detrimental effect on aging, dystrophic, or atrophic muscle tissue.In aging muscle tissue, PGE2 is detected at relatively low levels, a phenomenon that has not previously been associated with aging.Furthermore, the elevated levels of the PGE2-degrading enzyme 15-PGDH in aging or dystrophic muscle, due in part to the accumulation of senescent cells, result in a decrease in muscle tissue PGE2 levels.Therefore, the present disclosure provides compositions and methods based on the use of 15-PGDH activity as a therapeutic target in aging and / or dystrophic muscle, for example, to improve muscle atrophy and increase muscle mass, muscle function, and muscle strength.In particular, in aging and muscular dystrophy, reducing or inhibiting 15-PGDH (e.g., activity or level, e.g., mRNA and / or protein) can improve skeletal muscle function. In one embodiment, the methods provided herein include administering a 15-PGDH inhibitor to treat aging and / or dystrophic muscles. In some cases, the methods include increasing the level of PGE2 in aging, atrophy, or dystrophic muscles (e.g., by inhibiting the PGE2-degrading enzyme 15-PGDH).

[0031] For example, in the absence of injury, exercise or regeneration, the elevation, increase or restoration of PGE2 levels in aging, atrophy or dystrophic muscle can improve muscle wasting, thereby revealing the previously unrecognized role of PGE2 degrading enzyme 15-PGDH in muscle wasting diseases such as muscular dystrophy and aging.In particular, PGE2 can act on mature muscle fibers in homeostasis in the absence of injury.Therefore, 15-PGDH inhibitors (such as SW033291) can restore the PGE2 levels in aging, atrophy and / or dystrophic skeletal muscle, along with reducing the levels of inactive PGE2 metabolites such as PGEM.In some cases, the use of 15-PGDH inhibitors described herein can enhance or strengthen muscle mass, muscle strength, muscle exercise capacity and / or muscle function. The PGE2 signaling pathway can occur via the EP4 receptor in differentiated muscle cells and muscle fibers and directly regulate muscle mass through inhibition of atrogin-1 expression, a key mediator of muscle atrophy. 15-PGDH inhibition, achieved through local or systemic strategies, overcomes the detrimental effects of the microenvironment in aging, atrophic, and dystrophic muscles, resulting in robust increases in muscle mass, strength, and endurance in aging and dystrophic muscles.

[0032] The present disclosure is further based in part on the discovery that the PGE2-degrading enzyme 15-PGDH or its transcripts are elevated in a range of aging tissues, particularly non-skeletal muscle tissues.Therefore, 15-PGDH protein or transcripts can be used as a biomarker of aging in non-skeletal muscle tissues, such as in subjects with age-related disorders or age-related diseases.Furthermore, inhibiting 15-PGDH can reverse or delay the aging and aging-related processes of non-skeletal muscle tissues, thereby improving their function.Without being bound by the following theory, it is believed that elevated 15-PGDH levels in non-skeletal muscle tissues, such as the colon, brain, skin, spleen, or liver, of subjects with age-related pathologies or age-related diseases, lead to the degradation of PGE2 and / or PGD2 in these tissues, thereby reducing the levels of PGE2 and / or PGD2 and PGE2 and / or PGD2 signaling, which have adverse effects on tissue function, as manifested in aging. Therefore, the present disclosure provides compositions and methods based on the use of 15-PGDH activity as a therapeutic target in non-skeletal muscle tissues of subjects with age-related diseases or age-related pathologies.Inhibiting 15-PGDH in these tissues can restore or increase the levels of PGE2 and / or PGD2 in these tissues, improving their function, health, and / or physiological activity.Therefore, reducing 15-PGDH can improve quality of life and the outcome of age-related diseases.

[0033] A non-limiting list of non-skeletal muscle tissues that can be treated using the methods and compositions of the present invention includes, for example, epidermis, blood vessels, cardiac muscle, brain, bone, cartilage, smooth muscle, brown fat, spleen, liver, etc. Elevated 15-PGDH levels can occur in diseases of aging tissues, including cardiovascular diseases (e.g., atrial fibrillation, stroke, ischemic heart disease, cardiomyopathy, endocarditis, intracerebral hemorrhage), chronic respiratory diseases (e.g., chronic obstructive pulmonary disease, asbestosis, silicosis), nutritional diseases (trachoma, diarrheal diseases, encephalitis), renal diseases (e.g., chronic kidney disease), gastrointestinal and digestive diseases (e.g., NASH, pancreatitis, ulcers, intestinal obstruction), neurological disorders (e.g., Alzheimer's disease, dementia, Parkinson's disease), sensory disorders (e.g., hearing loss, macular degeneration, glaucoma), skin and subcutaneous diseases (e.g., cellulitis, ulcers, fungal skin diseases, pyoderma), osteoporosis, osteoarthritis, rheumatoid arthritis, etc. In addition, genetic disorders of these tissues that cause premature aging syndromes, such as Bloom's syndrome, Cockayne's syndrome, Hutchinson-Gilford progeria syndrome, mandibular dysplasia, progeria, progeria-like syndromes, Rothmund-Thomson syndrome, Seip syndrome, Werner syndrome, Down's syndrome, acroprogeria, and Rothmund-Thomson syndrome, as well as immune deficiencies of these tissues that cause premature aging syndromes, such as ataxia-telangiectasia, and infectious diseases of these tissues that cause premature aging syndromes, such as human immunodeficiency virus (HIV), may also benefit from 15-PGDH inhibition.

[0034] Treating non-skeletal muscle tissue with inhibitors of 15-PGDH may offer many advantages, such as the ability to localize treatment to specific cell types that express high levels of this enzyme (e.g., diseased or aging non-skeletal muscle tissue), the ability to restore endogenous levels of PGE2 and / or PGD2 to achieve physiological, "youthful" levels of PGE2 and / or PGD2, the ability to target non-skeletal muscle tissues (e.g., colon, skin, spleen) that have high senescent cell infiltration that is thought to have deleterious effects in aging and aging-related pathologies, and the possibility of targeting 15-PGDH with molecules with relatively long half-lives or by using gene therapy to provide sustained, systemic PGE2 and / or PGD2 effects.

[0035] 2. Overview The implementation of the methods disclosed herein uses routine techniques in the field of molecular biology. Basic textbooks that disclose the general methods used herein include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0036] For nucleic acids, sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and / or single-stranded RNA can be given in nucleotides. These are estimates obtained from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[0037] Non-commercially available oligonucleotides can be chemically synthesized, for example, using an automated synthesizer as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984) according to the solid-phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981). Oligonucleotide purification can be performed using any art-recognized strategy, such as native acrylamide gel electrophoresis or anion-exchange high-performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255:137-149 (1983).

[0038] 3. Definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0039] As used herein, the terms "a," "an," or "the" not only include aspects having one member, but also aspects having multiple members. For example, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, reference to an "agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0040] As used herein, the terms "about" and "approximately" generally refer to an acceptable degree of error in the measured quantity, given the nature or precision of the measurement. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to "about X" includes at least the value X, 0.8X, 0.81X, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X are specifically indicated. Thus, "about X" is intended to teach and provide a statement supporting a claim limitation of, for example, "0.98X."

[0041] An "age-related condition" or "age-related disease" refers to any disease, condition, or disorder that exhibits or potentially exhibits any sign or characteristic associated with aging or the passage of time in non-skeletal muscle tissue, including, for example, loss or decrease in tissue function, loss or decrease in tissue health, loss or decrease in one or more physiological activities of the tissue, decreased protein synthesis in cells of the tissue, increased protein degradation in cells of the tissue, decreased survival or viability of the tissue, decreased proliferation of cells in the tissue, shortened telomeres in cells of the tissue, mitochondrial dysfunction in cells of the tissue, increased presence of senescent cells in the tissue, decreased levels of PGE2 and / or PGD2 in the tissue, etc. The condition or disease may be the result of the natural aging process over time, the result of other factors, such as lifestyle factors or diseases, e.g., infectious diseases, or the result of a genetic condition that causes premature aging.

[0042] As used herein, "non-skeletal muscle" tissue refers to tissue other than skeletal muscle (e.g., pectoralis musculi complex, latissimus dorsi, teres major and subscapularis, brachioradialis, biceps, brachialis, pronator teres, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, flexor pollicis brevis, opponens pollicis, adductor pollicis brevis, flexor pollicis brevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteus maximus, gluteus medius, medial hamstring, gastrocnemius, lateral hamstring, quadriceps mechanism, adductor longus, adductor brevis, adductor magnus, gastrocnemius medial, gastrocnemius lateral). lateral, soleus, tibialis posterior, tibialis anterior, flexor digitorum longus, flexor digitorum brevis, flexor pollicis longus, extensor hallucis longus, eye muscles, pharyngeal muscles, sphincter muscles, hand muscles, arm muscles, foot muscles, leg muscles, chest muscles, stomach muscles, back muscles, gluteal muscles, shoulder muscles, and head and neck muscles), and can encompass organs containing multiple tissue types and specific cell types within an organ or tissue. For example, "non-skeletal muscle tissue" can include any of the following: epithelial tissue, nervous tissue, connective tissue, smooth muscle, cardiac muscle, epidermal tissue, vascular tissue, heart, kidney, brain, bone, cartilage, brown fat, spleen, liver, colon, sensory organs, thyroid gland, lung, blood, small intestine, dental tissue, ovary or other reproductive tissue or organs, hair, cochlea, oligodendrocytes, and combinations thereof.

[0043] " Sarcopenia " refers to the loss of muscle mass, muscle strength, and / or physical performance associated with aging.Sarcopenia is a progressive process that can occur at different rates in different individuals, and there is no minimum age for diagnosis.For example, when a person is, for example, at least 20 years old, at least 25 years old, at least 30 years old, at least 35 years old, at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old or older, for the purpose of the method provided herein, can be considered to have sarcopenia.

[0044] "Aging muscle" or "senescent muscle" refers to any muscle (e.g., skeletal muscle) that exhibits or potentially exhibits any signs or characteristics associated with aging or the passage of time in developed muscle, including, for example, loss of muscle mass or strength, decreased protein synthesis, accumulation of lipids within and outside of muscle cells, mitochondrial dysfunction, expression of muscle atrophy-related genes (e.g., atrogin 1, Murf, and MuSA), increased presence of senescent cells, increased levels of PGE2 metabolites (e.g., PGEM), etc. In some embodiments, aging muscle or senescent muscle refers to muscle in a subject with sarcopenia.

[0045] "Muscle atrophy" or "atrophied muscle" refers to any loss or wasting of muscle tissue for any reason, e.g., any amount of reduction in muscle size, muscle mass, or muscle function, for example, with respect to conditions such as sarcopenia, diabetes, muscular dystrophy, sarcopenic obesity, neuropathy, cancer or HIV cachexia, frailty, or muscle atrophy due to immobility or disuse.

[0046] The terms "prostaglandin E2," "PGE2," and "dinoprostone" refer to prostaglandins that can be synthesized from arachidonic acid via cyclooxygenase (COX) enzymes and terminal prostaglandin E synthase (PGES). PGE2 plays a role in many biological functions, including vasodilation, inflammation, and sleep / wake cycle regulation. Structural and functional information regarding PGE2 can be found, for example, under "dinoprostone" at PubChem: pubchem.ncbi.nlm.nih.gov / compound / Dinoprostone, the contents of which are incorporated herein by reference in their entirety.

[0047] The term "prostaglandin D2" or "PGD2" refers to a prostaglandin that can be synthesized from arachidonic acid via cyclooxygenase (COX) enzyme and PGD2 synthase (PTDS). PGD2 is a structural isomer of PGE2, and the 9-keto and 11-hydroxy groups on PGE2 are reversed on PGD2. PGD2 plays a role in many biological functions, including vasoconstriction, inflammation, regulation of body temperature during sleep, chemotaxis, and male sexual development. Structural and functional information about PGD2 can be found, for example, in the "Prostaglandin D2" section of PubChem: pubchem.ncbi.nlm.nih.gov / compound / 448457, the contents of which are incorporated herein by reference in their entirety.

[0048] "15-PGDH" (15-hydroxyprostaglandin dehydrogenase) is an enzyme involved in the inactivation of several active prostaglandins, for example, by catalyzing the oxidation of PGE2 to 15-keto-prostaglandin E2 (15-keto-PGE2) or PGD2 to 15-keto-prostaglandin D2 (15-keto-PGD2). The human enzyme is encoded by the HPGD gene (Gene ID: 3248). This enzyme is a member of the short-chain non-metalloenzyme alcohol dehydrogenase protein family. For example, in humans, multiple isoforms of this enzyme exist, any of which can be targeted using the present methods. For example, any of the human isoforms 1 to 6 (e.g., GenBank accession numbers NP_000851.2, NP_001139288.1, NP_001243236.1, NP_001243234.1, NP_001243235.1, NP_001350503.1, NP_001243230.1) can be targeted. The amino acid sequence of any of 139288.1, NP_001243236.1, NP_001243234.1, NP_001243235.1, NP_001350503.1, NP_001243230.1, or any isoform having 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity to the amino acid sequence of any other 15-PGDH enzyme can be targeted.

[0049] "15-PGDH inhibitor" refers to any agent that can inhibit, reduce, decrease, attenuate, eliminate, eliminate, delay, or counteract in any way any aspect of the expression, stability, or activity of 15-PGDH. A 15-PGDH inhibitor can, for example, reduce any aspect of expression, e.g., transcription, RNA processing, RNA stability, or translation, of a gene encoding 15-PGDH, e.g., the human HPGD gene, by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, e.g., in the absence of the inhibitor, in vitro or in vivo, compared to a control. Similarly, a 15-PGDH inhibitor can, for example, reduce the activity, e.g., enzymatic activity, of the 15-PGDH enzyme by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, compared to a control, e.g., in the absence of the inhibitor, in vitro or in vivo. Additionally, a 15-PGDH inhibitor can, for example, reduce the stability of the 15-PGDH enzyme by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to a control, e.g., in the absence of the inhibitor, in vitro or in vivo.A "15-PGDH inhibitor," also referred to herein as an "agent" or "compound," can be any molecule, either natural or synthetic, such as a peptide, protein, oligopeptide (e.g., about 5 to about 25 amino acids in length, e.g., about 5, about 10, about 15, about 20, or about 25 amino acids in length), a small molecule (e.g., an organic molecule having a molecular weight of less than about 2500 daltons, e.g., less than 2000 daltons, less than 1000 daltons, or less than 500 daltons), an antibody, a nanobody, a polysaccharide, a lipid, a fatty acid, an inhibitory RNA (e.g., siRNA, shRNA, microRNA), a modified RNA, a polynucleotide, an oligonucleotide, e.g., an antisense oligonucleotide, an aptamer, an affimer, a drug compound, or other compound.

[0050] A "senolytic agent" refers to any agent that can induce the death of senescent cells, e.g., that can induce the death of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of a population of senescent cells in vitro or in vivo. A non-limiting list of senolytic agents that can be used in the present methods includes Bcl2 inhibitors (e.g., navitoclax (ABT-263), ABT-737), pan-tyrosine kinase inhibitors (e.g., dasatinib), flavonoids (e.g., quercetin), peptides that interfere with FOXO4-p53 interaction (e.g., FOXO4-DRI), selective targeting systems for senescent cells using galactooligosaccharide-coated nanoparticles, HSP90 inhibitors (e.g., 17-DMAG), and combinations thereof. In certain embodiments, the senolytic agent can induce the death of, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more senescent cells, e.g., macrophages and / or fibroadipogenic progenitor (FAP) cells in aged and / or atrophied muscle, and / or macrophages and / or fibroadipocytes in non-skeletal muscle tissue.

[0051] The terms "expression" and "expressed" refer to the production of a transcription and / or translation product, e.g., the production of a nucleic acid sequence encoding a protein (e.g., 15-PGDH). In some embodiments, the terms refer to the production of a transcription and / or translation product encoded by a gene (e.g., the human HPGD gene) or a portion thereof. The expression level of a DNA molecule in a cell can be assessed based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell.

[0052] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The term includes antibody fragments and fusion products thereof that have the same antigen specificity.

[0053] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. Hence the terms "variable heavy chain," "V" and "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab, whereas the term "variable light chain," "V L" or "VL" refers to the variable region of an immunoglobulin light chain, including Fv, scFv, dsFv, or Fab. Equivalent molecules include antigen-binding proteins with the desired antigen specificity obtained, for example, by modifying antibody fragments or by selection from a phage display library.

[0054] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., 15-PGDH protein). Examples of antibody-binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains), F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region), single-chain Fvs (scFvs), disulfide-linked Fvs (dsFvs), complementarity-determining regions (CDRs), VL (light chain variable regions), VH (heavy chain variable regions), nanobodies, and any combination thereof, or any other functional portion of an immunoglobulin peptide that can bind to a target antigen (see, for example, Fundamental Immunology (Paul ed., 4th ed. 2001)).

[0055] The phrase "specifically binds" refers to a molecule (e.g., a 15-PGDH inhibitor, e.g., a small molecule or antibody) that binds to its target in a sample with higher affinity, avidity, more readily, and / or for a longer period than it binds to non-target compounds. In some embodiments, a molecule that specifically binds to a target (e.g., 15-PGDH) binds to the target with at least twice the affinity of the non-target compound, e.g., at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 25 times, at least 50 times, or more. For example, in some embodiments, a molecule that specifically binds to 15-PGDH typically binds to 15-PGDH with at least twice the affinity of a non-15-PGDH target.

[0056] In the context of chemical compounds, the term "derivative" includes, but is not limited to, amide, ether, ester, amino, carboxyl, acetyl, and / or alcohol derivatives of a given compound.

[0057] The term "treating" or "treatment" refers to any one of the following: ameliorating one or more symptoms of a disease or condition; preventing the onset of such symptoms before they occur; slowing or completely preventing the progression of a disease or condition (which may be manifested by an increase in the time between recurrent episodes, a delay in worsening of symptoms, or prevention of worsening of symptoms, etc.); enhancing the onset of periods of remission; slowing irreversible damage caused in the progression-chronic stages (both primary and secondary) of a disease or condition; delaying the onset of said progression stages; or any combination thereof.

[0058] The terms "administer," "administering," or "administration" refer to methods that can be used to deliver an agent or composition, such as the compounds described herein, to a desired biological site of action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial, intravascular, intracardiac, intrathecal, intranasal, intradermal, intravitreal, etc.), transmucosal injection, oral administration, administration as a suppository, and topical administration. Those skilled in the art will know additional methods for administering a therapeutically effective amount of the compounds described herein to prevent or alleviate one or more symptoms associated with a disease or condition.

[0059] The term "therapeutically effective amount" or "therapeutically effective dose" or "effective amount" refers to an amount of a compound (e.g., a 15-PGDH inhibitor) sufficient to produce a beneficial or desired clinical effect. The therapeutically effective amount or dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease or condition, stage of disease or condition, route of administration, type or extent of replacement therapy used, ongoing disease process, and type of treatment desired (e.g., conventional treatment versus aggressive treatment). The therapeutically effective amount of the pharmaceutical compounds or pharmaceutical compositions described herein can be initially estimated from cell culture and animal models. For example, IC determined by cell culture methods. 50 IC values ​​determined in animal models can serve as a starting point, but 50 The values ​​can be used to find a therapeutically effective dose in humans.

[0060] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound.

[0061] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, mice, rats, apes, humans, domestic or livestock for human consumption, such as pigs, cattle, and sheep, as well as sport animals and pets. Subjects also include vertebrates such as fish and poultry.

[0062] The term "acute regimen," in the context of compound administration, refers to the temporary or short-term application of a compound to a subject, e.g., a human subject, or the repeated application of a compound to a subject, e.g., a human subject, with a desired period (e.g., one day) elapsing between applications. In some embodiments, an acute regimen involves acute exposure of a compound to a subject (e.g., a single dose) over the course of treatment or over an extended period. In other embodiments, an acute regimen involves intermittent exposure of a compound to a subject (e.g., repeated administration), with a desired period elapsing between each exposure.

[0063] The term "chronic regimen," in the context of administration of a compound, refers to the repeated chronic application of a compound to a subject, e.g., a human subject, over an extended period of time, such that the amount or level of the compound remains substantially constant over a selected period of time. In some embodiments, a chronic regimen involves continuous exposure of a subject to a compound over an extended period of time.

[0064] An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct that has a set of specific nucleic acid elements that allow for transcription of a particular polynucleotide sequence in a host cell. An expression cassette can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette comprises a polynucleotide to be transcribed and operably linked to a promoter. The promoter can be a heterologous promoter. In the context of a promoter operably linked to a polynucleotide, a "heterologous promoter" refers to a promoter that is not operably linked to the same polynucleotide as found in the natural product (e.g., wild-type organism).

[0065] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. In certain embodiments, modified RNA molecules are used, such as mRNAs with specific chemical modifications that allow for increased stability and / or translation when introduced into cells, as described in more detail below. It is understood that any of the RNAs used in the present methods, including nucleic acid inhibitors such as siRNA or shRNA, can be used with chemical modifications, e.g., to enhance stability and / or efficacy, as described, for example, in Dar et al. (2016) Scientific Reports 6:article no. 20031 (2016) and presented in the database accessible at crdd.osdd.net / servers / sirnamod / .

[0066] " Polypeptide ", " peptide " and " protein " are used interchangeably herein to refer to polymers of amino acid residues.All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, and to natural amino acid polymers and unnatural amino acid polymers.As used herein, these terms encompass any length of amino acid chain, including full-length protein, in which amino acid residues are linked by covalent peptide bonds.

[0067] As used herein, the term "identical" or percent "identity," in the context of describing two or more polynucleotide or amino acid sequences, refers to two or more sequences or specified subsequences that are the same. Two sequences that are "substantially identical" have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity when compared and aligned for maximum correspondence over a comparison window or specified region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection where no specific region is specified. With respect to polynucleotide sequences, this definition also refers to the complement of a test sequence. With respect to amino acid sequences, identity optionally exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

[0068] In sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to the reference sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.For nucleic acid and protein sequence comparison, BLAST 2.0 algorithm and default parameters are used.

[0069] 4. Methods for enhancing muscle mass, muscle endurance, muscle strength, or muscle function in atrophied and / or aging muscles In one aspect, provided herein is a method for enhancing muscle function of aging skeletal muscle in a subject, the method comprising administering to the aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels (e.g., mRNA and / or protein levels) in senescent cells (e.g., present near or within the skeletal aging muscle, e.g., within the aging skeletal muscle microenvironment), thereby enhancing muscle function of the aging skeletal muscle.

[0070] In another aspect, provided herein is a method for increasing muscle mass, strength, and / or endurance of aging skeletal muscle of a subject, the method comprising administering to aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels (e.g., mRNA and / or protein levels) in senescent cells (e.g., present near or within the aging skeletal muscle, e.g., within the aging skeletal muscle microenvironment), thereby increasing muscle mass, strength, and / or endurance of the aging skeletal muscle.

[0071] In another aspect, a method for increasing the level of PGE2 in aging skeletal muscle of a subject is provided, the method comprising administering to skeletal aging muscle (e.g., having reduced levels of PGE2) an amount of a 15-PGDH inhibitor effective to increase the level of PGE2 in the aging skeletal muscle (e.g., by inhibiting 15-PGDH activity or by reducing 15-PGDH expression levels), thereby increasing the level of PGE2 in the aging skeletal muscle.

[0072] In another aspect, a method of rejuvenating aging skeletal muscle in a subject having one or more biomarkers of aging is provided, the method comprising administering to a subject having one or more biomarkers of aging a 15-PGDH inhibitor in an amount effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels (e.g., mRNA and / or protein levels) in the subject, thereby rejuvenating the aging skeletal muscle.

[0073] The methods provided herein can be used to enhance the function of aging skeletal muscle. The methods provided herein can be used to rejuvenate aging skeletal muscle. The methods provided herein can be used to increase muscle mass, muscle strength, muscle force, and / or muscle endurance of aging skeletal muscle.

[0074] In various aspects, aging skeletal muscle may have one or more senescent cells (e.g., present within or near skeletal muscle tissue). In some cases, aging skeletal muscle may have multiple senescent cells (e.g., present within or near skeletal muscle tissue). In some cases, aging skeletal muscle may have an increased accumulation of senescent cells (in or near skeletal muscle tissue) (e.g., compared to young skeletal muscle). In some cases, aging skeletal muscle may have a greater (e.g., substantially greater) number of senescent cells than typically found in young skeletal muscle. Senescent cells may express one or more senescent markers. Senescent cells may have increased levels of one or more senescent markers compared to non-senescent cells. The one or more senescent markers may be, but are not limited to, p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6. In various aspects, a subject can be selected for treatment (for example, by any method disclosed herein) based on the level of senescent cells present in skeletal muscle and / or the presence or level of one or more senescence markers.In some cases, the presence of senescent cells in skeletal muscle (for example, in a number greater than that typically found in young muscle), and / or the presence and / or level of one or more senescence markers, can indicate that treatment (for example, any of those disclosed herein) is likely to provide therapeutic benefit.In some cases, senescent cells can express 15-PGDH (for example, at a level effective for reducing the level of PGE2 in aged skeletal muscle).In some cases, senescent cells can be macrophages.

[0075] In various aspects, the subject may express one or more biomarkers of aging. Biomarkers of aging can include, but are not limited to, increased 15-PGDH levels (e.g., compared to levels present in young skeletal muscle), decreased PGE2 levels (e.g., compared to levels present in young skeletal muscle), increased PGE2 metabolites (e.g., compared to levels present in young skeletal muscle), increased or increased accumulation of senescent cells (e.g., compared to levels present in young skeletal muscle), increased expression of one or more muscle atrophy-related genes (e.g., atrogin 1 (MAFbx1), MuSA (Fbxo30), and Trim63 (MuRF1)) (e.g., compared to levels present in young skeletal muscle), decreased mitochondrial biogenesis and / or mitochondrial function (e.g., compared to levels present in young skeletal muscle), and increased transforming growth factor pathway signaling (e.g., increased expression of one or more genes involved in the transforming growth factor signaling pathway, e.g., activin receptors, myostatin, SMAD proteins, and one or more bone morphogenetic proteins) (e.g., compared to levels present in young skeletal muscle). In some cases, the biomarker of aging can include an increase in the level or activity of 15-PGDH (e.g., in aging skeletal muscle) (e.g., compared to the level present in young skeletal muscle). In some cases, the biomarker of aging can include a decrease in the level of PGE2 (e.g., in aging skeletal muscle) (e.g., compared to the level present in young skeletal muscle). In some cases, the biomarker of aging can include an increase in the level of PGE2 metabolites (e.g., 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2) (e.g., compared to the level present in young skeletal muscle). In some cases, the presence of the biomarker of aging can indicate that the subject can benefit from treatment by any method disclosed herein. In some cases, the subject is selected for treatment by the method disclosed herein (e.g., using a 15-PGDH inhibitor) based on the presence of one or more biomarkers of aging.

[0076] In various aspects, the level of PGE2 present in aging skeletal muscle can be increased (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein) compared to the level present in aging skeletal muscle before treatment (e.g., using a 15-PGDH inhibitor).The level of PGE2 present in aging skeletal muscle can be increased (e.g., by any method disclosed herein) by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more) compared to the level present in aging skeletal muscle before treatment (e.g., using a 15-PGDH inhibitor).In various aspects, the level of PGE2 present in aging skeletal muscle can be increased to a level substantially equivalent to the level present in young skeletal muscle (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein). PGE2 levels in aging skeletal muscle can be increased (e.g., by any method disclosed herein) to within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, or within about 1%) of levels present in young skeletal muscle.

[0077] In various aspects, the level of PGE2 metabolites present in aging skeletal muscle can be reduced (for example, after treatment with 15-PGDH inhibitors, for example, according to the methods provided herein) compared to the level present in aging skeletal muscle before treatment (for example, using 15-PGDH inhibitors).The level of PGE2 metabolites present in aging skeletal muscle can be reduced (for example, by any method disclosed herein) by at least 10% (for example, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more) compared to the level present in aging skeletal muscle before treatment (for example, using 15-PGDH inhibitors).In various aspects, the level of PGE2 metabolites present in aging skeletal muscle can be reduced to a level substantially equivalent to the level present in young skeletal muscle (for example, after treatment with 15-PGDH inhibitors, for example, according to the methods provided herein). The PGE2 metabolite levels in aged skeletal muscle can be reduced to within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, or within about 1%) of the levels present in young skeletal muscle (e.g., by any method disclosed herein). The PGE2 metabolite can be 15-ketoPGE2, 13,14-dihydro-15-ketoPGE2, or both.

[0078] In some cases, treatment (e.g., according to the methods provided herein, e.g., using a 15-PGDH inhibitor) can result in an increase in the cross-sectional area and / or diameter of muscle fibers and / or myotubes (e.g., compared to aged skeletal muscle before treatment and / or to a level substantially equivalent to the level of young skeletal muscle (or within 50% or less of the level of young skeletal muscle)). In some cases, treatment (e.g., according to the methods provided herein, e.g., using a 15-PGDH inhibitor) can result in an increase in the cross-sectional area and / or diameter of oxidative (type IIa) fibers and / or glycolytic (type IIb) fibers (e.g., compared to aged skeletal muscle before treatment and / or to a level substantially equivalent to the level of young skeletal muscle (or within about 50% or less of the level of young skeletal muscle)).

[0079] In some cases, treatment (e.g., according to the methods provided herein, e.g., using a 15-PGDH inhibitor) can result in a decrease in the expression level (e.g., in aged skeletal muscle) of one or more muscle atrophy-related genes selected from the group consisting of atrogin1 (MAFbx1), MuSA (Fbxo30), and Trim63 (MuRF1) (e.g., compared to aged skeletal muscle before treatment and / or to a level substantially equivalent to that of young skeletal muscle (or within about 50% or less of that of young skeletal muscle)). In some cases, treatment (e.g., according to the methods provided herein, e.g., using a 15-PGDH inhibitor) can result in an increase in the expression level (e.g., in aged skeletal muscle) of one or more components of a mitochondrial complex (e.g., compared to aged skeletal muscle before treatment and / or to a level substantially equivalent to that of young skeletal muscle (or within about 50% or less of that of young skeletal muscle)). The one or more components of the mitochondrial complex may be selected from the group consisting of Ndufal 1, Ndufal 2, Ndufal 3, Ndufa2, Ndufa3, Ndufa4, Ndufa5, Ndufal O, Ndufb5, Ndufc1, Ndufs4, Ndufs8, Ndufvl, Ndufv2, Uqcrb, Uqcrc1, Uqcrh, Uqcrq, Ucqr10, Cox8b, Cox7a1, Cox7a2, Cox7b, Cox6c, Cox5a, Cox5b, Atp5f1, Atp5g1, Atp5h, Atp5j2, Atp5o, Atp5e, and Atp5k. In some cases, treatment (e.g., according to the methods provided herein, e.g., with a 15-PGDH inhibitor) may result in an increase in the expression level of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (Pgc1α) (e.g., compared to aged skeletal muscle before treatment and / or to a level substantially equivalent to (or within about 50% or less of) the level of young skeletal muscle).In some cases, treatment (e.g., according to the methods provided herein, e.g., with a 15-PGDH inhibitor) may result in a decrease in the expression level of one or more genes selected from the group consisting of Tnfaip1, Klhdc8a, Fbxw11, Tnfaip3, Herc3, Herc2, Hdac4, Traf6, Ankib1, Mib1, Pja2, Ubr3, Thbs1, Smad3, Acvr2a, Rgmb, Tgfb2, and Mstn (e.g., compared to aged skeletal muscle before treatment and / or to a level substantially equivalent to that of young skeletal muscle (or within about 50% or less of that of young skeletal muscle)).

[0080] In various aspects, the muscle function of aged skeletal muscle can be enhanced (e.g., after treatment with a 15-PGDH inhibitor according to the methods provided herein, for example) compared to aged skeletal muscle before treatment (e.g., using a 15-PGDH inhibitor).The muscle function of aged skeletal muscle can be enhanced (e.g., by any method disclosed herein) by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more) compared to aged skeletal muscle before treatment (e.g., using a 15-PGDH inhibitor).In various aspects, the muscle function of aged skeletal muscle can be enhanced to a level substantially equivalent to that present in young skeletal muscle (e.g., after treatment with a 15-PGDH inhibitor according to the methods provided herein, for example). Muscle function of aged skeletal muscle can be enhanced (e.g., by any method disclosed herein) to levels within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, or within about 1%) of levels present in young skeletal muscle. Muscle function can include increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof.

[0081] In various aspects, the muscle mass, strength, and / or endurance of aged skeletal muscle can be increased (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein) compared to the aged skeletal muscle before treatment (e.g., using a 15-PGDH inhibitor).The muscle mass, strength, and / or endurance of aged skeletal muscle can be increased (e.g., by any method disclosed herein) by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more) compared to the aged skeletal muscle before treatment (e.g., using a 15-PGDH inhibitor).In various aspects, the muscle mass, strength, and / or endurance of aged skeletal muscle can be increased to a level substantially equivalent to that of young skeletal muscle (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein). The muscle mass, strength, and / or endurance of aged skeletal muscle can be increased (e.g., by any method disclosed herein) to levels that are within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, or within about 1%) of young skeletal muscle.

[0082] In a further aspect, the present disclosure provides a method for enhancing the function of a subject's skeletal muscle, the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in skeletal muscle, thereby enhancing the function of the subject's skeletal muscle. In some cases, the skeletal muscle is healthy skeletal muscle. In some cases, the skeletal muscle is not damaged, has not undergone or has not undergone regeneration, and / or has not undergone or has not undergone significant or substantial exercise. In some cases, the skeletal muscle is not dystrophic, atrophic, or aging. In some cases, the method is unrelated to increasing the proliferation of muscle stem cells in the subject. In some cases, the skeletal muscle is young skeletal muscle. In some cases, the subject is under 30 years old (e.g., 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year old). In various aspects, the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof (e.g., compared to skeletal muscle before treatment, e.g., with a 15-PGDH inhibitor). In various aspects, the method results in increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof (e.g., compared to skeletal muscle before treatment, e.g., with a 15-PGDH inhibitor).

[0083] The present disclosure further provides a method for increasing the function of aged and / or atrophied muscles in a subject, e.g., a human subject, comprising administering a 15-PGDH inhibitor to the subject. Administration of the 15-PGDH inhibitor can be systemic or local, e.g., via intramuscular injection, and can enhance any of several aspects of aged and / or atrophied muscles, including enhancing the subject's muscle mass, muscle function, muscle strength, muscle endurance, athletic performance, or any other measure of muscle function. In certain embodiments, administration of the 15-PGDH inhibitor results in an increase in the size of muscle fibers and / or myotubes in the subject's aged and / or atrophied muscles, e.g., an increase in their diameter or cross-section. In other embodiments, administration of the 15-PGDH inhibitor results in protection against muscle cell death, particularly of mature muscle cells, in the subject.

[0084] In certain embodiments, inhibiting 15-PGDH in a subject results in an increase in PGE2, for example, an increase, increase, or restoration of PGE2 levels in the subject's muscles, and a decrease in PGE2 metabolites, such as 15-keto-PGE2 or 13,14-dihydro-15-keto-PGE2 (PGEM). In some embodiments, inhibition also results in an increase in EP4 activity in the subject's atrophied and / or aging muscles. In some embodiments, inhibition also results in a decrease in the level or activity of atrogin 1 in the subject's atrophied and / or aging muscles.

[0085] In certain embodiments, the benefits described herein of administering 15-PGDH inhibitor, such as the enhancement of muscle strength, muscle mass, athletic performance, muscle endurance, muscle fiber or myotube size, etc., occur independently of the increase in the number or proliferation of muscle stem cells (MuSCs) in the atrophied muscle and / or aging muscle of the subject.In other words, although the number or proliferation of MuSCs can increase in the subject, the effects described herein do not require MuSCs and occur even if the number or proliferation of MuSCs does not increase.In certain embodiments, the aging muscle and / or atrophied muscle has not been injured and has never experienced exercise or regeneration.

[0086] In some embodiments, administration of a 15-PGDH inhibitor inhibits 15-PGDH activity or reduces 15-PGDH levels in senescent cells, such as macrophages and / or fibroadipogenic precursor (FAP) cells, within aging and / or atrophied muscles. In some embodiments, the method further comprises administering a senolytic agent to the subject. Examples of senolytic agents that can be used include, among others, Bcl2 inhibitors, such as navitoclax (also known as ABT-263) and ABT-737, flavonoids such as quercetin, pan-tyrosine kinase inhibitors, such as dasatinib, peptides that interfere with FOXO4-p53 interaction, such as FOXO4-DRI, selective targeting systems for senescent cells using galactooligosaccharide-coated nanoparticles, combination therapies including dasatinib and quercetin, and HSP90 inhibitors, such as 17-DMAG. It is understood that the senolytic agent can be administered together with the 15-PGDH inhibitor, for example, in a single pharmaceutical formulation, or separately.

[0087] subject The subject may be any subject, e.g., a human or other mammal, that has aging and / or atrophied skeletal muscle or is at risk of having aging and / or atrophied skeletal muscle. In some embodiments, the subject is a human. In some embodiments, the subject is an adult (e.g., an adult with age-related sarcopenia). In some embodiments, the subject is a child (e.g., a child with muscular dystrophy, e.g., Duchenne muscular dystrophy). In some embodiments, the subject is a female (e.g., an adult female). In some embodiments, the subject is a male (e.g., an adult male).

[0088] In some embodiments, the subject is a human, and the method further comprises selecting the human for treatment with a 15-PGDH inhibitor based on its age.For example, the human can be selected for treatment based on age being 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 years old or older, or any age at which the human has or may have sarcopenia or aging muscle.In some embodiments, the subject is determined to have aging muscle and / or atrophy muscle, as determined, for example, in a muscle biopsy taken from the subject by a physician or other qualified medical professional, using any method for assessing muscle strength or muscle function, such as grip strength testing, walking speed, muscle strength testing, functional testing, resistance testing, or treadmill, by imaging-based testing, by assessing muscle mass, and / or by molecular or cellular analysis.

[0089] In some embodiments, the subject has a condition or disease associated with muscle atrophy, such as diabetes, frailty, muscular dystrophy, sarcopenic obesity, neuropathy, cachexia, such as cancer cachexia or HIV cachexia, or muscle atrophy due to immobility or muscle disuse. In some embodiments, the subject has a muscular dystrophy selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy (MDD), and oculopharyngeal muscular dystrophy. In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0090] In certain embodiments, the muscle is a skeletal muscle. In some embodiments, the muscle is undamaged and / or has not undergone exercise or regeneration. Muscles include, but are not limited to, the pectoralis complex, latissimus dorsi, teres major and subscapularis, brachioradialis, biceps, brachialis, pronator teres, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, flexor pollicis brevis, opponens pollicis, adductor pollicis, flexor pollicis brevis, iliopsoas, psoas, rectus abdominis, rectus femoris, gluteus maximus, gluteus medius, medial flexor thigh, and gastrocnemius. The muscle may be any muscle of the body, including: lateral hamstrings, quadriceps mechanism, adductor longus, adductor brevis, adductor magnus, medial gastrocnemius, lateral gastrocnemius, soleus, tibialis posterior, tibialis anterior, flexor digitorum longus, flexor digitorum brevis, flexor pollicis longus, extensor hallucis longus, eye muscles, pharyngeal muscles, sphincter muscles, hand muscles, arm muscles, foot muscles, leg muscles, chest muscles, abdominal muscles, back muscles, gluteal muscles, shoulder muscles, head and neck muscles, etc.

[0091] In some embodiments, the subject is identified for treatment based on the diagnosis of muscle atrophy-related pathology or disease, based on the determination of the existence or possibility of muscle atrophy, based on the subject's age, for example, the age associated with sarcopenia or the age of possible sarcopenia, or based on the detection of any of the characteristics described herein of aging muscle and / or atrophied muscle.For example, the detection of high levels of PGE2 metabolites in muscle, such as 15-keto-PGE2 or PGEM, the detection of decreased protein synthesis in muscle, the detection of decreased size of muscle fiber and / or myotube, the detection of decreased muscle mass, the detection of decreased muscle strength, muscle function or muscle endurance, the detection of increased atrogin1 level or activity, the detection of decreased EP4 activity, the detection of increased expression of genes related to aging phenotype, for example, Ptges, Cox2, the detection of increased number of senescent cells, the detection of the presence of one or more senescent markers, particularly the detection of increased level or activity of 15-PGDH in senescent cells, for example, macrophages and / or fibroadipogenic progenitor cells, can indicate that the subject is a candidate for treatment with 15-PGDH inhibitors. In certain embodiments, such detection occurs when the muscle has never been injured and has never experienced exercise or regeneration.

[0092] Assessment of a subject's muscle function, strength, endurance, mass, or any of the characteristics described herein can be assessed using any of a wide variety of methods known to those of skill in the art, for example, by analysis of muscle function such as grip strength testing, walking speed, strength testing, functional testing, resistance testing, or treadmill testing on a muscle biopsy taken from the subject, by imaging-based testing, by assessment of muscle mass, and / or by molecular or cellular analysis.

[0093] In some embodiments, the subject is a livestock animal, e.g., a livestock animal intended for human consumption, e.g., a pig, cattle, sheep, poultry, or fish, and the method is used to enhance muscle mass, muscle function, or muscle strength, for example, in an aging animal, e.g., an animal with aging and / or atrophied muscles. In some such embodiments, the animal is administered a small molecule inhibitor of 15-PGDH. In some embodiments, a vector or expression cassette containing a nucleic acid inhibitor of 15-PGDH, e.g., an shRNA, is introduced into the animal so that the nucleic acid inhibitor is expressed in the animal's cells, e.g., muscle cells. In some embodiments, a vector or expression cassette containing a polynucleotide encoding a polypeptide inhibitor of 15-PGDH, e.g., an antibody or peptide, is introduced into the animal so that the polypeptide inhibitor is expressed in the animal's cells, e.g., muscle cells. In some embodiments, gene therapy is used, for example, to replace all or part of the endogenous 15-PGDH-encoding gene with a form of the gene that is less active, less stable, or not highly expressed in the animal's cells, e.g., muscle cells. In some embodiments, modified RNA, e.g., a chemically modified RNA inhibitor such as an shRNA, or a chemically modified mRNA encoding a polypeptide 15-PGDH inhibitor, is introduced into an animal so that the RNA inhibitor or expressed protein inhibitor is present in the muscle cells of the animal.

[0094] 5. Methods for enhancing tissue function in subjects with age-related pathologies In another aspect, a method is provided for rejuvenating aging non-skeletal muscle tissue in a subject, comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH, thereby rejuvenating the aging non-skeletal muscle tissue.

[0095] In various aspects, aged non-skeletal muscle tissue may have one or more senescent cells (e.g., present within or near aged tissue). In some cases, aged non-skeletal muscle tissue may have multiple senescent cells (e.g., present within or near aged tissue). In some cases, aged non-skeletal muscle tissue may have an increased accumulation of senescent cells (e.g., within or near aged non-skeletal muscle tissue) (e.g., compared to young non-skeletal muscle tissue). In some cases, aged non-skeletal muscle tissue may have a greater (e.g., substantially greater) number of senescent cells than the number typically found in young non-skeletal muscle tissue. Senescent cells may express one or more senescence markers. Senescent cells may have increased levels of one or more senescence markers compared to non-senescent cells. The one or more senescence markers may be, but are not limited to, p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6. In various aspects, a subject may be selected for treatment (e.g., by any method disclosed herein) based on the level of senescent cells present in aged non-skeletal muscle tissue and / or based on the presence or level of one or more senescence markers. In some cases, the presence of senescent cells in aged non-skeletal muscle tissue (e.g., in a number greater than that typically found in young non-skeletal muscle tissue) and / or the presence and / or level of one or more senescence markers may indicate that a treatment (e.g., any of those disclosed herein) is likely to provide therapeutic benefit. In some cases, senescent cells may express 15-PGDH (e.g., at a level effective to reduce the level of PGE2 in aged non-skeletal muscle tissue). In some cases, senescent cells may be macrophages.

[0096] In various aspects, the subject may express one or more biomarkers of aging. Biomarkers of aging can include, but are not limited to, increased 15-PGDH levels (e.g., compared to levels present in young non-skeletal muscle tissue), decreased PGE2 levels (e.g., compared to levels present in young non-skeletal muscle tissue), increased PGE2 metabolites (e.g., compared to levels present in young non-skeletal muscle tissue), increased or greater accumulation of senescent cells (e.g., compared to levels present in young non-skeletal muscle tissue), increased expression of one or more muscle atrophy-related genes (e.g., atrogin 1 (MAFbx1), MuSA (Fbxo30), and Trim63 (MuRF1)) (e.g., compared to levels present in young non-skeletal muscle tissue), decreased mitochondrial biogenesis and / or mitochondrial function (e.g., compared to levels present in young non-skeletal muscle tissue), and increased transforming growth factor pathway signaling (e.g., increased expression of one or more genes involved in the transforming growth factor signaling pathway, e.g., activin receptors, myostatin, SMAD proteins, and bone morphogenetic proteins) (e.g., compared to levels present in young non-skeletal muscle tissue). In some cases, the biomarker of aging can include an increase in the level or activity of 15-PGDH (e.g., in aged non-skeletal muscle tissue) (e.g., compared to the level present in young non-skeletal muscle tissue). In some cases, the biomarker of aging can include a decrease in the level of PGE2 (e.g., in aged non-skeletal muscle tissue) (e.g., compared to the level present in young non-skeletal muscle tissue). In some cases, the biomarker of aging can include an increase in the level of PGE2 metabolites (e.g., 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2 in aged non-skeletal muscle tissue) (e.g., compared to the level present in young non-skeletal muscle tissue). In some cases, the presence of the biomarker of aging can indicate that a subject is likely to benefit from treatment by any of the methods disclosed herein.Young non-skeletal muscle can include non-skeletal muscle from a subject under 30 years of age (e.g., 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year old).

[0097] In various aspects, the level of PGE2 present in aged non-skeletal muscle tissue can be increased (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein) compared to the level present in aged non-skeletal muscle tissue before treatment (e.g., using a 15-PGDH inhibitor).The level of PGE2 present in aged non-skeletal muscle tissue can be increased (e.g., by any method disclosed herein) by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or more) compared to the level present in aged non-skeletal muscle tissue before treatment (e.g., using a 15-PGDH inhibitor).In various aspects, the level of PGE2 present in aged non-skeletal muscle tissue can be increased to a level substantially equivalent to the level present in young non-skeletal muscle tissue (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein). PGE2 levels in aged non-skeletal muscle tissue can be increased (e.g., by any method disclosed herein) to levels within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, or within about 1%) of levels present in young non-skeletal muscle tissue.

[0098] In various aspects, the level of PGE2 metabolites present in aged non-skeletal muscle tissue can be reduced (for example, after treatment with a 15-PGDH inhibitor according to the methods provided herein) compared to the level present in aged non-skeletal muscle tissue before treatment (for example, using a 15-PGDH inhibitor).The level of PGE2 metabolites present in aged non-skeletal muscle tissue can be reduced (for example, by any method disclosed herein) by at least 10% (for example, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more) compared to the level present in aged non-skeletal muscle tissue before treatment (for example, using a 15-PGDH inhibitor).In various aspects, the level of PGE2 metabolites present in aged non-skeletal muscle tissue can be reduced to a level substantially equivalent to the level present in young non-skeletal muscle tissue (for example, after treatment with a 15-PGDH inhibitor according to the methods provided herein). The PGE2 metabolite level in aging non-skeletal muscle tissue can be reduced to within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, or within about 1%) of the level present in young non-skeletal muscle tissue (e.g., by any method disclosed herein).PGE2 metabolite can be 15-ketoPGE2, 13,14-dihydro-15-ketoPGE2, or both.PGE2 metabolite can be 15-ketoPGE2, 13,14-dihydro-15-ketoPGE2, or both.

[0099] In various aspects, the function of aged non-skeletal muscle tissue can be enhanced (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein) compared to aged non-skeletal muscle tissue before treatment (e.g., using a 15-PGDH inhibitor). The function of aged non-skeletal muscle tissue can be enhanced (e.g., by any method disclosed herein) by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or more) compared to the level present in aged non-skeletal muscle tissue before treatment (e.g., using a 15-PGDH inhibitor). In various aspects, the function of aged non-skeletal muscle tissue can be enhanced to a level substantially equivalent to the level present in young non-skeletal muscle tissue (e.g., after treatment with a 15-PGDH inhibitor, for example, according to the methods provided herein). The function of aged non-skeletal muscle tissue can be enhanced (e.g., by any method disclosed herein) to a level that is within about 50% or less (e.g., within about 40%, within about 35%, within about 30%, within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, within about 1%) of the level present in young non-skeletal muscle tissue. Function can include increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof.

[0100] In some cases, treatment (e.g., according to the methods provided herein, e.g., with a 15-PGDH inhibitor) may result in rejuvenation of aging non-skeletal muscle tissue (e.g., an increase in one or more functions of aging non-skeletal muscle tissue).

[0101] The present disclosure provides a method for enhancing the function, health, and other characteristics of non-skeletal muscle tissue in a subject, such as a human subject, with an age-related condition or disease, comprising administering a 15-PGDH inhibitor to the subject. Administration of the 15-PGDH inhibitor can be systemic or local and can enhance any aspect of the tissue, including enhancing function in any assay for assessing tissue function, physiological activity, endurance, performance, or any other measure of tissue function or health. In some embodiments, administration of the 15-PGDH inhibitor results in protection against cell death in the subject's non-skeletal muscle tissue. In some embodiments, administration of the 15-PGDH inhibitor results in reduced protein degradation in the subject's non-skeletal muscle tissue. In some embodiments, administration of the 15-PGDH inhibitor results in increased protein synthesis in the subject's non-skeletal muscle tissue. In some embodiments, administration of the 15-PGDH inhibitor can result in increased endurance (e.g., as measured during exercise, e.g., using a treadmill). In some cases, an increase in a subject's endurance (eg, during exercise) may be due to increased function and / or rejuvenation of aging non-skeletal muscle tissue (eg, heart, lungs, bone, etc.).

[0102] The present disclosure also provides a method for measuring 15-PGDH levels in non-skeletal muscle tissues of subjects with age-related pathologies.Such methods are useful, for example, for the use of 15-PGDH as a biomarker for aging or aging non-skeletal muscle tissues and / or loss or decline in the function of non-skeletal muscle tissues, for example, an increased level of 15-PGDH level or activity, for example, an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the control level of a subject without age-related pathologies, indicates aging of tissue or loss or decline in function.In such methods, 15-PGDH can be evaluated by any of several methods, for example, by detecting the level of transcripts encoding 15-PGDH protein, by detecting the level of 15-PGDH polypeptide, or by detecting 15-PGDH enzyme activity.

[0103] In certain embodiments, inhibition of 15-PGDH in a subject results in an increase in PGE2 and / or PGD2, e.g., an increase, increase, or restoration of PGE2 and / or PGD2 levels, and a decrease in PGE2 and / or PGD2 metabolites, such as 15-keto-PGE2, 13,14-dihydro-15-keto-PGE2 (PGEM), 15-keto-PGD2, and 13,14-dihydro-15-keto-PGD2, in the non-skeletal muscle tissue of the subject. In some embodiments, inhibition also results in increased signaling through PGE2 receptors, e.g., EP1, EP2, EP3, and / or EP4 (also known as Ptger1, Ptger2, Ptger3, and Ptger4), in the non-skeletal muscle tissue. In some embodiments, inhibition also results in increased signaling through PGD2 receptors, e.g., DP1 and / or DP2 (also known as PTGDR1, PTGDR2 / CRTH2).

[0104] In certain embodiments, the benefits described herein of administering a 15-PGDH inhibitor to non-skeletal muscle tissue, such as enhancing tissue health, function, physiological activity, etc., occur regardless of the regeneration of any of the target tissue.In other words, for example, if a tissue has been damaged or injured, the target tissue may regenerate, but the effects described herein do not require regeneration and occur even without regeneration.In certain embodiments, the non-skeletal muscle tissue has not been damaged or injured, and has not undergone or has not undergone regeneration.

[0105] In some embodiments, administration of a 15-PGDH inhibitor inhibits 15-PGDH activity or reduces 1 or 5-PGDH levels in senescent cells in non-skeletal muscle tissue of the subject, e.g., macrophages, fibroadipocytes, other mononuclear interstitial tissue resident cells including other immune cells, fibroblasts, endothelial cells, preadipocytes, and / or adipocytes. In some embodiments, the method further comprises administering to the subject a senolytic agent. Examples of senolytic agents that can be used include, among others, Bcl2 inhibitors such as navitoclax (also known as ABT-263) and ABT-737, pan-tyrosine kinase inhibitors such as dasatinib, peptides that interfere with FOXO4-p53 interaction such as FOXO4-DRI, selective targeting systems for senescent cells using galactooligosaccharide-coated nanoparticles, combination drug therapies including dasatinib and quercetin, and HSP90 inhibitors such as 17-DMAG, for example. It will be understood that the senolytic agent can be administered together with the 15-PGDH inhibitor, for example, in a single pharmaceutical formulation, or separately.

[0106] subject The subject can be any subject, e.g., a human or other mammal, having or at risk of having an age-related condition. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is a child (e.g., a child with progeria). In some embodiments, the subject is a female (e.g., an adult female). In some embodiments, the subject is a male (e.g., an adult male).

[0107] In some embodiments, the subject is a human, and the method further comprises selecting the human for treatment with a 15-PGDH inhibitor based on the diagnosis of an age-related condition or age-related disease, or based on the possibility or risk of developing an age-related condition or age-related disease.In some such embodiments, the human is selected based on its age.For example, the human can be selected for treatment based on the age of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 years old or older, or any age at which the human has or may have an age-related condition or age-related disease. In some embodiments, the human is selected based on the likelihood of an age-related condition or age-related disease based on the presence or potential presence of environmental, lifestyle, or medical factors associated with premature aging of one or more non-skeletal muscle tissues, e.g., smoking, alcohol use, diet, lack of physical activity, lack of sleep, drug use, exposure to ultraviolet light, exposure to extreme temperatures, stress, excess weight, or health-related factors, e.g., infectious disease, psychiatric illness, cancer, diabetes, etc. In some embodiments, the subject has an age-related condition caused by premature aging of one or more tissues, e.g., a genetic disorder, e.g., osteogenesis imperfecta, Bloom's syndrome, Cockayne's syndrome, Hutchinson-Gilford progeria syndrome, mandibular dysplasia, progeria, progeria-like syndrome, Rothmund-Thomson syndrome, Seip syndrome, Werner syndrome, Down syndrome, acroprogeria, Rothmund-Thomson syndrome, or premature aging syndrome, an immune deficiency in these tissues, e.g., ataxia-telangiectasia, or an infectious disease of these tissues, e.g., human immunodeficiency virus (HIV), causing a premature aging syndrome.

[0108] In some embodiments, a subject is determined to have aging tissue or to have age-related pathology or age-related disease, as determined by any method that evaluates the function, performance, health, strength, endurance, physiological activity, or any other characteristic of non-skeletal muscle tissue, such as performance-based assays, image-based assays, physiological assays, molecular assays, cellular assays, or functional assays.For example, the heart can be evaluated by any method that evaluates cardiac function or health, such as angiograms, electrocardiograms, treadmill tests, echocardiograms, etc.In some embodiments, a subject is selected for treatment based on the detection of increased levels of 15-PGDH transcripts, proteins, or enzyme activity in non-skeletal muscle-related tissues, or the detection of decreased levels of PGE2 and / or PGD2 in tissues.

[0109] In some embodiments, the method includes an additional step comprising assessing the health, function, performance, or any other characteristic of the subject's non-skeletal muscle tissue after administration of a 15-PGDH inhibitor, or assessing levels of 15-PGDH (e.g., 15-PGDH protein, transcript, or activity) and / or PGE2 and / or PGD2 in the subject's non-skeletal muscle tissue, e.g., to ascertain potential effects of prior administration of a 15-PGDH inhibitor on the tissue. In some such embodiments, the health, function, performance, 15-PGDH levels, PGE2 levels, PGD2 levels or other characteristic of the tissue is detected or examined and compared to the health, function, performance, 15-PGDH levels, PGE2 levels, PGD2 levels or other characteristic of the tissue before administration of the 15-PGDH inhibitor, or a control value, and a determination that the health, function or performance of the tissue is improved, or that 15-PGDH levels are reduced, or that PGE2 and / or PGD2 levels are increased in the tissue after administration of the inhibitor, compared to the value obtained before administration of the 15-PGDH inhibitor or the control value, indicates that the 15-PGDH inhibitor had a beneficial effect in the non-skeletal muscle tissue of the subject.

[0110] In some embodiments, the subject is suffering from an age-related condition, disorder, or disease, such as a cardiovascular disease or condition (e.g., atrial fibrillation, stroke, ischemic heart disease, cardiomyopathy, endocarditis, intracerebral hemorrhage, hypertension), a chronic respiratory disease or condition (e.g., chronic obstructive pulmonary disease, asbestosis, silicosis), a nutritional disease or condition (e.g., trachoma, diarrheal disease, encephalitis), a renal disease or condition (e.g., chronic kidney disease), a gastrointestinal disease or condition (e.g., NASH, pancreatitis, ulcer, intestinal obstruction), a neurological disorder (e.g., Alzheimer's disease, dementia, Parkinson's disease, cognitive decline), a sensory disorder (e.g., hearing loss, loss of vision, loss of smell or taste, macular degeneration, retinitis pigmentosa, glaucoma), or a skin disease. or subcutaneous disease or condition (e.g., cellulitis, ulcers, fungal skin disease, pyoderma), osteoporosis, osteoarthritis, rheumatoid arthritis, a genetic disorder causing premature aging in one or more non-skeletal muscle tissues (e.g., progeria, osteogenesis imperfecta, Bloom's syndrome, Cockayne's syndrome, Hutchinson-Gilford progeria syndrome, mandibular dysplasia, progeria-like syndrome, Rothmund-Thomson syndrome, Seip syndrome, Werner's syndrome, Down's syndrome, acroprogeria, Rothmund-Thomson syndrome), an immune deficiency of these tissues causing a premature aging syndrome (e.g., ataxia-telangiectasia), or an infectious disease of these tissues causing a premature aging syndrome (e.g., human immunodeficiency virus (HIV)).

[0111] Administration of a 15-PGDH inhibitor can result in improvement of any of these symptoms, for example, osteoporosis, hair loss, aging skin, cognitive impairment, sensory impairment, aging hematopoietic stem cell function, and gastrointestinal function.

[0112] The methods and compositions of the present invention can be used to treat any non-skeletal muscle tissue, or organ containing such tissue, or cells within such tissue, including epithelial tissue, nervous tissue, connective tissue, smooth muscle, cardiac muscle, epidermal tissue, vascular tissue, heart, kidney, brain, bone, cartilage, brown fat, spleen, liver, colon, sensory organs, thyroid, lung, blood, small intestine, dental tissue, ovary or other reproductive tissue, hair, cochlea, oligodendrocytes, etc.

[0113] In some embodiments, the subject is monitored for muscle weakness based on a diagnosis of an age-related condition, disorder, or disease, based on a determination of the presence or likelihood of age-related loss of function, health, or performance of non-skeletal muscle tissue, based on the age of the subject, e.g., an age associated with an age-related condition or disease, or based on detection of any of the characteristics described herein of aging non-skeletal muscle tissue, e.g., detection of elevated levels of PGE2 and / or PGD2 metabolites, e.g., 15-keto-PGE2, PGEM, 15-keto-PGD2, or 13,14-dihydro-15-PGD2, elevated levels of PGE2 and / or PGD2. senescent cells may be identified for treatment based on detecting decreased expression of 15-PGDH, e.g., decreased mitochondrial activity, decreased signaling through the EP1, EP2, EP3, EP4, DP1 and / or DP2 receptors, increased expression of genes associated with the senescent phenotype, e.g., p16 (Ink4a) or p21 (Cdkn1a), shortened telomere length in cells of the tissue, increased number of senescent cells in non-skeletal muscle tissue, or increased levels or activity of 15-PGDH, particularly in senescent cells, e.g., macrophages, fibroadipocytes, fibroblasts, endothelial cells, etc.

[0114] In some embodiments, the subject is a pet or livestock animal, such as a pig, cow, sheep, poultry, or fish, and the method is used, for example, to enhance the function or health of non-skeletal muscle tissues of aging animals. In some such embodiments, the animal is administered a small molecule inhibitor of 15-PGDH. In some embodiments, a vector or expression cassette containing a nucleic acid inhibitor of 15-PGDH, such as an shRNA, is introduced into the animal so that the nucleic acid inhibitor is expressed in the animal's cells, for example, in cells of non-skeletal muscle tissues. In some embodiments, a vector or expression cassette containing a polynucleotide encoding a polypeptide inhibitor of 15-PGDH, such as an antibody or peptide, is introduced into the animal so that the polypeptide inhibitor is expressed in the animal's cells, for example, in cells of non-skeletal muscle tissues. In some embodiments, gene therapy is used, for example, to replace all or part of the endogenous 15-PGDH-encoding gene with a form of the gene that is less active, less stable, or not highly expressed in the animal's cells, for example, in cells of non-skeletal muscle tissues. In some embodiments, modified RNA, e.g., a chemically modified RNA inhibitor such as an shRNA, or a chemically modified mRNA encoding a polypeptide 15-PGDH inhibitor, is introduced into an animal so that the RNA inhibitor or expressed protein inhibitor is present in the animal's cells.

[0115] 6. Assessment of 15-PGDH levels For example, when using 15-PGDH as a biomarker or when evaluating the effects of 15-PGDH inhibitors, the level of 15-PGDH in non-skeletal muscle tissue or skeletal muscle tissue can be assessed using any of several methods. For example, the level of 15-PGDH can be assessed by examining the transcription of a gene encoding 15-PGDH (e.g., the Hpgd gene), by examining the level of 15-PGDH protein in a tissue (e.g., skeletal muscle tissue or non-skeletal muscle tissue), or by measuring 15-PGDH enzyme activity in a tissue (e.g., skeletal muscle tissue or non-skeletal muscle tissue). Such methods can be performed on the entire tissue or on a subset of cells within the tissue, such as senescent cells.

[0116] In some embodiments, the method includes measuring 15-PGDH enzyme activity using standard methods, such as, for example, incubating a candidate compound in the presence of 15-PGDH enzyme, NAD(+), and PGE2 in an appropriate reaction buffer and monitoring the production of NADH (see, e.g., Zhang et al., (2015) Science 348:1224), or by using any of several available kits, such as the fluorometric PicoProbe 15-PGDH Activity Assay Kit (BioVision), or by using any of the methods and / or indicators described, for example, in publication EP 2838533.

[0117] In some embodiments, the methods involve detection of polynucleotide (e.g., mRNA) expression encoding 15-PGDH, which may be analyzed using routine techniques such as RT-PCR, real-time RT-PCR, semi-quantitative RT-PCR, quantitative polymerase chain reaction (qPCR), quantitative RT-PCR (qRT-PCR), multiplexed branched DNA (bDNA) assay, microarray hybridization, or sequence analysis (e.g., RNA sequencing ("RNA-Seq")). Methods for quantifying polynucleotide expression are described, for example, in Fassbinder-Orth, Integrative and Comparative Biology, 2014, 54: 396-406; Thellin et al., Biotechnology Advances, 2009, 27: 323-333; and Zheng et al., Clinical Chemistry, 2006, 52: 7 (doi: 10 / 1373 / clinchem. 2005. 065078). In some embodiments, real-time PCR or quantitative PCR or RT-PCR is used to measure the level of polynucleotide (e.g., mRNA) in biological samples. For example, see Nolan et al., Nat. Protoc, 2006, 1: 1559-1582; ​​Wong et al., BioTechniques, 2005, 39: 75-75. Additionally, quantitative PCR and RT-PCR assays for measuring gene expression are commercially available (eg, TaqMan® Gene Expression Assays, ThermoFisher Scientific).

[0118] In some embodiments, the method includes detecting the expression or stability of 15-PGDH protein using routine techniques known to those skilled in the art, such as immunoassays, two-dimensional gel electrophoresis, and quantitative mass spectrometry. Protein quantification techniques are generally described in "Strategies for Protein Quantitation," Principles of Proteomics, 2nd Edition, R. Twyman, ed., Garland Science, 2013. In some embodiments, protein expression or stability is detected by immunoassays, including, but not limited to, enzyme immunoassays (EIAs), such as enzyme-amplified immunoassay technology (EMIT), enzyme-linked immunosorbent assays (ELISAs), IgM antibody capture ELISAs (MAC ELISAs), and microparticle enzyme immunoassays (MEIAs); capillary electrophoresis immunoassays (CEIAs); radioimmunoassays (RIAs); immunoradiometric assays (IRMAs); immunofluorescence (IF); fluorescence polarization immunoassays (FPIAs); and chemiluminescence assays (CLs). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser-induced fluorescence (see, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-80 (1997)).

[0119] 7. 15-PGDH as a biomarker In some embodiments, 15-PGDH can be used as a biomarker for aging skeletal muscle and / or non-skeletal muscle tissue, or for the presence or possibility of age-related pathology or age-related disease.For example, detecting the increase in 15-PGDH level in skeletal muscle and / or non-skeletal muscle tissue, for example, in the whole tissue, or in specific cells in tissue, for example, in senescent cells, indicates the aging of tissue, the loss or decline of tissue function or health associated with aging, or the presence of age-related pathology or age-related disease.For example, detecting the increase in 15-PGDH of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more in skeletal muscle and / or non-skeletal muscle tissue compared with the control tissue from a subject without age-related pathology or age-related disease can indicate the aging of tissue, the loss or decline of tissue function or health associated with aging, or the presence of age-related pathology or age-related disease.

[0120] 8. 15-PGDH Inhibitors In the present method, any agent that reduces, decreases, counteracts, attenuates, inhibits, blocks, down-regulates, or eliminates the expression, stability, or activity, e.g., enzymatic activity, of 15-PGDH in any manner can be used. The inhibitor can be a small molecule compound, a peptide, a polypeptide, a nucleic acid, an antibody, e.g., a blocking antibody or a nanobody, or any other molecule that reduces, decreases, counteracts, attenuates, inhibits, blocks, down-regulates, or eliminates the expression, stability, and / or activity, e.g., the enzymatic activity of 15-PGDH in any manner.

[0121] In some embodiments, a 15-PGDH inhibitor reduces the activity, stability or expression of 15-PGDH by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more compared to control levels, e.g., in vivo or in vitro in the absence of the inhibitor.

[0122] The effectiveness of inhibitors can be assessed, for example, by measuring 15-PGDH enzyme activity using standard methods, such as incubating a candidate compound in the presence of 15-PGDH enzyme, NAD(+), and PGE2 in an appropriate reaction buffer and monitoring the production of NADH (see, e.g., Zhang et al., (2015) Science 348:1224), or by using any of several available kits, such as the fluorometric PicoProbe 15-PGDH Activity Assay Kit (BioVision), or by using any of the methods and / or indicators described, for example, in publication EP 2838533.

[0123] The effect of inhibitors can also be evaluated by detecting reduced polynucleotide (e.g., mRNA) expression, which can be analyzed using conventional techniques such as RT-PCR, real-time RT-PCR, semi-quantitative RT-PCR, quantitative polymerase chain reaction (qPCR), quantitative RT-PCR (qRT-PCR), multiplex branched DNA (bDNA) assay, microarray hybridization or sequence analysis (e.g., RNA sequencing ("RNA-Seq")).Methods for quantifying polynucleotide expression are described, for example, in Fassbinder-Orth, Integrative and Comparative Biology, 2014, 54: 396-406; Thellin et al., Biotechnology Advances, 2009, 27: 323-333; and Zheng et al., Clinical Chemistry, 2006, 52: 7 (doi: 10 / 1373 / clinchem. 2005. 065078). In some embodiments, real-time PCR or quantitative PCR or RT-PCR is used to measure the level of polynucleotide (e.g., mRNA) in biological samples.See, for example, Nolan et al., Nat. Protoc, 2006, 1:1559-1582; ​​Wong et al., BioTechniques, 2005, 39:75-75.In addition, quantitative PCR assays and RT-PCR assays for measuring gene expression are commercially available (e.g., TaqMan® Gene Expression Assay, ThermoFisher Scientific).

[0124] In some embodiments, a 15-PGDH inhibitor is considered effective when the level of expression of a polynucleotide encoding 15-PGDH is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more in vitro or in vivo compared to a reference value, e.g., the value in the absence of the inhibitor. In some embodiments, a 15-PGDH inhibitor is considered effective when the level of expression of a polynucleotide encoding 15-PGDH is reduced by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more compared to the reference value.

[0125] The effect of a 15-PGDH inhibitor can also be evaluated by detecting protein expression or stability using routine techniques known to those skilled in the art, such as immunoassays, two-dimensional gel electrophoresis, and quantitative mass spectrometry. Protein quantification techniques are generally described in "Strategies for Protein Quantitation," Principles of Proteomics, 2nd Edition, R. Twyman, ed., Garland Science, 2013. In some embodiments, protein expression or stability is detected by immunoassays, such as, but not limited to, enzyme immunoassays (EIAs), such as enzyme-amplified immunoassay technology (EMIT), enzyme-linked immunosorbent assays (ELISAs), IgM antibody capture ELISAs (MAC ELISAs), and microparticle enzyme immunoassays (MEIAs); capillary electrophoresis immunoassays (CEIAs); radioimmunoassays (RIAs); immunoradiometric assays (IRMAs); immunofluorescence (IF); fluorescence polarization immunoassays (FPIAs); and chemiluminescence assays (CLs). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser-induced fluorescence (see, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-80 (1997)).

[0126] To determine whether 15-PGDH protein levels are reduced in the presence of a 15-PGDH inhibitor, the method includes comparing the level of the protein (e.g., 15-PGDH protein) in the presence of the inhibitor with a reference value, e.g., the level in the absence of the inhibitor. In some embodiments, if the level of 15-PGDH protein is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to the reference value, the 15-PGDH protein is reduced in the presence of an inhibitor. In some embodiments, if the level of 15-PGDH protein is reduced by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more compared to the reference value, the 15-PGDH protein is reduced in the presence of an inhibitor.

[0127] small molecule In certain embodiments, 15-PGDH is inhibited by administering a small molecule inhibitor. Any small molecule inhibitor that reduces the expression, stability, or activity of 15-PGDH compared to a control, for example, the expression, stability, or activity in the absence of an inhibitor, by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more can be used. In certain embodiments, small molecule inhibitors that can reduce the enzymatic activity of 15-PGDH in vitro or in vivo can be used. Non-limiting examples of small molecule compounds that can be used in this method include the small molecules disclosed in EP2838533, the entire disclosure of which is incorporated herein by reference. Small molecules include, inter alia, small molecules disclosed in Table 2 of publication EP2838533, namely SW033291, SW033291 isomer B, SW033291 isomer A, SW033292, 413423, 980653, 405320, SW208078, SW208079, SW033290, SW208080, SW208081 , SW206976, SW206977, SW206978, SW206979, SW206980, SW206992, SW208064, SW208065, SW208066, SW208067, SW208068, SW208069, SW208070, and combinations, derivatives, isomers, or tautomers thereof. In certain embodiments, the 15-PGDH inhibitor used is SW033291 (2-(butylsulfinyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-amine; PubChem CID: 3337839).

[0128] In some embodiments, the 15-PGDH inhibitor is a thiazolidinedione derivative (e.g., a benzylidene thiazolidine-2,4-dione derivative), such as (5-(4-(2-(thiophen-2-yl)ethoxy)benzylidene)thiazolidine-2,4-dione), 5-(3-chloro-4-phenylethoxybenzylidene)thiazolidine-2,4-dione, 5-(4-(2-cyclohexylethoxy)benzylidene)thiazolidine-2,4-dione, 5-(3-chloro-4-(2-cyclohexylethoxy)benzyl)thiazolidine-2,4-dione, (Z)-N-benzyl-4-((2,4-dioxothiazolidin-5-ylidene)methyl)benzamide, or a derivative thereof, the entire disclosure of which is incorporated herein by reference. 21:4477-4484; Wu et al. (2010) Bioorg. Med. Chem. 18(2010)1428-1433; Wu et al. (2011) J. Med. Chem. 54:5260-5264; or Yu et al. (2019) Biotechnology and Bioprocess Engineering 24:464-475. In some embodiments, the 15-PGDH inhibitor is a COX inhibitor or chemopreventive agent, such as ciglitazone (CID:2750), or any of the compounds disclosed in Cho et al. (2002) Prostaglandins, Leukotrienes and Essential Fatty Acids 67(6):461-465, the entire disclosure of which is incorporated herein by reference.

[0129] In some embodiments, the 15-PGDH inhibitor is a compound containing a benzimidazole group, e.g., (1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperidin-1-yl)methanone (CID: 3474778), or a compound containing a triazole group, e.g., 3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine (CID: 71307851), or "Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandins" as published in Probe Reports by Duveau et al. (2015) (NIH Molecular Libraries Program [Internet]), the entire disclosure of which is incorporated herein by reference. dehydrogenase''). In some embodiments, the 15-PGDH inhibitor is 1-(3-methylphenyl)-1H-benzimidazol-5-yl)(piperidin-1-yl)methanone (CID: 4249877) or any of the compounds disclosed in Niesen et al. (2010) PLoS ONE 5(11):e13719, the entire disclosure of which is incorporated herein by reference.In some embodiments, the 15-PGDH inhibitor is 2-((6-bromo-4H-imidazo[4,5-b]pyridin-2-ylthio)methyl)benzonitrile (CID: 3245059), piperidin-1-yl(1-m-tolyl-1H-benzo[d]imidazol-5-yl)methanone (CID: 3243760), or 3-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine (CID: 2331284), or “Potent and selective inhibitors of NAD+-dependent 15-hydroxyprostaglandins” as published in Probe Reports by Jadhav et al. (2011) (NIH Molecular Libraries Program [Internet]), the entire disclosure of which is incorporated herein by reference. dehydrogenase (HPGD)'').

[0130] In some embodiments, the 15-PGDH inhibitor is TD88 or any of the compounds disclosed in Seo et al. (2015) Prostaglandins, Leukotrienes and Essential Fatty Acids 97:35-41 or Shao et al. (2015) Genes & Diseases 2(4):295-298, the entire disclosures of which are incorporated herein by reference. In some embodiments, the 15-PGDH inhibitor is EEAH (ethanol extract of jackfruit (Artocarpus heterophyllus)) or any of the compounds disclosed in Karna (2017) Pharmacogn Mag. 2017 Jan;13(Suppl 1):S122-S126, the entire disclosures of which are incorporated herein by reference.

[0131] inhibitory nucleic acid In some embodiments, the agent comprises an inhibitory nucleic acid, e.g., antisense DNA or antisense RNA, small interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA). In some embodiments, the inhibitory RNA is identical or substantially identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 100 contiguous nucleotides) to a target sequence within a 15-PGDH polynucleotide, e.g., a portion comprising 20-500, 20-250, 20-100, 50-500, or 50-250 contiguous nucleotides of a polynucleotide sequence encoding 15-PGDH. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to a sequence (e.g., the human HPGD gene, including any of its transcript variants as described in GenBank Accession Nos. NM_000860.6, NM_001145816.2, NM_001256301.1, NM_001256305.1, NM_001256306.1, NM_001256307.1, or NM_001363574.1, Gene ID: 3248).

[0132] In some embodiments, the method described herein comprises using shRNA or siRNA to treat a subject, for example, a subject with sarcopenia, aging muscle, or atrophy muscle, or a subject with age-related pathology, age-related disorder, or age-related disease.shRNA is an artificial RNA molecule with a hairpin turn, which can be used to silence target gene expression through the siRNA it produces in cells.See, for example, Fire et al., Nature 391:806-811, 1998; Elbashir et al., Nature 411:494-498, 2001; Chakraborty et al., Mol Ther Nucleic Acids 8:132-143, 2017; and Bouard et al., Br. J. Pharmacol. 157:153-165, 2009. In some embodiments, for example, a method for treating a subject with aging and / or atrophied muscles, or a subject with an age-related condition, disorder, or disease, comprises administering to the subject a therapeutically effective amount of a modified RNA or vector comprising a polynucleotide encoding an shRNA or siRNA capable of hybridizing to a portion of 15-PGDH mRNA (e.g., a portion of the polynucleotide sequence encoding human 15-PGDH set forth in any of GenBank Accession Nos. NM_000860.6, NM_001145816.2, NM_001256301.1, NM_001256305.1, NM_001256306.1, NM_001256307.1, or NM_001363574.1). In some embodiments, the vector further comprises appropriate expression control elements known in the art, including, for example, a promoter (e.g., an inducible promoter or a tissue-specific promoter), an enhancer, and a transcription terminator.

[0133] In some embodiments, the agent is 15-PGDH-specific microRNA (miRNA or miR). MicroRNA is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNA base pairs with the complementary sequence in mRNA transcript. As a result, mRNA transcript can be silenced by one or more of the following mechanisms: mRNA strand cleavage, mRNA destabilization due to shortening of its poly(A) tail, and reducing the efficiency of ribosome translation of mRNA transcript into protein.

[0134] In some embodiments, the agent can be an antisense oligonucleotide, such as an RNase H-dependent antisense oligonucleotide (ASO). ASOs are single-stranded chemically modified oligonucleotides that bind to complementary sequences in target mRNA and reduce gene expression by RNase H-mediated cleavage of the target RNA and inhibiting translation by steric blocking of ribosomes. In some embodiments, the oligonucleotide can hybridize to a portion of 15-PGDH mRNA (e.g., a portion of the polynucleotide sequence encoding human 15-PGDH described in any of GenBank accession numbers NM_000860.6, NM_001145816.2, NM_001256301.1, NM_001256305.1, NM_001256306.1, NM_001256307.1, or NM_001363574.1). In some embodiments, the oligonucleotide has a length of about 10-30 nucleotides (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides). In some embodiments, the oligonucleotide has 100% complementarity to the portion of the mRNA transcript to which it binds. In other embodiments, the DNA oligonucleotide has less than 100% complementarity (e.g., 95%, 90%, 85%, 80%, 75%, or 70% complementarity) to the portion of the mRNA transcript to which it binds, but is still able to form a stable RNA:DNA duplex for RNase H to cleave the mRNA transcript.

[0135] Suitable antisense molecules, siRNA, miRNA, and shRNA can be produced by standard methods of oligonucleotide synthesis, or by ordering such molecules from a contract research organization or supplier by providing the targeted polynucleotide sequence. The production and deployment of such antisense molecules in general terms can be found in modern reference textbooks, for example, Gene and Cell Therapy: Therapeutic Mechanisms and Strategies by N.S. Templeton, 4 th edition;Translating Gene Therapy to the Clinic: Techniques and Approaches,1 by J. Laurence and M. Franklin st High-Throughput RNAi Screening: Methods and Protocols (Methods in Molecular Biology) by D.O. Azorsa and S. Arora; and Oligonucleotide-Based Drugs and Therapeutics: Preclinical and Clinical Considerations by N. Ferrari and R. Segui.

[0136] Inhibitory nucleic acids can also include RNA aptamers, which are short synthetic oligonucleotide sequences that bind to proteins (see, for example, Li et al., Nuc. Acids Res. (2006), 34:6416-24). They are notable for both high affinity and specificity for target molecules, with the added advantage of being smaller than antibodies (usually less than 6 kD). RNA aptamers with the desired specificity are generally selected from combinatorial libraries and can be modified to reduce their vulnerability to ribonucleases using methods known in the art.

[0137] antibody In some embodiments, the agent is an anti-15-PGDH antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a blocking antibody (e.g., an antibody that binds to a target and directly interferes with the function of the target, such as 15-PGDH enzyme activity). In some embodiments, the antibody is a neutralizing antibody (e.g., an antibody that binds to a target and neutralizes the downstream cellular effects of the target). In some embodiments, the antibody binds to human 15-PGDH.

[0138] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an antigen-binding fragment, e.g., F(ab')2, Fab', Fab, scFv, etc. The term "antibody or antigen-binding fragment" can also encompass multispecific antibodies and hybrid antibodies having dual or multiple antigen or epitope specificities.

[0139] In some embodiments, the anti-15-PGDH antibody comprises a heavy chain sequence or a portion thereof and / or a light chain sequence or a portion thereof of the antibody sequence disclosed herein. In some embodiments, the anti-15-PGDH antibody comprises one or more complementarity determining regions (CDRs) of the anti-15-PGDH antibody disclosed herein. In some embodiments, the anti-15-PGDH antibody is a nanobody or single domain antibody (sdAb) comprising a single monomeric variable antibody domain, for example, a single VHH domain.

[0140] Many techniques known in the art can be used to prepare antibodies that bind to 15-PGDH.See, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed. 1986)).In some embodiments, antibodies are prepared by immunizing one or more animals (e.g., mice, rabbits, or rats) with an antigen to induce antibody responses. In some embodiments, the antigen is administered in conjugation with an adjuvant (e.g., Freund's adjuvant). In some embodiments, after the initial immunization, one or more subsequent booster injections of the antigen can be administered to improve antibody production. After immunization, antigen-specific B cells are harvested, for example, from the spleen and / or lymphoid tissue. To generate monoclonal antibodies, the B cells are fused with myeloma cells, which are then screened for antigen specificity.

[0141] Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells; for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can be produced from hybridomas or plasma cells. Furthermore, phage display technology or yeast display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992); Lou et al. m PEDS 23:311 (2010); and Chao et al., Nature Protocols, 1:755-768 (2006)). Alternatively, antibodies and antibody sequences can be isolated and / or identified using yeast-based antibody display systems, such as those disclosed in Xu et al., Protein Eng Des Sel, 2013, 26:663-670; WO 2009 / 036379; WO 2010 / 105256; and WO 2012 / 009568. Random combination of heavy chain and light chain gene products generates a large pool of antibodies with different antigen specificities (see, for example, Kuby, Immunology (3rd ed. 1997)). Techniques for producing single-chain antibodies or recombinant antibodies (U.S. Patent No. 4,946,778, U.S. Patent No. 4,816,567) can also be adapted to produce antibodies.

[0142] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell, such as a hybridoma, or a CHO cell. Many such systems are widely available from commercial suppliers. In embodiments in which an antibody includes both a VH region and a VL region, the VH region and the VL region can be expressed using a single vector, for example, in a dicistronic expression unit, or can be under the control of different promoters. In other embodiments, the VH region and the VL region can be expressed using separate vectors.

[0143] In some embodiments, the anti-15-PGDH antibody comprises one or more affinity-matured CDR sequences, heavy chain sequences, and / or light chain sequences. For chimeric antibodies, methods for producing chimeric antibodies are known in the art. For example, chimeric antibodies can be produced in which the antigen-binding region (heavy chain variable region and light chain variable region) from one species, such as mouse, is fused with the effector region (constant domain) from another species, such as human. As another example, "class-switched" chimeric antibodies can be produced in which the effector region of an antibody is replaced with the effector region of a different immunoglobulin class or subclass.

[0144] In some embodiments, the anti-15-PGDH antibody comprises one or more humanized CDR sequences, heavy chain sequences, and / or light chain sequences. Regarding humanized antibodies, methods for producing humanized antibodies are known in the art. See, for example, U.S. Patent No. 8,095,890. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. As an alternative to humanization, human antibodies can be produced. As a non-limiting example, transgenic animals (e.g., mice) can be produced that, upon immunization, produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that in chimeric and germline mutant mice, homozygous deletion of the antibody heavy-chain joining region (JH) gene results in complete inhibition of endogenous antibody production. In such germline mutant mice, transfer of the human germline immunoglobulin gene array results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immun., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807.

[0145] In some embodiments, antibody fragments (such as Fab, Fab', F(ab')2, scFv, nanobody, or diabody) are produced. Various techniques for producing antibody fragments have been developed, such as proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Meth., 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), and the use of recombinant host cells to produce the fragments. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab')2 fragments (see, e.g., Carter et al., BioTechnology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled artisan.

[0146] Methods for measuring binding affinity and binding kinetics are known in the art. These methods include, but are not limited to, solid-phase binding assays (e.g., ELISA assays), immunoprecipitation, surface plasmon resonance (e.g., Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA™), flow cytometry, fluorescence-activated cell sorting (FACS), biolayer interferometry (e.g., Octet™ (ForteBio, Inc., Menlo Park, CA)) and Western blot analysis.

[0147] peptide In some embodiments, the agent is a peptide, for example, a peptide that binds to 15-PGDH and / or inhibits the enzymatic activity or stability of 15-PGDH. In some embodiments, the agent is a peptide aptamer. Peptide aptamers are artificial proteins selected or engineered to bind to specific target molecules. Typically, peptides contain one or more peptide loops of variable sequence displayed by a protein scaffold. Peptide aptamers can be selected using various systems, including the yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies, such as mRNA display, ribosome display, bacterial display, and yeast display. See, for example, Reverdatto et al., 2015, Curr. Top. Med. Chem. 15:1082-1101.

[0148] In some embodiments, the agent is an affimer. Affimers are small, highly stable proteins, typically with a molecular weight of approximately 12-14 kDa, that bind to their target molecules with specificity and affinity similar to that of antibodies. Generally, affimers present two peptide loops and an N-terminal sequence that can be randomized to bind to various target proteins with high affinity and specificity in a manner similar to monoclonal antibodies. Stabilization of the two peptide loops by the protein backbone constrains the possible conformations that the peptide can adopt, thereby increasing binding affinity and specificity compared to libraries of free peptides. Affimers and methods for producing them have been described in the art. See, for example, Tiede et al., eLife, 2017, 6:e24903. Affimers are also commercially available, for example, from Avacta Life Sciences.

[0149] Vectors and modified RNA In some embodiments, a polynucleotide that exerts 15-PGDH inhibitory activity, for example, a nucleic acid inhibitor such as siRNA or shRNA, or a polynucleotide encoding a polypeptide that inhibits 15-PGDH, is introduced into cells, for example, muscle cells, non-skeletal muscle tissue cells, using a suitable vector. Examples of delivery vectors that can be used with the present disclosure include viral vectors, plasmids, exosomes, liposomes, bacterial vectors, or nanoparticles. In some embodiments, a polynucleotide encoding any of the 15-PGDH inhibitors described herein, for example, a nucleic acid inhibitor or a polypeptide inhibitor, is introduced into cells, for example, muscle cells, non-skeletal muscle tissue cells, using a vector such as a viral vector. Suitable viral vectors include, but are not limited to, adeno-associated viruses (AAV), adenoviruses, and lentiviruses. In some embodiments, a polynucleotide encoding a 15-PGDH inhibitor, for example, a nucleic acid inhibitor or a polypeptide inhibitor, is typically provided in the form of a recombinantly produced expression cassette, with a promoter operably linked to the polynucleotide sequence encoding the inhibitor. In some cases, the promoter is a universal promoter that directs gene expression in all or most tissue types. In other cases, the promoter is one that specifically directs gene expression in cells of the targeted tissue.

[0150] In some embodiments, the nucleic acid inhibitor or protein inhibitor of 15-PGDH is introduced into a subject using modified RNA, for example, into the subject's skeletal muscle or non-skeletal muscle tissue.It is known in the art that various modifications of RNA enhance the translation, efficacy and / or stability of RNA, for example, shRNA, or mRNA encoding 15-PGDH polypeptide inhibitors, when introduced into a subject's cells, for example.In certain embodiments, modified mRNA (mmRNA), for example, mmRNA encoding 15-PGDH polypeptide inhibitors, is used.In other embodiments, modified RNA, for example, siRNA, shRNA, or miRNA, comprising the RNA inhibitor of 15-PGDH expression is used. Non-limiting examples of RNA modifications that can be used include anti-reverse cap analogs (ARCAs), e.g., polyA tails of 100 to 250 nucleotides in length, replacement of AU-rich sequences in the 3'UTR with sequences derived from known stable mRNAs, and the inclusion of modified nucleosides and structures, such as pseudouridines, e.g., N1-methylpseudouridine, 2-thiolysine, 4'thioRNA, 5-methylcytidine, 6-methyladenosine, amide 3' linkages, thioate linkages, inosine, 2'-deoxyribonucleotides, 5-bromo-uridine, and 2'-O-methylated nucleosides. A non-limiting list of chemical modifications that can be used can be found, for example, in the online database crdd.osdd.net / servers / sirnamod / . RNA can be introduced into cells in vivo using any known method, including, inter alia, physical disruption, generation of RNA endocytosis by cationic carriers, electroporation, gene guns, ultrasound, nanoparticles, conjugates, or high-pressure injection. Modified RNA can also be introduced, for example, by direct injection into citrate-buffered saline. RNA can also be delivered using self-assembled lipoplexes or polyplexes, which are spontaneously generated by charge-charge interactions between negatively charged RNA and cationic lipids or polymers, such as lipoplexes, polyplexes, polycations, and dendrimers.Polymers such as poly-L-lysine, polyamidoamine and polyethyleneimine, chitosan and poly(β-aminoester) can also be used.See, for example, Youn et al. (2015) Expert Opin Biol Ther, Sep 2;15(9):1337-1348; Kaczmarek et al. (2017) Genome Medicine 9:60,; Gan et al. (2019) Nature comm. 10:871; Chien et al. (2015) Cold Spring Harb Perspect Med. 2015;5:a014035, the disclosures of each of which are incorporated herein by reference in their entirety.

[0151] 9. Administration Method The compound described herein can be administered to the subject locally or systemically.In some embodiments, compound can be administered, for example, intraperitoneally, intramuscularly, intraarterially, orally, intravenously, intracranially, intrathecally, intraspinal, intralesional, intranasally, subcutaneously, intracerebroventricularly, locally, and / or by inhalation.In one example, compound is administered intramuscularly, for example, by intramuscular injection.

[0152] In some embodiments, the compound is administered according to an acute regimen. In certain cases, the compound is administered to the subject once. In other cases, the compound is administered at one time point and then again at a second time point. In yet other examples, the compound is repeatedly administered to the subject (e.g., once a day or twice a day) as intermittent doses over a short period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or more). In some cases, the time between compound administrations is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, or more. In other embodiments, the compound is administered continuously or chronically according to a chronic regimen for a desired period of time. For example, the compound can be administered so that the amount or level of the compound is substantially constant over a selected period of time.

[0153] The administration of compound to a subject can be achieved by methods commonly used in the art.The amount of compound to be introduced can take into account factors such as sex, age, weight, type of disease or disorder, stage of disorder, and the amount required to produce desired results.Generally, when administering a compound for therapeutic purposes, cells are administered at a pharmacologically effective dose.A "pharmacologically effective amount" or "pharmacologically effective dose" is an amount sufficient to produce the desired physiological effect or physiological amount that can achieve the desired result, particularly for treating a pathology or disease, including reducing or eliminating one or more symptoms or manifestations of the pathology or disease.

[0154] The compounds described herein may be directly injected or otherwise administered to any number of muscles in the body, such as the biceps; triceps; brachioradialis; brachialis muscle (brachialis anticus); superficial compartment wrist flexor; deltoid; thigh flexors biceps femoris, gracilis, semitendinosus, and semimembranosus; quadriceps rectus femoris, vastus lateralis, vastus medialis, and vastus intermedius; gastrocnemius (lateral and medial), tibialis anterior, and soleus muscles in the calf; pectoralis major and pectoralis minor in the chest; latissimus dorsi in the upper back; rhomboid muscles (rhomboid major and rhomboid minor); trapezius muscles across the neck, shoulders, and back; rectus abdominis in the abdomen; gluteus maximus, gluteus medius, and gluteus minimus in the buttocks; muscles of the hand; sphincter muscles; eye muscles; and pharyngeal muscles.

[0155] The compounds described herein may be administered locally by injection into the targeted non-skeletal muscle tissue or by administration in close proximity to the targeted tissue.

[0156] 10. Pharmaceutical Compositions The pharmaceutical composition of the compound described herein can include pharmaceutically acceptable carrier.In certain aspects, pharmaceutically acceptable carrier is determined in part according to the specific composition to be administered and according to the specific method used to administer the composition.Therefore, there are a wide variety of suitable formulations of the pharmaceutical composition described herein (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990)).

[0157] As used herein, " pharmaceutically acceptable carrier " includes any of the standard pharmaceutically acceptable carriers known to those skilled in the art in the formulation of pharmaceutical compositions.Therefore, the compound itself, for example, as pharmaceutically acceptable salt or as conjugate, can be prepared as a solution in pharmaceutically acceptable diluent, for example, saline, phosphate buffered saline (PBS), aqueous ethanol solution, or glucose, mannitol, dextran, propylene glycol, oil (for example, vegetable oil, animal oil, synthetic oil, etc.), microcrystalline cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80, etc., or as a solid preparation in suitable excipient.

[0158] Pharmaceutical compositions often further comprise one or more buffering agents (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds as needed.

[0159] The pharmaceutical compositions described herein are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.The amount to be administered depends on various factors, including, for example, the age, weight, physical activity and diet of the individual, the condition or disease to be treated, and the stage or severity of the condition or disease.In certain embodiments, the size of the dose can also be determined by the existence, nature and extent of any adverse side effects associated with the administration of the therapeutic agent to a specific individual.

[0160] It should be understood, however, that the specific dose level and frequency of administration for any particular patient may vary and will depend upon a variety of factors including the activity of the particular compound used, the metabolic stability and length of action of that compound, age, body weight, genetic characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host being treated.

[0161] In certain embodiments, the compound dose may take the form of a solid, semi-solid, lyophilized powder or liquid dosage form, such as a tablet, pill, pellet, capsule, powder, solution, suspension, emulsion, suppository, retention enema, cream, ointment, lotion, gel, aerosol, foam, etc., preferably in a unit dosage form suitable for simple administration of a precise dosage.

[0162] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a single dosage for humans and other mammals, each unit containing a predetermined amount of a therapeutic agent calculated to achieve a desired onset, tolerability, and / or therapeutic effect in combination with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, highly concentrated dosage forms may be prepared, from which further diluted unit dosage forms may be produced. Thus, highly concentrated dosage forms may substantially contain, for example, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more of the therapeutic compound.

[0163] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, supra). Dosage forms typically contain a conventional pharmaceutical carrier or excipient and may further contain other medicinal agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, supra).

[0164] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acid, such as Carbopol, for example, Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage form can further include lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as methyl hydroxybenzoate, ethyl hydroxybenzoate, and propyl hydroxybenzoate (e.g., parabens); pH adjusters, such as inorganic and organic acids and bases; sweeteners; and flavoring agents. The dosage form can also include biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0165] For oral administration, the therapeutically effective dosage can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders and sustained-release preparations.Excipients suitable for oral administration include pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0166] The therapeutically effective dose can also be provided in a lyophilized form. Such dosage forms may contain a buffer, such as bicarbonate, for reconstitution before administration, or a buffer may be included in the lyophilized dosage form for reconstitution, for example, with water. The lyophilized dosage form may further contain a suitable vasoconstrictor, such as epinephrine. The lyophilized dosage form may be provided in a syringe packaged, optionally combined with a buffer for reconstitution, so that the reconstituted dosage form can be immediately administered to an individual.

[0167] In some embodiments, additional compounds or drugs can be co-administered to the subject.Such compounds or drugs can be co-administered for the purpose of alleviating the signs or symptoms of the disease being treated, reducing the side effects caused by the induction of immune response, etc.In some embodiments, for example, the 15-PGDH inhibitor described herein is administered with a senolytic agent, a compound that enhances PGE2 level or PGD2 level, a compound that reduces the level or activity of Atrogin 1, a compound that increases the signal transduction through EP1, EP2, EP3, EP4, DP1 and / or DP2 receptors, and / or any other compound that is intended to enhance muscle mass, muscle strength, or muscle function, or the function, health, or any other desired characteristics of targeted non-skeletal muscle tissue.

[0168] 11. Kit Another embodiment of the composition described herein is a kit containing 15-PGDH inhibitor.Kit typically includes a container that can be made of various materials such as glass or plastic, and can include, for example, bottles, vials, syringes and test tubes.Labels are typically attached to kits, and include any written or recorded material, which can be in electronic or computer-readable form, that provides instructions or other information for using the contents of the kit.

[0169] In some embodiments, the kit includes one or more reagents for treating aging and / or atrophied muscle. In some embodiments, the kit includes one or more reagents for treating non-skeletal muscle tissue of a subject with an age-related condition, disorder, or disease. In some embodiments, the kit includes an agent that antagonizes 15-PGDH expression or activity. In some embodiments, the kit includes an inhibitory nucleic acid (e.g., antisense RNA, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA)) or polynucleotide encoding a 15-PGDH inhibitory polypeptide that inhibits or suppresses the expression or activity, e.g., enzymatic activity, of 15-PGDH mRNA or protein. In some embodiments, the kit includes modified RNA, e.g., modified shRNA or modified siRNA, or modified mRNA encoding a polypeptide 15-PGDH inhibitor. In some embodiments, the kit further includes one or more plasmids, bacterial vectors, or viral vectors for expressing an inhibitory nucleic acid or polynucleotide encoding a 15-PGDH inhibitory polypeptide. In some embodiments, the kit includes an antisense oligonucleotide capable of hybridizing to a portion of the mRNA encoding 15-PGDH. In some embodiments, the kit includes an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a humanized antibody, a bispecific antibody, a chimeric antibody, a blocking antibody, or a neutralizing antibody) or an antibody-binding fragment thereof that specifically binds to and inhibits 15-PGDH protein. In some embodiments, the kit includes a blocking peptide. In some embodiments, the kit includes an aptamer (e.g., a peptide aptamer or a nucleic acid aptamer). In some embodiments, the kit includes an affimer. In some embodiments, the kit includes modified RNA. In certain embodiments, the kit includes a small molecule inhibitor, e.g., SW033291, that binds to or inhibits the enzymatic activity of 15-PGDH. In some embodiments, the kit further includes one or more additional therapeutic agents, e.g., agents for administration in combination therapy with agents that antagonize 15-PGDH expression or activity.

[0170] In some embodiments, the kits can further include instructional materials containing directions (e.g., protocols) for practicing the methods described herein (e.g., instructions for using the kit to enhance muscle mass, strength, or function of aging and / or atrophied muscles; and / or instructions for using the kit to enhance the function, health, or other characteristics of non-skeletal muscle tissue). The instructional materials typically include, but are not limited to, written or printed material. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROMs), and the like. Such media may include addresses to internet sites that provide such instructional materials. [Example]

[0171] The present disclosure will be further described in detail with specific examples.The following examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any way.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to essentially achieve the same results.

[0172] Example 1. Targeting prostaglandin E2 degrading enzyme improves sarcopenia and muscular dystrophy summary Sarcopenia is an aging-related muscle-wasting syndrome for which no effective treatment exists to date. Here, we have identified that loss of PGE2 levels contributes to muscle atrophy in aging skeletal muscle. We demonstrate that the accumulation of senescent cells in aging muscle contributes to elevated levels of the PGE2-degrading enzyme (15-PGDH). We observed increases in muscle mass, strength, and exercise capacity in aging mice using the pharmacological agent SW033291 to inhibit the 15-PGDH enzyme or gene therapy to knock down 15-PGDH. A similar decrease in 15-PGDH and increased muscle strength were observed in mice with Duchenne muscular dystrophy (mdxcv4 / mTRKO(G2)). We demonstrated that systemic senolytic treatment (ABT-263) reduced 15-PGDH levels in muscle tissue. Using genetic and cell culture models, we have demonstrated the role of prostaglandin E2 (PGE2) signaling via the EP4 receptor in differentiated cells and muscle fibers as a regulator of muscle mass. PGE2 signaling inhibits atrogin-1 expression, a key mediator of muscle atrophy. Here, we have identified inhibition of the prostaglandin E2-degrading enzyme 15-PGDH as an effective target for reversing muscle mass and strength loss and combating aging and muscular dystrophy.

[0173] Introduction Atrophy results from the rapid loss of muscle mass and strength, primarily due to excessive protein breakdown, often accompanied by a decrease in protein synthesis. This loss of muscle function reduces quality of life and increases morbidity and mortality. While much is known about how muscle atrophy occurs, current therapeutic strategies to effectively prevent or delay atrophy are limited to exercise. Experimental approaches currently under investigation are primarily directed at increasing muscle mass by altering protein balance, for example, via myostatin inhibitors (1).

[0174] Here, we tested whether modulation of the PGE2 pathway could increase the function of atrophied muscle in aging mice. We unexpectedly found that PGE2 catabolism is dysregulated and has detrimental effects on aging mouse muscle tissue. We demonstrate that PGE2 is detected at lower levels in aged muscle tissue than in young mice, a finding not previously associated with aging muscle. We uncover the cellular and molecular basis for the dysregulation of PGE2 synthesis, catabolism, and signaling in aging muscle. We designed a strategy to increase PGE2 levels by inhibiting 15-PGDH, a catabolic enzyme that inactivates PGE2 and is detectable as a PGE2 metabolite (PGEM) in aging muscle tissue. 15-PGDH inhibition overcomes the detrimental effects of the aging muscle microenvironment, resulting in robust increases in muscle strength, muscle mass, and muscle endurance in aging mice.

[0175] Discovery of decreased PGE2 levels in aging muscle tissue A progressive decline in muscle strength accompanies aging, as shown here for mouse gastrocnemius (GA) muscles assessed by plantar flexion torque at different ages (Figure 1A). PGE2 is catabolized by a two-step process: the first step is mediated by the rate-limiting enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and involves the conversion of PGE2 to the unstable 15-keto-PGE2, and the second step is mediated by prostaglandin reductase 2 and involves the conversion of 15-keto-PGE2 to the relatively stable 13,14-dihydro-15-keto-PGE2 metabolite (3, 4) (Figure 1B). In aged muscle tissue, 15-PGDH activity dramatically increased in line with the decrease in PGE2 (Figure 1C). Further analysis of the PGE2 signaling pathway during aging revealed lower levels of PGE2 in aged muscle, as shown by mass spectrometry (Figure 1D). Taken together, this suggests that PGE2 is catabolized in the aging muscle microenvironment or niche.

[0176] In senescent cells within aging tissues, PGE2 catabolism is mediated by upregulation of 15-PGDH Senescent cells have been reported to accumulate and adversely affect tissue function with aging. PGE2 has been hypothesized to be a component of the senescence-associated secretory phenotype (SASP) (5, 6). We hypothesized that 15-PGDH expression and PGE2 inactivation are due to senescent cells in aging muscle. To investigate this possibility, we treated aged mice (20 months old) with the senolytic agent ABT-263, also known as navitoclax, which acts by inhibiting Bcl-2, Bcl-w, and Bcl-xL, to induce apoptosis in senescent cells (7) (Figure 2A). After 2 months of ABT-263 treatment, the level of PGE2-degrading enzyme (15-PGDH) mRNA significantly decreased (Figure 2A), indicating that the main cellular source of PGE2 in aging muscle is senescent cells, which are eliminated from the tissue by senolytic treatment. These results suggest that PGE2 inactivation is partly mediated by senescent cells within aging muscle tissue, contributing to the muscle-wasting phenotype associated with aging.

[0177] 15-PGDH inhibition improves muscle function in aged mice We sought to determine whether PGE2 inactivation is a major component of muscle wasting and decline in muscle function in aging mice. We treated aging mice daily for one month with the 15-PGDH inhibitor SW033291 (SW) and found that 15-PGDH inhibition resulted in significant increases in muscle mass, strength, and endurance in aging mice (Figure 3A). We performed histological analysis and found that SW-treated aged mice had larger muscle fiber cross-sectional areas (Figure 3B-D). To confirm that the phenotype was due to increased PGE2 levels, we performed mass spectrometry on muscle samples and found that SW treatment resulted in elevated PGE2 levels in muscle comparable to those in young muscle (Figure 3E). To confirm that this effect was due to 15-PGDH inhibition, we used an independent method via enzyme knockdown using shRNA (sh15PGDH) delivered to aging muscle by adeno-associated virus AAV9 (Figure 4A). We confirmed by qPCR that AAV9-mediated sh15PGDH knockdown reduced Hpgd(15-PGDH) mRNA levels compared with AAV9-mediated shRNA-scrambled (shscr) controls (Fig. 4B). Muscle mass and strength were found to be increased compared with AAV(shscr)-infected control muscles (Fig. 4C, D).

[0178] 15-PGDH inhibition improves muscle function in Duchenne mice To extend our findings to other muscle-wasting diseases characterized by muscle atrophy and high senescent cell infiltration, we analyzed the mdx4cv / mTRKO(G2) Duchenne muscular dystrophy (DMD) mouse model, which has "humanized" telomere length and recapitulates skeletal muscle and cardiac DMD phenotypes (8, 9). We analyzed the levels of senescence and senescence-associated secretory phenotype (SASP) markers by qPCR and found that they were significantly elevated in mdx4cv / mTRKO(G2) mice (10) (Figure 5A). Importantly, we found that the degradative enzyme 15-PGDH was significantly increased in mdx4cv / mTRKO(G2) compared with mTRKO(G2) controls (Figure 5A). To elucidate whether PGE2 inactivation contributes to the muscle wasting seen in DMD, we treated 8-month-old mdx4cv / mTRKO(G2) and mTRKO(G2) controls with SW and observed a 22% increase in muscle strength in these mice compared to vehicle-treated controls after 4 weeks of treatment (Figure 5B).

[0179] PGE2 prevents atrophy via EP4 receptors in muscle fibers To understand the downstream mechanisms by which 15-PGDH inhibition improves muscle atrophy, we performed qPCR analysis of aged muscles treated with SW or AAV-sh15PGDH. We hypothesized that PGE2 stimulation of the EP4 receptor may be involved in improving the atrophy phenotype via inhibition of atrogin-1 (11-14). Our data confirm that SW treatment and knockdown of 15-PGDH by AAV9-sh15PGDH delivery result in decreased expression of Fbxo32 (atrogin-1) at the mRNA level (Figure 6A).

[0180] To further delineate the mechanism of action of PGE2 in muscle, we tested whether PGE2 signals through the EP4 receptor in differentiated myotubes. We analyzed the levels of all PGE2 receptors, EP1–EP4 (Ptger1–4), and found that EP4 is highly expressed in muscle stem cells (MuSCs), as previously described (15). However, in differentiated myoblasts and myotubes, EP4 was also expressed, but at lower levels than in MuSCs (Figure 6B, C). To mimic atrophy in vitro and elucidate the effects of PGE2 signaling, we treated starved myotubes with either vehicle, PGE2, or PGE2 in the presence of an EP4 antagonist (ONO-AE3-208). PGE2 was found to significantly reduce atrogin-1 expression in starved myotubes (Figure 6D). Furthermore, PGE2 was found to increase myotube diameter in starved or non-starved cultured myotubes (Figure 6D). In the presence of an EP4 antagonist (ONO-AE3-208), this effect was abolished, providing evidence that PGE2 promotes myotube hypertrophy via the EP4 receptor (Figure 6D). To confirm whether SW can mediate its effects on myotubes independently of PGE2, its effect on cultured myotubes was assessed. In the absence of senescent cells or other cells expressing 15-PGDH, we found that starved myofibers treated with SW did not exhibit an increase in myotube diameter (Figure 6D), in contrast to the increase in myofiber cross-sectional area observed after SW treatment in vivo (Figure 3B-D). To confirm the role of the EP4 receptor in myotubes, we used EP4flox / flox myoblasts, in which the receptor has been genetically ablated after infection with a cre-expressing lentivirus with an empty vector, which served as a control. In the absence of the EP4 receptor, relatively small myotubes were observed, suggesting that the EP4 receptor plays an important role in myotube differentiation (Figure 6E). These results highlight the role of PGE2 signaling via the EP4 receptor in muscle atrophy.Furthermore, we demonstrate that, similar to muscle tissue, PGE2 treatment of cultured myotubes inhibits the atrophy-related ubiquitin ligase, atrogin-1 (Figure 6F).

[0181] Consideration We identify 15-PGDH as a therapeutic target for aging and dystrophic muscle, where its reduction ameliorates muscle atrophy. We previously demonstrated the importance of PGE2 signaling in muscle stem cell (MuSC) function in the context of young muscle regeneration (15). This required transplantation of PGE2-treated MuSCs into injured muscle or local intramuscular delivery of PGE2 to injured muscle. Previous studies have implicated 15-PGDH inhibition in regeneration in young mice, showing that systemic delivery of the small molecule inhibitor of 15-PGDH, SW033291, is a potent inducer of endogenous PGE2, improving regeneration of hematopoietic, liver, and colonic tissues (16). Here, we demonstrate a previously unrecognized role for 15-PGDH in muscle aging. 15-PGDH levels are expressed only at low levels in young muscle tissue and increase as senescent cells accumulate. Furthermore, we demonstrate that inhibition of 15-PGDH improves skeletal muscle function in aging mice. Systemic reconstitution of endogenous PGE2 levels by preventing its degradation in muscle ameliorates muscle atrophy and leads to increases in muscle mass and strength. Our findings provide unexpected evidence for the role of PGE2-degrading enzymes in muscle-wasting diseases, such as DMD and aging, and indicate that they constitute a potent therapeutic target.

[0182] References TIFF2025148416000002.tif25150TIFF2025148416000003.tif217150

[0183] material and method mouse We conducted all experiments and protocols in accordance with the institutional guidelines of Stanford University and the Administrative Panel on Laboratory Animal Care (APLAC). For the aging muscle studies, middle-aged (18 months) and aged (>24 months) C57BL / 6 mice were obtained from the National Institute on Aging (NIA), and young (2-4 months) wild-type C57BL / 6 mice were obtained from the Jackson Laboratory. Mice were housed in specific pathogen-free enclosures with a 12-hour dark / light cycle throughout the study period.

[0184] For ABT-263 treatment, 20-month-old C57 / Bl6 mice were treated by oral gavage with vehicle (ethanol:polyethylene glycol 400:Phosal 50 PG) or ABT-263 (in ethanol:polyethylene glycol 400:Phosal 50 PG) for two 1-week cycles with a 2-week rest period between cycles as previously described (1). For the Duchenne muscular dystrophy (DMD) mouse model, 8- to 10-month-old mdx4cv / mTRKO (G2) mice were used, generated as previously described (2).

[0185] Mice were treated with SW033291 (SW) (Cayman Chemicals) or vehicle for 1 month as previously described (3). Time to exhaustion and distance were measured in SW-treated mice and their controls as previously described (4) (Fig. 3A).

[0186] Mouse transgenic lines were purchased from Jackson Laboratory (EP4flox / flox) No. 028102. Their genotypes were verified by appropriate PCR-based strategies. Unless specified, studies were performed using female and male mice.

[0187] Immunofluorescence staining and imaging Recipient tibialis anterior (TA) or gastrocnemius (GA) muscle tissue was collected and prepared for histological examination as previously described (5). Transverse sections of the muscle were fixed using 4% PFA, blocked and permeabilized using PBS / 1% BSA / 0.1% Triton X-100, and incubated with anti-laminin (Millipore, clone A5, catalog number 05-206, 1:200) followed by an AlexaFluor secondary antibody (Jackson ImmunoResearch Laboratories, 1:200) or wheat germ agglutinin-Alexa647 conjugate (WGA, Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Invitrogen).

[0188] Myotubes were fixed with 4% PFA, blocked and permeabilized with PBS / 1% BSA / 0.1% Triton X-100, and stained with the primary antibody anti-MyHC (Thermo Fisher Scientific, catalog no. 14-6503-82, clone MF-20, 1:500) followed by an AlexaFluor secondary antibody (Jackson ImmunoResearch Laboratories, 1:500). Nuclei were counterstained with DAPI (Invitrogen).

[0189] Images were acquired using a Zeiss 510 laser scanning confocal microscope (Carl Zeiss Microimaging) with a 40x / 0.9 NA objective to capture multiple contiguous focal planes, or a KEYENCE BZ-X700 all-in-one fluorescence microscope (Keyence) with a 20x / 0.75 NA objective. Myofiber area was analyzed using Keyence Advanced Analysis Software. For cross-sectional area, the maximum cross-sectional area of ​​the muscle was quantified, or at least 10 fields of laminin-stained myofiber cross-sections encompassing more than 400 myofibers were captured for each mouse as described above. Data analysis was blinded. The researchers who performed image acquisition and scoring were unaware of the treatment conditions given to the sample groups analyzed.

[0190] cell culture Primary myoblasts were grown in myogenic cell culture medium containing DMEM / F10 (50:50), 15% FBS, 2.5 ng ml-1 fibroblast growth factor-2, and 1% penicillin-streptomycin. For differentiation experiments, confluent myoblasts were grown in medium containing 5% horse serum and DMEM. Day 4 differentiated myotubes were supplemented with 10 ng / ml prostaglandin E2 (Cayman Chemicals), 1 μM SW033291 (ApexBio), or 1 μM ONO-AE3-208 (Cayman Chemicals). Myoblasts were cultured at EP4. fl / fl were isolated from mice and administered mCherry / Cre lentivirus or mock infected as previously described ( 5 ).

[0191] Quantitative RT-PCR RNA was isolated from MuSCs using the RNeasy Micro Kit (Qiagen). For muscle samples, tissue was snap-frozen in liquid nitrogen, homogenized in Trizol (Invitrogen) using a FastPrep FP120 homogenizer (MP Biomedicals), and then RNA was isolated. cDNA was reverse-transcribed from total mRNA from each sample using the SensiFAST™ cDNA Synthesis Kit (Bioline). cDNA was subjected to RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems) or TaqMan Assays (Applied Biosystems) on an ABI 7900HT Real-Time PCR System (Applied Biosystems). Samples were cycled at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. To quantify relative transcript levels, treated and untreated samples were compared using 2-ΔΔCt and results were expressed relative to Gapdh.

[0192] For SYBR Green qRT-PCR, the following primer sequences were used: TIFF2025148416000004.tif66154. For mouse aging and aging-associated markers, previously described primers (6) were used.

[0193] TaqMan Assays (Applied Biosystems) were used to quantify Pax7, Myh, p21, Ptger3, and Ptger4 in samples using the TaqMan Universal PCR Master Mix reagent kit (Applied Biosystems) according to the manufacturer's instructions. Transcript levels were expressed relative to Gapdh levels. For SYBR Green qPCR, Gapdh qPCR was used to normalize input cDNA samples. For TaqMan qPCR, multiplex qPCR allowed target signals (FAM) to be individually normalized by their internal Gapdh signal (VIC).

[0194] 15-PGDH kinetic assay 15-PGDH activity in muscle lysates was analyzed using the BioVision PicoProbe 15-PGDH Activity Assay Kit (catalog no. K562) according to the manufacturer's protocol.

[0195] mass spectrometry Analyte: Prostaglandin standards (PGF2α; PGE2; PGD2; 15-keto PGE2; 13,14-dihydro-15-keto PGE2; PGE2-D4; and PGF2α-D9) were all purchased from Cayman Chemical. The PGE2-D4 internal standard was labeled with four deuterium atoms at positions 3 and 4. The PGF2α-D9 internal standard was labeled with nine deuterium atoms at positions 17, 18, 19, and 20.

[0196] Creating a calibration curve: Analyte stock solutions (5 mg / mL) were prepared in DMSO. These stock solutions were serially diluted with acetonitrile / water (1:1 v / v) to obtain a series of standard working solutions, which were used to generate calibration curves. Calibration curves were generated by adding 10 μL of each standard working solution to 200 μL of homogenization buffer (acetone / water 1:1 v / v; 0.005% BHT to prevent oxidation), followed by 10 μL of internal standard solution (3000 ng / mL each of PGF2α-D9 and PGE2-D4). Calibration curves were generated anew for each set of samples. Calibration curve ranges were: PGE2 and 13,14-dihydro-15-keto-PGE2, 0.05 ng / mL to 500 ng / mL; PGD2 and PGF2α, 0.1 ng / mL to 500 ng / mL; and 15-keto-PGE2, 0.025 ng / mL to 500 ng / mL.

[0197] Extraction Procedure: The extraction procedure was modified from that of Prasain et al. (7) to include acetone protein precipitation followed by a two-step liquid-liquid extraction, the latter step enhancing LC-MS / MS sensitivity. Oxidation was prevented using butylated hydroxytoluene (BHT) and evaporation under nitrogen (N2) gas.

[0198] Solid tissue samples were harvested, weighed, and flash-frozen using liquid nitrogen. Muscle tissue samples were combined with homogenization beads and 200 μL of homogenization buffer in a polypropylene tube and processed in a FastPrep 24 homogenizer (MP Biomedicals) at a speed of 6 m / s for 40 seconds. After homogenization, 10 μL of internal standard solution (3000 ng / mL) was added to the tissue homogenate, followed by sonication and shaking for 10 minutes. The sample was centrifuged, and the supernatant was transferred to a clean Eppendorf tube. 200 μL of hexane was added to the sample, which was then shaken for 15 minutes and then centrifuged. The sample was frozen at -80°C for 40 minutes. The hexane layer was poured off from the frozen lower aqueous layer and discarded. After thawing, 25 μL of 1 N formic acid was added to the bottom aqueous layer, and the sample was vortexed. For the second extraction, 200 μL of chloroform was added to the aqueous phase. The samples were shaken for 15 minutes to ensure complete extraction. The layers were separated by centrifugation. The lower chloroform layer was transferred to a new Eppendorf tube and evaporated to dryness under nitrogen at 40°C. The dry residue was reconstituted in 100 μL of acetonitrile / 10 mM ammonium acetate (2:8 v / v) and analyzed by LC-MS / MS.

[0199] LC-MS / MS: Chromatographic separation is important because many prostaglandins are positional isomers with identical masses and similar fragmentation patterns. Two SRM transitions were carefully selected for each analyte: one quantitative ion and one qualitative ion. A distinct qualitative ion intensity ratio and retention time were essential for authenticating the target analyte. All analyses were performed by negative electrospray LC-MS / MS using an LC-20 ADXR Prominence liquid chromatograph and an 8030 triple quadrupole mass spectrometer (Shimadzu). HPLC conditions: An Acquity UPLC BEH C18 2.1 x 100 mm, 1.7 μm particle size column was operated at 50 °C with a flow rate of 0.25 mL / min. The mobile phase consisted of A: 0.1% acetic acid in water and B: 0.1% acetic acid in acetonitrile. The elution profile was: initial hold at 35% B for 5 min, followed by a gradient from 35% to 40% B in 3 min, then 40% to 95% B in 3 min; total run time was 14 min. The injection volume was 20 μL. Using these HPLC conditions, baseline separation of the analytes of interest was achieved.

[0200] Selected reaction monitoring (SRM) was used for quantification. The mass transitions were as follows: PGD2: m / z 351.10 → m / z 315.15 (quantitative ion) and m / z 351.10 → m / z 233.05 (qualitative ion); PGE2: m / z 351.10 → m / z 271.25 (quantitative ion) and m / z 351.10 → m / z 315.20 (qualitative ion); PGF2α: m / z 353.10 → m / z 309.20 (quantitative ion) and m / z 353.10 → m / z 193.20 (qualitative ion); 15-keto-PGE2: m / z 349.30 → m / z 331.20 (quantitative ion) and m / z 349.30 → m / z 113.00 (qualifying ion); 13,14-dihydro-15-keto PGE2: m / z 351.20 → m / z 333.30 (quantitative ion) and m / z 351.20 → m / z 113.05 (qualifying ion); PGE2-D4: m / z 355.40 → m / z 275.20; and PGF2α-D9: m / z 362.20 → m / z 318.30. The dwell time was 20–30 ms.

[0201] Quantitative analysis was performed using a LabSolutions LCMS (Shimadzu). An internal standard method was used for quantification. PGE2-D4 was used as the internal standard for quantification of PGE2, 15-ketoPGE2, and 13,14-dihydro15-ketoPGE2. For quantification of PGD2 and PGF2α, PGF2α-D9 was the internal standard. Calibration curves were linear (R > 0.99) across the concentration range using a weighting factor of 1 / X2, where X is the concentration. Back-calculated standard concentrations were ±15% from the nominal value and ±20% at the lower limit of quantification (LLOQ).

[0202] In vivo and in situ muscle force measurements Peak isometric torque (N·mm) of the ankle plantarflexors was assessed as previously described (8, 9). Briefly, the foot of an anesthetized mouse was placed on a footplate attached to a servomotor (Model 300C-LR; Aurora Scientific). Two Pt-Ir electrode needles (Aurora Scientific) were inserted percutaneously and subcutaneously onto the tibial nerve just posterior / medial to the knee. The ankle joint was immobilized at a 90° angle. Peak isometric torque was achieved by varying the current delivered to the tibial nerve with a 200 Hz frequency and 0.1-ms square-wave pulse duration. Three tetanic measurements were performed for each muscle, with a 1-minute recovery between measurements. Data were collected using Aurora Scientific Dynamic Muscle Data Acquisition and Analysis Software.

[0203] statistical analysis Cell culture experiments were performed in at least three independent experiments, each pooled with three biological replicates. Paired t-tests were used for experiments in which control samples were derived from the same experiment in vitro or from the contralateral limb muscle in vivo. A nonparametric Mann-Whitney test was used to determine significant differences between untreated and treated groups using α = 0.05. ANOVA or multiple t-tests were performed for multiple comparisons, and the significance level was determined using the Bonferroni correction, as indicated in the figure legends. Data are presented as mean ± SEM unless otherwise noted.

[0204] References TIFF2025148416000005.tif135150

[0205] Example 2. Inhibition of the prostaglandin-degrading enzyme 15-PGDH increases muscle strength in aging mice Introduction With aging, loss of muscle function throughout the body reduces quality of life and increases morbidity and mortality (1, 2). This widespread muscle atrophy and loss of muscle strength, or sarcopenia, accounts for $18 billion in annual healthcare costs in the United States alone (2). Identifying therapeutic agents for sarcopenia would be of major clinical benefit (1, 2).

[0206] During aging, skeletal muscle undergoes structural and functional changes. The most obvious is muscle loss, which can decline by 50–80% in lower-body muscles in aging humans. This loss of muscle strength is accompanied by decreases in muscle fiber cross-sectional area, muscle mass, and strength (3). This functional loss results from disrupted cell-cell interactions and abnormal cell signaling pathways, particularly those related to inflammation, protein turnover, and mitochondrial function (1, 4–6). Due to this multifactorial etiology, it has proven challenging to unravel the causative molecular pathways in order to identify therapeutic targets to prevent, delay, or reverse sarcopenia.

[0207] Previously, we determined that PGE2 stimulates muscle stem cells (MuSCs) and is essential for the regeneration of damaged muscle in young mice (7), consistent with findings regarding the function of PGE2 in bone, colon, liver, and blood regeneration (8-10). We reasoned that prostaglandin signaling may be impaired with aging. Using liquid chromatography coupled with atmospheric pressure ionization tandem mass spectrometry (LC-MS / MS) to distinguish closely related members of the prostaglandin family (11), we found that PGE2 and PGD2 levels decreased in aging skeletal muscle.

[0208] We hypothesized that the decline in prostaglandins in aging muscle may be due to increased prostaglandin catabolism by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Here, we demonstrate that elevated 15-PGDH is a hallmark of aging muscle and certain other aging tissues. Furthermore, we show that inhibition of 15-PGDH enhances muscle mass and strength in aging mice. Genetic experiments demonstrate that the beneficial effects of 15-PGDH inhibition are specific to increased PGE2 signaling. Our findings provide new insights into sarcopenia and suggest innovative therapeutic strategies.

[0209] Increased prostaglandin-degrading enzyme (15-PGDH) in aging tissues We previously demonstrated the importance of PGE2 signaling in stimulating stem cells to regenerate damaged tissue in young mice (7). We reasoned that PGE2 may also act on mature muscle myofibers and play an important role in maintaining muscle tissue homeostasis. We hypothesized that aging may result in a decrease in PGE2 and other endogenous eicosanoids, which are lipid metabolites generated from membrane fatty acids, and may have a detrimental effect on muscle tissue function. To analyze the eicosanoid composition of aging skeletal muscle, we used LC-MS / MS. This method overcomes the cross-reactivity of antibody-based assays, such as ELISA, and exceeds other mass spectrometry methods in resolving the related eicosanoids, PGE2 and PGD2, as well as PGF2α, at the same mass (Figures 8A-C, 9A-C, and 10A). This is achieved by isolating hindlimb muscles from young and aged mice, homogenizing them, and then acetone precipitation to eliminate proteins. A two-step liquid-liquid extraction is then performed to enhance LC-MS / MS sensitivity. A significant decrease in PGE2 and PGD2 levels was observed in aging muscle (Figures 8A-C, 9A-C, and 10A). PGE2 and PGD2 are degraded by a multistep process initiated by the rate-limiting enzyme 15-PGDH to produce unstable 15-keto-PGE2 and 15-keto-PGD2 metabolites, which are then converted to multiple downstream metabolites, including 13,14-dihydro-15-keto-PGE2 metabolite (PGEM) (12, 13). These intermediates were not detected at all or only at low levels by LC-MS / MS due to their instability (Figures 8C and 9C). MS spectral plots demonstrate that this method easily distinguishes closely related eicosanoids.

[0210] We hypothesized that an increase in the degradative enzyme 15-PGDH may be a major factor in the observed decrease in PGE2 and PGD2 in muscle and may constitute a general feature of aging tissue. Consistent with this, we found that the specific activity of this enzyme was elevated not only in aging skeletal muscle but also in aging cardiac, skin, spleen, and colonic tissues (Figures 8D and 11). Accordingly, 15-PGDH mRNA and protein significantly increased in aging muscle (Figures 8E, 8F, 12A, and 12B). To determine the relevance of this finding to human aging, we reanalyzed publicly available microarray data on young and aging human muscle samples (14). We found that 15-PGDH expression was significantly increased in aged human (78 ± 6 years) biopsies from the vastus lateralis muscle compared with those from a young population (25 ± 3 years) (Figure 13A). Together, these data identify 15-PGDH as a potential driver of the decline in prostaglandin levels seen in aging muscle.

[0211] Increased muscle mass and strength in aged individuals after 15-PGDH inhibition We hypothesized that inhibition of 15-PGDH could result in increased levels of PGE2 and PGD2, thereby ameliorating muscle wasting in aging mice. Similar to humans, aging mice exhibit sarcopenia, a generalized loss of muscle strength (1). We first used a genetic approach to reduce enzyme levels, involving adeno-associated viral (AAV9) intramuscular (IM) delivery of either GFP and shRNA to 15-PGDH or a control AAV9 encoding GFP and a scrambled (scr) shRNA under the control of a ubiquitous promoter (U6) (Figure 8G). The resulting local intramuscular gene therapy delivery strategy resulted in a significant decrease in 15-PGDH mRNA levels and specific activity and increased levels of PGE2 and PGD2, as assessed by mass spectrometry (Figures 8H-J and 14A). Muscle targeting of these vectors was confirmed by immunofluorescence analysis of the GFP reporter in transduced tibialis anterior (TA) and gastrocnemius (GA) muscles (Figure 14B). Genetic knockdown of 15-PGDH in aged, but not young, muscles was accompanied by a significant increase in cross-sectional muscle fiber area in aged muscles treated with 15-PGDH shRNA compared with controls (Figure 8K-M). Furthermore, in contrast to young, knockdown of 15-PGDH in aged muscles resulted in significant increases in both muscle mass and strength after 1 month of treatment (Figure 8N-P and Figure 14C).

[0212] To test whether the widespread muscle wasting seen in sarcopenia could be overcome by systemic delivery of a small-molecule inhibitor of 15-PGDH, aged and young control mice were treated intraperitoneally with SW033291 (SW) or vehicle (10) (Figure 15A). SW has previously been extensively characterized as a specific inhibitor of 15-PGDH that is noncompetitive with PGE2 with an apparent K i of 0.1 nM (10). In vivo, SW increased PGE2 levels twofold in bone marrow, colon, lung, and liver, and to a lesser extent, PGD2 levels, which was previously shown to enhance regeneration of these tissues after injury in young mice (10). We found that after 1 month of daily intraperitoneal SW treatment, 15-PGDH specific activity was significantly reduced in aged muscle, and concomitant increases in PGE2 and PGD2 levels were detected by LC-MS / MS, comparable to those in young muscle (Figures 15B, 15C, 16A, and 16B). Histological analysis revealed a significant increase in muscle fiber cross-sectional area in SW-treated aged mice, but not in young mice, indicating attenuated muscle atrophy in aged mice (Figures 15D–F). Fiber type analysis revealed that SW treatment promoted an increase in the cross-sectional area of ​​both oxidative (type IIa) and glycolytic (type IIb) fibers (Figures 15G–J). SW-treated young mice showed a trend toward increased muscle mass and absolute muscle strength, but this was not statistically significant (Figures 15K, 15L, and 16C). In contrast, SW-treated aged mice showed significant increases in muscle mass in the TA, GA, and soleus muscles (Fig. 15K) and plantar flexion strength (Fig. 15L and Fig. 16C). Furthermore, endurance (time to exhaustion on a treadmill) increased, suggesting an overall systemic beneficial effect in addition to muscle strength (Fig. 15M). Collectively, our studies using the small molecule inhibitor SW corroborate our findings using local shRNA-mediated genetic loss-of-function, demonstrating that a 1-month systemic reduction in 15-PGDH activity is sufficient to attenuate skeletal muscle atrophy and enhance muscle function in aged mice.

[0213] 15-PGDH expression by senescent interstitial cells in the aging muscle microenvironmentWe sought to identify the cellular source of 15-PGDH in aging muscle tissue. To this end, we analyzed Hpgd (15-PGDH) mRNA levels in cells isolated by fluorescence-activated cell sorting from dissociated young and aging muscle tissue. We detected a significant increase in 15-PGDH transcript levels in FACS-purified macrophages (Cd11b+ / Cd11c- / F4 / 80+ / Cd31-), but not in endothelial cells (Cd31+ / Cd11b- / Cd11c- / F4 / 80-) or myogenic stem and progenitor cells (α7+ / Cd11b- / Cd45- / Cd31- / Sca1-) isolated from aging muscle (Figures 17A, 18A, and 18B). Furthermore, aged macrophages and endothelial cells expressed high levels of the cell cycle regulators p16 (Ink4a, Cdkn2a) and p21 (Cdkn1a), markers of senescent cells, which have been reported to accumulate with aging and adversely affect tissue function, including muscle (15) (Figures 17B and 19C). To determine whether senescent cells are the source of 15-PGDH in aging muscle, we used two strategies to eliminate these cells: genetic modeling and senolytic drug treatment. First, we analyzed muscle from INK-ATTAC transgenic mice, in which senescent cells are eliminated by expression of a minimal Ink4a promoter (p16) under the control of the fusion protein in response to treatment with AP20187 (AP), a dimerizer that activates FK506-binding protein-caspase 8 fusion proteins, leading to cell death (16) (Figures 17C and 19A). After 16 months of AP treatment in aged INK-ATTAC mice, 15-PGDH transcript levels were significantly reduced (Fig. 17D), leading to increased PGE2 levels analyzed by LC-MS / MS (Fig. 17E and Fig. 19B). To determine the cellular source of 15-PGDH in this mouse model, macrophages were isolated by FACS from control and AP-treated INK-ATTAC muscles. Reduced levels of 15-PGDH were found in these cells after removal of senescent cells (Fig. 17F), consistent with reduced expression of p16 and p21 (Fig. 19C).In contrast, FACS-isolated senescent endothelial cells did not show significant 15-PGDH levels (Figures 17F and 19C). Notably, muscle fibers did not die, and their function improved. In aged mice, elimination of senescent cells resulted in increases in hindlimb muscle mass (TA and GA), muscle strength assessed as grip strength, and endurance assessed as a composite measure of treadmill distance run to exhaustion and body weight (Figure 17G). These aged mice from which senescent cells had been removed not only ran longer distances, but also gained weight, indicating a relatively high work capacity (Figure 17G).

[0214] As a second approach, we induced apoptosis in senescent cells by treating aged mice with the senolytic agent ABT-263, also known as the pan-Bcl inhibitor navitoclax (17) (Figure 19D). After 2 months of treatment, the percentage of cells expressing 15-PGDH, as detected by immunohistochemistry, and the overall 15-PGDH gene expression level, as detected by qRT-PCR, were significantly reduced in muscle tissue (Figures 19D–G). Muscle-resident interstitial cells, which showed the highest 15-PGDH staining, were eliminated by this senolytic treatment (Figures 19E and 19F), but myofibers were not. These results suggest that PGE2 is partially degraded by a paracrine mechanism, whereby senescent 15-PGDH-expressing interstitial cells, such as macrophages, near muscle fibers degrade PGE2, contributing to the dysfunction of the aged myogenic niche or microenvironment.

[0215] Muscle weakness after ectopic expression of 15-PGDH in young muscle We reasoned that if 15-PGDH plays a major role in the loss of muscle function seen with aging, ectopic expression of PGE2-degrading enzymes in the muscles of young mice would have a detrimental effect on muscle function. To test this hypothesis, we used AAV9 to deliver and overexpress the 15-PGDH gene (Hpgd) under the control of the ubiquitous cytomegalovirus (CMV) promoter (Figure 20A). We confirmed by qRT-PCR that 15-PGDH expression increased upon intramuscular injection of AAV9-CMV-15-PGDH (Figure 20B). Furthermore, LC-MS / MS analysis revealed a significant decrease in the prostaglandins PGE2 and PGD2 in young muscles expressing 15-PGDH, similar to the decrease in these prostaglandins seen in aged muscles (Figure 20C). Reduction of these prostaglandins for just 1 month resulted in a significant decrease in the mean cross-sectional area of ​​individual muscle fibers (Figures 20D and 20E) and an acute loss of muscle function, assayed as muscle mass and strength, in young adult mice (Figures 20F and 20G). We analyzed markers of muscle atrophy by qRT-PCR and found that in acute models of atrophy, in accordance with findings by others (18-21), the muscle atrophy-related genes Trim63 (MuRF1) and Fbxo32 (atrogin-1) and the autophagy genes p62, Lc3b, Atg4, and Atg6 were upregulated in 15-PGDH-overexpressing muscles (Figure 20H). These data provide strong evidence that 15-PGDH overexpression plays a causal role in reducing intramuscular PGE2 and PGD2 levels, which in turn leads to decreased muscle mass and strength. Furthermore, these data indicate that 15-PGDH activity has a profound effect on muscle homeostasis, inducing an atrophy phenotype.

[0216] To determine the specificity of SW for its target 15-PGDH, rescue experiments were performed in young mice overexpressing this enzyme after intramuscular AAV9-mediated gene delivery. We reasoned that inhibition of the overexpressed enzyme with SW should overcome the deleterious effects seen with 15-PGDH overexpression. Therefore, control and 15-PGDH-overexpressing young mice were systemically treated with vehicle or SW (Figure 20I). Treatment with SW was found to increase muscle mass (Figure 20J) and strength (Figure 20K) in 15-PGDH-overexpressing young muscles. These data demonstrate that 15-PGDH inhibition using small molecule SW specifically targets 15-PGDH and leads to improved muscle function.

[0217] Increased muscle strength in aging mice is mediated by PGE2 but not PGD2 15-PGDH degrades both PGE2 and PGD2 in aging muscle. Notably, these two prostaglandins have different receptors and downstream signaling cascades (22). To determine which prostaglandin is responsible for promoting the improvement of aging muscle function, we increased their levels by inhibiting 15-PGDH using SW and inhibited the expression of the PGD2 synthase PTGDS. This was achieved by intramuscularly injecting AAV9 viruses encoding either shRNA targeting PTGDS or a scrambled control shRNA into aging muscle and treating mice with the 15-PGDH inhibitor SW or vehicle for 1 month (Figure 21A). Knockdown of PTGDS in transduced aging muscle was verified by confirming reduced Ptgds mRNA levels by qRT-PCR and reduced PGD2 levels by mass spectrometry (Figures 21B and 21C). Knockdown of PTGDS resulted in increased muscle mass, strength, and endurance after SW treatment (Figures 21D-G). These results suggest that PGE2, but not PGD2, is the mediator of the increased muscle function observed in aging muscle upon 15-PGDH inhibition.

[0218] We conducted additional experiments to demonstrate the specific role of PGE2 in attenuating muscle atrophy in aging mice. Because three enzymes, cPGES, PGES1, and PGES2, are responsible for PGE2 synthesis (22), targeting the PGE2 synthesis pathway would involve triple knockdown, which would be technically challenging. As an alternative approach, we focused on the PGE2 receptor in muscle. qRT-PCR revealed that the PGE2 receptor EP4 (Ptger4) was the most highly expressed eicosanoid receptor in differentiated myotubes (Figure 22A). To conclusively determine whether the observed muscle hypertrophy was due to PGE2-mediated EP4 signaling in mature muscle myofibers in vivo, we created a mouse model in which the receptor was genetically ablated exclusively in the myofibers of GA muscles. This was the case in aging EP4f / f mice (MCK-EP4). Δ / Δ This was achieved by intramuscular AAV9-mediated delivery of muscle creatine kinase (MCK) promoter-driven Cre into GA muscle fibers of aged mice. Surprisingly, loss of EP4 expression within muscle fibers of aged mice abolished the beneficial effects on muscle mass and strength induced by a 1-month SW-mediated 15-PGDH inhibition treatment (Figure 21H-K). These data demonstrate that the observed effects of SW treatment are primarily mediated by PGE2 signaling via the EP4 receptor on aged muscle fibers.

[0219] Increased mitochondrial function and mitochondrial biogenesis after 15-PGDH inhibition PGE2 signaling through the G-coupled protein receptor EP4 is known to be mediated by cyclic AMP (cAMP) (12, 22, 23). We confirmed that PGE2 activates cyclic AMP response element-binding protein (CREB) in skeletal muscle (Figures 23A and 23B). To identify the downstream signaling pathways through which PGE2 exerts its effects in aging muscle, we performed unbiased transcriptomic analysis of vehicle-treated and SW-treated aging muscles. Most notable was the strong enrichment of mitochondrial pathways, including mitochondrial oxidative phosphorylation, ATP synthesis, and other metabolic and energy-generating processes (Figure 24A). In SW-treated aging muscles, numerous components of mitochondrial complexes I, II, IV, and V of the electron transport chain were significantly increased (Figure 24B). When we assayed the mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-α (Pgc1α), a key cofactor for mitochondrial biogenesis that contains a CREB-binding motif in its promoter (24), we found that its levels were restored to those seen in young muscle (Figure 24C). Overall mitochondrial content increased, as reflected by an increased ratio of mitochondrial DNA to nuclear DNA, after SW treatment of aged muscle (Figure 24D). Together, these data provide strong evidence that PGE2 induces a robust increase in mitochondrial number to meet the energetic requirements of muscle growth.

[0220] Gene expression analysis also revealed a decrease in signaling pathways associated with age-related muscle atrophy. Among the top down-regulated genes upon SW treatment of aged muscle were members of the ubiquitin signaling pathway (Figures 24A and 24E). PGE2 signaling has previously been linked to activation of the AKT / FOXO pathway in non-muscle cells (12, 25, 26). Therefore, we sought to determine whether this pathway could function in muscle to regulate the expression of E3 ubiquitin ligases, which are known to play a role in muscle atrophy (27-29). To this end, muscle cells were acutely exposed to PGE2 in the absence of other cell types. As shown by Western blot analysis, differentiated myotubes derived from human donor muscle cells treated with PGE2 for 15 or 30 minutes showed increased levels of pAKT, which inactivated FOXO (pFOXO3a) (Figure 24F). Furthermore, myotubes treated with PGE2 activated the downstream target phospho-S6 ribosomal protein (pS6rp), indicating increased protein synthesis (Figure 24F), and showed a significant increase in diameter that was not observed when a PGE2 antagonist (ONO-AE3-208) was added (Figures 25A-C). In support of this finding, an increase in protein synthesis, as quantified by puromycin uptake, was observed after PGE2 treatment of myotubes (Figure 25D). Treatment with SW did not affect the diameter of cultured myotubes (Figures 25A and 25B), consistent with its indirect mechanism of inhibiting 15-PGDH expression by resident stromal cells in aging muscle tissue. These in vitro data demonstrate that PGE2 can directly act on myotubes to activate AKT signaling and enhance myotube growth and protein synthesis, providing evidence for the previously investigated role of PGE2 in combating muscle atrophy.

[0221] Decreased protein degradation and TGF-β signaling after 15-PGDH inhibition in aging muscle We sought to determine in vivo in aging muscle tissue whether elevated PGE2 by 15-PGDH inhibition results in signaling through the AKT / FOXO pathway, as seen in myotubes in vitro. We found that pFOXO increased in SW-treated aging muscle compared with vehicle-treated controls (Figure 24G). FOXO has previously been shown to play a role in reducing the expression of the muscle-specific atrophy-associated E3 ubiquitin ligases atrogin-1 (Fbxo32), MuRF1 (Trim63), Musa1, and Smart (30-32). RT-qPCR analysis revealed that expression of all of these muscle atrophy-related genes, as well as the E3 ubiquitin ligase Traf6 (33), was decreased in SW-treated aged muscle compared with vehicle-treated controls (Figures 24E, 24H, and 26A), suggesting that regulation of protein degradation contributes to the attenuation of muscle atrophy. This finding is consistent with our transcriptome analysis of aged compared with young muscle, which showed that genes in the ubiquitin ligase pathway were among the top enriched upregulated genes in aged muscle (Figures 12A–D), and is consistent with findings by others that muscle atrophy-related gene expression increases with aging (34–36). We observed a similar decrease in E3 ubiquitin ligase expression after genetic inhibition of the 15-PGDH enzyme in aging muscle, mediated by intramuscular delivery of shRNA to 15-PGDH, compared with scr shRNA controls (Figure 24I). Interestingly, expression of the histone deacetylase Hdac4, another mediator of muscle atrophy that deacetylates proteins such as MyHC and PGC1α, leading to their ubiquitination, and increases the expression of the muscle atrophy-related genes atrogin-1 and MuRF1 (37, 38), was decreased in SW-treated muscle (Figure 24E). These results indicate that PGE2 suppresses the increased protein degradation seen in aging muscle and regulates muscle atrophy-related gene expression, contributing to the observed improvement in muscle atrophy in aging muscle.

[0222] Our transcriptome analysis revealed a decrease in a second signaling pathway, the TGF-β pathway, after 1 month of SW treatment, providing evidence of another synergistically beneficial effect of 15-PGDH inhibition on aging muscle. Expression of key TGF-β pathway genes (Mstn, Tgfb2, Acrv2a, Smad3), such as myostatin, known to be detrimental to muscle function and associated with age-related muscle atrophy in aging (27), was decreased, likely contributing to the observed attenuation of muscle atrophy (Figure 24E). Notably, no significant changes were observed in other assayed markers of aging, inflammation, and autophagy in the muscles of SW-treated aging mice (Figure 26B-D). Together, these results indicate that 1 month of 15-PGDH inhibition and the resulting elevation of PGE2 in aging muscle stimulate several synergistic signaling pathways that lead to improved muscle function and attenuated atrophy in aging mice.

[0223] Consideration Skeletal muscle constitutes 40% of body mass. After age 50, humans lose an average of 15% of muscle mass per decade (39), leading to the dramatic loss of muscle strength characteristic of sarcopenia. There is currently no cure for sarcopenia, and its healthcare burden is high (2). Here, we discover that elevated expression of the prostaglandin-degrading enzyme 15-PGDH is a novel marker of aging muscle in both mice and humans. We find that increased 15-PGDH activity is not limited to muscle but is a characteristic of many aging tissues, including the aging heart, skin, colon, and spleen. The crucial role of 15-PGDH in aging is highlighted by the finding that overexpression of this enzyme causes muscle wasting in young mice. In aging mice, inhibition of 15-PGDH, either by gene knockdown or small molecules, counteracts muscle atrophy and significantly increases muscle mass, strength, and endurance. Using mass spectrometry and targeted loss-of-function experiments, we demonstrate that improved muscle function is due to increased PGE2 levels. We previously demonstrated the importance of PGE2 signaling in stimulating stem cells to regenerate damaged tissue in young mice (7-10). Here, we demonstrate that PGE2 also acts on mature muscle myofibers and plays a critical role in maintaining muscle tissue homeostasis. Importantly, our data suggest that 15-PGDH constitutes a therapeutic target for combating the debilitating muscle atrophy characteristic of sarcopenia.

[0224] To our knowledge, there have been no previous reports demonstrating that increased 15-PGDH activity leads to decreased PGE2 levels in aging tissues. Our study benefited from an LC-MS / MS method capable of reliably resolving and quantifying highly similar members of the prostaglandin family in skeletal muscle. Thus, we were able to demonstrate the magnitude of the decline in PGE2 in aging muscle and link 15-PGDH to that decline. The importance of this enzyme in the atrophy phenotype is underscored by the finding that overexpression of this enzyme in young muscle leads to significant loss of muscle mass and strength within one month. Collectively, our data highlight the causal role of 15-PGDH in the decline of muscle mass and function. Given that we have detected increased 15-PGDH in many other aging tissues, this finding may have broad implications for age-related pathologies.

[0225] Our data suggest that intercellular signaling mechanisms play a role in the decrease in PGE2 in aging muscle. After either senolytic treatment or genetic ablation of senescent cells in aging muscle, 15-PGDH levels decrease and a concomitant increase in PGE2 is observed. These results implicate senescent interstitial cells within the aging muscle environment as the primary site of PGE2 catabolism. Among the senescent inflammatory cell types present in the aging muscle niche, macrophages appear to be the primary cell type expressing 15-PGDH and degrading PGE2. These cells appear to act indirectly through paracrine mechanisms to contribute to the muscle-wasting phenotype termed "inflammation" (43). This adverse microenvironment could be overcome by eliminating senescent interstitial cells through senolytic treatment or by inhibiting 15-PGDH expression in aging muscle, both of which would increase endogenous PGE2 levels sufficiently to attenuate muscle atrophy. Future studies are needed to investigate this paracrine mechanism in detail. We hypothesize that similar tissue-resident senescent stromal cells are responsible for the elevation of 15-PGDH that we have detected in other aging tissues.

[0226] Previous studies on the role of PGE2 in muscle protein homeostasis suggested that PGE2 induces protein degradation, but these studies were performed on denervated, excised muscles, which undergo rapid muscle protein catabolism caused by muscle removal from the body (44, 45). In contrast, we provide evidence here that in live mice, inhibition of 15-PGDH prevents PGE2 degradation and regulates endogenous PGE2 levels within a physiological range sufficient to ameliorate muscle atrophy. Our data are consistent with previous studies in which perturbation of COX enzyme levels revealed the role of prostaglandins in muscle hypertrophy and recovery from muscle atrophy (22, 46, 47). However, COX2 is not an ideal therapeutic target because it is important for the synthesis of antagonistic prostaglandins. Here, we uncover a previously unrecognized link between PGE2 signaling and muscle atrophy through multiple signaling pathways (TGF-β, cAMP / CREB, AKT / FOXO, and mitochondrial function) that synergize to enhance muscle function and attenuate muscle atrophy.

[0227] Sarcopenia is a multifactorial disease that brings together dysregulated signaling pathways that result in chronic inflammation, muscle denervation, mitochondrial defects, and disrupted proteostasis (4, 48, 49). In particular, mitochondrial function is impaired (50). To elucidate the mechanisms underlying the beneficial effects of 15-PGDH inhibition on muscle function, we chose an unbiased approach. Transcriptome analysis comparing aging muscles after 1 month of treatment with a small-molecule inhibitor of 15-PGDH with vehicle-treated controls revealed that mitochondrial function was among the top upregulated pathways. PGE2 signaling via the EP4 receptor via cAMP / CREB may be a key factor in the observed increase in mitochondrial number and function, consistent with previous reports (12, 22, 23). Similar to the beneficial effects on skeletal muscle previously demonstrated for other cAMP inducers, such as β-adrenergic receptor (β-AR) agonists or corticotropin-releasing factor receptor 2 (CRFR2) agonists, PGE2 induction of cAMP may enhance mitochondrial function by activating downstream transcription factors with cAMP response elements (CREB-binding motifs) that promote mitochondrial biogenesis, including the key mitochondrial regulator Pgc1α and other oxidative genes (51-53). This signaling cascade leads to increased mitochondrial mass and a significant improvement in muscle atrophy.

[0228] Our transcriptome analysis also revealed key signaling pathways that were downregulated after 1 month of 15-PGDH inhibition, including ubiquitin-proteasome pathway genes. Supporting this finding, transcriptome analysis of aged muscle compared to young muscle showed enrichment for this pathway. Consistent with this, others have reported elevated levels of the E3 ubiquitin ligases atrogin-1 and MuRF1 in aged rat muscle (34, 35) and human muscle (36). Whether ubiquitin ligase expression plays a causal role in sarcopenia remains controversial. Knockout models of specific E3 ubiquitin ligases, including atrogin-1 and MuRF1, have shown detrimental effects on muscle function (54, 55), but beneficial effects in the setting of acute denervation atrophy (27). Notably, these genetic models have not been investigated in the context of aging. Indeed, interventions such as rapalogs, sestrins, and apelin, which reduced muscle atrophy-related gene expression (atrogin-1 and MuRF1) in aging muscle (21, 56, 57), improved muscle mass and function, and ameliorated sarcopenia. Consistent with this, we observed decreased expression of multiple E3-ubiquitin ligases upon 15-PGDH inhibition in aging muscle. Collectively, these data suggest that modulating muscle atrophy-related gene expression is beneficial for aging muscle function. In addition to muscle atrophy-related genes, we observed downregulation of Hdac4, which promotes atrophy, by modulating the levels of E3 ubiquitin ligases MuRF1 and atrogin-1, MyHC, and Pgc1a (37, 38, 48), as well as downregulation of Traf6, an adaptor protein and nonconventional E3 ubiquitin ligase previously associated with muscle atrophy (33). In addition to regulating muscle atrophy-related gene expression, enhanced autophagy has been associated with reversal of aging phenotypes downstream of AKT / FOXO signaling (21, 30), which was not evident in our transcriptome analysis. Here, we show that partial inhibition of 15-PGDH in aging mice results in a reduction in a number of these atrophy markers and improves muscle mass and function.

[0229] We also observed a significant downregulation of a second pathway, the TGF-β signaling pathway, in the transcriptome of SW-treated aging muscles. Myostatin, a prominent member of this family, has a pronounced inhibitory effect on muscle growth, and its loss in knockout animals is associated with dramatic hypertrophy (58). Myostatin signals through activin receptors and downstream Smad transcription factors, turning off the AKT pathway and protein synthesis while triggering the expression of ubiquitin ligases that regulate muscle protein degradation (59). Several genes in the TGF-β pathway, including myostatin, transforming growth factor β-2 (TGFβ-2), and activin receptor type 2A, were significantly reduced in the transcriptome of SW-treated aging muscles.

[0230] In summary, we now identify 15-PGDH as a previously unrecognized marker and therapeutic target for strategies aimed at ameliorating muscle wasting associated with aging and sarcopenia. Our intervention is advantageous because it involves physiologically restoring homeostatic levels of PGE2 in aging mice to those found in young mice. The resulting modest increase in PGE2 levels regulates several signaling pathways to promote mitochondrial biogenesis and function, while inhibiting the TGF-β and ubiquitin proteosome pathways, leading to increased muscle function. Because 15-PGDH activity is elevated in a range of tissues, we hypothesize that its partial inhibition may have beneficial effects extending beyond skeletal muscle during aging.

[0231] References TIFF2025148416000006.tif224151TIFF2025148416000007.tif231151TIFF2025148416000008.tif231151TIFF2025148416000009.tif231151

[0232] material and method. mouse We conducted all experiments and protocols in accordance with the institutional guidelines of Stanford University and the Administrative Panel on Laboratory Animal Care (APLAC). For the aging muscle studies, middle-aged (18-20 months) and aged (>24 months) C57BL / 6 mice were obtained from the National Institute on Aging (NIA), and young (2-4 months) wild-type C57BL / 6 mice were obtained from the Jackson Laboratory. INK-ATTAC mice were generated as previously described (1). INK-ATTAC lifespan assessment: INK-ATTAC mice were bred onto a C57BL6 / J genetic background and housed in a specific-pathogen-free enclosure with a 12-h dark / light cycle for the duration of the study. Male mice were randomized to receive either vehicle or AP20187 (2 mg / kg intraperitoneal injection; B / B homodimerizer, Clontech) twice weekly for baseline lifespan assessment and terminal muscle collection at 12 months of age until assessment and sacrifice at 28 months of age (2). Mice were treated once daily for 1 month by intraperitoneal injection of 5 mg / kg SW033291 (SW) (Cayman Chemicals) or vehicle (10% ethanol, 5% Cremophor EL, 85% D5W (dextrose 5% water)) as previously described (3). Time to exhaustion and distance were measured for SW-treated mice and their controls as previously described (4).

[0233] For ABT-263 treatment, 20-month-old C57 / B16 mice were treated by oral gavage with vehicle (ethanol:polyethylene glycol 400:Phosal 50 PG) or 50 mg / kg / day ABT-263 (in ethanol:polyethylene glycol 400:Phosal 50 PG) for two 1-week cycles with a 2-week rest period between cycles as previously described (5). Young mice received intramuscular injections of PGE2 using either 13 nmol PGE2 (Cayman Chemicals) or vehicle control (PBS) in the TA muscle. The Jackson Laboratory (EP4 flox / flox ;EP4 f / f Transgenic mouse strains were purchased from the Ministry of Health, Labour and Welfare (MHLW) No. 028102 (6). Their genotypes were verified by a suitable PCR-based strategy. Male mice were used for the study.

[0234] Primary cell isolation using FACs Myogenic cells were isolated and enriched as previously described (6-9). Briefly, hindlimb muscles were minced and digested with a MACs Dissociator (Miltenyi) using collagenase and dispase solutions. FACs were used to identify myogenic stem and progenitor cells, as well as cells negative for hematopoietic lineages and non-myogenic cells (CD45). - / CD11b - / CD31 - / Sca1 - ) and α7-integrin + Cell markers were selected. For macrophage isolation, a7 - / Cd11b + / Cd11c - / F4 / 80 + For endothelial cells, a7 - / Cd11b - / Cd11c - / CD31 + Flow cytometry scatter plots were generated and analyzed using FlowJo v10.0.

[0235] Intramuscular AAV9 delivery of shRNA and MCK-Cre An shRNA (NM_008278) against Hpgd (15-PGDH) was integrated into AAV9 under the U6 promoter with eGFP (AAV9-eGFP-U6-sh15PGDH) (Vector Biolabs). Control mice were treated with a similar construct containing a scrambled peptide sequence instead of sh15PGDH (AAV9-eGFP-U6-shscr). Cre was integrated into AAV9 under the muscle-specific tMCK promoter with eGFP (AAV9-tMCK-eGFP-WPRE) (Vector Biolabs). Overexpression of Hpgd (15-PGDH) was achieved by integrating AAV9 under the CMV promoter with eGFP (Vector Biolabs) under the IRES (AA9-CMV-m-HPGD-IRES-eGFP). The control virus was AAV9-tMCK-eGFP-WPRE. Knockdown of Ptdgs was achieved using AAV9 integrated under U6 promoter dependency (AAV9-GFP-U6-m-PTGDS-shRNA), and control mice were injected with scramble (AAV9-GFP-U6-scrmb-shRNA) (Vector Biolabs) at a final concentration of 2 × 10 11 GC / GA. C57Bl / 6 mice aged 3–4 months or >24 months were injected with GC / GA at a final concentration of 2 × 10 11 Two intramuscular injections into the gastrocnemius (GA) of a 20 µl dilution of the above AAV9 particles in PBS to a final concentration of 2 x 10 particles / GA. 11 A single intramuscular injection into the tibialis anterior (TA) muscle was administered to achieve GC / TA.

[0236] Immunofluorescence staining and imaging Recipient tibialis anterior (TA) or gastrocnemius (GA) muscle tissue was collected and prepared for histological examination as previously described ( 6 ). Cross sections or isolated myofibers obtained from muscles were fixed using 4% PFA, blocked and permeabilized using PBS / 1% BSA / 0.1% Triton X-100, and incubated with biotin-anti-CD11b (BD Biosciences, catalog no. 553309, 1:100), anti-15-PGDH (Novus Biologicals, catalog no. NB200-179SS, 1:100), or anti-laminin (Millipore, clone A5, catalog no. 05-206, 1:200). Subsequently, AlexaFluor secondary antibodies (Jackson ImmunoResearch Laboratories, 1:200), streptavidin-Cy3 (Biolegend, 1:500), or wheat germ agglutinin-Alexa647 conjugate (WGA, Thermo Fisher Scientific) were used. Nuclei were counterstained with DAPI (Invitrogen).

[0237] Fiber typing was performed by immunohistochemistry on frozen 10 μM sections mounted on glass slides. Air-dried sections were immediately blocked in PBS / 1% goat serum for 1 h at room temperature and immunostained overnight at 4°C using antibodies against MHC2a (DSHB SC71, 1:1000), MHC2b (DSHB BF-F3, 1:100) (10, 11), and laminin (Millipore, clone A5, catalog no. 05-206, 1:200) diluted in PBS / 1% goat serum. Secondary antibodies against IgG1 Alexa 488, IgM Alexa 405, and IgG2b Alexa 647 (Jackson ImmunoResearch Laboratories, 1:500) diluted in PBS / 1% BSA were applied for 1 h at room temperature, followed by nuclear counterstaining with DAPI (Invitrogen). Images were acquired using a KEYENCE BZ-X700 all-in-one fluorescence microscope with a 20× / 0.75 NA objective, and individual fields were stitched and analyzed using Keyence Advanced Analysis Software.

[0238] Cultured myotubes were fixed with 4% PFA, blocked and permeabilized with PBS / 1% BSA / 0.1% Triton X-100, and stained with a primary antibody, anti-MYH (Thermo Fisher Scientific, catalog number 14-6503-82, clone MF-20, 1:500), followed by an AlexaFluor secondary antibody (Jackson ImmunoResearch Laboratories, 1:500). Nuclei were counterstained with DAPI (Invitrogen). Images were acquired using a KEYENCE BZ-X700 all-in-one fluorescence microscope (Keyence) with a 20x / 0.75 NA objective. Fiber area was analyzed using Keyence Advanced Analysis Software. For fiber area, either the entire maximal cross-sectional area of ​​the muscle was quantified, or at least 10 fields encompassing more than 400 myofibers, laminin-stained or WGA-stained myofiber cross-sections, were captured for each mouse as described above. For fiber typing analysis, we used the MATLAB application SMASH (Semi-Automatic Muscle Analysis Using Segmentation of Histology) as previously described (12). Data analysis was blinded; the investigators who performed image acquisition and scoring were unaware of the treatment conditions given to the analyzed sample groups.

[0239] cell culture DMEM / F10(50:50), 15%FBS, 2.5ng ml -1Primary mouse myoblasts were grown in myogenic cell culture medium containing fibroblast growth factor-2 and 1% penicillin-streptomycin. Primary human progenitor cells derived from the pectoral muscles of two 59-year-old women were grown using SkGM-2 skeletal muscle growth medium (Lonza, CC-3245) as previously described (13). For differentiation experiments, confluent myoblasts were grown in medium containing 5% horse serum and DMEM. Day 4 differentiated mouse myotubes or day 7 differentiated human myotubes were supplemented with 10 ng / ml prostaglandin E2 (Cayman Chemicals), 1 μM SW033291 (ApexBio), or 1 μM ONO-AE3-208 (Cayman Chemicals).

[0240] Protein synthesis by in vitro SUnSET. Protein synthesis rates were monitored using the SUnSET assay as previously described (4). Briefly, puromycin was added to the culture medium at 1 μg / ml 10 min before cell harvest. As a control, cycloheximide was added to block protein translation. Cell extracts were then processed for Western blotting using anti-puromycin 12D10 antibody (Millipore).

[0241] Quantitative RT-PCR RNA was isolated from MuSCs, myoblasts, and myotubes using the RNeasy Kit (Qiagen). Muscle samples were quickly frozen in liquid nitrogen and then homogenized in Trizol (Invitrogen) using a FastPrep FP120 homogenizer (MP Biomedicals) before RNA isolation. cDNA was reverse transcribed from total mRNA from each sample using the SensiFAST™ cDNA Synthesis Kit (Bioline). cDNA was subjected to RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems) or TaqMan Assays (Applied Biosystems) on an ABI 7900HT Real-Time PCR System (Applied Biosystems). Samples were cycled at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. To quantify relative transcript levels, treated and untreated samples were compared using 2-ΔΔCt and results were expressed relative to Gapdh.

[0242] For SYBR Green qRT-PCR, the following primer sequences were used: TIFF2025148416000010.tif121131TIFF2025148416000011.tif120128.

[0243] For mouse senescence and senescence-associated markers, we used previously described primers (2). TaqMan Assays (Applied Biosystems) were used to quantify p21, Mstn, Ptger3, and Ptger4 in samples using the TaqMan Universal PCR Master Mix reagent kit (Applied Biosystems) according to the manufacturer's instructions. Transcript levels were expressed relative to Gapdh levels. For SYBR Green qPCR, Gapdh qPCR was used to normalize input cDNA samples. For TaqMan qPCR, multiplex qPCR allowed target signals (FAM) to be individually normalized by their internal Gapdh signal (VIC). Mitochondrial copy number was quantified using previously described methods and primers (14).

[0244] Microarray data The publicly available repository Gene Expression Omnibus ( ncbi.nlm.nih.gov / geo / ) Microarray gene expression profiles were collected. Microarray data from GSE25941 (15) were analyzed for Hpgd expression.

[0245] RNA-Seq For RNA-seq, RNA was isolated from muscle lysates using Trizol reagent (Thermoscientific) and purified using the Qiagen RNAEasy kit from the University of California, San Diego, Calif. Libraries were constructed from the RNA using the TruSEQ RNA Library Prep Kit v2 (Illumina) and analyzed at 30-40 × 10 bp using a NextSeq 550 at the Stanford Functional Genomics Facility. 6 × 75-bp reads / sample.

[0246] RNA-seq analysis For RNA-Seq analysis, sequences were aligned to the house mouse (Mus musculus) genome (mm9) using STAR (16). Transcripts were called using RSEM, and transcripts per million (TPM) values ​​and total counts were calculated (17). A count matrix containing the number of counts for each gene and each sample was obtained. This matrix was analyzed using DESeq2 to calculate statistical analysis of gene significance across samples (18). Up- or down-regulated genes with a p-value cutoff of <0.05 were used for pathway analysis using DAVID (19). Heatmaps were created with normalized counts and plotted as row-wide Z-scores using Python's Seaborn data visualization library. The data reported in this editorial have been deposited in the Gene Expression Omnibus (GEO) database GSE149924.

[0247] Protein extraction and immunoblotting Total lysates were prepared using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors, and micrococcal nuclease). For tissue extracts, lysates were homogenized in a FastPrep 24 homogenizer (MP Biomedicals) at a speed of 6 m / s for 40 s. The following antibodies were used: 15-PGDH (Santa Cruz Biotechnology, catalog number sc-271418); phospho-AKT (Ser473) (Cell Signaling catalog number 4060), AKT (Cell Signaling catalog number 2920); phospho-FoxO1 (Thr24) / FoxO3a (Thr32) antibody (Cell Signaling catalog number 9464T); Foxo3a (Cell Signaling catalog number 2497); phospho-CREB (Ser133) (Cell Signaling catalog number 9198S); phospho-S6 ribosomal protein (Ser235 / 236) (Cell Signaling catalog number 4858); SMC1 (Bethyl Laboratories catalog number A300-055A-T). HRP-conjugated secondary antibodies were used, and the membranes were incubated with ECL Western blotting substrate (Nacalai USA) and developed by imaging using a ChemiDoc imaging system (BioRad).

[0248] 15-PGDH kinetic assay 15-PGDH activity in tissue lysates was analyzed using the BioVision PicoProbe 15-PGDH Activity Assay Kit (catalog no. K562) according to the manufacturer's protocol.

[0249] Determination of PGE2 and related prostaglandins in mouse tissues by LC-MS / MS analyte standard Prostaglandin standards (PGF2α; PGE2; PGD2; 15-keto PGE2; 13,14-dihydro15-keto PGE2; PGD2-D4; PGA2; 13,14-dihydro15-keto PGA2; PGE2-D4; and PGF2α-D9) were all purchased from Cayman Chemical. The PGE2-D4 and PGD2-D4 internal standards were labeled with four deuterium atoms at positions 3 and 4. PGF2α-D9 was labeled with nine deuterium atoms at positions 17, 18, 19, and 20.

[0250] Creating a calibration curve Analyte stock solutions (5 mg / mL) were prepared in DMSO. These stock solutions were serially diluted with acetonitrile / water (1:1 v / v) to obtain a series of standard working solutions, which were used to generate calibration curves. Calibration curves were generated by adding 10 μL of each standard working solution to 200 μL of homogenization buffer (acetone / water 1:1 v / v; 0.005% BHT to prevent oxidation), followed by 10 μL of internal standard solution (3000 ng / mL each of PGF2α-D9, PGD2-D4, and PGE2-D4). A new calibration curve was generated for each set of samples. Calibration curve range: 0.05 ng / mL to 500 ng / mL for PGA2, PGD2, and 13,14-dihydro15-ketoPGE2; 0.1 ng / mL to 500 ng / mL for PGE2, 13,14-dihydro15-ketoPGA2, and PGF2α; 0.25 ng / mL to 500 ng / mL for 15-ketoPGE2.

[0251] Sample preparation procedure The extraction procedure was modified from that of Prasain et al. (20) and included acetone protein precipitation followed by a two-step liquid-liquid extraction, the latter step enhancing LC-MS / MS sensitivity. Oxidation was prevented using butylated hydroxytoluene (BHT) and evaporation under nitrogen (N2) gas. Solid tissue was harvested, weighed, and flash-frozen using liquid nitrogen. Muscle tissue was combined with homogenization beads and 200 μL of homogenization buffer in a polypropylene tube and processed in a FastPrep 24 homogenizer (MP Biomedicals) at a speed of 6 m / s for 40 s. After homogenization, 10 μL of internal standard solution (3000 ng / mL) was added to the tissue homogenate, followed by shaking for 2 min (Multi-Tube Vortexer, Thermo Scientific). The sample was centrifuged, and the supernatant was transferred to a clean Eppendorf tube. 200 μL of hexane was added to the sample, which was shaken for 15 minutes (Vortex Mixer, Thermo Scientific) and then centrifuged. The sample was frozen at -80°C for 40 minutes. The hexane layer was poured off the frozen lower aqueous layer and discarded. After thawing, 25 μL of 1 N formic acid was added to the bottom aqueous layer, and the sample was vortexed. For the second extraction, 200 μL of chloroform was added to the aqueous phase. The sample was shaken for 15 minutes to ensure complete extraction. The layers were separated by centrifugation. The lower chloroform layer was transferred to a new Eppendorf tube and evaporated to dryness under nitrogen at 40°C. The dried residue was reconstituted in 100 μL of acetonitrile / 10 mM ammonium acetate (2:8 v / v) and analyzed by LC-MS / MS.

[0252] LC-MS / MS Chromatographic separation is important because many prostaglandins are positional isomers with identical masses and similar fragmentation patterns. At least two SRM transitions, one quantitative ion and one qualitative ion, were carefully selected for each analyte. Clear qualitative-to-quantitative ion intensity ratios and retention times were essential for authenticating the target analytes. All analyses were performed on an LC-20 AD XRAnalysis was performed by negative electrospray LC-MS / MS using a Prominence liquid chromatograph and an 8030 triple quadrupole mass spectrometer (Shimadzu). HPLC conditions: An Acquity UPLC BEH C18 2.1 x 100 mm, 1.7 um particle size column was operated at 50 °C with a flow rate of 0.25 mL / min. The mobile phase consisted of A: 0.1% acetic acid in water and B: 0.1% acetic acid in acetonitrile. The elution profile was an initial 5-minute hold at 35% B, followed by a gradient from 35% to 40% in 3 minutes, then 40% to 95% in 3 minutes; the total run time was 14 minutes. The injection volume was 20 μL. Using these HPLC conditions, baseline separation of the analytes of interest was achieved. Selected reaction monitoring (SRM) was used for quantitation.The mass transitions were as follows: PGD2: m / z 351.10 → m / z 271.3 (quantitative ion); m / z 351.10 → m / z 233.05 (qualitative ion) and m / z 351.10 → m / z 189.15 (qualitative ion); PGE2: m / z 351.20 → m / z 271.10 (quantitative ion); m / z 351.20 → m / z 333.15 (qualitative ion) and m / z 351.20 → m / z 315.20 (qualitative ion); PGF2α: m / z 353.10 → m / z 3193.3 (quantitative ion) and m / z 353.10 → m / z 309.20 (qualifying ion); 15-keto-PGE2: m / z 349.30 → m / z 331.20 (quantifying ion) and m / z 349.30 → m / z 113.00 (qualifying ion); 13,14-dihydro-15-keto-PGE2: m / z 351.20 → m / z 333.30 (quantifying ion) and m / z 351.20 → m / z 113.05 (qualifying ion); PGE2-D4: m / z 355.40 → m / z 275.20 (quantifying ion); PGF2α-D9: m / z 362.20 → m / z 318.30; PGD2-D4: m / z 355.10 → m / z 275.40; PGA2: m / z 332.90 → m / z 271.25 (quantitative ion) and m / z 332.90 → m / z 189.10 (qualitative ion); and 13,14-dihydro-15-keto PGA2: m / z 332.90 → m / z 235.15 (quantitative ion) and m / z 332.90 → m / z 113.00 (qualitative ion). The dwell time was 20–30 ms.

[0253] Quantitative data analysis was performed using LabSolutions LCMS (Shimadzu). An internal standard method was used for quantification. PGE2-D4 was the internal standard for the quantification of PGE2, 15-keto PGE2, and 13,14-dihydro 15-keto PGE2, PGA2; 13,14-dihydro 15-keto PGA2. PGF2α-D9 was the internal standard for the quantification of PGF2α. PD2-D4 was the internal standard for the quantification of PGD2. Calibration curves were plotted as 1 / X, where X is the concentration. 2The concentration range was linear (R>0.99) using a weighting factor of . Back-calculated standard concentrations were ±15% from the nominal value and ±20% at the lower limit of quantitation (LLOQ).

[0254] In vivo muscle force measurement Peak isometric torque (N·mm) of the ankle plantarflexors was assessed as previously described (21, 22). Briefly, the foot of an anesthetized mouse was placed on a footplate attached to a servomotor (Model 300C-LR; Aurora Scientific). Two Pt-Ir electrode needles (Aurora Scientific) were inserted percutaneously and subcutaneously onto the tibial nerve just posterior / medial to the knee. The ankle joint was immobilized at a 90° angle. Peak isometric torque was achieved by varying the current delivered to the tibial nerve with a 200 Hz frequency and 0.1-ms square-wave pulse duration. Three tetanic measurements were performed for each muscle, with a 1-minute recovery between measurements. Data were collected using Aurora Scientific Dynamic Muscle Data Acquisition and Analysis Software.

[0255] statistical analysis The nonparametric Mann-Whitney test was used to determine significant differences between untreated and treated groups using α = 0.05. ANOVA or multiple t-tests were performed for multiple comparisons, and significance levels were determined using Bonferroni correction as indicated in the figure legends, or by Fisher's test. Data are presented as mean ± SEM unless otherwise noted.

[0256] References TIFF2025148416000012.tif39150TIFF2025148416000013.tif231151TIFF2025148416000014.tif45150

[0257] Example 3. Targeting prostaglandin E2 degrading enzymes to improve non-skeletal muscle tissue function in age-related diseases and conditions As we age, quality of life declines and mortality increases. Age-related diseases are a group of conditions that occur more frequently in people as they age and directly correlate with decreased life expectancy (1). These conditions include cardiovascular disease (atrial fibrillation, stroke, ischemic heart disease, cardiomyopathy, endocarditis, intracerebral hemorrhage), chronic respiratory disease (chronic obstructive pulmonary disease, asbestosis, silicosis), nutritional diseases (trachoma, diarrheal diseases, encephalitis), kidney disease (chronic kidney disease), gastrointestinal and digestive diseases (NASH, pancreatitis, ulcers, intestinal obstruction), neurological disorders (Alzheimer's disease, dementia, Parkinson's disease), sensory disorders (hearing loss, macular degeneration, glaucoma), skin and subcutaneous diseases (cellulitis, ulcers, fungal skin diseases, pyoderma), osteoporosis, osteoarthritis, and rheumatoid arthritis (2).

[0258] We previously determined that PGE2 stimulates muscle stem cells (MuSCs) to regenerate damaged muscle in young mice (3), in agreement with findings regarding its function in the regeneration of other tissues, including bone, colon, liver, and blood (4-6). We reasoned that PGE2 signaling may be impaired during aging. Here, we demonstrate a previously unrecognized role for the PGE2-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), in aging tissues. Partial inhibition of 15-PGDH can restore PGE2 and / or PGD2 to youthful levels, thereby rejuvenating tissue function. Our findings provide new insights into aging and reveal innovative therapeutic strategies.

[0259] We hypothesized that the decrease in PGE2 is due to increased degradation by 15-PGDH in aging tissues (Figure 27A). We found that the specific activity of this enzyme actually increases in aging tissues, including the heart, skin, spleen, and colon (Figures 27B and 28). Therefore, inhibition of 15-PGDH may help ameliorate age-related diseases and pathologies by restoring or increasing PGE2 and / or PGD2 levels in aging tissues.

[0260] We identify 15-PGDH as a novel marker of aging, detectable upon elevated activity in multiple tissues, including the heart, skin, colon, and spleen. Thus, restoring PGE2 and / or PGD2 to youthful levels may have pleiotropic ameliorative effects, as 15-PGDH is upregulated in various tissues with aging.

[0261] References TIFF2025148416000015.tif93151

[0262] material and method mouse All experiments and protocols were conducted in accordance with the institutional guidelines of Stanford University and the Administrative Panel on Laboratory Animal Care (APLAC). For aging muscle studies, aged (>24 months) C57BL / 6 mice were obtained from the National Institute on Aging (NIA), and young (2-4 months) wild-type C57BL / 6 mice were obtained from the Jackson Laboratory.

[0263] 15-PGDH kinetic assay 15-PGDH activity in tissue lysates was analyzed using the BioVision PicoProbe 15-PGDH Activity Assay Kit (Cat. No. K562) according to the manufacturer's protocol. Briefly, tissues were isolated and flash-frozen in liquid nitrogen. Total lysates were prepared using lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM CaCl, 1.5% Triton X-100, protease inhibitors, and micrococcal nuclease) and homogenized for 40 seconds at 6 m / s using a FastPrep 24 homogenizer (MP Biomedicals).

[0264] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

[0265] Sequence information SEQUENCE LISTING <110> THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY <120> METHODS OF REJUVENATING AGED TISSUE BY INHIBITING 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE (15-PGDH) <150> US 62 / 883,025 <151> 2019-08-05 <150> US 62 / 882,981 <151> 2019-08-05 <150> US 62 / 875,915 <151> 2019-07-18 <150> US 62 / 860,180 <151> 2019-06-11 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 ttcaccacca tggagaaggc 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 cccttttggc tccaccct 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 3 tccagtgtga tgtggctgac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 4 attgttcacg cctgcattgt 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 5 gtggtgtcgt gcatctgct 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 ccgctgcagg gagttagagt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 accttcgcca tatgctcctt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 ggaccggtgg cctaagtatg 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 aacccagggg atcgagtgt 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 cgcagctcag tgtttgggat 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 tagtaaggct gttggagctg atag 24 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 ctgcaccagt gtgcataagg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400>...

Claims

1. A method for enhancing the function of aging skeletal muscle in a subject, comprising administering to the aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in one or more senescent cells within the aging skeletal muscle, thereby enhancing the function of the aging skeletal muscle.

2. A method for increasing muscle mass, strength, and / or endurance of aging skeletal muscle in a subject, comprising administering to the aging skeletal muscle an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in one or more senescent cells within the aging skeletal muscle, thereby increasing muscle mass, strength, and / or endurance of the aging skeletal muscle.

3. A method for increasing the level of PGE2 in aged skeletal muscle of a subject, the method comprising the step of administering to the aged skeletal muscle an amount of a 15-PGDH inhibitor effective to increase the level of PGE2 in the aged skeletal muscle, thereby increasing the level of PGE2 in the aged skeletal muscle.

4. The method of any one of claims 1 to 3, wherein the subject has one or more biomarkers of aging.

5. A method for rejuvenating aging skeletal muscle in a subject having one or more biomarkers of aging, comprising administering to the subject having one or more biomarkers of aging an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the subject, thereby rejuvenating the aging skeletal muscle.

6. 6. The method of claim 4 or 5, wherein the one or more biomarkers of aging are selected from the group consisting of an increased level of 15-PGDH compared to the level present in young skeletal muscle, a decreased level of PGE2 compared to the level present in young skeletal muscle, an increased level of PGE2 metabolites compared to the level present in young skeletal muscle, an increased or greater accumulation of senescent cells compared to the level present in young skeletal muscle, an increased expression of one or more muscle atrophy-related genes (atrogenes) compared to the level present in young skeletal muscle, a decrease in mitochondrial biogenesis and / or mitochondrial function compared to the level present in young skeletal muscle, and an increased transforming growth factor pathway signaling compared to the level present in young skeletal muscle.

7. The method of claim 6, wherein the one or more muscle atrophy-related genes are selected from the group consisting of Atrogin 1 (MAFbx1), MuSA (Fbxo30), and Trim63 (MuRF1).

8. The method of claim 6, wherein the increased transforming growth factor pathway signaling comprises increased expression of one or more genes selected from the group consisting of activin receptors, myostatin, SMAD proteins, and bone morphogenetic proteins.

9. The method of any one of claims 1 to 8, wherein the accumulation of senescent cells is increased in aged skeletal muscle compared to young skeletal muscle.

10. 10. The method of any one of claims 1, 2, or 9, wherein said senescent cells express one or more senescence markers.

11. 11. The method of any one of claims 1, 2, 9, or 10, wherein the senescent cells have increased levels of one or more senescence markers compared to non-senescent cells.

12. 12. The method of claim 10 or 11, wherein the one or more senescence markers are selected from the group consisting of p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6.

13. 13. The method of any one of claims 1, 2, or 19-12, wherein the senescent cells are macrophages.

14. 14. The method of any one of claims 1 to 13, wherein the aged skeletal muscle has not been injured and / or has not undergone exercise and / or has not undergone regeneration.

15. The method of any one of claims 1 to 14, further comprising administering a senolytic agent to the aged skeletal muscle.

16. 16. The method of claim 15, wherein the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a combination therapy of dasatinib and quercetin, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof.

17. 17. The method of any one of claims 1 to 16, wherein the 15-PGDH inhibitor is selected from the group consisting of a small molecule compound, a blocking antibody, a nanobody, and a peptide.

18. The method of any one of claims 1 to 17, wherein the 15-PGDH inhibitor is SW033291.

19. The method of any one of claims 1 to 16, wherein the 15-PGDH inhibitor is selected from the group consisting of an antisense oligonucleotide, a microRNA, an siRNA, and an shRNA.

20. The method of any one of claims 1 to 19, wherein the subject is a human.

21. 21. The method of any one of claims 1 to 20, wherein the subject is at least 30 years old.

22. 22. The method of any one of claims 1 to 21, wherein said administering comprises systemic administration or local administration.

23. 23. The method of any one of claims 1 to 22, wherein the level of PGE2 is increased in aged skeletal muscle compared to the level of PGE2 present in the aged skeletal muscle before administration of the 15-PGDH inhibitor.

24. 24. The method of any one of claims 1 to 23, wherein the level of PGE2 is increased by at least 10% compared to the level of PGE2 present in aged skeletal muscle before administration of the 15-PGDH inhibitor.

25. 25. The method of any one of claims 1 to 24, wherein the level of PGE2 is increased to a level substantially equivalent to that present in young skeletal muscle.

26. 26. The method of any one of claims 1 to 25, wherein the level of PGE2 is increased to a level that is within about 50% or less of the level present in young skeletal muscle.

27. 27. The method of any one of claims 1 to 26, which results in an increase in the cross-sectional area and / or diameter of muscle fibers and / or myotubes.

28. 28. The method of any one of claims 1 to 27, which results in an increase in the cross-sectional area and / or diameter of oxidative (type IIa) and / or glycolytic (type IIb) fibers.

29. 29. The method of any one of claims 1 to 28, wherein the 15-PGDH inhibitor reduces or blocks 15-PGDH expression.

30. 30. The method of any one of claims 1 to 29, wherein the 15-PGDH inhibitor reduces or blocks the enzymatic activity of 15-PGDH.

31. 31. The method of any one of claims 1 to 30, wherein the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, of said aged skeletal muscle.

32. 32. The method of any one of claims 1 to 31, wherein the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, in aged skeletal muscle compared to aged skeletal muscle before administration of the 15-PGDH inhibitor.

33. 33. The method of any one of claims 1-32, wherein the method results in an increase in muscle mass, muscle strength, muscle endurance, or any combination thereof, in said aged skeletal muscle to a level substantially equivalent to that present in young skeletal muscle.

34. 34. The method of any one of claims 1-33, wherein the method results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, in said aged skeletal muscle to a level that is within about 50% or less of the level present in young skeletal muscle.

35. 35. The method of any one of claims 1 to 34, which results in enhanced function of said aged skeletal muscle.

36. 36. The method of any one of claims 1 to 35, which results in enhanced function of aged skeletal muscle compared to aged skeletal muscle before administration of the 15-PGDH inhibitor.

37. 37. The method of any one of claims 1 to 36, resulting in an enhancement of the function of said aged skeletal muscle to a level substantially equivalent to that present in young skeletal muscle.

38. 38. The method of any one of claims 1-37, resulting in an enhancement of the function of said aged skeletal muscle to a level that is within about 50% or less of the level present in young skeletal muscle.

39. 39. The method of any one of claims 35 to 38, wherein said function is increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof.

40. 40. The method of any one of claims 1 to 39, wherein the method results in a reduction in the level of PGE2 metabolites in aged skeletal muscle compared to aged skeletal muscle prior to administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle.

41. 41. The method of claim 40, wherein the PGE2 metabolite is selected from the group consisting of 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2.

42. The method of any one of claims 1 to 41, wherein the subject has sarcopenia due to aging.

43. 43. The method of any one of claims 1 to 42, wherein the expression level of one or more muscle atrophy-related genes is reduced compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle.

44. 44. The method of any one of claims 1 to 43, wherein the expression level of one or more components of a mitochondrial complex is increased compared to aged skeletal muscle prior to administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle.

45. 45. The method of claim 44, wherein said one or more components of a mitochondrial complex are selected from the group consisting of Ndufal 1, Ndufal 2, Ndufal 3, Ndufa2, Ndufa3, Ndufa4, Ndufa5, Ndufal O, Ndufb5, Ndufc1, Ndufs4, Ndufs8, Ndufvl, Ndufv2, Uqcrb, Uqcrc1, Uqcrh, Uqcrq, Ucqr10, Cox8b, Cox7a1, Cox7a2, Cox7b, Cox6c, Cox5a, Cox5b, Atp5f1, Atp5g1, Atp5h, Atp5j2, Atp5o, Atp5e, and Atp5k.

46. 46. ​​The method of any one of claims 1 to 45, wherein the expression level of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (Pgc1α) is increased compared to aged skeletal muscle prior to administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle.

47. The method of any one of claims 1 to 46, wherein the expression level of one or more genes selected from the group consisting of Tnfaip1, Klhdc8a, Fbxw11, Tnfaip3, Herc3, Herc2, Hdac4, Traf6, Ankib1, Mib1, Pja2, Ubr3, Thbs1, Smad3, Acvr2a, Rgmb, Tgfb2, and Mstn is reduced compared to aged skeletal muscle before administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young skeletal muscle.

48. 48. The method of any one of claims 1 to 47, wherein the method is independent of increasing proliferation of muscle stem cells (MuSCs) in the subject.

49. 49. The method of any one of claims 1-48, wherein said administering comprises daily, twice-daily, weekly, or monthly administration.

50. A method for rejuvenating aged non-skeletal muscle tissue in a subject, the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the subject, thereby rejuvenating the aged non-skeletal muscle tissue.

51. 51. The method of claim 50, wherein said administration increases the level of PGE2 in aged non-skeletal muscle tissue of said subject.

52. 52. The method of claim 50 or 51, wherein the level of PGE2 in aged non-skeletal muscle tissue is increased compared to aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor.

53. 53. The method of any one of claims 50 to 52, wherein the level of PGE2 in aged non-skeletal muscle tissue is increased by at least 10% compared to aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor.

54. 54. The method of any one of claims 50 to 53, wherein the level of PGE2 in said aged non-skeletal muscle tissue is increased to a level substantially equivalent to that present in young non-skeletal muscle tissue.

55. 55. The method of any one of claims 50-54, wherein the level of PGE2 in the aged non-skeletal muscle tissue is increased to a level that is within about 50% or less of the level present in young non-skeletal muscle tissue.

56. 56. The method of any one of claims 50-55, wherein said aging non-skeletal muscle tissue is selected from the group consisting of epidermal tissue, epithelial tissue, vascular tissue, cardiac muscle, brain, bone, cartilage, sensory organs, kidney, thyroid, lung, smooth muscle, brown fat, spleen, liver, heart, small intestine, colon, skin, ovaries and other reproductive tissues, hair, dental tissue, blood, cochlea, and any combination thereof.

57. 57. The method of any one of claims 50-56, wherein the subject has one or more biomarkers of aging.

58. 58. The method of claim 57, wherein the one or more biomarkers of aging are selected from the group consisting of increased 15-PGDH levels compared to young non-skeletal muscle tissue, decreased PGE2 levels compared to young non-skeletal muscle tissue, increased PGE2 metabolites compared to young non-skeletal muscle tissue, increased or greater accumulation of senescent cells compared to young non-skeletal muscle tissue, increased expression of one or more muscle atrophy-associated genes compared to young non-skeletal muscle tissue, decreased mitochondrial biogenesis and / or mitochondrial function compared to young non-skeletal muscle tissue, and increased transforming growth factor pathway signaling compared to young non-skeletal muscle tissue.

59. 59. The method of any one of claims 50 to 58, wherein said aged non-skeletal muscle tissue has an increased accumulation of senescent cells compared to young non-skeletal muscle tissue.

60. 60. The method of claim 58 or 59, wherein said senescent cells express one or more senescence markers.

61. 61. The method of any one of claims 58-60, wherein the senescent cells have increased levels of one or more senescence markers compared to non-senescent cells.

62. 62. The method of claim 60 or 61, wherein the one or more senescence markers are selected from the group consisting of p15Ink4b, p16Ink4a, p19Arf, p21, Mmp13, Il1a, Il1b, and Il6.

63. The method of any one of claims 60 to 62, wherein the senescent cells are macrophages.

64. 64. The method of any one of claims 50-63, further comprising administering a senolytic agent to said aged non-skeletal muscle tissue.

65. 65. The method of claim 64, wherein the senolytic agent is selected from the group consisting of a Bcl2 inhibitor, a pan-tyrosine kinase inhibitor, a combination therapy of dasatinib and quercetin, a flavonoid, a peptide that interferes with FOXO4-p53 interaction, a selective targeting system for senescent cells using galactooligosaccharide-coated nanoparticles, an HSP90 inhibitor, and combinations thereof.

66. 66. The method of any one of claims 50 to 65, wherein the 15-PGDH inhibitor is selected from the group consisting of a small molecule compound, a blocking antibody, a nanobody, and a peptide.

67. 67. The method of any one of claims 50 to 66, wherein the 15-PGDH inhibitor is SW033291.

68. 66. The method of any one of claims 50 to 65, wherein the 15-PGDH inhibitor is selected from the group consisting of an antisense oligonucleotide, a microRNA, an siRNA, and an shRNA.

69. The method of any one of claims 50 to 68, wherein the subject is a human.

70. 70. The method of any one of claims 50 to 69, wherein the subject is at least 30 years old.

71. 71. The method of any one of claims 50 to 70, wherein the 15-PGDH inhibitor reduces or blocks 15-PGDH expression.

72. 72. The method of any one of claims 50 to 71, wherein the 15-PGDH inhibitor reduces or blocks the enzymatic activity of 15-PGDH.

73. 73. The method of any one of claims 50 to 72, wherein the function of aged non-skeletal muscle is enhanced compared to the function of aged non-skeletal muscle before administration of the 15-PGDH inhibitor.

74. 74. The method of any one of claims 50 to 73, wherein the function of aged non-skeletal muscle tissue is enhanced by at least 10% compared to the function of aged non-skeletal muscle tissue before administration of the 15-PGDH inhibitor.

75. 75. The method of any one of claims 50-74, wherein the function of said aged non-skeletal muscle tissue is enhanced to a level substantially equivalent to that present in young non-skeletal muscle tissue.

76. 76. The method of any one of claims 50-75, wherein the function of said aged non-skeletal muscle tissue is enhanced to a level that is within about 50% or less of the level present in young non-skeletal muscle tissue.

77. 77. The method of any one of claims 73-76, wherein said function comprises increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof.

78. 78. The method of any one of claims 50-77, wherein the method results in a reduction in the level of PGE2 metabolites in aged non-skeletal muscle tissue compared to aged non-skeletal muscle tissue prior to administration of the 15-PGDH inhibitor and / or to a level substantially equivalent to that present in young non-skeletal muscle.

79. 79. The method of claim 78, wherein the PGE2 metabolite is selected from the group consisting of 15-ketoPGE2 and 13,14-dihydro-15-ketoPGE2.

80. 1. A method for enhancing skeletal muscle function in a subject, comprising: the method comprising administering to the subject an amount of a 15-PGDH inhibitor effective to inhibit 15-PGDH activity and / or reduce 15-PGDH levels in the skeletal muscle, thereby enhancing skeletal muscle function in the subject; wherein the skeletal muscle is healthy, and the method is independent of increasing proliferation of muscle stem cells (MuSCs) in the subject; The method.

81. 81. The method of claim 80, wherein the skeletal muscle is undamaged.

82. 82. The method of claim 80 or 81, wherein the skeletal muscle has not undergone regeneration.

83. 83. The method of any one of claims 80-82, wherein said skeletal muscle has not experienced significant or substantial exercise.

84. The method of any one of claims 80 to 83, wherein the function is enhanced compared to skeletal muscle before administration of the 15-PGDH inhibitor.

85. 85. The method of any one of claims 80-84, wherein said function is increased protein synthesis, increased cell proliferation, increased cell survival, decreased protein degradation, or any combination thereof.

86. 86. The method of any one of claims 80-85, which results in increased muscle mass, increased muscle strength, increased muscle endurance, or any combination thereof, compared to skeletal muscle before administration of the 15-PGDH inhibitor.

87. The method of any one of claims 80 to 86, wherein the skeletal muscle is young skeletal muscle.

88. 88. The method of claim 87, wherein the subject is under 30 years of age.

89. 87. The method of any one of claims 80 to 86, wherein the skeletal muscle is aged skeletal muscle.

90. 90. The method of claim 89, wherein the subject is over 30 years of age.

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