Compositions and methods for modulating muscle contraction

Abstract

The present disclosure features compositions and methods for modulating mammalian muscle contraction. The present disclosure also features compositions and methods for the treatment of a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure) in a subject. The methods involve administering flightin polypeptides and / or WYR polypeptides, or polynucleotides encoding the same, as described herein.

Description

[0001] COMPOSITIONS AND METHODS FOR MODULATING MUSCLE CONTRACTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application serial number 63 / 656,807, filed June 6, 2024, and U.S. Provisional Patent Application serial number 63 / 656,796, filed June 6, 2024, each of which is incorporated herein by reference in their entireties.

[0004] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant Nos. MCB- 1050834, IOS-0718417, MCB-0315865, MCB-0090768, 9728868, 9613759, and 9253045, awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Heart failure and cardiomyopathies are commonly associated with underlying conditions that cause contraction and / or relaxation dysfunction. This contraction-relaxation dysfunction often takes the form of abnormal cardiac output by the heart. However, the majority of available treatments for heart failure and cardiomyopathies are aimed at ameliorating the symptoms of these diseases. Therefore, there is a need for treatments which address the underlying conditions, such as undesirable alterations in cardiac muscle contraction and / or relaxation, or abnormal force production or cessation by the heart muscle, directly.

[0008] SUMMARY OF THE INVENTION

[0009] As described herein, the present invention features compositions and methods for modulating mammalian cardiac muscle contraction and / or relaxation featuring a flightin or a WYR polypeptide, and polynucleotides encoding same. In some embodiments, the compositions described herein are useful for the treatment of a cardiac disease or disorder (e.g., a cardiac disorder characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic or mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic or post-infarction cardiac remodeling, or cardiac failure) in a subject.

[0010] In one aspect, the present disclosure provides a method of modifying muscle contraction and / or relaxation. The methods involves contacting a muscle cell with a flightin polypeptide, a WYR polypeptide, e.g., including or consisting of a peptide having at least about 85% identity to the following amino acid sequence or a sequence shown in FIGs. 6A-6C:

[0011] HWVRPKFLQ Y KYMYNYRTNX1YDDVID YIDK KQTX2VAREIP_RPQTWAX3RVLRT, where the WYR peptide includes a W at position 2, R at position 4, P at position 40, R at position 48, and Y at positions 11 and 21, where each of the specified positions is identified by bold and underlining, or a corresponding amino acid position in another WYR peptide, and where X1is Y or C; where X2is G, S, or K; and where X3is E, D, Q, or a polynucleotide encoding said polypeptide or peptide, thereby modifying muscle contraction and / or relaxation function.

[0012] In another aspect, the present disclosure provides a method of modifying a stress response to quick stretch in a muscle cell. The method involves contacting a muscle cell with a flightin polypeptide, a WYR polypeptide, or polynucleotide encoding said polypeptides, thereby modifying stress response to quick stretch in said muscle cell.

[0013] In another aspect, the present disclosure provides a vector, the vector including a polynucleotide encoding a flightin polypeptide, a WYR polypeptide, or a fragment thereof.

[0014] In another aspect, the present disclosure provides a cell expressing a heterologous polypeptide. The heterologous polypeptide is a flightin polypeptide or a WYR polypeptide.

[0015] In another aspect, the present disclosure provides a recombinant cell produced by transforming a cell with any of the vectors provided in any of the above aspects, or embodiments thereof.

[0016] In another aspect, the present disclosure provides a method of treating cardiac hypertrophy in a subject in need thereof or reducing the propensity of a subject to acquire cardiac hypertrophy. The method involves administering to the subject a flightin polypeptide, a WYR polypeptide, or a polynucleotide encoding said polypeptides, thereby treating the cardiac hypertrophy. In another aspect, the present disclosure provides a method of treating cardiac hypertrophy in a subject in need thereof or reducing the propensity of a subject to acquire cardiac hypertrophy. The method involves administering to the subject a mammalian cell expressing a flightin polypeptide or a WYR polypeptide, thereby treating the cardiac hypertrophy.

[0017] In another aspect, the present disclosure provides a method of treating a cardiac disease or disorder in a subject in need thereof, or reducing the propensity of a subject to acquire a cardiac disease or disorder. The method involves administering to the subject a mammalian cell expressing a flightin polypeptide or a WYR polypeptide, thereby treating the cardiac hypertrophy, where the cardiac disease or disorder is characterized by abnormal force production and / or cessation in cardiac muscle.

[0018] In another aspect, the present disclosure provides a method of reducing the risk of hypertrophic cardiomyopathy in a selected subject. The method involves administering a polynucleotide encoding a flightin polypeptide or a WYR polypeptide to the subject, thereby preventing the hypertrophic cardiomyopathy, where the selected subject is selected by detecting in a biological sample of the subject a marker selected from the group consisting of: MYH7 (0- myosin heavy chain 7), MYBPC3 (myosin- binding protein C), TNNT2 (cardiac muscle troponin T), TNNI3 (cardiac troponin I), TPM1 (a-tropomyosin), ACTC1 (cardiac a-actin), MYL2 (myosin lightchain 2), MYL3 (myosin light chain 3), CSRP3 (cysteine and glycine-rich protein 3), TTN (titin), TCAP (telethonin), MY0Z2 (myozenin 2), TRIM63 (ubiquitin E3 ligase tripartite motif protein 63 or MuRFl), and FHL1 (four-and-a-half LIM domains 1), and selecting the subject if mutations in the one or more markers are found.

[0019] In another aspect, the present disclosure provides a kit for use in any of the methods provided in aspect above, or embodiments thereof. The kit includes a flightin polypeptide, a WYR polypeptide, polynucleotide encoding said polypeptides, any of the vectors disclosed in any of the above aspects, or embodiments thereof, or any of the cells disclosed in any of the above aspects, or embodiments thereof, and instructions for using the kit.

[0020] In any of the above aspects, or embodiments thereof, the muscle cell is a myocyte or progenitor thereof.

[0021] In any of the above aspects, or embodiments thereof, the myocyte or progenitor thereof is a cardiac myocyte or progenitor thereof.

[0022] In any of the above aspects, or embodiments thereof, the muscle cell is in vivo or in vitro. In any of the above aspects, or embodiments thereof, the polynucleotide is DNA or RNA. In any of the above aspects, or embodiments thereof, the DNA is provided in a vector. In any of the above aspects, or embodiments thereof, the vector is an adeno-associated vector. In any of the above aspects, or embodiments thereof, the vector is AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, or AAVHSC17. In any of the above aspects, or embodiments thereof, the vector is AAV1, AAV2, AAV8, AAV9, AAV6, AAV2i8, Anc80, or Anc82. In any of the above aspects, or embodiments thereof, the vector is a pseudotyped vector comprising a capsid protein. In any of the above aspects, or embodiments thereof, the capsid protein is a capsid protein from AAV4, AAV6, AAV7, AAV8, or AAV9.

[0023] In any of the above aspects, or embodiments thereof, the RNA is mRNA.

[0024] In any of the above aspects, or embodiments thereof, the vector is a mammalian expression vector.

[0025] In any of the above aspects, or embodiments thereof, the vector comprises a promoter. In any of the above aspects, or embodiments thereof, the promoter is constitutive or inducible. In any of the above aspects, or embodiments thereof, the promoter is a myosin promoter. In any of the above aspects, or embodiments thereof, the promoter is a cardiac tissue-specific promoter. In any of the above aspects, or embodiments thereof, the promoter is a cardiomyocyte-specific promoter. In any of the above aspects, or embodiments thereof, the promoter is cardiac troponin T promoter, a-myosin heavy chain (a-MHC) promoter, myosin light chain-2v (MLC-2v) promoter, cardiac NCX1 promoter, beta-myosin heavy chain isoform 7 (MHC7) promoter, alpha-myosin heavy chain isoform 6 (MHC6) promoter, actin alpha cardiac muscle 1 (ACTC1) promoter, cardiac myosin-binding protein-C (MYBPC3) promoter, alpha-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, or sarco / endoplasmic reticulum Ca2+ ATPase (SERCA) promoter.

[0026] In any of the above aspects, or embodiments thereof, the cell is an induced pluripotent stem (iPS) cell. In any of the above aspects, or embodiments thereof, the cell is a cardiomyocyte derived from an iPS cell.

[0027] In any of the above aspects, or embodiments thereof, the cardiac disease or disorder is one or more of cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure.

[0028] In any of the above aspects, or embodiments thereof, the subject is a human subject.

[0029] In any of the above aspects, or embodiments thereof, the administering is by local or systemic injection.

[0030] In any of the above aspects, or embodiments thereof, the WYR polypeptide is a polypeptide with a sequence shown in FIGs. 6A-6C.

[0031] Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

[0032] Definitions

[0033] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0034] By "agent" is meant any small molecule chemical compound, nucleic acid molecule, polypeptide, or fragments thereof.

[0035] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0036] By "alteration" is meant a change in the structure, expression levels or activity of a polynucleotide, polypeptide, cell, or tissue (e.g., muscle) as detected by standard art known methods such as those described herein. The alteration can be an increase or a decrease. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels. In some embodiments, the alteration is in the force of contraction. In some embodiments, the alteration is in the cessation of the force of contraction. Methods for measuring contraction are known in the art and described herein.

[0037] By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. In embodiments, an analog of WYR might have increased ligand binding (e.g., an increased affinity for a ligand). An analog may include an unnatural amino acid.

[0038] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of’ or “consisting essentially of’ the particular component(s) or element(s) in some embodiments.

[0039] By “cardiac function” is meant the biological function of cardiac tissue or heart (e.g., contraction and / or relaxation function). Methods for measuring the biological function of the heart are standard in the art (e.g., Textbook of Medical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co., 2000) and are also described herein.

[0040] By “increasing in cardiac function” is meant an increase in a biological function of the heart by at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to the biological function present in a naturally-occurring, corresponding cardiac tissue or heart.

[0041] By “consist essentially” it is meant that the ingredients include only the listed components along with the normal impurities present in commercial materials and with any other additives present at levels which do not affect the operation of the disclosure, for instance at levels less than 5% by weight or less than 1% or even 0.5% by weight. By “decrease” is meant to alter negatively relative to a reference. A decrease may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, -fold 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more.

[0042] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

[0043] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0044] By “cardiac disorder characterized by undesirable changes in muscle contraction and / or relaxation” is meant disorders involving an increase or decrease in muscle contraction and / or relaxation that adversely effects the health of the subject.

[0045] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include cardiac disorders characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production or cessation by cardiac muscle. Exemplary cardiac disorders characterized by undesirable changes in muscle contraction and / or relaxation include, but are not limited to, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure.

[0046] By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. By “engraftment” is meant the integration of an exogenous cell into a tissue of a subject. In one embodiment, a cell (e.g., cardiac myocyte or myocyte progenitor cell) is engrafted into the heart of a subject in need thereof.

[0047] The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

[0048] By “flightin polypeptide” is meant a protein or fragment thereof having at least 85% amino acid identity to GenBank Accession No.: CAA79309.1, and that has myosin binding activity. In some embodiments, the flightin polypeptide is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the flightin protein in Drosophila melanogaster. In one embodiment, a flightin polypeptide fragment comprises a WYR peptide. An exemplary flightin full-length amino acid sequence from D. melanogaster is provided below: >CAA79309.1 flightin [Drosophila melanogaster] MADEEDPWGFDDGGEEEKAASTQAGTPAPPSKAPSVASDHKADSWAGTPANEEAAPEEVEEIK APPPPPEDDGYRKPVQLYRHWVRPKFLQYKYMYNYRTNYYDDVIDYIDKKQTGVAREIPRPQTW AE RVL RTRNISGSDIDS YAP AKRDKQ L I QT L AAS I RT YN YH T KAY I NQR YAS VL

[0049] By “flightin polynucleotide” is meant a nucleic acid molecule encoding a flightin polypeptide. An exemplary flightin polynucleotide is provided at GenBank Accession No.: Z18858.1, and is reproduced below:

[0050] >Z18858.1 D. melanogaster mRNA for flightin CCTTTATATCAGTATAAACACTATAGTAAAGTAATATGGCAGACGAAGAAGATCCAT GGGGTTTCGACGACGGCGGCGAGGAGGAGAAGGCGGCATCCACCCAGGCGGGCAC TCCGGCCCCACCCTCAAAAGCACCGAGTGTGGCATCCGATCACAAGGCTGATAGCG TCGTGGCTGGCACTCCGGCCAACGAGGAGGCTGCTCCGGAAGAGGTAGAAGAAATC AAAGCACCGCCGCCTCCGCCAGAAGACGATGGTTACAGGAAACCGGTGCAGCTCTA CCGCCACTGGGTCAGGCCGAAGTTTCTGCAATACAAATACATGTACAACTACAGAA CCAACTATTACGATGATGTAATTGACTATATCGACAAAAAACAAACTGGAGTGGCC CGGGAAATACCAAGACCACAGACGTGGGCGGAACGTGTCCTGCGCACACGGAATAT CAGTGGCAGTGACATTGATTCGTATGCACCGGCAAAGAGGGACAAACAACTAATTC AAACACTGGCGGCCTCGATAAGAACTTATAATTACCACACCAAGGCATATATCAAC CAAAGGTATGCCAGTGTCCTTTAGTAAGCGCACCTAAATGTACCAATAGAACCGTCG GACGAACGGATGAGCACTCGTAGTTATATAAAGGGTAACACCATTGTCAAAAGATC CATACGTACATATGTGTATAGTCAGTCAGTAAGCGTTTGCATATCTGCGCATCTCAA AACTCTGTATCCTGAATCTGCCTTATGGAGTGGAGACCTTTCAAGAAAACGAATATT GTATATTTTTTCTTACATGCGTAATTTCTGTTAATATATTGCCATATGTACTTATTCAT ATATATTAACACAACTTACCTCTCAATACATGGTATTTTTAAGGTTATTTACTTCATT TTTAGCAAAGTGAGTAAATAAAACAAACCTATTTATATTGTAAAGCTTAAAATAAAA ACTGTGCAATAAATAAAACTAAAT

[0051] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. In embodiments, portion contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0052] By “increase” is meant to alter positively relative to a reference. An increase may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, -fold 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more.

[0053] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0054] By "isolated polynucleotide" is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0055] By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. In embodiments, the preparation is at least 75%, at least 90%, and or at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0056] By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a developmental state, condition, disease, or disorder. Exemplary markers include MYH7 (P-myosin heavy chain 7), MYBPC3 (myosin- binding protein C), TNNT2 (cardiac muscle troponin T), TNNI3 (cardiac troponin I),TPM1 (a- tropomyosin), ACTC1 (cardiac a-actin), MYL2 (myosin lightchain 2), MYL3 (myosin light chain 3), CSRP3 (cysteine and glycine-rich protein 3), TTN (titin), TCAP (telethonin), MY0Z2 (myozenin 2), TRIM63 (ubiquitin E3 ligase tripartite motif protein 63 or MuRFl), FHL1 (four- and-a-half LIM domains 1), high sensitivity cardiac troponin I, N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and C-reactive protein (CRP). By “modulating” is meant increasing or decreasing. In embodiments, the disclosure provides compositions and methods for increasing or decreasing relaxation or contraction function of a cell, such as a muscle cell).

[0057] As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

[0058] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. In some embodiments, contact or expression of a WYR peptide with a cardiac tissue reduces the propensity of the tissue to become hypertrophic.

[0059] By "polypeptide" or “amino acid sequence” is meant any chain of amino acids, regardless of length or post-translational modification. In various embodiments, the post-translational modification is glycosylation or phosphorylation. In various embodiments, conservative amino acid substitutions may be made to a polypeptide to provide functionally equivalent variants, or homologs of the polypeptide. In some aspects the invention embraces sequence alterations that result in conservative amino acid substitutions. In some embodiments, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequence of the proteins and polypeptides disclosed herein.

[0060] By “reduce” is meant to alter negatively relative to a reference. A reduction may be by 1%, 5%, 10%, 25%, 30%, 50%, 75%, 100%, or more, or by 1.5-fold, -fold 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more. In some embodiments, a composition comprising a WYR peptide reduces muscle contraction (e.g., cardiac muscle contraction) by at least about 1%, 3%, 5%, 10%, 30%, 33%, 35%, 70%, 75%, or 80%.

[0061] By “reference” is meant a standard or control condition. In some cases, the reference is a healthy cell or a healthy subject, or the reference is a cell or subject that does not have or is not associated with a disease (e.g., a cardiac disorder characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production in cardiac muscle). Exemplary cardiac disorders include, but are not limited to, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post- infarction cardiac remodeling, or cardiac failure. In some instances, the reference is the degree of force exerted by a muscle (e.g., cardiac muscle) of an untreated subject or a subject at an earlier time point during treatment. In other embodiments, the reference is a measure of contraction and / or relaxation (e.g., speed, amplitude) as measured, for example, as shown in FIG. 1

[0062] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer thereabout or therebetween.

[0063] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a doublestranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a doublestranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0064] For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, about less than about 500 mM NaCl and 50 mM trisodium citrate, or about less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, of at least about 37° C, or of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0065] For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0066] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In embodiments, such a sequence is at least 60%, at least 80% or 85%, or at least about 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0067] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0068] By "subject" is meant an animal. The animal can be a mammal. The mammal can be a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline.

[0069] By “tissue” is meant a collection of cells having a similar morphology and function. As provided herein a “WYR polypeptide” or “WYR peptide” or “WYR” is meant an amino acid sequence having at least 85% amino acid identity to any WYR sequence disclosed in FIGs. 6A-6C, herein, or a fragment thereof and having myosin binding activity. An exemplary WYR peptide from D. melanogaster is found below: HWVRPKFLQYKYMYNYRTNYYDDVIDYIDKKQTGVAREI PRPQTWAERVLRT .

[0070] In embodiments, a WYR peptide comprises at least about 10, 20, 30, 35, 40, or 45 amino acids of the above referenced WYR sequences. In embodiments, the WYR peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations in said sequence.

[0071] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0072] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0073] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0074] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art. In some cases, a range of normal tolerance in the art is within 1 or 2 standard deviations of the mean. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0075] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0076] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG. 1 provides a diagram and a graph showing stress responses recorded after a 1% quick stretch applied to demembranated rat cardiac muscle. Rat heart muscle was prepared by removing cell membranes, activating force production by exposure to calcium, and then recording stress response to a quick stretch in the absence and presence of WYR. The effect of WYR was to reduce the early and late amplitudes of the response and increase the myosin detachment rate.

[0078] FIG. 2A-2B provide graphs showing the effects of WYR peptide on the viscoelastic characteristics of rat cardiac muscle. FIG. 2A. The magnitude of the elastic modulus at high frequencies was depressed by WYR and partially recovered by partial removal of WYR. FIG. 2B. A similar depression of magnitude was also observed in the viscous modulus. The WYR peptide also extended the frequency range of negative-valued viscous modulus, which indicates enhanced myosin kinetics due to WYR.

[0079] FIG. 3A-3B provide images showing that WYR peptide binds to the thick filament in a cardiac myocyte. FIG. 3A. The WYR peptide included a HIS-tag which allowed immunostaining and visualization of WYR within the sarcomere. The dark gray channel demonstrates a WYR band 1.2 pm thick followed by 0.6 pm absence repeated along the longitudinal axis of the myocyte. FIG. 3B. The light gray channel shows placement of a-actinin, which is in the Z-line of the sarcomere. The WYR is clearly localized to the region of the sarcomere between the Z-lines, i.e., the thick filament.

[0080] FIG. 4A-4B provide X-ray diffraction images showing the effects of the WYR peptide on thick filament structure of mouse skeletal muscle. FIG. 4A shows that, without WYR peptide, the usual meridional reflections Ml, M2, and M3 are present and identifiable. FIG. 4B shows that, with WYR peptide, these reflections become more distinct due to increased regularity of structural repeats. An additional unknown reflection below Ml, indicated by “?” also appears and suggests enhanced periodicity of a myosin structure currently not identified.

[0081] FIGs. 5A-5B are graphs showing that sinusoidal analysis provides a frequency response of the muscle in the presence of WYR. FIG. 5 A shows the effects of WYR on the frequency response of the muscle was to reduce the amplitudes at low and high frequencies, which correspond to the late and early amplitudes of the quick stretch, respectively. FIG. 5B shows that the myosin detachment rate is also detected as the frequency of maximum viscous modulus. WYR peptide increased the myosin detachment rate. N= 4 fibers. In each panel, the graph lines are ordered as follows, from top to bottom: 100 nM, 250 nM, 1 pM, 2.5 pM, 5 pM, 10 pM.

[0082] FIG. 6 provides a multiple sequence alignment for the conserved WYR amino acid sequence and the two related forms found in decapods (CpW) and chelicerates (QpW). Numbers across the top refer to amino acid position for D. melanogaster (putative indels are not numbered). Numbers in brackets are the length in amino acid for each sequence. The top row(bold letters) shows strictly conserved sites (upper case, no exceptions; lower case, some exceptions, see below) in WYR. Dots represent identities with the sequence of D. melanogaster. Site gl 17 is conditionally conserved (depending on indel placement) in WYR, but is not conserved in CpW or QpW; site yl03 is conserved in all species listed except Agrotis segetum; site 130 is e in all WYR and CpW sequences except for Diaprepes abbreviatus, and it is replaced by D in all QpW except in Limulus polyphemus. The letters A and B after Culex quinquefasciatus and Gammarus pulex identify different isoforms. Sequences for the alternative isoforms found in Aedes, Anopheles, and Phlebotomus are not included because the differences are found outside the WYR domain. The bold letters at the bottom are positions conserved in WYR, CpW, and QpW.

[0083] FIGs. 7A-7B are graphs illustrating the generation of contractile stress with increasing concentrations of calcium ion in mouse cardiac muscle. The effect of WYR was to reduce the maximal contractile stress without affecting calcium sensitivity compared to a control Scramble peptide, which possesses the same amino acids in a randomized sequence. FIG. 7A shows the stress response to calcium in the WYR and Scramble treated muscle. FIG. 7B shows the EC 50 of the stress response to calcium in the WYR and Sramble treated muscle.

[0084] FIGs. 8A-8B illustrates effects of WYR on the mechanical response to a 1% stretch in mouse cardiac muscle. FIG. 8A illustrates example raw data traces. FIG. 8B illustrates the results of the calculated rate of relaxation rate after a quick stretch, kreiease, which was enhanced by the presence of WYR peptide compared to Scramble control.

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] The present disclosure features compositions and methods that are useful for modifying muscle contraction. In embodiments, the disclosure provides compositions useful for treating cardiac disorders characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production in cardiac muscle.

[0087] The present disclosure is based, at least in part, on the discovery that a fragment of the Drosophila flightin protein can be used to modulate contraction of mammalian muscle cells, including cardiac myocytes. As detailed below, protein interactions in the thick filament backbone affect thick filament mechanics and force production. Further, the present disclosure is based, at least in part, on the discovery that flightin modulates muscle function, particularly cardiac muscle function, and is therefore useful in treating cardiac disorders characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production and / or cessation in cardiac muscle. The majority of current heart failure treatments are aimed at ameliorating the symptoms and do not address the underlying condition. Without intending to be bound by theory, the present disclosure includes a novel approach to treating one of the main underlying conditions that lead to heart failure, namely abnormal force production by the heart muscle. In some embodiments, the present disclosure includes compositions and methods useful for reducing the risk of cardiac disorders characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or abnormal force production in cardiac muscle.

[0088] Flightin, a muscle protein widespread in hexapods and crustaceans, influences muscle function (including force production and power output) through its interaction with the coiled- coil “rod” region of myosin that forms the backbone of the thick filament. In particular, a peptide fragment of flightin, which may be denoted as WYR, was discovered and characterized herein that binds the myosin rod and imparts mechanical strength to the thick filament. In some embodiments, WYR, and discoverable peptide mimics and / or analogs (e.g., peptides from other proteins that bind the myosin rod or similar coiled-coil structures, including but not limited to: myomesin, myosin binding protein C, myofilin, paramyosin, and M protein), are used as a treatment for human heart failure.

[0089] Current treatments for heart failure rely on small molecule regulation of myosin motor activity. Current basic research into other treatment strategies includes gene therapy (gene replacement or gene fixing), RNA therapy (virus with therapeutic vectors), and cell therapy (cell replacement via stem cells). In some embodiments, flightin, peptide fragments thereof (e.g., WYR), and / or peptide mimics or analogs thereof (e.g., peptides from other proteins that bind the myosin rod or similar coiled-coil structures, including but not limited to: myomesin, myosin binding protein C, myofilin, paramyosin, and M protein) may be used in gene therapy, RNA therapy, or cell therapy for treating cardiac disorders (e.g., cardiac disorders characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production in cardiac muscle).

[0090] Flightin and WYR

[0091] Flightin is a myosin binding protein essential to the structure and function of the indirect flight muscle in Drosophila melanogaster. In Drosophila, flightin imparts stiffness to the thick filament and is essential for their length determination and structural integrity. Flightin is a 20 kDa protein that binds to myosin II light meromyosin. The analyses of flightin mutants in D. melanogaster have revealed its important role in flight and courtship. Specifically, mutants that express a truncated flightin missing the C-terminal region (flnAC44) are incapable of generating a courtship song or a wing beat to propel flight, similar to the flightin null mutant, (fin0). In contrast, mutants that express a truncated flightin missing the N-terminal region (flnAN62) have impaired flight mechanics and produce an abnormal courtship song that lessens the male’s mating success.

[0092] WYR is a conserved protein domain characteristic of the muscle protein flightin, estimated to have originated in the ancestor to hexapods and crustaceans -500 MYA. WYR has been shown to harbor an essential myosin binding site. The examples provided herein indicate that modulates cardiac muscle function, and alters myosin kinetics in cardiac muscle by enhancing the stiffness of the thick filament.

[0093] WYR is a peptide approximately 50 amino acids in length, that has been shown to mechanically reinforce the packing of myosin molecules into the thick filaments of insect flight muscles (Chakravorty et al., Proc Biol Sci. 2017 May 17;284(1854):20170431; Soto-Adames et al., J Mol Evol. 2014 Jan;78(l):24-37). WYR peptides are not expressed in mammals, but the specific binding sites in the tails of the force-producing myosin molecules - the heptad repeats - are present in mammalian heart muscle and are remarkably conserved among species. The functional effect of these peptides is to induce a cessation in myosin force specifically when the muscle is lengthening. Without intending to be bound by theory, such a functional effect in mammalian heart muscle would address poor ventricular filling that underlies about half of all cases of heart failure.

[0094] Cardiac Hypertrophy

[0095] Cardiac hypertrophy is an adaptive response to pressure or volume stress, mutations of sarcomeric (or other) proteins, or loss of contractile mass from prior infarction. Hypertrophic growth accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease. In these types of cardiac pathology, pressure overload- induced concentric hypertrophy is believed to have a compensatory function by diminishing wall stress and oxygen consumption. At the same time, ventricular hypertrophy is associated with significantly increased risk of heart failure and malignant arrhythmia. Hypertrophic transformation due to pressure overload of the heart can be divided into 3 stages: (1) developing hypertrophy, in which load exceeds output, (2) compensatory hypertrophy, in which the workload / mass ratio is normalized and resting cardiac output is maintained, and (3) overt heart failure, with ventricular dilation and progressive declines in cardiac output despite continuous activation of the hypertrophic program.

[0096] At the cellular level, cardiomyocyte hypertrophy is characterized by an increase in cell size, enhanced protein synthesis, and heightened organization of the sarcomere. Classically, two different hypertrophic phenotypes can be distinguished: (1) concentric hypertrophy due to pressure overload, which is characterized by parallel addition of sarcomeres and lateral growth of individual cardiomyocytes, and (2) eccentric hypertrophy due to volume overload or prior infarction, characterized by addition of sarcomeres in series and longitudinal cell growth.

[0097] In some instances, such as in endurance athletes, cardiac hypertrophy is generally accepted to be physiological and not associated with adverse sequelae. Little is known, however, about the specific molecular events that lead to physiological hypertrophy and how these pathways differ from pathological hypertrophy. The morphological phenotypes differ significantly: Exercise-induced hypertrophy is typically not accompanied by myocardial accumulation of collagen, and increases in wall thickness are modest. Hypertrophic Cardiomyopathy

[0098] Hypertrophic cardiomyopathy (HCM) is a genetic disorder of cardiac myocytes that is characterized by cardiac hypertrophy unexplained by the loading conditions; a nondilated left ventricle; and a normal or increased ejection fraction. Cardiac hypertrophy is usually asymmetrical with greatest involvement most commonly of the basal interventricular septum subjacent to the aortic valve. It is occasionally restricted to other myocardial regions, such as the apex, the midportion, and the posterior wall of the left ventricle. At the cellular level, cardiac myocytes are hypertrophied, disorganized, and separated by areas of interstitial fibrosis.

[0099] HCM is a disorder without a distinct geographic, ethnic, or sex pattern of distribution. Prevalence of HCM has been estimated at 0.16% to 0.29% (~ 1:625-1:344 individuals) in the general adult population. In adults, HCM may be diagnosed by the presence of left ventricular end diastolic wall thickness >13 mm on an echocardiogram or other imaging technique. The European Society of Cardiology guidelines recommend using a left ventricular wall thickness of >15 mm in the diagnostic criteria. Estimating the prevalence of HCM based on detection of cardiac hypertrophy, although clinically valuable, has many limitations. Notable among them is the age-dependent expression of cardiac hypertrophy; the latter is present in approximately one half of patients with the underlying causal mutations by the third decade of life and in approximately three fourths by the sixth decade. Given the age-dependent expression of HCM mutations, its prevalence is expected to be higher in older subjects. Indeed, HCM has been recognized in 0.29% (1:333) of 60-year-old individuals undergoing echocardiography for cardiovascular evaluation in the United States. A similar prevalence has been reported in other parts of the world. When more sensitive imaging methods are used, when more family members are evaluated, and when genetic testing is more widely used (see below), a much higher estimate of 0.6% (1 : 167) has been suggested.

[0100] Cardiac hypertrophy may be of late onset, and thus HCM may be underdiagnosed in such individuals. Moreover, the presence of concomitant conditions that may cause myocardial hypertrophy, such as arterial hypertension or aortic stenosis, may make the differentiation of primary (HCM) from secondary hypertrophy challenging. However, the asymmetrical shape of the ventricle with greatest hypertrophy of the basal interventricular septum and genetic testing of the subjects and their families may be helpful in this population. The histological features of HCM include myocyte hypertrophy and disarray, as well as interstitial fibrosis. The hypertrophy is also frequently associated with left ventricular diastolic dysfunction. In the majority of patients, HCM has a relatively benign course. However, HCM is also an important cause of sudden cardiac death, particularly in adolescents and young adults. Nonsustained ventricular tachycardia, syncope, a family history of sudden cardiac death, and severe cardiac hypertrophy are major risk factors for sudden cardiac death. This complication can usually be averted by implantation of a cardioverter-defibrillator in appropriate high-risk patients. Atrial fibrillation is also a common complication and is not well tolerated. Mutations in over a dozen genes encoding sarcomere-associated proteins cause HCM. MYH7 and MYBPC3, encoding P-myosin heavy chain and myosin-binding protein C, respectively, are the 2 most common genes involved, together accounting for >50% of the HCM families.

[0101] HCM is an archetypical single gene disorder with an autosomal dominant pattern of inheritance, whereby a single mutation is usually sufficient to cause the disease, albeit with variable penetrance and expression. The variability of the phenotype is due, at least in part, to the causal mutation acting in concert with many other genetic and of nongenetic influences. Approximately 60% of patients with HCM have a clearly recognizable familial disease.

[0102] The identification of multiple separate mutations in the principal causal genes, all encoding sarcomere proteins, established HCM as a genetically heterogeneous disease. Among the known causal genes, MYH7 (P-myosin heavy chain 7) and MYBPC3 (myosin- binding protein C) are the 2 most common, together being responsible more than half of the patients with familial HCM. Mutations TNNT2, TNNI3 (cardiac troponin I), and TPM1 (a-tropomyosin) are relatively uncommon causes of HCM and together are responsible for <10% of cases. Mutations in ACTC1 (cardiac a-actin), MYL2 (myosin lightchain 2), MYL3 (myosin light chain 3), and CSRP3 (cysteine and glycine-rich protein 3) are also established, albeit uncommon, causes of HCM. Mutations in TTN (titin), TCAP (telethonin), MY0Z2 (myozenin 2), TRIM63 (ubiquitin E3 ligase tripartite motif protein 63 or MuRFl), and FHL1 (four-and-a-half LIM domains 1) also have been implicated as causes of HCM but occur typically in sporadic cases and small families.

[0103] Methods of Treatment, Delivery, and Administration

[0104] The present disclosure provides methods of treating disease and / or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a flightin polypeptide, WYR peptide, or a polynucleotide encoding a flightin polypeptide or a WYR peptide. Thus, one embodiment is a method of treating a subject suffering from or susceptible to a cardiac disease or disorder (e.g., a cardiac disorder characterized by undesirable alterations in cardiac muscle contraction and / or relaxation, or involving abnormal force production and / or cessation in cardiac muscle. Exemplary cardiac disorders include, but are not limited to, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post- infarction cardiac remodeling, or cardiac failure. ) or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or a pharmaceutical composition comprising a nucleotide encoding a flightin polypeptide or a WYR polypeptide, as disclosed herein, sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

[0105] The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or a pharmaceutical composition comprising a nucleotide encoding a flightin polypeptide or a WYR polypeptide, described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

[0106] The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the polynucleotides disclosed herein, such as a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or a pharmaceutical composition comprising a nucleotide encoding a flightin polypeptide or a WYR polypeptide disclosed herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, marker, family history, and the like). The polynucleotides and compositions herein may be also used in the treatment of any other disorders in which abnormal force production in cardiac muscle may be implicated. In some embodiments, a subject in need of such treatment may be identified through the use of one or more markers (e.g., MYH7 (P-myosin heavy chain 7), MYBPC3 (myosin- binding protein C), TNNT2 (cardiac muscle troponin T), TNNI3 (cardiac troponin I),TPM1 (a- tropomyosin), ACTC1 (cardiac a-actin), MYL2 (myosin lightchain 2), MYL3 (myosin light chain 3), CSRP3 (cysteine and glycine-rich protein 3), TTN (titin), TCAP (telethonin), MY0Z2 (myozenin 2), TRIM63 (ubiquitin E3 ligase tripartite motif protein 63 or MuRFl), and FHL1 (four-and-a-half LIM domains 1)). In some embodiments, mutations in the markers indicate a subject having, or at risk for a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post- infarction cardiac remodeling, or cardiac failure), and / or suitable for treatment by the polynucleotides or compositions disclosed herein.

[0107] In another embodiment, a method of monitoring the progress of a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof, or monitoring treatment of the disease is provided. The method includes a diagnostic measurement (e.g., CT scan, screening assay or detection assay) in a subject suffering from or susceptible to disease or symptoms thereof, in which the subject has been administered an amount (e.g., a therapeutic amount) of a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or a pharmaceutical composition thereof, as described herein, sufficient to treat the disease or symptoms thereof. The diagnostic measurement in the method can be compared to samples from healthy, normal controls; in a pre-disease sample of the subject; or in other afflicted / diseased patients to establish the treated subject’s disease status. For monitoring, a second diagnostic measurement may be obtained from the subject at a time point later than the determination of the first diagnostic measurement, and the two measurements can be compared to monitor the course of disease or the efficacy of the therapy / treatment. In certain embodiments, a pre-treatment measurement in the subject (e.g., in a sample or biopsy obtained from the subject or CT scan) is determined prior to beginning treatment as described; this measurement can then be compared to a measurement in the subject after the treatment commences and / or during the course of treatment to determine the efficacy of (monitor the efficacy of) the disease treatment.

[0108] The polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or pharmaceutical compositions thereof, can be administered to a subject by any of the routes normally used for introducing a recombinant protein or composition containing the recombinant protein into a subject. Routes and methods of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral, such as intravenous (IV) or subcutaneous (SC), vaginal, rectal, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection (immunization). Injectables can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, solid forms (e.g., lyophilized forms) suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.

[0109] The polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or pharmaceutical compositions thereof, can be administered in any suitable manner, such as with pharmaceutically acceptable carriers, diluents, or excipients as described supra. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, a pharmaceutical composition comprising the polynucleotide encoding a flightin polypeptide or a WYR polypeptide, can be prepared using a wide variety of suitable and physiologically and pharmaceutically acceptable formulations.

[0110] Administration of the polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or pharmaceutical compositions thereof, can be accomplished by single or multiple doses. The dose administered to a subject should be sufficient to induce a beneficial therapeutic response in a subject over time, such as to inhibit, block, reduce, ameliorate, protect against, or prevent a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof. The dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, by the severity of the cancer being treated, by the particular composition being used and by the mode of administration. An appropriate dose can be determined by a person skilled in the art, such as a clinician or medical practitioner, using only routine experimentation. One of skill in the art is capable of determining therapeutically effective amounts of the polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or pharmaceutical compositions thereof, that provide a therapeutic effect or protection against a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof, suitable for administering to a subject in need of treatment or protection.

[0111] In some embodiments, a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or a pharmaceutical composition thereof, is administered as a maximum-tolerated dose (MTD). In some embodiments, MTD is the dose with estimated probability of dose limiting toxicity (DLT) closest to the target toxicity rate of 20%. In some embodiments, a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, or a pharmaceutical composition thereof, is administered in a therapeutically effective dose for a mammal. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0112] Polynucleotide Therapy

[0113] Polynucleotide therapy featuring a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, analog, variant, or fragment thereof is another therapeutic approach for treating a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof. Expression of such proteins in a cardiac cell is expected to modulate function of the cardiac cell, tissue, or organ, for example, by modulating cardiac muscle function, altering myosin kinetics, and / or otherwise preventing abnormal force production in cardiac muscle. Such nucleic acid molecules can be delivered to cells of a subject having a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post- infarction cardiac remodeling, or cardiac failure), or symptom thereof. The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of a flightin polypeptide or a WYR polypeptide can be produced.

[0114] Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). For example, a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). In one embodiment, a viral vector is used to administer a polynucleotide encoding a flightin polypeptide or a WYR polypeptide to a cardiac tissue.

[0115] Transducing viral vectors have tissue tropisms that permit selective transduction of one cell type compared to another. For instance, while expression of a flightin polypeptide or a WYR polypeptide in cardiomyocytes will be therapeutic for cardiac hypertrophy, hypertrophic cardiomyopathy or other forms of heart disease, its inhibition in other tissues, such as the brain, may not be desirable. In some embodiments, vectors that target cardiomyocytes with high specificity compared to other cell types are used. This would allow specific cardiac targeting of the expression of the flightin polypeptide or WYR polypeptide. This is because alteration of myosin kinetics in other non-cardiac cells can be deleterious. Among potential adeno-associated virus candidates are AAV1, AAV2, AAV8, AAV9, AAV6, AAV2i8, Anc80, and Anc82. Adeno- associated virus transduction efficiency is enhanced when the genome is “self-complimentary.” In some embodiments, self-complementary adeno-associated virus is used to increase the cardiac transduction by the gene therapy vector.

[0116] In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type. For example, the AAV2 genome can be packaged into the capsid of another AAV serotype sych as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2 / 5, AAV2 / 7, and AAV2 / 8 respectively, as described in Balaji et al. J. Surg. Res. September; 184(1): 691-698 (2013). In some embodiments, the AAV expression vector comprises a capsid protein. Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the AAV expression vector. Capsid proteins are classified by the serotype. Wild type capsid serotypes can be, for example, AA1, AAV2AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al. BioDrugs 31:317-334 (2017)). Engineered capsid types include chimeric capsids and mosaic capsids (Choi et al. Curr Gene Ther. 5: 299-310 (2005)). Capsid proteins used in AAV expression vectors for delivery to cardiac cells that result in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mai. Ther. 16:P1073-1080 (2008)). Artificial capsids, such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery to cardiac cells that results in high expression. AAV vectors and capsids are provided in U.S. Pat. Pub. Nos. US10011640B2, U.S. Pat. No. 7,892,809B2, U.S. Pat. No. 8,632,764B2, US8889641132, U.S. Pat. No.

[0117] 9,475, 845B2, US10889833132, U.S. Pat. No.10,480,01182, and U.S. Pat. No. 10,894,949B2, the contents of which are herein incorporated by reference; and Int'l Pat. Pub. Nos.

[0118] W02020198737A1, W02019028306A2, WO2016054554A1, WO201.8152333A1, WO2017106236A1, W02008124724 Al, W02017212019A1, W02020117898A1, WO2017192750A, the entireties of each of which are hereby incorporated by reference.

[0119] In some embodiments, the AAV capsid protein is an AAVHSC capsid protein. AAVHSC capsid proteins are members of clade F and are closely related to AAV9. AAVHSC capsid proteins were originally isolated from CD34+hemopoietic system cells (Smith LJ et al. Mol Ther. 2014 Sep;22(9): 1625-34.; Chatterjee et al. Hum Gene Ther. 2020 May;31(9-10):542- 552.) and were designated AAVHSC to reflect their origin. Exemplary AAVHSCs include AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, AAVHSC17. AAVHSCs are described in more detail in IntT Pat. Pub. No. WO2019079437A1, the entirety of which is hereby incorporated by reference.

[0120] Non-viral approaches can also be employed for the introduction of therapeutic to a cardiac cell of a patient requiring treatment of a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

[0121] Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), the CMV- chicken b-actin hybrid promoter (“CAG”), or metallothionein promoters, and regulated by any appropriate mammalian regulatory element. For treatment of a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure), or symptom thereof, it is desirable to selectively express the flightin polypeptide or WYR polypeptide in cardiomyocytes and to minimize expression in other cell types. In some embodiments, cardiomyocyte-selective promoters are used for the expression of the flightin polypeptide or WYR polypeptide. The promoters or enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. For example, the cardiac troponin T promoter, the a-myosin heavy chain (a-MHC) promoter, the myosin light chain- 2v (MLC-2v) promoter, the cardiac NCX1 promoter, the beta-myosin heavy chain isoform 7 (MHC7) promoter, the alpha-myosin heavy chain isoform 6 (MHC6) promoter, the actin alpha cardiac muscle 1 (ACTC1) promoter, the cardiac myosin-binding protein-C (MYBPC3) promoter, the alpha-tropomyosin promoter, the cardiac troponin C promoter, the cardiac troponin I promoter, and the sarco / endoplasmic reticulum Ca2+ATPase (SERCA) promoter can be used to direct expression in cardiomyocytes. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0122] Exemplary vectors and promoters may be found, for example, in U.S. Patent No. 11,446,397, and U.S. Patent App. Pub. Nos. 2022 / 0154217 and 2023 / 0133924, the disclosures of each of which are hereby incorporated by reference.

[0123] Cell Therapy

[0124] Cells comprising a heterologous polynucleotide encoding a flightin protein or WYR polypeptide can be conveniently provided to a subject in sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Cells comprising a heterologous polynucleotide encoding a flightin protein or WYR polypeptide may be provided in liquid or viscous formulations. For some applications, liquid formations are desirable because they are convenient to administer, especially by injection. Where prolonged contact with a tissue is desired, a viscous composition may be preferred. Such compositions are formulated within the appropriate viscosity range. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0125] Sterile injectable solutions are prepared by compositions comprising a cardiac myocyte or progenitor cells in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0126] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the cells.

[0127] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent, such as methylcellulose. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form). Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert.

[0128] Compositions containing cells comprising a heterologous polynucleotide encoding a flightin protein or WYR polypeptide are administered to a cardiac tissue in an amount required to achieve a therapeutic or prophylactic effect. Such an amount will vary depending on the conditions. In some embodiments, the cells comprising a heterologous polynucleotide encoding a flightin protein or WYR polypeptide are induced pluripotent cells (iPS cells) or cardiomyocytes derived from iPS cells. In some embodiments, the iPS cells or cardiomyocytes derived from iPS cells are engrafted and / or seeded onto a cardiac tissue. Without intending to be bound by theory, after engraftment and / or seeding of the iPS cells or cardiomyocytes derived from iPS cells comprising a heterologous polynucleotide encoding a flightin protein or WYR polypeptide, the flightin protein or WYR polypeptide would be expected to stabilize the thick filament backbone and thereby augment myosin force production and transmission across the sarcomere.

[0129] The precise determination of what would be considered an effective dose is based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0130] Optionally, the methods of the invention provide for the administration of a composition of the invention to a suitable animal model to identify the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which modulates cardiac contract. Such determinations do not require undue experimentation, but are routine and can be ascertained without undue experimentation.

[0131] Methods of Delivery

[0132] Compositions comprising cells comprising a heterologous polynucleotide encoding a flightin protein or WYR polypeptide may be delivered to a subject in need thereof. Modes of administration include intramuscular, intra-cardiac, intra-arterial, within / on implants, or by parenteral routes. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal, or infusion.

[0133] The compositions can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). Compositions of the invention can be introduced by injection, catheter, or the like. Compositions of the invention include pharmaceutical compositions comprising cells disclosed herein and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous.

[0134] If desired, biologically active agents present in conditioned media are incorporated into a polymer scaffold to promote tissue repair, cell survival, proliferation in a tissue in need thereof. Polymer scaffolds can comprise, for example, a porous, non-woven array of fibers. The polymer scaffold can be shaped to maximize surface area, to allow adequate diffusion of nutrients and growth factors to a cell of the invention. Polymer scaffolds can comprise a fibrillar structure. The fibers can be round, scalloped, flattened, star-shaped, solitary or entwined with other fibers. Branching fibers can be used, increasing surface area proportionately to volume.

[0135] Unless otherwise specified, the term “polymer” includes polymers and monomers that can be polymerized or adhered to form an integral unit. The polymer can be non-biodegradable or biodegradable, typically via hydrolysis or enzymatic cleavage. The term “biodegradable” refers to materials that are bioresorbable and / or degrade and / or break down by mechanical degradation upon interaction with a physiological environment into components that can be metabolized or excreted, over a period of time from minutes to three years, preferably less than one year, while maintaining the requisite structural integrity. As used in reference to polymers, the term “degrade” refers to cleavage of the polymer chain, such that the molecular weight stays approximately constant at the oligomer level and particles of polymer remain following degradation.

[0136] Materials suitable for polymer scaffold fabrication include polylactic acid (PLA), poly-L- lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, and shape memory materials, such as poly(styrene-block- butadiene), polynorbornene, hydrogels, metallic alloys, and oligo(£-caprolactone)diol as switching segment / oligo(p-dioxyanone)diol as physical crosslink. Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N.Y., 1989).

[0137] Pharmaceutical Compositions

[0138] Compositions comprising a flightin polypeptide, a WYR polypeptide, or polynucleotides encoding a flightin polypeptide or a WYR polypeptide, vectors comprising such polynucleotides, and / or transgenic cells expressing such flightin polypeptides or WYR polypeptides, as described herein are provided. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In some embodiments, an adjuvant (a pharmacological or immunological agent that modifies or boosts an immune response, e.g., to produce more antibodies that are longer-lasting) is also employed. For example, without limitation, the adjuvant can be an inorganic compound, such as alum, aluminum hydroxide, or aluminum phosphate; mineral or paraffin oil; squalene; detergents such as Quil A; plant saponins; Freund's complete or incomplete adjuvant, a biological adjuvant (e.g., cytokines such as IL-1, IL-2, or IL- 12); bacterial products such as killed Bordetella pertussis, or toxoids; or immunostimulatory oligonucleotides (such as CpG oligonucleotides). In some embodiments, the adjuvant is conjugated to an amphiphile as previously described (H. Liu etal., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 5199522 (2014)). In some embodiments, the amphiphile is N-hydroxy succinimidyl ester-end- functionalized poly(ethylene gly col)-lipid (NHS-PEG2KDa-DSPE)

[0139] Compositions and preparations (e.g., physiologically or pharmaceutically acceptable compositions) containing polynucleotides encoding a flightin polypeptide or a WYR polypeptide, vectors comprising such polynucleotides, and / or transgenic cells expressing such flightin polypeptides or WYR polypeptides for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Nonlimiting examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and canola oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present in such compositions and preparations, such as, for example, antimicrobials, antioxidants, chelating agents, colorants, stabilizers, inert gases, and the like.

[0140] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl amines and substituted ethanolamines.

[0141] Provided herein are pharmaceutical compositions which include a therapeutically effective amount of an isolated polynucleotide encoding a flightin polypeptide or a WYR polypeptide, vector comprising such polynucleotides, and / or transgenic cell expressing such flightin polypeptides or WYR polypeptides, as described herein, alone, or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid or aqueous solution, suspension, emulsion, dispersion, tablet, pill, capsule, powder, or sustained release formulation. A liquid or aqueous composition can be lyophilized and reconstituted with a solution or buffer prior to use. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the commonly known pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives, and the like. Other media that can be used in the compositions and administration methods as described are normal saline and sesame oil.

[0142] Combination Therapies

[0143] Optionally, a cardiac therapeutic described herein (e.g., a flightin polypeptide, a WYR polypeptide, or polynucleotides encoding a flightin polypeptide or a WYR polypeptide, vectors comprising such polynucleotides, and / or transgenic cells expressing such flightin polypeptides or WYR polypeptides) may be administered in combination with any other standard therapy useful for regulating cardiac function; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.

[0144] In some embodiments, the polynucleotides encoding a flightin polypeptide or a WYR polypeptide, vectors comprising such polynucleotides, and / or transgenic cells expressing such flightin polypeptides or WYR polypeptides, are administered in combination with contractile inhibitors. Examples of contractile inhibitors include, but are not limited to, Ca2+inhibitors (e.g., Epigallocatechin-3 -gallate (EGCg), epicatechin gallate (ECg), or nebivolol), beta blockers (e.g., Acebutolol, Atenolol, Bisoprolol, Metoprolol, Nadolol, Nebivolol, or Propranolol), recouplers (small molecules that demonstrate the ability to reverse the uncoupling of troponin I phosphorylation and Ca2+ sensitivity, e.g., EGCg, Silybin B, Dehydrosilybin B, resveratrol, or novobiocin), or cardiac myosin inhibitors (e.g., 2,3-butanedione monoimine (BDM), N-benzyl-p- toluene sulphonamide (BTS), mavacamten, CK-274, or blebbistatin).

[0145] Methods for Evaluating Cardiac Function

[0146] Compositions of the invention may be used to enhance cardiac function in a subject having reduced cardiac function. Methods for measuring the biological function of the heart (e.g., contractile function) are standard in the art and are described, for example, in the Textbook of Medical Physiology, Tenth edition, (Guyton et al., W.B. Saunders Co., 2000). In the invention, cardiac function is increased by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to the cardiac function present in a naturally-occurring, corresponding tissue or organ. Most advantageously, cardiac function is enhanced or damage is reversed, such that the function is substantially normal (e.g., 85%, 90%, 95%, or 100% of the cardiac function of a healthy control subject). Enhanced and reduced cardiac function may result from conditions such as cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy (e.g., hypertrophic cardiomyopathy originating from a genetic or a secondary cause), post ischemic and post-infarction cardiac remodeling and cardiac failure.

[0147] Any number of standard methods are available for assaying cardiovascular function. Preferably, cardiovascular function in a subject (e.g., a human) is assessed using non-invasive means, such as measuring net cardiac ejection (ejection fraction, fractional shortening, and ventricular end-systolic volume) by an imaging method such echocardiography, nuclear or radiocontrast ventriculography, or magnetic resonance imaging, and systolic tissue velocity as measured by tissue Doppler imaging. Systolic contractility can also be measured non-invasively using blood pressure measurements combined with assessment of heart outflow (to assess power), or with volumes (to assess peak muscle stiffening). Measures of cardiovascular diastolic function include ventricular compliance, which is typically measured by the simultaneous measurement of pressure and volume, early diastolic left ventricular filling rate and relaxation rate (can be assessed from echo Doppler measurements). Other measures of cardiac function include myocardial contractility, resting stroke volume, resting heart rate, resting cardiac index (cardiac output per unit of time [L / minute], measured while seated and divided by body surface area [m2])) total aerobic capacity, cardiovascular performance during exercise, peak exercise capacity, peak oxygen (O2) consumption, or by any other method known in the art or described herein. Measures of vascular function include determination of total ventricular afterload, which depends on a number of factors, including peripheral vascular resistance, aortic impedance, arterial compliance, wave reflections, and aortic pulse wave velocity, Methods for assaying cardiovascular function include any one or more of the following: Doppler echocardiography, 2- dimensional echo-Doppler imaging, pulse-wave Doppler, continuous wave Doppler, oscillometric arm cuff, tissue Doppler imaging, cardiac catheterization, magnetic resonance imaging, positron emission tomography, chest X-ray, X ray contrast ventriculography, nuclear imaging ventriculography, computed tomography imaging, rapid spiral computerized tomographic imaging, 3-D echocardiography, invasive cardiac pressures, invasive cardiac flows, invasive cardiac cardiac pressure-volume loops (conductance catheter), non-invasive cardiac pressure-volume loops.

[0148] Kits

[0149] Also provided are kits containing the polynucleotides encoding a flightin polypeptide or a WYR polypeptide, vectors comprising such polynucleotides, and / or transgenic cells expressing such flightin polypeptides or WYR polypeptides, or a pharmaceutically acceptable composition thereof, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient, for administering to a subject, for example. In some embodiments, the kit is provided for treating a cardiac disease or disorder (e.g., a cardiac disorder involving abnormal force production and / or cessation in cardiac muscle, cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure) in a subject (e.g., human). In some embodiments, the kit is provided for making polynucleotides encoding a flightin polypeptide or a WYR polypeptide, vectors comprising such polynucleotides, and / or transgenic cells expressing such flightin polypeptides or WYR polypeptides as provided herein. In some embodiments, the kit will contain one or more of a polynucleotide encoding a flightin polypeptide or a WYR polypeptide, vector comprising such polynucleotides, and / or transgenic cell expressing such flightin polypeptides or WYR polypeptides, as disclosed herein. As will be appreciated by the skilled practitioner in the art, such a kit may contain one or more containers, labels, carriers, diluents or excipients, as necessary, and instructions for use.

[0150] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0151] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

[0152] Example 1: Maximal Contractile Stress was Dramatically Reduced with WYR

[0153] Maximal contractile stress (i.e., force normalized to cross-sectional area) was dramatically reduced with WYR in demembranated rat cardiac muscle at maximal calcium activation (pCa 4.5) and 5 mM inorganic phosphate (Pi). 100 pm thick rat cardiac muscle samples were provided. Under conditions without WYR, contractile stress was recorded in two muscle samples as 27.38 and 29.12 mN / mm2. With addition of WYR at 2.56 pM and 5.12 pM, contractile stress was reduced to 18.40 and 7.44 mN / mm2, respectively. In one muscle sample, partial removal of WYR by >5 min incubation in the original solution resulted in a partial restoration of contractile force from 7.44 to 9.89 mN / mm2. The results are provided in Table 1, below.

[0154] Table 1: WYR Peptide Functional Effect

[0155] Relax = relaxing solution, pCa 8.0

[0156] Active = activating solution, pCa 4.5

[0157] DMSO = control condition (no WYR)

[0158] WYR = WYR peptide in DMSO 1% condition = 2.56 pM 2% condition = 5.12 pM

[0159] Pre / Post = isometric tension pre and post Multi and Step measures at this condition

[0160] Example 2: WYR Alters Stretch Response of Cardiac Muscle

[0161] A rapid 1% stretch was applied to muscle already calcium-activated and producing maximum contractile force in demembranated rat cardiac muscle at maximal calcium activation (pCa 4.5) and 5 mM inorganic phosphate (Pi). The stress response to a quick stretch (FIG. 1) demonstrates the sensitivity of the force-producing myosin molecules to the additional mechanical load caused by the stretch. The control response illustrated the typical sharp rise in force followed by dramatic drop and eventual recovery to a new elevated stress. After application of WYR peptide, these three characteristics, sharp rise in force, dramatic drop, and recovery, are significantly blunted compared to the control response. After partial removal of WYR, these characteristics are partially recovered. These recordings demonstrate that the WYR peptide was able to affect the force-producing myosin and was at least partially reversible

[0162] The force response quantifies the stiffness due to the presence of myosin crossbridges as well as the myosin detachment rate. The addition of the WYR peptide significantly reduced the stiffness as indicated by reduced amplitudes in the early and late phases of the response (FIG. 1). Furthermore, the myosin detachment rate was accelerated by WYR.

[0163] Without intending to be bound by theory, both of these attributes would benefit relaxation function in the heart. Example 3: WYR Alters Viscoelastic Characteristics of Cardiac Muscle

[0164] Sinusoidal length perturbations were applied to demembranated rat cardiac muscle at maximal calcium activation (pCa 4.5) and 5 mM inorganic phosphate (Pi) to record the effects of WYR on viscoelastic characteristics of the muscle sample (FIGs. 2A and 2B). The WYR peptide was found to reduce the magnitude of both the elastic and viscous moduli at high frequencies. In addition, the WYR peptide appears to enhance the frequency characteristics, which would be consistent with the faster temporal characteristics observed with the quick stretch assay.

[0165] Example 4: Immunostaining and Visualization of WYR

[0166] The WYR peptide was examined for its binding to thick filaments in a rat heart muscle cell. As shown in FIGs. 3A-3B, the WYR peptide appears in bands ~1.5 mm wide, about the length of the thick filament. The protein a-actinin is not in the thick filament and was found between WYR bands. Therefore, the WYR peptide localizes to the thick filament of rat heart as expected.

[0167] Example 5: X-Ray Diffraction of Mouse Skeletal Muscle Thick Filament Structures

[0168] The WYR peptide affects insect thick filament by mechanically reinforcing the thick filament backbone. To test whether this is the case in mammalian thick filament, the WYR peptide was exposed to mouse skeletal muscle, which was then examined by X-ray diffraction. The diffraction patterns of interest are the meridionals reflections (Ml, M2, M3, etc.) that run vertically in the examples of FIGs. 4A-4B. The WYR peptide enhances the sharpness of the reflections, indicating a more regular periodicity of the thick filament due to peptide reinforcement of the thick filament.

[0169] Example 6: Sinusoidal Analysis and Dose Response

[0170] An especially sensitive assay to detect the effects of a compond on muscle function is sinusoidal analysis, which detects the frequency response of the muscle. FIGs. 5A-5B show the effects of WYR at concentrations of 250-500 nM. The results are consistent with the force response after a quick stretch and verify that WYR peptide reduces stiffness and increased myosin detachment rate - both of which would benefit relaxation function in the heart. It was found that the WYR peptide binds specifically to mammalian thick filament and promotes a more regular periodicity of myosin arrangement within the thick filament. Therefore, in terms of structural effects, the WYR peptide mechanically reinforces the mammalian thick filament as it was shown to do in insect thick filaments. It was also found that the presence of WYR peptide accelerates the myosin crossbridge detachment over a range of peptide concentrations that are low enough to further demonstrate their utility as a therapeutic agent.

[0171] Example 7: WYR Reduces Maximal Contractile Stress without Affecting Calcium Sensitivity

[0172] Contractile stress (i.e., force normalized to cross-sectional area) was recorded in demembranated mouse cardiac muscle for concentrations of 100 nM to 10 pM calcium ion in the presence of 1 pM WYR or 1 pM Scramble, i.e., a peptide of the exact same amino acid composition of WYR, but with a randomized sequence. The contractile stress rose in response to calcium concentration (FIG. 7A), but the maximum calcium activated stress was significantly (p<0.05) reduced in the WYR treatment by -55% compared to the Scramble treatment. Importantly, there was no differential effect on the concentration of calcium (EC 50 of calcium sensitivity) which induced contractile stress (FIG. 7B) in the WYR and Scramble treated muscle.

[0173] Without intending to be bound by theory, these attributes would benefit relaxation function in the heart.

[0174] Example 8: WYR Specifically Accelerates Relaxation of Cardiac Muscle after Stretch

[0175] Further examination of the phenomenon shown in Example 3 was performed comparing the effects of the 1 pM WYR peptide against 1 pM Scramble peptide. A rapid 1% stretch was applied to muscle calcium-activated and producing sub-maximum contractile force in demembranated mouse cardiac muscle at sub-maximal calcium activation (pCa 6.0). There was no inorganic phosphate (Pi) present. The exponential rate of the initial relaxation, denoted kreiease as in FIG. 8A, was found to be statistically significantly (p<0.05) faster in the WYR treatment compared to Scramble treatment (FIG. 8B).

[0176] Without intending to be bound by theory, this attribute would benefit relaxation function in the heart. Example 9: Multiple Sequence Alignment for Conserved WYR Amino Acid Sequence and

[0177] Related Isoforms

[0178] Sequence alignment was performed relative to D. melanogaster (FIG. 6). The numbers across the top refer to amino acid position for D. melanogaster, where putative indels are not numbered. The top row shows those positions strictly conserved in WYR. The dots represent identities with the sequence of D. melanogaster. Site gl 17 is conditionally conserved depending on indel placement in WYR, but is not conserved in CpW or QpW; site yl03 is conserved in all species listed except Agrotis segetum; site 130 is e in all WYR and CpW sequences except for Diaprepes abbreviatus, and it is replaced by D in all QpW except in Limulus polyphemus. Sequences for the alternative isoforms found in Aedes, Anopheles, and Phlebotomus are not included because the differences are found outside the WYR domain.

[0179] METHODS OF THE EXAMPLES

[0180] The following methods were employed in the above examples.

[0181] Solutions

[0182] Chemicals and reagents were obtained from Sigma- Aldrich unless otherwise noted.

[0183] Krebs solution contained (in mmol / liter) 140 NaCl, 5.4 KC1, 1.8 CaC12, 0.6 MgSO4, 10 HEPES, and 10 glucose, pH 7.4; nonactivating relaxing solution: calcium concentration (pCa) 8.0, 5 EGTA, 5 MgATP, 1 Mg2+, 5 Pi, 35 phosphocreatine, and 300 U / ml creatine kinase, ionic strength 200, pH 7.0; activating solution: same as nonactivating, with pCa 4.0; skinning solution: same as nonactivating relaxing solution without creatine kinase and with 1% Triton X-100 wt / vol, 10 pl / ml E-64, 1.25 pl / ml PMSF, 1 tablet / 10 ml PhosStop phosphatase inhibitor cocktail, 40 mM 2,3-butanedione 2-monoxime (BDM) and 50% glycerol wt / vol; storage solution: same as skinning, without Triton X-100. Where indicated, the myosin crossbridge inhibitor BDM was added at 40 mM.

[0184] Rodent myocardium

[0185] All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Vermont College of Medicine and complied with Guide for the Use and Care of Laboratory Animals published by the National Institutes of Health. Animals were fully anesthetized with isoflurane and hearts were removed. Cardiac slices of the left ventricle were produced at 100 pm thick as described (Palmer and Bell, 2022) and placed in preoxygenated (100% 02) Krebs-Ringer solution at room temperature and demembranated overnight at 4°C in skinning solution. Demembranated myocardial strips were prepared and studied as previously described (Donaldson, C., et al. 2012. Myosin crossbridge dynamics in patients with hypertension and concentric left ventricular remodeling. Circ. Heart Fail. 5 : 803— 811.; Zile et al., 2015. Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin. Circulation. 131 :1247-1259.). Strips were mounted between a piezoelectric motor (P841.60, Physik Instrumente) and a strain gauge (AE801, Kronex), lowered into a 90-pl droplet of relaxing solution maintained at room temperature, and stretched to 2.2 pm sarcomere length (lonOptix). Strips were subjected to activating solution with WYR peptide and / or Scramble peptide and / or DMSO to induce distinct experimental conditions. Force responses were recorded after step-length changes of 1.0% resting muscle length (ML). Viscoelastic responses were measured using 0.1% length changes over a range of perturbation frequencies.

[0186] Other Embodiments

[0187] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0188] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0189] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method of modifying muscle contraction, the method comprising contacting a muscle cell with a flightin polypeptide or a WYR peptide comprising or consisting of a peptide having at least about 85% identity to the following amino acid sequence or a sequence shown in FIGs. 6A- 6C:HWVRPKFLQ Y KYMYNYRTNX1YDDVID YIDK KQTX2VAREIP_RPQTWAX3RVLRT, wherein the WYR peptide comprises a W at position 2, R at position 4, P at position 40, R at position 48, and Y at positions 11 and 21, wherein each of the specified positions is identified by bold and underlining, or a corresponding amino acid position in another WYR peptide, and wherein X1is Y or C; wherein X2is G, S, or K; and wherein X3is E, D, Q., or a polynucleotide encoding said polypeptide or peptide, thereby modifying muscle contraction and / or relaxation function.

2. A method of modifying a stress response to quick stretch in a muscle cell, the method comprising contacting a muscle cell with a flightin polypeptide, a WYR polypeptide, or polynucleotide encoding said polypeptides, thereby modifying stress response to quick stretch in said muscle cell.

3. The method of claim 1 or 2, wherein the muscle cell is a myocyte or progenitor thereof.

4. The method of claim 3, wherein the myocyte or progenitor thereof is a cardiac myocyte or progenitor thereof.

5. The method of claims 1 or 2, wherein the muscle cell is in vivo or in vitro.

6. The method of claims 1 or 2, wherein the polynucleotide is DNA or RNA.

7. The method of claim 6, wherein the DNA is provided in a vector.

8. The method of claim 7, wherein the vector is an adeno-associated vector.

9. The method of claim 7 or 8, wherein the vector is AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, or AAVHSC17.

10. The method of claim 7 or 8, wherein the vector is AAV1, AAV2, AAV8, AAV9, AAV6, AAV2i8, Anc80, or Anc82.

11. The method of claim 7 or 8, wherein the vector is a pseudotyped vector comprising a capsid protein.

12. The method of claim 11, wherein the capsid protein is a capsid protein from AAV4, AAV6, AAV7, AAV8, or AAV9.

13. The method of claim 1 or 2, wherein the RNA is mRNA.

14. A vector comprising a polynucleotide encoding a flightin polypeptide, a WYR polypeptide, or a fragment thereof.

15. The vector of claim 14, wherein the vector is an adeno-associated vector.

16. The vector of claim 14 or 15, wherein the vector is AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, or AAVHSC17.

17. The vector of claim 14 or 15, wherein the vector is AAV1, AAV2, AAV8, AAV9, AAV6, AAV218, Anc80, or Anc82.

18. The vector of claim 14 or 15, wherein the vector is a pseudotyped vector comprising a capsid protein.

19. The vector of claim 18, wherein the capsid protein is a capsid protein from AAV4, AAV6, AAV7, AAV8, or AAV9.

20. The vector of claim 14, wherein the vector is a mammalian expression vector.

21. The vector of any of claims 14-20, wherein the vector comprises a promoter.

22. The vector of claim 21, wherein the promoter is constitutive or inducible.

23. The vector of claim 22, wherein the promoter is a myosin promoter.

24. The vector of claim 21, wherein the promoter is a cardiac tissue-specific promoter.

25. The vector of claim 21, wherein the promoter is a cardiomyocyte-specific promoter.

26. The vector of claim 24 or 25, wherein the promoter is cardiac troponin T promoter, a- myosin heavy chain (a-MHC) promoter, myosin light chain-2v (MLC-2v) promoter, cardiac NCX1 promoter, beta-myosin heavy chain isoform 7 (MHC7) promoter, alpha-myosin heavy chain isoform 6 (MHC6) promoter, actin alpha cardiac muscle 1 (ACTC1) promoter, cardiac myosin-binding protein-C (MYBPC3) promoter, alpha-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, or sarco / endoplasmic reticulum Ca2+ATPase (SERCA) promoter.

27. A cell expressing a heterologous polypeptide, wherein the heterologous polypeptide is a flightin polypeptide or a WYR polypeptide.

28. A recombinant cell produced by transforming a cell with the vector of any of claims 14- 26.

29. The cell of claim 27 or 28, wherein the cell is an induced pluripotent stem (iPS) cell.

30. The cell of claim 27 or 28, wherein the cell is a cardiomyocyte derived from an iPS cell.

31. A method of treating cardiac hypertrophy in a subject in need thereof or reducing the propensity of a subject to acquire cardiac hypertrophy, the method comprising administering to the subject a flightin polypeptide, a WYR polypeptide, or a polynucleotide encoding said polypeptides, thereby treating the cardiac hypertrophy.

32. A method of treating cardiac hypertrophy in a subject in need thereof or reducing the propensity of a subject to acquire cardiac hypertrophy, the method comprising administering to the subject a mammalian cell expressing a flightin polypeptide or a WYR polypeptide, thereby treating the cardiac hypertrophy.

33. A method of treating a cardiac disease or disorder in a subject in need thereof, or reducing the propensity of a subject to acquire a cardiac disease or disorder, the method comprising administering to the subject a mammalian cell expressing a flightin polypeptide or a WYR polypeptide, thereby treating the cardiac hypertrophy, wherein the cardiac disease or disorder is characterized by abnormal force production and / or cessation in cardiac muscle.

34. The method of claim 33, wherein the cardiac disease or disorder is one or more of cardiac hypertrophy, reduced systolic function, reduced diastolic function, maladaptive hypertrophy, heart failure with preserved systolic function, diastolic heart failure, systolic heart failure, hypertensive heart disease, aortic and mitral valve disease, pulmonary valve disease, hypertrophic cardiomyopathy, post ischemic and post-infarction cardiac remodeling, or cardiac failure.

35. The method of any of claims 31-34, wherein the subject is a human subject.

36. The method of any of claims 31-34, wherein the administering is by local or systemic injection.

37. A method of reducing the risk of hypertrophic cardiomyopathy in a selected subject, the method comprising administering a polynucleotide encoding a flightin polypeptide or a WYR polypeptide to the subject, thereby preventing the hypertrophic cardiomyopathy, wherein the selected subject is selected by detecting in a biological sample of the subject a marker selected from the group consisting of: MYH7 (P-myosin heavy chain 7), MYBPC3 (myosin- binding protein C), TNNT2 (cardiac muscle troponin T), TNNI3 (cardiac troponin I),TPM1 (a- tropomyosin), ACTC1 (cardiac a-actin), MYL2 (myosin lightchain 2), MYL3 (myosin light chain 3), CSRP3 (cysteine and glycine-rich protein 3), TTN (titin), TCAP (telethonin), MY0Z2 (myozenin 2), TRIM63 (ubiquitin E3 ligase tripartite motif protein 63 or MuRFl), and FHL1 (four-and-a-half LIM domains 1), and selecting the subject if mutations in the one or more markers are found.

38. The methods of any one of claims 1-13 or 31-34, the vector of any one of claims 14-26, or the cell of any one of claims 27-30, wherein the WYR polypeptide is a polypeptide with a sequence shown in FIGs. 6A-6C.

39. A kit for use in the methods of any one of claims 1-13, 31-34, or 38, the kit comprising a flightin polypeptide, a WYR polypeptide, polynucleotide encoding said polypeptides, the vector of any one of claims 14-26 or 38, or the cell of any one of claims 27-30 or 38, and instructions for using the kit.

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