Use of follistatin-like protein 1 (FSTL1) in cardiac metabolic heart failure

Abstract

Provided herein, inter alia, are compositions and kits comprising epicardium-derived paracrine factors, such as follicstatin-like protein 1 (FSTL1), and methods of using the same, for adjuvant treatment of cardiac metabolic heart failure.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 324,018 filed on March 25, 2022 and U.S. Provisional Patent Application Serial No. 63 / 326,718 filed on April 1, 2022, which are incorporated herein by reference in their entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with U.S. Government support under Grant No. R44HL140649 from the National Heart and Lung Institute of the National Institutes of Health. The U.S. Government has certain rights in this invention. Incorporation of Sequence Listing

[0003] This application contains a sequence listing, which is hereby incorporated by reference in its entirety. The attached sequence listing text file is named "Sequence_Listing_ST26 048722-505001WO", created on March 8, 2023, and is 7.4KB. Background Art

[0004] Heart failure is a complex clinical syndrome that can result from functional or structural impairment of the ventricles, leading to symptomatic left ventricular dysfunction. Symptoms result from inadequate cardiac output to keep up with the body's metabolic demands. Despite advances in treatment and prevention, heart failure remains the leading cause of cardiovascular morbidity and mortality worldwide. It can result from disorders of the pericardium, myocardium, endocardium, heart valves, great vessels, or from several metabolic abnormalities.

[0005] Cardiometabolic syndrome is a prevalent metabolic disorder. Epidemiological studies have associated cardiometabolic syndrome with an increased risk of coronary heart disease, ischemic stroke, cardiovascular death, and total mortality. There is also evidence that cardiometabolic syndrome is a risk factor for abnormal myocardial metabolism, cardiac dysfunction, and arrhythmias. Diabetic cardiomyopathy is increasingly problematic clinically, in which the combination of metabolic syndrome and chronic inflammation greatly increases the risk of coronary and peripheral artery disease.

[0006] There is a need for therapies that can address and / or treat cardiometabolic heart failure (eg, diabetic cardiomyopathy). The invention disclosed herein addresses these needs and also provides additional benefits. Summary of the invention

[0007] The present invention particularly provides a method for assisting the treatment of heart failure, the method comprising contacting cardiac tissue with a non-glycosylated or low-glycosylated follistatin-like protein 1 (FSTL1) polypeptide, wherein the heart failure may be cardiometabolic heart failure. The present invention also provides a method for treating cardiometabolic heart failure, the method comprising contacting cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide. The present invention further provides a method for repairing cardiac tissue after cardiometabolic heart failure, the method comprising contacting the cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide.

[0008] In any of the methods described herein, the cardiometabolic heart failure may be heart failure with reduced ejection fraction (HFrEF). In some embodiments, the cardiometabolic heart failure may be diabetic cardiomyopathy. In some embodiments, the FSTL1 may reduce heart failure mortality. In some embodiments, the FSTL1 may attenuate the development of coronary and cerebrovascular dysfunction. In some embodiments, the FSTL1 may improve cardiac function. In some embodiments, the FSTL1 may reduce heart rate. In some embodiments, the FSTL1 does not change left ventricular volume and systolic or diastolic volume. In some embodiments, the FSTL1 may prevent left ventricular diastolic dysfunction. In some embodiments, the FSTL1 may improve ventricular-vascular interaction. In some embodiments, the FSTL1 may reduce pulmonary congestion. In some embodiments, the FSTL1 may prevent mismatch between ventricular function and arterial load that varies with reduced contractility. In some embodiments, the FSTL1 may prevent pulse pressure narrowing. In some embodiments, the FSTL1 may improve coronary and / or peripheral vascular function. In some embodiments, the FSTL1 can increase blood flow to the heart and skeletal muscles. In some embodiments, the FSTL1 can attenuate the damaged BK Ca In some embodiments, the FSTL1 can attenuate the loss of TXA2-mediated vasoconstriction. In some embodiments, the FSTL1 can improve mitochondrial function. In some embodiments, the FSTL1 can reduce necrosis in the infarct area. In some embodiments, the FSTL1 can reduce lung weight.

[0009] In some embodiments, the FSTL1 can be delivered systemically. In some embodiments, the FSTL1 can be delivered endocardially. In some embodiments, the endocardial delivery can be performed via a catheter. In some embodiments, the FSTL1 can be delivered epicardially. In some embodiments, the cardiac tissue can be contacted from one or more of the epicardial site, the endocardial site, and / or by direct injection into the myocardium. In some embodiments, the FSTL1 can be delivered using a drug eluting stent. In some embodiments, the FSTL1 can be delivered by a hydrogel embedded or inoculated with the FSTL1. In some embodiments, the FSTL1 can be delivered by a collagen patch embedded or inoculated with the FSTL1. In some embodiments, the FSTL1 can be delivered via an osmotic pump. In some embodiments, the FSTL1 can be delivered as a single or several subcutaneous boluses. In some embodiments, the FSTL1 can be delivered by coronary infusion.

[0010] In some embodiments, the FSTL1 can be expressed in the heart by using modified RNA (modRNA). In some embodiments, the FSTL1 can be expressed by genome editing.

[0011] In some embodiments, the method further comprises an inhibitor of FSTL1 glycosylation. In some embodiments, the inhibitor of FSTL1 glycosylation comprises tunicamycin.

[0012] Each of the aspects and embodiments described herein can be used together unless expressly or clearly excluded from the context of an embodiment or aspect.

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments utilizing the principles of the present disclosure, in which:

[0015] Figure 1is a schematic summary of the experimental design of Example 1. Intact female Ossabaw pigs (2 months old) were fed a Western diet for 4 months to develop metabolic syndrome. At 6 months of age, the animals were subjected to 90 minutes of ischemia followed by reperfusion (I / R) to induce MI. One month after MI, ALZET osmotic pumps were implanted and delivered vehicle (MI group) or FSTL1 (MI+FSTL1 group) for two weeks, and terminal in vitro vascular experiments were performed 2 months after MI.

[0016] Figure 2A-2B Together, it illustrates the effects of FSTL1 treatment on insulin resistance and contractile function in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0017] Figure 2A is a graph illustrating the effect of FSTL1 treatment on insulin resistance.

[0018] Figure 2B is a graph illustrating the effect of FSTL1 treatment on ejection fraction.

[0019] Figure 3 is a graph illustrating the reduction in mortality after treatment with FSTL1.

[0020] Figure 4A-4B Together, they explain the difference in infarct size between the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0021] Figure 4A It is a graph illustrating the relative infarct size between the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0022] Figure 4B The difference in infarct size between the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1) is shown.

[0023] Figure 5A-Figure 5B Together, these results suggest that heart rate can be reduced after FSTL1 treatment in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0024] Figure 5A is a graph illustrating the effect of FSTL1 treatment on heart rate.

[0025] Figure 5B is a graph illustrating the effect of FSTL1 treatment on left ventricular stroke volume.

[0026] Figure 6is a graph illustrating that FSTL1 treatment does not change left ventricular end-systolic volume or end-diastolic volume. Heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1).

[0027] Figure 7A-7C Together, these results suggest that FSTL1 protects against left ventricular diastolic dysfunction in heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1).

[0028] Fig. 7A Graphs illustrating the effect of FSTL1 treatment on left ventricular end-systolic pressure.

[0029] Figure 7B Graphs illustrating the effect of FSTL1 treatment on left ventricular end-diastolic pressure.

[0030] Figure 7C is a graph illustrating the effect of FSTL1 treatment on the end-diastolic pressure-volume relationship.

[0031] Figure 8 It is a graph illustrating that FSTL1 treatment can reduce lung congestion. Heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1).

[0032] Figure 9A-9C Together, these results suggest that FSTL1 prevents the mismatch between ventricular function and arterial load that is associated with reduced contractility. Heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1).

[0033] Fig.9A is a graph illustrating the effect of FSTL1 treatment on ventriculoarterial coupling ratio.

[0034] Fig. 9B is a graph illustrating the effect of FSTL1 treatment on the end-systolic pressure-volume relationship.

[0035] Fig. 9C is a graph illustrating the effect of FSTL1 treatment on preload recruitable stroke work.

[0036] Figure 10A-10B Together, these results suggest that FSTL1 prevents pulse narrowing in heart failure (HF) and HF treated with FSTL1 (HF+FSTL1).

[0037] Fig. 10A is a graph illustrating the effect of FSTL1 treatment on pulmonary artery pressure.

[0038] Fig. 10B is a graph illustrating the effect of FSTL1 treatment on pulmonary pulse pressure / pulmonary systolic pressure.

[0039] Figure 11A-11B Together, these results suggest that FSTL1 treatment increases blood flow to the heart and skeletal muscles.

[0040] Fig.11A is a graph illustrating the effect of FSTL1 treatment on skeletal muscle blood flow.

[0041] Fig. 11B is a graph illustrating the effect of FSTL1 treatment on coronary blood flow.

[0042] Figure 12A-12C Together, these results suggest that FSTL1 treatment has an effect on coronary arteriolar function. FSTL1 attenuates impaired BK in distal coronary vessels. Ca Channel-mediated dilatation of coronary arterioles in heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1).

[0043] Fig. 12A is a graph illustrating the effect of FSTL1 treatment on the % possible dilation in the distal myocardium. The coronary arteriole diameter was 139.5 ± 4.9 μm.

[0044] Fig. 12B is a graph illustrating the effect of FSTL1 treatment on the % possible dilation in the marginal zone. The coronary arteriole diameter was 139.5 ± 4.9 μm.

[0045] Fig. 12C is a graph illustrating the effect of FSTL1 treatment on the % dilation possible in infarcts. The coronary arteriole diameter was 139.5 ± 4.9 μm.

[0046] Figure 13A-13C Together, we demonstrate the effects of FSTL1 treatment on cerebrovascular function. Functional capacity is lost in secondary pial arteries in HFrEF. FSTL1 attenuates the loss of TXA2-mediated vasoconstriction in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0047] Fig.13A Graph illustrating the effect of FSTL1 treatment on the % contraction possible with U46619 (thromboxane A2 agonist). The diameter of cerebral vessels was 385.3 ± 18.7 μm.

[0048] Fig. 13B is a graph illustrating the effect of FSTL1 treatment on the % dilation possible with sodium nitroprusside. The diameter of the cerebral vessels was 385.3 ± 18.7 μm.

[0049] Fig. 13Cis a graph illustrating the effect of FSTL1 treatment on the % dilation possible with NS-1619 (large conductance calcium-activated potassium channel activator; BKCa). The diameter of cerebral vessels was 385.3 ± 18.7 μm.

[0050] Figures 14A-14D Together, we show that FSTL1 treatment improves mitochondrial function, particularly in the infarcted region. Mitochondrial function was measured by high-resolution respirometry in diabetic pigs with subcutaneous FSTL1-treated and FSTL1-untreated myocardial infarction. Excised hearts were analyzed in three sections, depending on the distance from the infarct lesion. Oxygen flux (Y-axis) is shown as basal or substrate-saturated respiration before ADP addition, ADP-stimulated coupled respiration with octanoylcarnitine, glutamate, and succinate (state III), and maximal uncoupled respiration after addition of the uncoupling factor FCCP.

[0051] Fig.14A is a schematic summary of the experimental design of Example 6.

[0052] Fig. 14B is a diagram illustrating mitochondrial function in the distal region.

[0053] Fig. 14C is a diagram illustrating the function of mitochondria in the marginal zone.

[0054] Fig.14D is a diagram illustrating mitochondrial function in the infarcted area.

[0055] Figure 15A-15E Together, these results suggest that FSTL1 treatment reduces necrosis in the infarcted area.

[0056] Fig.15A is a graph illustrating the quantification of infarct size per treatment in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0057] Fig. 15B is a graph illustrating quantification of infarct area normalized to heart size in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0058] Fig. 15C is a graph illustrating the quantification of necrotic core volume in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0059] Fig.15D is a graph illustrating the quantification of necrotic core volume normalized to infarct size in the heart failure (HF) group and the HF group treated with FSTL1 (HF+FSTL1).

[0060] Fig.15ERepresentative images of gross morphological cardiac sections used for analysis in Example 7.

[0061] Fig.16 is a graph illustrating that FSTL1 treatment reduces lung weight. Heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1).

[0062] Figures 17A-17F Together, this indicates the effect of FSTL1 treatment. Heart failure (HF) group and HF group treated with FSTL1 (HF+FSTL1)

[0063] Fig.17A is a graph illustrating that FSTL1 treatment improves contractile function.

[0064] Fig. 17B is a graph illustrating that FSTL1 treatment improves diastolic function.

[0065] Fig. 17C is a graph illustrating that FSTL1 treatment restores PA proportional pulse pressure (PAPPP).

[0066] Fig.17D is a graph illustrating that FSTL1 treatment improves ventriculoarterial coupling.

[0067] Fig.17E is a graph illustrating that FSTL1 treatment reduces heart rate.

[0068] Fig.17F is a graph illustrating that FSTL1 treatment improves coronary blood flow. DETAILED DESCRIPTION

[0069] The present disclosure relates in particular to the use of human recombinant non-glycosylated or hypoglycosylated FSTL1 protein for both coronary and cerebrovascular function in cardiometabolic heart failure. Provided herein is a method of assisting the treatment of heart failure, the method comprising contacting cardiac tissue with a non-glycosylated or hypoglycosylated follistatin-like protein 1 (FSTL1) polypeptide, wherein the heart failure may be cardiometabolic heart failure.

[0070] The following description and examples detail embodiments of the disclosure.Although the disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0071] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0072] Although the various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided individually or in any suitable combination. On the contrary, although for clarity, the present disclosure may be described herein in the context of a separate embodiment, the present disclosure may also be implemented in a single embodiment. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore be changed. Those skilled in the art will recognize that variations and modifications of the present disclosure are encompassed within the scope of the present disclosure.

[0073] Each maximum numerical limit given throughout this specification is intended to include each numerical lower limit, as if such numerical lower limits were explicitly written herein. Each minimum numerical limit given throughout this specification will include each numerical upper limit, as if such numerical upper limits were explicitly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as if such narrower numerical ranges were explicitly written herein.

[0074] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated to be incorporated by reference. If different versions of a sequence are associated with accession numbers at different times, it means that the version is associated with the accession number at the effective filing date of the present application. The effective filing date means the earlier of the actual filing date or the filing date of the priority application with respect to the accession number (if applicable). Similarly, if different versions of a publication, website, etc. are published at different times, unless otherwise stated, it refers to the version published on the effective filing date closest to the application. Unless otherwise specifically stated, any feature, step, element, embodiment or aspect of the present disclosure may be used in combination with any other. definition

[0075] All terms are intended to be understood as understood by those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates.

[0076] The following definitions supplement those of the art and relate to the present application and are not attributed to any related or unrelated circumstances, e.g., any commonly owned patent or application. Although any methods and materials similar or equivalent to the methods and materials described herein may be used in the practice of testing the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting.

[0077] In this application, unless otherwise expressly stated, the use of the singular includes the plural meaning. It must be noted that, as used in the specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0078] In this application, the use of "or" means "and / or" unless otherwise stated. As used herein, the terms "and / or" and "any combination thereof," and their grammatical equivalents, may be used interchangeably. These terms may convey that any combination is specifically intended. For purposes of illustration only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C." The term "or" may be used in conjunction or disjunction, unless the context specifically dictates disjunction.

[0079] Furthermore, use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting.

[0080] Reference in the specification to "some embodiments," "embodiments," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

[0081] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. In addition, the compositions of the disclosure can be used to implement the methods of the disclosure.

[0082] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which error range depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice of the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" may include any amount of 10 and 9 to 11. In yet another embodiment, the term "about" associated with a reference value may also include a range of values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude of a value, preferably within 5 times of a value, and more preferably within 2 times of a value. Where particular values ​​are described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean that the particular value is within an acceptable error range.

[0083] As used herein, the phrase "cardiac tissue" refers to any tissue of the heart. Cardiac tissue includes myocardial tissue, epicardial tissue, and endocardial tissue. Cardiac tissue includes any cell type found within the heart.

[0084] As used herein, the phrase "epicardium-derived paracrine factors" may refer to any protein, polypeptide, or fragment thereof produced by cells of the outer epithelial layer of the heart that is capable of inducing a physiological, protective, proliferative, and / or reparative response in cardiac (e.g., myocardial) tissue following injury due to cardiovascular disease, myocardial infarction, or other ischemic events. In one embodiment, the epicardium-derived paracrine factors may be components of a conditioned medium obtained from epicardial cell cultures.

[0085] As used in the context of the present disclosure, the term "hypoglycosylated" or "non-glycosylated" may refer to a protein that may be post-translationally modified with a minimal number of carbohydrate moieties or may be completely lacking in carbohydrate moieties. In some embodiments, hypoglycosylation may refer to a protein that may be completely lacking in any carbohydrate modification, regardless of the modification (e.g., N-linked glycans, O-linked glycans, or phosphoglycans). In another embodiment, the term may refer to a protein that has reduced carbohydrate modifications (e.g., any of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells. In another embodiment, the term can refer to a protein with reduced carbohydrate modifications (e.g., about any of up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in a mammalian cell. In another embodiment, the term can refer to a protein with reduced carbohydrate modifications (e.g., reduced carbohydrate modifications by about at least any of 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%) relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in a mammalian cell. In another embodiment, the term can refer to a protein with reduced carbohydrate modifications (e.g., a reduction of about up to 1-100%, 5-95%, 10-90%, 20-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%) relative to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells. In yet other embodiments, the hypoglycosylated protein can be engineered so that all amino acid residues that can undergo glycosylation (e.g., amino acid residues that can undergo N-linked glycans, O-linked glycans, or phospho-glycans) can be substituted with amino acid residues that cannot undergo glycosylation.

[0086] A "subject" or "individual" can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, farm animals, sports animals, pets, primates, rodents, mice, and rats. In one aspect, the subject can be a human.

[0087] It should be understood that certain features of the disclosure described in the context of a separate embodiment for clarity may also be provided in combination in a single embodiment. On the contrary, various features of the disclosure described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to the disclosure are exactly covered by the disclosure and disclosed herein, as each combination is individually and clearly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also exactly covered by the disclosure and disclosed herein as each and every such sub-combination is individually and clearly disclosed herein. Overview of Cardiometabolic Heart Failure

[0088] Heart failure is a heterogeneous disease with an age-related increase in prevalence. The increased prevalence of heart failure in the elderly population may be attributed to the increasing number of long-term survivors after myocardial infarction, who are particularly susceptible to left ventricular dysfunction, the main cause of heart failure. In addition to age, obesity and diabetes have been identified as important risk factors for heart failure. Diabetes is one of the most relevant risk factors for heart failure, and its prevalence is increasing worldwide. Heart failure often presents as the first cardiovascular event in diabetic patients. Even individuals with prediabetes may have a greater risk of developing heart failure. In addition, heart failure itself is becoming a precursor to the development of diabetes. Therefore, diabetes and heart failure may be interrelated: diabetes increases the risk of heart failure, heart failure is very common in patients with diabetes, and heart failure may increase the risk of developing diabetes.

[0089] Three main phenotypes describe heart failure based on measurements of the left ventricular ejection fraction. Heart failure with reduced ejection fraction (HFrEF) has an ejection fraction of less than or equal to about 40%. Heart failure with preserved ejection fraction (HFpEF) has an ejection fraction greater than or equal to about 50%. Heart failure with moderate ejection fraction (HFmrEF) (other names: HFpEF-borderline and HFpEF-improved, when the ejection fraction in HFrEF improves to greater than 40%) has an ejection fraction of about 41% to about 49% according to European guidelines and about 40% to about 49% according to American guidelines. All patients with HFrEF have diastolic dysfunction; in contrast, diastolic dysfunction can occur in the absence of systolic dysfunction. Risk factors specific to HFrEF include a history of cardiovascular disease (such as myocardial infarction). Mortality is slightly higher in patients with HFrEF than in patients with HFpEF and is primarily caused by cardiovascular death.

[0090] Left ventricular dysfunction, the main cause of heart failure, can be divided into systolic dysfunction or abnormal myocardial relaxation (formerly known as diastolic dysfunction). The initial step in the clinical manifestation of systolic dysfunction is myocyte injury, for example due to myocardial infarction or ischemia. Injury-induced myocyte damage and thus progressive myocyte loss induce an inflammatory response and, thereby, ventricular remodeling. Remodeling produces an unbalanced cardiac wall structure with eccentric hypertrophy characterized by increased myocyte length. The inflammatory process triggers the excessive production of fibrotic tissue, which further disrupts cardiac function due to impaired transduction of myocyte contraction to cardiac force, ultimately leading to dysregulated contraction of myocyte bundles. In many cases, HFrEF presents with systolic dysfunction.

[0091] Diabetic cardiomyopathy is defined as cardiac dysfunction due to suppressed glucose metabolism and elevated fatty acid metabolism and as the presence of abnormal myocardial structure and appearance in individuals with diabetes who do not display any symptoms / signs of coronary artery disease, valvular disease, and other cardiovascular risk factors such as hypertension and dyslipidemia.

[0092] Diabetic cardiomyopathy can be divided into four stages. During the progression from stage I (impaired myocardial relaxation but normal ejection fraction) to the final stage IV (clinically significant ischemia and infarction leading to heart failure), muscle contraction decreases and fibrosis develops. In addition to and parallel to changes in cardiac structure, conditions associated with diabetes (such as hyperglycemia, hyperinsulinemia, inflammation, and hyperlipidemia) can also alter cardiac function. In diabetic cardiomyopathy, the myocardium shows impaired glucose metabolism due to insulin resistance, characterized by reduced glucose uptake, reduced glycolytic activity, and reduced pyruvate oxidation. As a result, glucose availability is limited, and there is an excess of circulating fatty acids, which are another major energy fuel for ATP production required to promote cardiac contraction after glucose, which is consumed primarily by myocardial cells in the case of diabetic cardiomyopathy. The resulting metabolic inflexibility and overactive fatty acid oxidation promote many secondary pathways, making the heart less able to cope with the increasing workload. In particular, fatty acid-rich cardiomyocytes produce ATP less efficiently, accumulate lipids and a range of toxic intermediates that are thought to promote proinflammatory and profibrotic responses, ultimately contributing to hypertrophy and diastolic dysfunction in diabetic cardiomyopathy. For example, ceramide is a toxic lipid that may be synthesized in the presence of excess fatty acids. Overloaded ceramide accumulates in cardiomyocytes and has profound effects on cell signaling, such as apoptosis and insulin resistance, and promotes ventricular remodeling, fibrosis, and macrophage infiltration after myocardial infarction. Compositions of the Disclosure

[0093] The present disclosure relates in particular to human recombinant non-glycosylated or hypoglycosylated follistatin-like 1 (FSTL1) protein for use in both coronary and cerebrovascular function in cardiometabolic heart failure.

[0094] FSTL1, also known as follistatin-related protein 1, is an epicardium-derived paracrine factor. FSTL1 is a protein that can be encoded by the FSTL1 gene in humans. The gene encodes a protein with similarity to follistatin, which can be an activin binding protein. FSTL1 contains an FS module (a follistatin-like sequence containing 10 conserved cysteine ​​residues), a Kazal-type serine protease inhibitor domain, two EF-hand domains, and a von Willebrand factor C-type domain (Entrez Gene: "FSTL1 follistatin-like protein 1").

[0095] FSTL1 can be a glycoprotein that exhibits regenerative properties when non-glycosylated or hypoglycosylated, including pro-angiogenic effects and prevention of abnormal vascular remodeling. The present disclosure relates in particular to the use of human recombinant non-glycosylated or hypoglycosylated FSTL1 protein for both coronary and cerebrovascular function in cardiometabolic heart failure. In some embodiments, FSTL1 can attenuate the development of coronary and cerebrovascular dysfunction in cardiometabolic heart failure with reduced ejection fraction (HFrEF). In some embodiments, the cardiometabolic heart failure can be diabetic cardiomyopathy. In some embodiments, heart failure can refer to a long-term condition in which the heart does not pump blood well to meet the needs of the body.

[0096] In one embodiment, FSTL1 can include the amino acid sequence of SEQ ID NO: 1 (NCBI Reference Sequence: NP_009016.1). Nucleic acids encoding FSTL1 are provided and encompassed within the scope of the present invention. In some embodiments, the nucleic acid can be a recombinant nucleic acid. In some embodiments, FSTL1 can be encoded by the nucleic acid of SEQ ID NO: 2 (NCBI Reference Sequence: NM_007085.4).

[0097] The polynucleic acid encoding FSTL1 can be incorporated into a vector such as an expression vector using standard techniques known to those skilled in the art. Methods for connecting a DNA construct comprising a nucleic acid of interest such as FSTL1, a promoter, a terminator and other sequences and inserting them into a suitable vector are well known in the art. In addition, known recombinant techniques (e.g., Invitrogen Life Technologies, Gateway Technology) can be used to construct vectors.

[0098] In some embodiments, it may be desirable to overexpress FSTL1 nucleic acids at levels far higher than currently found in naturally occurring cells. This result can be achieved by selectively cloning nucleic acids encoding these polypeptides into multicopy plasmids or placing these nucleic acids under strong inducible or constitutive promoters. Methods for overexpressing desired polypeptides are common and well known in the art of molecular biology, examples of which can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, 2001.

[0099] A variety of host cells can be used to prepare recombinant host cells that can express FSTL1. The host cell can be a cell that naturally produces FSTL1 or a cell that does not naturally produce FSTL1. For example, mammalian cells (such as, but not limited to, Chinese hamster ovary (CHO) cells) or epicardium-derived cell cultures can be used to produce FSTL1. However, in other embodiments, cells derived from organisms that do not post-translationally glycosylate proteins (i.e., cells that do not post-translationally modify proteins using one or more carbohydrate moieties) can be used to produce recombinant FSTL1.

[0100] Non-limiting examples of cells that do not post-translationally glycosylate proteins include bacterial cells. Thus, in one embodiment, the host cell may be a bacterial cell. In another embodiment, the bacterial cell may be a Gram-positive bacterial cell or a Gram-negative bacterial cell. In another embodiment, the bacterial cell may be selected from Escherichia coli (E. coli), Lactobacillus acidophilus (L. acidophilus), Pantoea citrea (P. citrea), Bacillus subtilis (B. subtilis), Bacillus licheniformis (B. licheniformis), Bacillus lentus (B. lentus), Bacillus brevis (B. brevis), Bacillus stearothermophilus (B. stearothermophilus), Bacillus alkalophilus (B. alkalophilus), Bacillus amyloliquefaciens (B. amyloliquefaciens), Bacillus clausii, Bacillus spp. Bacillus (B.clausii), Bacillus halodurans, B.megaterium, B.coagulans, B.circulans, B.lautus, B.thuringiensis, S.albus, S.lividans, S.coelicolor, S.griseus, Pseudomonas sp., Pseudomonas alcaligenes, Clostridium sp., Corynebacterium sp. and C.glutamicum cells.

[0101] Nucleic acids encoding FSTL1 or vectors containing them can be inserted into host cells (e.g., bacterial cells) using standard techniques for expression of the encoded FSTL1 polypeptide. Introduction of DNA constructs or vectors into host cells can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated or DEAE-dextrin-mediated transfection or transfection using recombinant bacteriophage viruses), incubation with calcium phosphate DNA precipitation, high-speed bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are well known in the art (see, for example, Current Protocols in Molecular Biology (FM Ausubel et al. (eds.) Chapter 9, 1987); Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, 2001; and Campbell et al., Curr Genet, 16:53-56, 1989, each of which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods). The introduced nucleic acid may be integrated into the chromosomal DNA of the host cell or maintained as an extrachromosomal replicating sequence. In yet another embodiment, the FSTL1 polypeptide may be produced in a host cell via delivery of a chemically modified mRNA encoding a mutant FSTL1 glycosylation-deficient polypeptide. (See Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Zangi L, et al. Nat Biotechnol. 2013 October; 31(10):898-907, incorporated herein by reference in its entirety). Chemically modified RNA (also referred to herein as modRNA) may include, for example, modification of phosphate to a phosphorothioate internucleotide bond, modification of the 2'-hydroxyl of ribose, or other modifications to the phosphate backbone or sugar moiety of the mRNA.

[0102] In some embodiments, FSTL1 may be non-glycosylated or under-glycosylated FSTL1. Non-glycosylated or under-glycosylated FSTL1 may be obtained by producing recombinant FSTL1 in host cells that naturally do not post-translationally modify proteins with carbohydrate moieties (such as bacteria, e.g., E. coli), or have been engineered so that they may not post-translationally modify proteins with carbohydrate moieties. Alternatively, non-glycosylated or under-glycosylated FSTL1 may be produced in mammalian cells or other eukaryotic cells that normally post-translationally modify proteins with carbohydrate moieties, but which have been treated with one or more glycosylation inhibitors. Suitable glycosylation inhibitors include, but are not limited to, tunicamycin (which blocks all N-glycosylation of proteins), streptovirin, sporicidin, amphomycin, jinzhimycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, pyroxetine, duimycin, 1-deoxymannonojirimycin, deoxynojirimycin, N-methyl-1-deoxymannonojirimycin, brefeldin A, glucose analogs, mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D-(+)-mannose, D-(+)-galactose, 2-deoxy-2-fluoro-D-glucose, 1,4-dideoxy-1,4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, hydroxymethylglutaryl coenzyme A reductase inhibitor, 25-hydroxycholesterol, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin ampicillin, m-chlorocarbonyl cyanide phenylhydrazone (CCCP), compatine, 2-deoxyglucose, N-acetyl-D-glucosamine, hygoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castamin, glutamine, bromoconduritol, bromoconduritol epoxide, bromoconduritol derivatives, aglycosylmethyl-p-nitrophenyltriazene, β-hydroxynorvaline, threo-β-fluoroasparagine, D-(+)-glucono delta-lactone, di(2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (diphenylmethyl)-phosphoric acid, [2-(diphenylphosphinyl)ethyl]trimethylammonium iodide, iodoacetate, 2-deoxy-D-glucose and fluoroacetate.

[0103] Alternatively, in other embodiments, recombinant FSTL1 may be engineered so that it may not be glycosylated when produced using eukaryotic host cells or other host cells capable of glycosylation. In most biological contexts, glycosylation may be N-linked or O-linked. The N-linked glycosylation process occurs in eukaryotes and widely occurs in archaea, but rarely occurs in true bacteria. In N-linked glycosylation, the glycan (i.e., the carbohydrate-containing moiety) may be attached to the nitrogen atom of the asparagine or arginine amino acid side chain. N-linked glycans can almost always be attached to the nitrogen atom of the asparagine (Asn) side chain, which may be present as part of the Asn-X-Ser / Thr consensus sequence, where X can be any amino acid except proline (Pro), serine (Ser), and threonine (Thr). O-linked glycosylation may be a form of glycosylation that occurs in the Golgi apparatus of eukaryotes. In O-linked glycosylation, the glycan can be attached to the hydroxyl oxygen of a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline amino acid side chain.

[0104] Thus, in some embodiments, recombinant FSTL1 may be engineered so that it may not be N-linked glycosylated. In this case, some or all arginine or asparagine amino acids in the polypeptide sequence that can be glycosylated may be replaced by amino acids that cannot be glycosylated (e.g., glutamine). In other embodiments, recombinant FSTL1 may be engineered so that it may not be O-linked glycosylated. In this case, all serine, threonine, tyrosine, hydroxylysine or hydroxyproline residues in the polypeptide sequence that can be glycosylated may be replaced by amino acids that cannot be glycosylated (e.g., alanine). In yet other embodiments, recombinant FSTL1 may be engineered so that it may not be O-linked glycosylated or N-linked glycosylated by replacing all amino acids that can be glycosylated with amino acids that cannot be glycosylated. In other embodiments, one or more asparagine (N) residues located at positions X144, X180, X175 and / or X223 in the FSTL1 amino acid sequence may be replaced by amino acids that cannot be glycosylated (e.g., but not limited to glutamine (Q)). The engineered FSTL1 that cannot be glycosylated can be produced in a host cell via transfection of a plasmid, viral vector, or chemically synthesized mRNA or mRNA mimics carrying a gene encoding the FSTL1 that cannot be glycosylated. Alternatively, the gene encoding the FSTL1 that cannot be glycosylated can be integrated into the chromosome of the host cell under the control of an inducible or constitutively expressed promoter. In yet another embodiment, the FSTL1 polypeptide that cannot be glycosylated can be produced in a host cell via delivery of a modified mRNA encoding the FSTL1 polypeptide that cannot be glycosylated. Methods of the present disclosure

[0105] The present disclosure relates in particular to the use of human recombinant non-glycosylated or under-glycosylated FSTL1 protein for both coronary and cerebrovascular function in cardiometabolic heart failure. The present disclosure also relates to a method for assisting the treatment of heart failure, the method comprising contacting cardiac tissue with a non-glycosylated or under-glycosylated follistatin-like protein 1 (FSTL1) polypeptide, wherein the heart failure may be cardiometabolic heart failure. The present disclosure also relates to a method for treating cardiometabolic heart failure, the method comprising contacting cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide. The present disclosure further relates to a method for repairing cardiac tissue after cardiometabolic heart failure, the method comprising contacting the cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide.

[0106] In any of the methods described herein, the cardiometabolic heart failure may be heart failure with reduced ejection fraction (HFrEF). In some embodiments, the cardiometabolic heart failure may be diabetic cardiomyopathy. In some embodiments, the FSTL1 may reduce heart failure mortality. In some embodiments, the FSTL1 may attenuate the development of coronary and cerebrovascular dysfunction. In some embodiments, the FSTL1 may improve cardiac function. In some embodiments, the FSTL1 may reduce heart rate. In some embodiments, the FSTL1 does not change left ventricular volume and systolic or diastolic volume. In some embodiments, the FSTL1 may prevent left ventricular diastolic dysfunction. In some embodiments, the FSTL1 may improve ventricular-vascular interaction. In some embodiments, the FSTL1 may reduce pulmonary congestion. In some embodiments, the FSTL1 may prevent mismatch between ventricular function and arterial load that varies with reduced contractility. In some embodiments, the FSTL1 may prevent pulse pressure narrowing. In some embodiments, the FSTL1 may improve coronary and / or peripheral vascular function. In some embodiments, the FSTL1 can increase blood flow to the heart and skeletal muscles. In some embodiments, the FSTL1 can attenuate the impaired large conductance voltage and Ca2 + Activated K +In some embodiments, FSTL1 can reduce the dilatation capacity of coronary arterioles mediated by the BKCa channel. BKCa can be an important regulator of membrane excitability in a variety of cells and tissues. In some embodiments, FSTL1 can attenuate the loss of thromboxane A2 (TXA2)-mediated vasoconstriction. TXA2 can be a known vasoconstrictor and is activated during tissue injury and inflammation. In some embodiments, the FSTL1 can improve mitochondrial function. In some embodiments, the FSTL1 can reduce necrosis in the infarct area. In some embodiments, the FSTL1 can reduce lung weight.

[0107] Damage to cardiac (e.g., myocardial) tissue may be associated with any number of diseases or conditions known to affect the heart or circulatory system, and include, but are not limited to, coronary heart disease, cardiomyopathy, ischemic heart disease, heart failure, inflammatory heart disease, valvular heart disease, and aneurysm. In one embodiment, the damage may be caused by myocardial infarction (MI; such as acute myocardial infarction (AMI)). In another embodiment, the damage may be caused by an ischemic event and subsequent reperfusion.

[0108] Repair of damaged cardiac (e.g., myocardial) tissue can include increasing the number of cardiomyocytes, which can be measured indirectly in living subjects by several imaging methods (e.g., delayed enhancement MRI, DE-MRI) as the size of myocardial infarction decreases. See, e.g., (Hendel RC et al., JACC 48(7); 1475-97) and (Sardella G et al., JACC 2009; 53(4): 309-15, incorporated herein by reference in their entirety). In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can result in about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or about any of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% recovery of damaged muscle and reduction of infarct size. In other embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can result in about at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or about any of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% recovery of damaged muscle and reduction of infarct size. In other embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can result in up to about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or about any of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% recovery of damaged muscle and reduction of infarct size.

[0109] The epicardium-derived paracrine factors (such as non-glycosylated FSTL1) can be delivered using a variety of methods known in the art. In some embodiments, the FSTL1 can be delivered systemically. In some embodiments, the FSTL1 can be delivered transendocardially. In some embodiments, the endocardial delivery can be performed via a catheter. In some embodiments, the FSTL1 can be delivered transepicardially. In some embodiments, the cardiac tissue can be contacted from one or more of the epicardial site, the endocardial site, and / or by direct injection into the myocardium. In some embodiments, the FSTL1 can be delivered using a drug eluting stent. In some embodiments, the FSTL1 can be delivered by a hydrogel embedded or seeded with the FSTL1. In some embodiments, the FSTL1 can be delivered by a collagen patch embedded or seeded with the FSTL1. In some embodiments, the FSTL1 can be delivered via an osmotic pump. In some embodiments, the FSTL1 can be delivered as a single or several subcutaneous boluses. In some embodiments, the FSTL1 can be delivered by coronary infusion. In some embodiments, the FSTL1 can be expressed in the heart by using modified RNA (modRNA). In some embodiments, the FSTL1 can be expressed by genome editing.

[0110] In some embodiments of any of the methods disclosed herein, epicardium-derived paracrine factors (such as non-glycosylated FSTL1) can be infused, seeded, or embedded in a 3D collagen-based patch. The collagen-based patch can then be directly contacted to the damaged area of ​​the epicardium or myocardium (such as an area of ​​the myocardium exposed to an ischemic event such as a myocardial infarction). The 3D collagen can be applied to the epicardium or myocardium by suturing or by any other means known in the art for contacting a patch with damaged tissue.

[0111] In yet other embodiments, epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can be components of a hydrogel that can be delivered to the epicardium, to the endocardium, or to damaged areas of the myocardium (via, for example, catheter technology; Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014) 7:232-241, herein incorporated by reference in its entirety).

[0112] In some embodiments, effects on cardiac (eg, myocardial) tissue can be achieved by transendocardial placement of non-glycosylated or underglycosylated FSTL1 into the heart using a percutaneous catheter delivery system, such as the system available from BioCardia (www.biocardia.com).

[0113] In some embodiments, effects on cardiac (eg, myocardial) tissue may be achieved by using a catheter device similar to those used in other applications (eg, the Epicardial Catheter System TM , St. Jude Medical) by placing non-glycosylated or low-glycosylated FSTL1 into the heart via the epicardium.

[0114] In some embodiments, effects on cardiac (eg, myocardial) tissue can be achieved by placement of non-glycosylated or hypoglycosylated FSTL1 when impregnated in a drug-diluting stent, such as those available from Abbott Laboratories or Biosensors International, among others.

[0115] In some embodiments, effects on cardiac (eg, myocardial) tissue can be achieved by systemically placing non-glycosylated or under-glycosylated FSTL1 using an approved formulation.

[0116] In some embodiments, the effect on cardiac (e.g., myocardial) tissue can be achieved by placing hypoglycosylated FSTL1, which can be achieved by using compounds or drugs that inhibit the glycosylation of endogenous glycosylated FSTL1 proteins, which may be readily available and known to those skilled in the art. Therefore, any method described herein may further include an FSTL1 glycosylation inhibitor. In some embodiments, the FSTL1 glycosylation inhibitor may be selected from: tunicamycin, streptovirin, sporidinin, amphomycin, jinzhimycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, pyroxetine, duromycin, 1-deoxymannojirimycin, deoxynojirimycin, N-methyl-1-deoxymannojirimycin, brefeldin A, glucose analogs, mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D-(+)-mannose, D-(+)-galactose, 2-deoxy-2-fluoro-D-glucose, 1,4-dideoxy-1,4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, hydroxymethylglutaryl coenzyme A reductase inhibitor, 25-hydroxycholesterol, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, Monensin, m-chlorocarbonyl cyanide phenylhydrazone (CCCP), Combetin, monol-phosphoryl-2-deoxyglucose, N-acetyl-D-glucosamine, hypoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castamin, glutamine, bromo-naphthoic acid, naphthoic acid epoxide, naphthoic acid derivatives, aglycosylmethyl-p-nitrophenyltriazene, β-hydroxynorvaline, threo-β-fluoroasparagine, D-(+)-glucono delta-lactone, di(2-ethylhexyl)phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (diphenylmethyl)-phosphoric acid, [2-(diphenylphosphinyl)ethyl]trimethylammonium iodide, iodoacetate, 2-deoxy-D-glucose and fluoroacetate. In some embodiments, the FSTL1 glycosylation inhibitor may be tunicamycin.

[0117] In some embodiments, effects on cardiac (eg, myocardial) tissue can be achieved by rendering FSTL1 non-glycosylated or hypoglycosylated, which can be achieved by introducing modRNA encoding specific mutagenesis targeting N-glycosylation sites in the FSTL1 mRNA sequence.

[0118] In some embodiments, the effect on cardiac (e.g., myocardial) tissue can be achieved by placing non-glycosylated or hypoglycosylated FSTL1, which can be achieved by genome editing using CRISPR / Cas9 technology or similar technology (see, e.g., Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Hao Yin, et al. Nature Biotechnology 32, 551-553 (2014) doi: 10.1038 / nbt.2884, incorporated herein by reference in its entirety).

[0119] In some other embodiments, the effects on the heart (eg, myocardium) can be achieved by delivering small molecule mimetics of non-glycosylated or hypoglycosylated FSTL1.

[0120] Injured cardiac (e.g., myocardial) tissue can be contacted with any epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) composition (e.g., pharmaceutical composition) disclosed herein before, during, or after cardiac (e.g., myocardial) tissue injury. In some embodiments, cardiac (e.g., myocardial) tissue can be contacted with an epicardium-derived paracrine factor composition in a subject who is considered to be at risk for cardiometabolic disease, cardiovascular disease, myocardial infarction, or another myocardial ischemic event to reduce or prevent damage to the myocardium caused by the event. In other embodiments, cardiac (e.g., myocardial) tissue can be contacted with an epicardium-derived paracrine factor composition immediately after the onset of an ischemic event caused by cardiovascular disease or myocardial infarction, such as about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes , 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours or 12 hours or more (including all time periods falling between these values). In some embodiments, the composition can be administered less than 1 minute after the cardiac injury. Alternatively, in other embodiments, cardiac (e.g., myocardial) tissue can be contacted with the epicardium-derived paracrine factor composition after injury, such as at least 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, three weeks, one month, 2 months, 3 months, 4 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or one or more years (including all time periods falling between these values) after the onset of an ischemic event caused by cardiovascular disease or myocardial infarction.

[0121] Any method for treating heart (e.g., myocardial) tissue damage disclosed herein can result in an increase in the survival rate of the subject after injury. As used herein, the survival rate increased includes, for example, compared with the relative survival rate of a subject who has not yet experienced methods described herein, the survival rate of the subject increases by at least about 5% (e.g., at least about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150% or more than 200% or greater). Survival time can be measured, for example, in days, weeks, months or years. In some embodiments, contacting the paracrine factors derived from epicardium with the heart (e.g., myocardial) tissue of damage according to any method described herein can extend the survival period of the subject by at least six months, seven months, eight months, nine months, 10 months, 12 months, 18 months, 24 months, 36 months or more.

[0122] In some embodiments, repair of damaged cardiac (e.g., myocardial) tissue may include a reduction or attenuation of fibrosis in cardiac (e.g., myocardial) tissue compared to the amount of fibrosis in cardiac (e.g., myocardial) tissue that may not have been contacted with epicardium-derived paracrine factors after injury. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (such as non-glycosylated FSTL1) may result in a reduction of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more of any one of (including all values ​​falling between these percentages) in fibrosis in cardiac (e.g., myocardial) tissue. In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (such as non-glycosylated FSTL1) can result in a reduction in fibrosis in cardiac (e.g., myocardial) tissue by up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages. In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) can result in a decrease in fibrosis in cardiac (e.g., myocardial) tissue by about at least 1%-100%, 5%-95%, 10%-90%, 20%-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, including all values ​​falling between these percentages. Assessment of cardiomyocyte fibrosis can be routine in the art and can be measured by DE-MRI or by histological examination (autopsy or biopsy) of cardiac (e.g., myocardial) tissue. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (such as non-glycosylated FSTL1) can result in a reduction in fibrosis in cardiac (e.g., myocardial) tissue by up to about any of 1%-100%, 5%-95%, 10%-90%, 20%-80%, 30-70%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%, including all values ​​falling between these percentages.Assessment of cardiomyocyte fibrosis is routine in the art and can be measured by DE-MRI or by histological examination (autopsy or biopsy) of cardiac (eg, myocardial) tissue.

[0123] In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (such as non-glycosylated FSTL1) can result in an improvement in cardiac function of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, including all values ​​falling between these percentages. In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) can result in a decrease in heart rate of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more (including all values ​​falling between these percentages) when compared to a subject not treated with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1). In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) does not alter left ventricular end-systolic volume or end-diastolic volume.

[0124] In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) prevents left ventricular diastolic dysfunction. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can result in an improvement of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more of left ventricular end-systolic pressure (including all values ​​falling between these percentages). In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) can result in a decrease in left ventricular end-diastolic pressure of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more, including all values ​​falling between these percentages, when compared to a subject not treated with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1). In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) can result in a decrease in the end-diastolic pressure-volume relationship by about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more, including all values ​​falling between these percentages, when compared to a subject not treated with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1).

[0125] In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) improves ventricular-vascular interactions. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can result in a decrease in pulmonary congestion of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more (including all values ​​falling between these percentages) when compared to subjects not treated with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1). In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) can prevent the mismatch between ventricular function and arterial load that varies with reduced contractility. In some embodiments, contacting cardiac (e.g., myocardial) tissue with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1) can result in a decrease in ventriculo-arterial coupling ratio of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more (including all values ​​falling between these percentages) when compared to a subject not treated with an epicardium-derived paracrine factor (e.g., non-glycosylated FSTL1). In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (such as non-glycosylated FSTL1) can result in an improvement in the end-systolic pressure-volume relationship of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more, including all values ​​falling between these percentages. In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can result in an improvement in preload-replenishing stroke work of about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more (including all values ​​falling between these percentages). In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) prevents pulse pressure narrowing.

[0126] In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can increase blood flow to cardiac and skeletal muscles by about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more (including all values ​​falling between these percentages). In some embodiments, contacting cardiac (e.g., myocardial) tissue with epicardium-derived paracrine factors (e.g., non-glycosylated FSTL1) can attenuate impaired BK in distal coronary vessels. Ca Channel-Mediated Coronary Arteriolar Dilatation Capacity In some embodiments, contacting the heart (eg, myocardium) with epicardium-derived paracrine factors (eg, non-glycosylated FSTL1) can attenuate the loss of TXA2-mediated vasoconstriction.

[0127] In one embodiment, treatment with FSTL1 of the present disclosure can prevent coronary and cerebrovascular dysfunction (Example 2). For example, in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction, treatment with non-glycosylated FSTL1 can increase the probability of survival and reduce infarct size. Figure 4A-4B The effect of treatment with non-glycosylated FSTL1 on infarct size is illustrated. Figure 3 The effect of treatment with non-glycosylated FSTL1 on the probability of survival is illustrated.

[0128] In one embodiment, treatment with FSTL1 of the present disclosure can increase the probability of survival and can reduce infarct size (Example 3). For example, in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction, treatment with non-glycosylated FSTL1 can increase the probability of survival and can reduce infarct size. Figure 4A-4B The effect of treatment with non-glycosylated FSTL1 on infarct size is illustrated. Figure 3 The effect of treatment with non-glycosylated FSTL1 on the probability of survival is illustrated.

[0129] In one embodiment, FSTL1 of the present disclosure can improve cardiac function (Example 4). For example, in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction, treatment with non-glycosylated FSTL1 can improve cardiac function. Figure 5A-Figure 5B This indicates that heart rate can be reduced after FSTL1 treatment. Figure 6 This indicates that FSTL1 treatment does not change the left ventricular end-systolic volume or end-diastolic volume. Figure 7A-7C This indicates that FSTL1 can prevent left ventricular diastolic dysfunction.

[0130] In one embodiment, treatment with FSTL1 of the present disclosure can improve ventricular-vascular interactions (Example 5). For example, in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction, treatment with non-glycosylated FSTL1 can improve ventricular-vascular interactions. Figure 8 This indicates that FSTL1 treatment can reduce lung congestion. Figure 9A-9C Together, these results suggest that FSTL1 may prevent the mismatch between ventricular function and arterial load that occurs with reduced contractility. Fig.9A To illustrate the effect of FSTL1 treatment on the ventricle-arterial coupling ratio. Fig. 9B To illustrate the effect of FSTL1 treatment on the end-systolic pressure-volume relationship. Fig. 9C To illustrate the effect of FSTL1 treatment on preload supplementary stroke work. Figure 10A-10B This indicates that FSTL1 can prevent pulse pressure from narrowing. Fig. 10A To illustrate the effect of FSTL1 treatment on pulmonary artery pressure. Fig. 10B Describe the effect of FSTL1 treatment on pulmonary pulse pressure / pulmonary systolic pressure.

[0131] In one embodiment, blood flow to the heart and skeletal muscles can be increased following FSTL1 treatment of the present disclosure. For example, Fig.11A Describe the effect of FSTL1 treatment on skeletal muscle blood flow. Fig. 11B The results suggest that FSTL1 treatment has an effect on coronary blood flow. In the experimental setting of cardiometabolic HFrEF, FSTL-1 can attenuate the loss of functional capacity of both coronary and cerebral arterioles, demonstrating its potential to improve vascular function. In addition, FSTL1 can improve coronary and / or peripheral vascular function.

[0132] In one embodiment, treatment with FSTL1 of the present disclosure can attenuate the loss of TXA2-mediated vasoconstriction. For example, NS-1619-induced vasodilation can be increased in MI+FSTL-1 animals compared to MI in distal regions of the heart, but not in the infarcted or border zones ( Figure 12A-12C In cerebral arterioles, the vasoconstrictor response to U46619 might also be dependent on the experimental group (interaction), with increased contractility observed in MI+FSTL-1 compared with MI animals ( Figure 13A-13C ).

[0133] In one embodiment, treatment with FSTL1 of the present disclosure can improve mitochondrial function in the infarcted area ( Figure 14B-Figure 14D In one embodiment, treatment with FSTL1 of the present disclosure can reduce necrosis in the infarcted area ( Figure 15C-Figure 15DIn one embodiment, treatment with FSTL1 of the present disclosure can reduce lung weight in diabetic pigs with myocardial infarction ( Fig.16 In one embodiment, treatment with FSTL1 of the present disclosure can improve contractile function ( Fig.17A ), can improve diastolic function ( Fig. 17B ), PAPPP can be restored ( Fig. 17C ), which can improve ventriculoarterial coupling ( Fig.17D ), can reduce heart rate ( Fig.17E ), and can improve coronary blood flow. Pharmaceutical composition / kit

[0134] The disclosure described herein contemplates incorporating FSTL1 into a pharmaceutical composition (e.g., a sterile pharmaceutical composition) containing one or more pharmaceutically acceptable carriers. As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" according to the disclosure may be a component of a composition such as a carrier, diluent, or excipient that is compatible with the other ingredients of the composition in that it can be combined with the agent and / or composition of the present invention without eliminating the biological activity of the agent or composition (e.g., FSTL1, such as non-glycosylated FSTL1), and may be suitable for use in a subject provided herein without excessive adverse side effects (e.g., toxicity, irritation, allergic reactions, and death). Side effects may be "excessive" when their risk outweighs the benefits provided by the pharmaceutical composition. Non-limiting examples of pharmaceutically acceptable components include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, sterile water, polyethylene glycol, polyvinyl pyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers, and various types of wetting agents. Additives such as water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, etc. may also be included in the composition along with the carrier, diluent, or excipient. In one embodiment, pharmaceutically acceptable carriers suitable for use in the compositions disclosed herein may be sterile, pathogen-free, and / or otherwise safe for administration to a subject without the risk of associated infection and other undue adverse side effects.

[0135] Any pharmaceutical composition containing FSTL1 (such as containing non-glycosylated FSTL1) disclosed herein can be formulated for administration using any number of administration methods available in the art. Administration can be performed by a variety of routes, including pumps, patches, catheters, stents, oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, intranasal, etc. In some embodiments, the above-mentioned administration methods can be used to deliver suspensions containing FSTL1 (e.g., non-glycosylated FSTL1 mixed with gel or gelatin sponge particles). These compositions can be effective as both injectable and oral compositions. Such compositions can be prepared in a manner well known in the pharmaceutical art and contain at least one active compound. When used as an oral composition, the polypeptide composition can be protected from acid digestion in the stomach by a pharmaceutically acceptable protective agent.

[0136] In some embodiments, any pharmaceutical composition containing FSTL1 (such as containing non-glycosylated FSTL1) disclosed herein can be incorporated into an engineered patch for direct application to damaged epicardial or myocardial tissue. In some embodiments, a highly hydrated collagen gel can be compressed to remove excess water and produce a dense biomaterial with improved biological and mechanical properties.

[0137] In some embodiments, any pharmaceutical composition containing FSTL1 (such as containing non-glycosylated FSTL1) disclosed herein can be delivered via an osmotic pump. In some embodiments, any pharmaceutical composition containing FSTL1 (such as containing non-glycosylated FSTL1) disclosed herein can be delivered as a single or several subcutaneous boluses.

[0138] Also provided herein are kits comprising (i) epicardium-derived paracrine factors (such as non-glycosylated FSTL1 polypeptides); and (ii) one or more pharmaceutically acceptable excipients. One or both of these kit components may be made sterile so that they can be administered to an individual in need (e.g., an individual with cardiac injury (such as MI)). The kit may optionally comprise a 3D collagen patch (such as any of those disclosed herein), which may be inoculated or infused with epicardium-derived paracrine factors prior to administration to a subject. Alternatively, a pre-inoculated or pre-infused 3D collagen patch may be included in the kit along with written instructions regarding the use and application of the patch to damaged cardiac (e.g., myocardial) tissue or epicardium of a subject in need. The kit may further include means for adhering the 3D collagen patch to the epicardium or damaged cardiac (e.g., myocardial) tissue, such as, but not limited to, suture material.

[0139] Any kit disclosed herein may also include a hydrogel (e.g., a self-polymerizing hydrogel) as a carrier of epicardium-derived paracrine factors (e.g., hypoglycosylated FSTL1 polypeptide). In one embodiment, the kit further includes one or more catheters for delivering the hydrogel (e.g., a hydrogel infused with hypoglycosylated FSTL1 polypeptide) to one or more damaged areas of the endocardium, epicardium, and / or myocardium.

[0140] The kit may also include written instructions for using the kit, such as instructions for infusing epicardium-derived paracrine factors into the 3D collagen patch, suturing the patch to the myocardium or epicardium, infusing epicardium-derived paracrine factors into a hydrogel (such as a self-polymerizing hydrogel), and delivering the hydrogel to one or more damaged areas of the epicardium or myocardium via catheter technology.

[0141] Each maximum numerical limit given throughout this specification is intended to include each numerical lower limit, as if such numerical lower limits were explicitly written herein. Each minimum numerical limit given throughout this specification will include each numerical upper limit, as if such numerical upper limits were explicitly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as if such narrower numerical ranges were explicitly written herein. Example

[0142] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. Example 1. Generation and general practice of Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction Test plan

[0143] The following experimental protocol was applied in all examples provided subsequently herein. Generation of a porcine model of cardiometabolic heart failure

[0144] Intact female Ossabaw pigs (2 months old) were fed a Western diet for 4 months to develop metabolic syndrome. At 6 months of age, animals were subjected to 90 minutes of ischemia followed by reperfusion (I / R) to induce MI. One month after MI, ALZET osmotic pumps were implanted and delivered vehicle (MI group) or FSTL1 (MI+FSTL1 group) for two weeks, and terminal in vitro vascular experiments were performed 2 months after MI ( Figure 1 ). Preoperative preparation

[0145] Sedation: I / R MI-Telazol (6-7 mg / kg IM) and buprenorphine (0.02 to 0.05 mg / kg). 1. Apply ophthalmic ointment to the eyes to prevent corneal drying and ulceration, followed by IM injection of clindamycin (if cefazolin is not available) or Excede, depending on the procedure. 2. Obtain baseline heart rate, respiratory rate, and body temperature. 3. Shave the incision site or sites (electric clippers), scrub with chlorhexidine until free of debris, and sponge with rubbing alcohol. 4. Place ear cannulas to deliver IV fluids and medications throughout the procedure. If the animal is not sufficiently sedated for ear cannulas to be placed, place a nose cone over the snout to deliver 100% isoflurane mixed with 3%-5% isoflurane. Oxygen until the animal is calm. 5. Administer propofol bolus for anesthesia induction. 6. The animal was intubated, placed on an X-ray fluoroscopic table, and ventilated at a rate of 8-12 breaths per minute, a tidal volume of 10-20 mL / kg, and a pressure of 20-25 cm H2 O. Anesthesia Methods - See below. 7. Connect patient monitoring leads and slowly administer cefazolin IV (if clindamycin or Excede was not given during preparation). 8. Intraoperative warmth was maintained by a warm water circulating jacket placed beneath the pig. The incision site was prepared using a 3-scrub regimen of chlorhexidine followed by a 70% alcohol rinse. Finally, the surgical field was draped sterilely.

[0146] Anesthesia: I / R MI-constant rate infusion (CRI) of propofol was started at approximately 10 mL / kg / h, determined by eyelid response and / or paw withdrawal reflex. Survival surgery

[0147] Animals were maintained under surgical anesthesia using isoflurane or propofol and ventilated with 100% oxygen. A room circulator trained in survival surgery procedures monitored adequate depth of anesthesia at least every 15 minutes throughout the procedure by means of jaw tone, foot reflex, and eyelid reflex. EKG, heart rate, blood pressure, SpO2, end-tidal CO2, respiratory rate, and body temperature were continuously monitored by members of the surgical team and / or by the circulator. If the pig's vital signs indicated the need for emergency treatment, the following medications could be used: epinephrine, lidocaine, phenylephrine (IV drip), and atropine. If the EKG indicated ventricular fibrillation or cardiac arrest, CPR was administered immediately. Defibrillation was performed at 200 joules biphasic. CPR, defibrillation, and medication administration were used to assist resuscitation when necessary. Surgery: Surgical Procedure

[0148] Survival Angioplasty I / R MI: After femoral artery access, a standard 6F-guiding catheter was connected to the manifold assembly to allow continuous central pressure monitoring, saline flushing, and Visipaque contrast injection. Based on fluoroscopic guidance, an intracoronary balloon catheter was advanced into the left anterior descending (LAD) coronary artery and inflated for 90 minutes to induce ischemia followed by reperfusion. During the duration of I / R, 1,000 mL of saline solution mixed with amiodarone and potassium acetate was administered at 300 mL / h IV CRI. Follistatin (FSTL1) delivery via Alzet pump

[0149] Placement of ALZET Osmotic Pumps (2-4): Two ±4-6 cm incisions were made on the right and left sides of the neck for placement of the FSTL1 and one or more EdU pumps. A subcutaneous pocket was created by blunt dissection and the pumps were implanted (approximately 5×1.5 cm). The dead space was adequately closed with dissolvable subcutaneous sutures. The skin was closed with 2-0 Ethilon in a simple interrupted pattern, followed by removal of the sutures within 7-10 days. Approximately 30 days after osmotic pump implantation, a non-surviving intercostal thoracotomy procedure was performed as above. Recovery after anesthesia

[0150] Animals are recovered in clean enclosures and kept warm with blankets and heating lamps if necessary. When animals lie flat and are intubated, animals are continuously monitored to evaluate capillary refill time, heart rate and respiratory rate, and body temperature to ensure full recovery. Once the pharyngeal reflex is restored, the animals are extubated. When the animals are semi-conscious and not in sternal recumbent position (sternally recumbent), the animals are monitored no less than once every 15 minutes. When consciousness is restored and the animals are in sternal recumbent position but cannot stand, they are monitored no less than once every 6 hours. Once standing and moving around but without normal eating / drinking, monitor twice a day. When active and alert and eating / drinking normally, monitor the animals daily for 3 days (if it is a minor operation) or 7-10 days until the sutures are removed. Surgery: Postoperative Care

[0151] The animals were recovered in clean enclosures and kept warm with blankets and heating lamps if necessary. The animals were continuously monitored while they were supine and intubated (i.e., semi-conscious). Capillary refill time, heart rate and respiratory rate, and body temperature were evaluated to ensure adequate recovery. Once the gag reflex was restored, the animals were extubated. While maintaining the sternal recumbency, the pigs were monitored every 15 minutes until they were alert and regained their motor function. If there were no complications associated with the surgery, postoperative recovery was considered complete at this time. Animal activity / temperament, food intake, and incisions were monitored daily after surgery until the sutures were removed. Surgery: Special Needs

[0152] Special needs of animals after surgery: All animals were housed individually following surgical procedures to prevent injury or discomfort. For the first 5-7 days, all animals were housed in elevated deck pens to help prevent infection. Example 2. Non-glycosylated FSTL1 protects against coronary and cerebrovascular dysfunction

[0153] Follistatin-like protein 1 (FSTL1) is a glycoprotein that, when non-glycosylated or hypoglycosylated, exhibits regenerative properties, including proangiogenic effects and prevention of abnormal vascular remodeling. Therefore, the goal of the current study was to evaluate the therapeutic benefits of human recombinant non-glycosylated FSTL1 protein on both coronary and cerebrovascular function in the preclinical Ossabaw pig model of myocardial infarction (MI). In the experimental setting of cardiometabolic heart failure with reduced ejection fraction (HFrEF), FSTL1 attenuates the development of coronary and cerebrovascular dysfunction.

[0154] In vitro assessment of isolated coronary arteriolar function (n=5-6 for infarct, rim, and distal regions of the left ventricle; 139.5±4.9 μm) and cerebral arteriolar function (n=2-6 for secondary pial middle cerebral artery; 385.3±18.7 μm) was performed using pressure myography according to dose-response (1e-9 to 1e-4) curves: (1) U46619 (thromboxane A2 agonist); and (2) NS-1619 (large conductance calcium-activated potassium channel activator; BKCa). 2x2 ANOVA (group x dose) was used to determine significance at the p<0.05 level ( Figure 12A-12C ; Figure 13A-13C Cardiometabolic HFrEF was indicated by a combined ejection fraction of 38% ± 2% and HOMA-IR of 3.1 ± 0.4 (compared to historical controls of 0.8 ± 0.1). Figure 2A-2B Coronary arteriolar vasodilator responses to NS-1619 were dependent on the experimental group (interaction). Specifically, in the distal regions of the heart, NS-1619-induced vasodilation was increased in MI+FSTL-1 animals compared with MI, but not in the infarcted or border zones ( Figure 12A-12C In cerebral arterioles, the vasoconstrictor response to U46619 was also dependent on the experimental group (interaction), and increased contractility was observed in MI+FSTL-1 compared with MI animals ( Figure 13A-13C ).

[0155] In addition, FSTL1 treatment increased blood flow to the heart and skeletal muscles ( Figure 11A-11B ). Fig.11A Describe the effect of FSTL1 treatment on skeletal muscle blood flow. Fig. 11BTo illustrate the effect of FSTL1 treatment on coronary blood flow.

[0156] In conclusion, in the experimental setting of cardiometabolic HFrEF, FSTL-1 attenuated the loss of functional capacity of both coronary and cerebral arterioles, demonstrating its potential to improve vascular function. Furthermore, FSTL1 improved coronary and / or peripheral vascular function. Example 3. Non-glycosylated FSTL1 increases survival probability and reduces infarct size

[0157] This example describes the results of experiments conducted to investigate the effect of treatment with non-glycosylated FSTL1 on infarct size and on the probability of survival in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction. The effect of treatment with non-glycosylated FSTL1 on infarct size is shown in Figure 4A-4B The effect of treatment with non-glycosylated FSTL1 on the probability of survival is shown in Figure 3 In conclusion, in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction, treatment with non-glycosylated FSTL1 increased the probability of survival and reduced infarct size. Example 4. Non-glycosylated FSTL1 improves cardiac function

[0158] This example describes the results of experiments performed to investigate the effects of treatment with non-glycosylated FSTL1 on cardiac function in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction. Figure 5A-Figure 5B Together, these results suggest that heart rate can be reduced after FSTL1 treatment. Figure 6 This indicates that FSTL1 treatment does not change the left ventricular end-systolic volume or end-diastolic volume. Figure 7A-7C Together, these results suggest that FSTL1 protects against left ventricular diastolic dysfunction. In conclusion, treatment with non-glycosylated FSTL1 improves cardiac function. Example 5. Non-glycosylated FSTL1 improves ventricular-vascular interaction

[0159] This example describes the results of experiments performed to investigate the effects of treatment with non-glycosylated FSTL1 on ventricular-vascular interactions in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction. Figure 8 This indicates that lung congestion was reduced after FSTL1 treatment. Figure 9A-9C Together, these results suggest that FSTL1 prevents the mismatch between ventricular function and arterial load that occurs with reduced contractility. Fig.9A To illustrate the effect of FSTL1 treatment on the ventricle-arterial coupling ratio. Fig. 9B To illustrate the effect of FSTL1 treatment on the end-systolic pressure-volume relationship. Fig. 9C To illustrate the effect of FSTL1 treatment on preload supplementary stroke work. Figure 10A-10B Together, these results suggest that FSTL1 prevents pulse narrowing. Fig. 10ATo illustrate the effect of FSTL1 treatment on pulmonary artery pressure. Fig. 10B The effect of FSTL1 treatment on pulmonary pulse pressure / pulmonary systolic pressure is illustrated. Data obtained by PV loops were recorded under conditions of reduced preload, which were achieved by transient occlusion of the inferior vena cava by inflation of a balloon catheter. In conclusion, in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction, non-glycosylated FSTL1 improves ventricular-vascular interactions. Example 6. Non-glycosylated FSTL1 improves mitochondrial function

[0160] This example describes the results of experiments performed to investigate the effects of treatment with non-glycosylated FSTL1 on mitochondrial function in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction.

[0161] The left ventricle (500 mg) was placed in ice-cold buffer A (100 mM KCl, 50 mM MOPS, 5 mM MgSO4, 1 mM EGTA, 1 mM ATP). The tissue was then minced and transferred to a glass homogenizer tube and homogenized with a Teflon pestle. One volume of mitochondrial isolation buffer (220 mM mannitol, 70 mM sucrose, 10 mM Tris base, 1 mM EDTA) supplemented with 0.5 mg / mL protease was added to the sample and slowly inverted. The sample was centrifuged at 800 g for 10 minutes at 4°C. The supernatant was transferred to a new tube, and one volume of mitochondrial isolation buffer supplemented with protease inhibitors was added to the supernatant to inactivate the protease. The supernatant was centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in mitochondrial isolation buffer containing 0.1% BSA and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in mitochondrial preservation buffer (0.5 mM EGTA, 3 mM MgCl2·6H2O, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 1 g / l BSA, 20 mM histidine, 20 μM vitamin E succinate, 3 mM glutathione, 1 μM leupeptin, 2 mM glutamate, 2 mM malate, 2 mM Mg-ATP).

[0162] Mitochondrial respiration was assessed using high-resolution respirometry (Oroboros Oxygraph-2k; Oroboros Instruments; Innsbruck, Austria). Briefly, oxygen flux (state 2-GM) was measured by adding glutamate (5 mM) and malate (2 mM) to the chamber in the absence of ADP to assess state 2 respiration. Oxidative phosphorylation (OXPHOS) with electron flux through complex I was then quantified by titrating ADP (25-125 μM) (GM+ADP: state 3-complex I) to assess state 3 respiration. The maximum ADP respiration (succinate: state 3-complex I+II) with electron flux through both complex I and complex II was assessed by adding succinate (10 mM). Finally, the maximum capacity (decoupled) of the electron transport system was assessed by adding FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, 0.25 μM) to decouple.

[0163] Four states were analyzed (state 2GM, state 3 complex 1GM+ADP, state 3 complex I+II succinate, and uncoupled FCCP). Figure 14B-Figure 14D As shown, oxygen flow was not affected by FSTL1 treatment in distal parts of the heart, areas not directly affected by the infarction ( Fig. 14B ). Likewise, there is no difference in the oxygen flux at the damage edge band in the four oxidation states measured ( Fig. 14C In contrast, in the infarcted area, FSTL1 treatment significantly increased oxygen flow (although it is normally reduced) ( Fig.14D In conclusion, FSTL1 SubQ treatment (120 mg / 14 days using an osmotic pump) of diabetic pigs with myocardial infarction clearly improved mitochondrial function in the infarcted area. Example 7. Non-glycosylated FSTL1 reduces necrosis in the infarct area

[0164] This example describes the results of experiments performed to investigate the effect of treatment with non-glycosylated FSTL1 on necrosis in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction.

[0165] The left ventricle is cut into five transverse slices from apex to base. The top and base photos of each slice are taken, and the digital images are analyzed using ImageJ software. For each slice, the left ventricle and infarct area are manually tracked from the photos of the top and base. Infarction is calculated as the left ventricle percentage of each slice, and the results of the top and base are averaged and multiplied by the weight of each individual slice weight to calculate the infarct area. The infarct area of ​​all slices is added up, and divided by the sum of the left ventricle.

[0166] Evaluation of infarct size showed no significant difference in infarct size between the different treatments, indicating that the performance of surgical coronary artery occlusion is consistent ( Figure 15A-15B Analysis of scar tissue showed a more than two-fold reduction in necrotic area in the FSTL1-treated group ( Fig. 15C ), even after correction for infarct size ( Fig.15D ). In conclusion, non-glycosylated FSTL1 reduces necrosis in the infarcted area. Example 8. Non-glycosylated FSTL1 reduces lung weight

[0167] This example describes the results of experiments performed to investigate the effects of treatment with non-glycosylated FSTL1 on lung weight in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction.

[0168] Direct lung weights were measured in FSTL1-treated and untreated pigs. FSTL1-treated animals showed statistically significant reduced lung weights compared to untreated animals, showing indirect evidence of improved cardiac function ( Fig.16 In conclusion, non-glycosylated FSTL1 reduces lung weight in diabetic pigs with myocardial infarction. Example 9. Non-glycosylated FSTL1 improves cardiac function

[0169] This example describes the results of experiments performed to investigate the effects of treatment with non-glycosylated FSTL1 on cardiac function in Ossabaw pigs with cardiometabolic heart failure with reduced ejection fraction.

[0170] PV loops were recorded under conditions of reduced preload, which were achieved by transient occlusion of the inferior vena cava by inflation of a balloon catheter. Indices of LV function were generated using a minimum of 10 consecutive cardiac cycles using Lab Scribe software (iWorx, Dover, NH), including HR, LV end-systolic and end-diastolic volumes (LVESV and LVEDV, respectively), LV end-systolic and end-diastolic pressures, ejection fraction (EF%), stroke volume, stroke work (SW), rate-pressure product, and cardiac output. Other previously published indices of LV function that have been proposed to be less sensitive to myocardial load and / or morphology were also determined using at least 15 consecutive cardiac cycles with decreasing preload, including the end-systolic PV relationship (ESPVR) and preload-supplemented SW (PRSW). In conclusion, treatment with nonglycosylated FSTL1 improved systolic function ( Fig.17A ), improve diastolic function ( Fig. 17B ), restore PAPPP( Fig. 17C ), improve ventricle-arterial coupling ( Fig.17D ), lower heart rate ( Fig.17E ), and improve coronary blood flow.

[0171] While the present disclosure has been particularly shown and described with reference to certain embodiments, some of which are preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein. sequence

Claims

1. A method for assisting the treatment of cardiometabolic heart failure, the method comprising contacting cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide.

2. A method for treating cardiometabolic heart failure, the method comprising contacting cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide.

3. A method for repairing cardiac tissue following cardiometabolic heart failure, the method comprising contacting the cardiac tissue with a non-glycosylated follistatin-like protein 1 (FSTL1) polypeptide.

4. The method according to any one of claims 1-3, wherein the cardiometabolic heart failure is heart failure with reduced ejection fraction (HFrEF).

5. The method according to any one of claims 1-4, wherein the cardiometabolic heart failure is diabetic cardiomyopathy.

6. The method according to any one of claims 1-5, wherein the FSTL1 reduces heart failure mortality.

7. The method according to any one of claims 1-5, wherein the FSTL1 attenuates the development of coronary and cerebrovascular dysfunction.

8. The method according to any one of claims 1-5, wherein the FSTL1 improves cardiac function.

9. The method of claim 8, wherein the FSTL1 reduces heart rate.

10. The method of claim 8, wherein the FSTL1 does not change left ventricular volume and systolic or diastolic volume.

11. The method of claim 8, wherein the FSTL1 prevents left ventricular diastolic dysfunction.

12. The method according to any one of claims 1-5, wherein the FSTL1 improves ventricular-vascular interaction.

13. The method of claim 12, wherein the FSTL1 reduces lung congestion.

14. The method of claim 12, wherein the FSTL1 prevents a mismatch between ventricular function and arterial load that varies with decreased contractility.

15. The method of claim 12, wherein the FSTL1 prevents pulse pressure narrowing.

16. The method according to any one of claims 1-5, wherein the FSTL1 improves coronary and / or peripheral vascular function.

17. The method of claim 16, wherein the FSTL1 increases blood flow to the heart and skeletal muscles.

18. The method of claim 16, wherein the FSTL1 attenuates impaired BK Ca Channel-mediated dilatation of coronary arterioles.

19. The method of claim 16, wherein the FSTL1 attenuates the loss of TXA2-mediated vasoconstriction.

20. The method of any one of claims 1-5, wherein the FSTL1 improves mitochondrial function.

21. The method of any one of claims 1-5, wherein the FSTL1 reduces necrosis in the infarct area.

22. The method of any one of claims 1-5, wherein the FSTL1 reduces lung weight.

23. The method of any one of claims 1-22, wherein the FSTL1 is delivered systemically.

24. The method of any one of claims 1-22, wherein the FSTL1 is delivered transendocardially.

25. The method of claim 24, wherein the endocardial delivery is performed via a catheter.

26. The method of any one of claims 1-22, wherein the FSTL1 is delivered transepicardially.

27. The method of any one of claims 1-22, wherein the FSTL1 is delivered using a drug eluting stent.

28. The method of any one of claims 1-22, wherein the FSTL1 is delivered via a hydrogel embedded or seeded with the FSTL1.

29. The method of any one of claims 1-22, wherein the FSTL1 is delivered via a collagen patch embedded or seeded with the FSTL1.

30. The method of any one of claims 1-22, wherein the FSTL1 is delivered via an osmotic pump.

31. The method of any one of claims 1-22, wherein the FSTL1 is delivered as a single or several subcutaneous boluses.

32. The method of any one of claims 1-22, wherein the FSTL1 is delivered by coronary infusion.

33. The method according to any one of claims 1-22, wherein the FSTL1 is expressed in the heart by using modified RNA (modRNA).

34. The method of any one of claims 1-22, wherein the FSTL1 is expressed by genome editing.

35. The method of any one of claims 24-34, wherein the cardiac tissue is contacted from one or more of an epicardial site, an endocardial site, and / or by direct injection into the myocardium.

36. The method of any one of claims 33-34, further comprising an inhibitor of FSTL1 glycosylation.

37. The method of claim 36, wherein the inhibitor of FSTL1 glycosylation comprises tunicamycin.

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