Epicardial-derived paracrine factors for repairing cardiac tissue

Abstract

To provide epicardial-derived paracrine factors for repairing cardiac tissue.SOLUTION: Provided herein is a composition and a kit comprising an epicardial-derived paracrine factor (e.g., hypoglycosylated follistatin-like 1 (FSTL1)) for treating and repairing damage to cardiac tissue caused by cardiovascular disease, myocardial infarction (MI), other ischemic events, or cardiac-growth deficiency, as well as a method for using them. In some aspects, a method for repairing a cardiac tissue (e.g., myocardial tissue) after injury in a subject in need thereof is provided, where the method comprises a step of contacting a cardiac tissue (e.g., myocardial tissue) with an epicardial-derived paracrine factor. In some embodiments, the epicardial-derived paracrine factor is hypoglycosylated follistatin-like 1 (FSTL1) polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 145,480, filed April 9, 2015, and U.S. Provisional Patent Application No. 62 / 196,766, filed July 24, 2015, the disclosures of which are incorporated herein by reference in their entireties.

[0002] In particular, the present invention relates to compositions comprising epicardium-derived paracrine factors and their use to treat or prevent damage to cardiac tissue (eg, myocardial tissue) following an ischemic event such as myocardial infarction. [Background technology]

[0003] Acute myocardial infarction (AMI) is one of the leading causes of death in the Western world, and many risk factors, both environmental and genetic, contribute to its pathogenesis. The heart generally does not have sufficient endogenous regenerative capacity for repair after injury. Left ventricular (LV) remodeling following myocardial infarction (MI) or other ischemic events leads to LV dilation and ultimately heart failure (Holmes et al., 2005, Annu Rev Biomed Eng., vol. 7, pp. 223-53). Immediately after coronary artery occlusion, ischemic myocytes downstream of the occlusion become necrotic and / or undergo apoptosis. Neutrophils immediately infiltrate the tissue, while leukocytes, primarily macrophages, arrive shortly thereafter and participate in the digestion of necrotic cellular debris. Neutrophils in the ischemic tissue can be toxic to the surrounding myocytes, as they release reactive oxygen species and proteolytic enzymes that further injure the surrounding myocytes (Nah and Rhee, Korean Journal of Cardiovascular Diseases, 2011, 144:145-146). Circ J., October, Vol. 39(10), 393-82009). Injury results in the formation of a hypocellular scar that leads to contractile dysfunction and, ultimately, heart failure. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Holmes et al., Annu Rev Biomed Eng. (2005) 7, 223-53 Summary of the Invention [Means for solving the problem]

[0005] In order to reduce the epidemiological and economic burden associated with ischemic events affecting the myocardium, there is an urgent need to develop novel compositions and strategies that maintain cardiomyocyte survival or stimulate cardiomyocyte growth after injury caused by ischemic events such as myocardial infarction. There is a need for therapies that can address and / or treat cardiac tissue (e.g., myocardial tissue) after injury. The invention disclosed herein addresses these needs and also provides additional benefits. Specifically provided herein are compositions and kits comprising epicardium-derived paracrine factors (e.g., underglycosylated follistatin-like 1 (FSTL1)) and methods for using the same to treat and repair damage to cardiac tissue (e.g., myocardial tissue) caused by cardiovascular disease, myocardial infarction (MI), or other ischemic events.

[0006] Thus, in some aspects, provided herein is a method for repairing cardiac tissue (e.g., myocardial tissue) following injury in a subject in need thereof, comprising contacting the cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor. In some embodiments, the epicardium-derived paracrine factor is an underglycosylated follistatin-like 1 (FSTL1) polypeptide. In some embodiments of any of the embodiments disclosed herein, the injury is due to ischemia-reperfusion cardiac injury (e.g., myocardial injury), ischemic heart disease, and / or due to a hypotrophic heart. In some embodiments of any of the embodiments disclosed herein, the injury is a myocardial infarction, and / or the heart contains scar tissue. In some embodiments of any of the embodiments disclosed herein, repairing the cardiac tissue (e.g., myocardial tissue) comprises increasing the number of cardiomyocytes in the cardiac tissue (e.g., myocardial tissue). In some embodiments, the number of cardiomyocytes is increased at least two-fold compared to the number of cardiomyocytes in the injured scar tissue not contacted by the epicardium-derived paracrine factor following injury. In some embodiments of any of the embodiments disclosed herein, the repair of the cardiac tissue (e.g., myocardial tissue) comprises an improvement in the fractional shortening of the cardiac tissue (e.g., myocardial tissue) compared to the amount of fractional shortening in cardiac tissue (e.g., myocardial tissue) not contacted by the epicardial-derived paracrine factor after injury. In some embodiments of any of the embodiments disclosed herein, the repair of the cardiac tissue (e.g., myocardial tissue) comprises an improvement in wall motion compared to the same subject before treatment. In some embodiments of any of the embodiments disclosed herein, the repair of the cardiac tissue (e.g., myocardial tissue) comprises an improvement in blood perfusion area compared to the same subject before treatment. In some embodiments of any of the embodiments disclosed herein, the repair of the cardiac tissue (e.g., myocardial tissue) comprises a reduction in cardiac (e.g., myocardial) incidents and hospitalizations compared to a similar subject without treatment.In some embodiments of any of the embodiments disclosed herein, repair of the cardiac tissue (e.g., myocardial tissue) comprises an increase in the amount of cytokinesis of cardiomyocytes in the cardiac tissue (e.g., myocardial tissue) compared to an amount of cytokinesis of cardiomyocytes in cardiac tissue (e.g., myocardial tissue) not contacted by epicardium-derived paracrine factors after injury. In some embodiments, the increase in the amount of cytokinesis of cardiomyocytes is determined by expression of Aurora B kinase. In some embodiments of any of the embodiments disclosed herein, repair of the cardiac tissue (e.g., myocardial tissue) comprises a decrease in apoptosis of cardiomyocytes. In some embodiments of any of the embodiments disclosed herein, the method results in an increase in the level of a transcript encoding a cardiac (e.g., myocardial) specific contractile protein in the cardiomyocyte. In some embodiments, the method results in a two-fold increase in the level of a transcript encoding a cardiac (e.g., myocardial) specific contractile protein in the cardiomyocyte. In some embodiments of any of the embodiments disclosed herein, the cardiac (e.g., myocardial) specific contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a. In some embodiments of any of the embodiments disclosed herein, the method comprises inducing rhythmic contractile Ca in cardiomyocytes. 2+ Actinin with +In some embodiments of any of the embodiments disclosed herein, the cardiac tissue (e.g., myocardial tissue) is contacted with the epicardium-derived paracrine factor immediately after the injury. In some embodiments of any of the embodiments disclosed herein, the method increases survival of the subject following injury. In some embodiments of any of the embodiments disclosed herein, the method attenuates fibrosis in the cardiac tissue (e.g., myocardial tissue) following the injury. In some embodiments of any of the embodiments disclosed herein, the method results in increased vascularization of the injured region of the cardiac tissue (e.g., myocardial tissue). In some embodiments, the increased vascularization is determined by expression of von Willebrand factor (vWF) or smooth muscle actin in vascular cells. In some embodiments of any of the embodiments disclosed herein, the method induces cell cycle entry of cardiomyocytes. In some embodiments, cell cycle entry of the cardiomyocytes is assessed by expression of phospho-histone H3. In some embodiments of any of the embodiments disclosed herein, the method results in at least a two-fold increase in cardiomyocyte cell cycle entry compared to the amount of cardiomyocyte cell cycle entry in a cardiac tissue (e.g., myocardial tissue) not contacted by epicardium-derived paracrine factors after injury. In some embodiments, the underglycosylated FSTL1 polypeptide is synthesized in a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the underglycosylated FSTL1 polypeptide is synthesized in a eukaryotic cell treated with a glycosylation inhibitor. In some embodiments, the inhibitor of glycosylation is tunicamycin. In some embodiments, the underglycosylated FSTL1 polypeptide is produced by replacing one or more glycosylated amino acids with one or more amino acids ineligible for glycosylation. In some embodiments, the one or more glycosylated amino acids are selected from the group consisting of N144, N175, N180, and N223.In some embodiments of any of the embodiments disclosed herein, the underglycosylated FSTL1 polypeptide does not protect cardiomyocytes from apoptosis after injury. In some embodiments of any of the embodiments disclosed herein, the underglycosylated FSTL1 polypeptide is directly injected into the injured myocardial tissue. In some embodiments of any of the embodiments disclosed herein, the underglycosylated FSTL1 polypeptide is delivered systemically. In some embodiments of any of the embodiments disclosed herein, the underglycosylated FSTL1 polypeptide is delivered endocardium. In some embodiments of any of the embodiments disclosed herein, the underglycosylated FSTL1 polypeptide is embedded or seeded in a three-dimensional collagen patch. In some embodiments of any of the embodiments disclosed herein, the underglycosylated FSTL1 polypeptide is embedded or seeded in a hydrogel. In some embodiments of any of the embodiments disclosed herein, the cardiac tissue (e.g., myocardial tissue) is contacted from one or more of an epicardial site, an endocardial site, and / or by direct injection into the myocardium.

[0007] In another aspect, provided herein is a pharmaceutical composition comprising an underglycosylated follistatin-like 1 (FSTL1) polypeptide and one or more pharma- ceutically acceptable excipients. In some embodiments, the underglycosylated FSTL1 polypeptide is synthesized in a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the underglycosylated FSTL1 polypeptide is synthesized in a eukaryotic cell treated with a glycosylation inhibitor. In some embodiments, the glycosylation inhibitor is tunicamycin. In some embodiments, the underglycosylated FSTL1 polypeptide is obtained by genome editing. In some embodiments, the underglycosylated FSTL1 polypeptide is obtained by insertion of a modified RNA. In some embodiments, the underglycosylated FSTL1 polypeptide is obtained by drug treatment of a subject (e.g., such that the treatment inhibits glycosylation of endogenous FSTL1 polypeptide). In some embodiments of any of the embodiments disclosed herein, the composition is formulated for direct injection into injured cardiac tissue (e.g., myocardial tissue). In some embodiments of any of the embodiments disclosed herein, the composition is formulated for systemic administration.In some embodiments of any of the embodiments disclosed herein, the low glycosylation FSTL1 polypeptide is embedded or seeded in a three-dimensional (3D) collagen patch.In some embodiments, the 3D collagen patch has an elastic modulus of 12±4 kPa.

[0008] In a further aspect, provided herein is a kit comprising: (i) a hypoglycosylated follistatin-like 1 (FSTL1) polypeptide; and (ii) one or more pharma-ceutically acceptable excipients. In some embodiments, the kit further comprises (iii) a three-dimensional (3D) collagen patch. In some embodiments of any of the embodiments disclosed herein, the hypoglycosylated FSTL1 polypeptide is embedded or seeded in a three-dimensional (3D) collagen patch. In some embodiments of any of the embodiments disclosed herein, the 3D collagen patch has an elastic modulus of 12±4 kPa. In some embodiments of any of the embodiments disclosed herein, the kit further comprises (iv) an adhesive means for attaching the 3D collagen patch to the epicardium or myocardium of the injured heart. In some embodiments, the adhesive means is a suture.

[0009] In yet another aspect, provided herein is a method for repairing cardiac tissue (e.g., myocardial tissue) after injury in a subject in need thereof, comprising contacting said cardiac tissue (e.g., myocardial tissue) with a three-dimensional (3D) collagen patch seeded or infused with recombinant underglycosylated follistatin-like 1 (FSTL1) polypeptide. In some embodiments, the injury is ischemia-reperfusion injury. In some embodiments, the injury is myocardial infarction. In some embodiments of any of the embodiments disclosed herein, the 3D collagen patch is sutured to the cardiac tissue (e.g., myocardial tissue).

[0010] In another aspect, provided herein is a three-dimensional (3D) collagen patch infused or seeded with recombinant underglycosylated follistatin-like 1 (FSTL1) polypeptide. In some embodiments, the recombinant underglycosylated FSTL1 polypeptide is synthesized in a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the recombinant underglycosylated FSTL1 polypeptide is synthesized in a eukaryotic cell treated with a glycosylation inhibitor. In some embodiments, the glycosylation inhibitor is tunicamycin. In some embodiments of any of the embodiments disclosed herein, the 3D collagen patch has an elastic modulus of 12±4 kPa.

[0011] Each of the aspects and embodiments described herein can be used together unless expressly or unambiguously excluded from the context of the embodiment or aspect.

[0012] Throughout this specification, various patents, patent applications and other types of publications are referenced (e.g., journal articles, electronic database entries, etc.). The disclosures of all patents, patent applications and other publications cited herein are incorporated herein by reference in their entireties for all purposes. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for repairing cardiac tissue after injury in a subject in need of repair of cardiac tissue after injury, comprising contacting the cardiac tissue with an underglycosylated follistatin-like 1 (FSTL1) polypeptide. (Item 2) 2. The method of claim 1, wherein the injury is ischemia-reperfusion cardiac injury, is due to ischemic heart disease, and / or is due to hypoplastic heart. (Item 3) 2. The method of claim 1, wherein the injury is a myocardial infarction and / or the heart contains scar tissue. (Item 4) 4. The method of any one of items 1 to 3, wherein repairing cardiac tissue comprises increasing the number of cardiomyocytes in the cardiac tissue. (Item 5) 4. The method of any one of items 1 to 3, wherein the repair of cardiac tissue comprises increasing the recovery of damaged cardiac tissue, including cardiomyocytes and / or the cardiovascular system. (Item 6) 6. The method according to item 4 or 5, wherein the number of cardiomyocytes is increased by at least three-fold compared to the number of cardiomyocytes in cardiac tissue not contacted by epicardium-derived paracrine factors after injury. (Item 7) 6. The method of any one of items 1 to 5, wherein the repair of the cardiac tissue comprises an improvement in the fractional shortening of the cardiac tissue compared to the amount of fractional shortening in cardiac tissue not contacted by the epicardium-derived paracrine factor after injury. (Item 8) 6. The method of any one of items 1 to 5, wherein the cardiac tissue repair comprises a reduction in scar area (fibrosis) of at least 2% compared to the amount of fibrosis in the same heart prior to treatment with epicardium-derived paracrine factor contact, delivery, or genome editing following injury. (Item 9) 6. The method of any one of items 1 to 5, wherein the cardiac tissue repair comprises an increase in vascular perfusion area of ​​at least 2% compared to the amount of perfusion area in the same heart prior to treatment with epicardium-derived paracrine factor contact, delivery, or genome editing following injury. (Item 10) 6. The method of any one of items 1 to 5, wherein the repair of the cardiac tissue comprises an increase in the amount of cytokinesis of cardiomyocytes in the cardiac tissue compared to the amount of cytokinesis of cardiomyocytes in cardiac tissue not contacted by epicardium-derived paracrine factors after injury. (Item 11) The method of item 10, wherein the increase in the amount of cytokinesis in cardiomyocytes is determined by expression of Aurora B kinase. (Item 12) 13. The method of any one of items 1 to 12, wherein the repair of cardiac tissue comprises a reduction in apoptosis of cardiomyocytes. (Item 13) 13. The method of any one of items 1 to 12, which results in an increase in the level of a transcript encoding a cardiac-specific contractile protein in cardiomyocytes. (Item 14) 14. The method of claim 13, which results in a two-fold increase in the level of a transcript encoding a cardiac-specific contractile protein in cardiomyocytes. (Item 15) The method of claim 13 or 14, wherein the cardiac-specific contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a. (Item 16) Rhythmic contraction of Ca in cardiac myocytes 2+ Actinin with + 11. The method of any one of items 1 to 10, which results in an increase in cells. (Item 17) 12. The method of any one of items 1 to 11, wherein the cardiac tissue is contacted with the epicardium-derived paracrine factor immediately after the injury. (Item 18) 13. The method of any one of items 1 to 12, wherein the cardiac tissue is contacted with the epicardium-derived paracrine factor at any time after the injury. (Item 19) 14. The method of any one of items 1 to 13, which reduces cardiac events and hospitalizations. (Item 20) 16. The method of any one of items 1 to 15, wherein the method attenuates fibrosis in the cardiac tissue following the injury. (Item 21) The method of claim 2 or 3, which results in increased vascularization of the injured area of ​​the cardiac tissue. (Item 22) 22. The method of claim 21, wherein the increased angiogenesis is determined by expression of von Willebrand factor (vWF) or smooth muscle actin in vascular cells. (Item 23) 23. The method according to any one of items 1 to 22, which induces cell cycle entry of cardiomyocytes. (Item 24) 24. The method of claim 23, wherein the cell cycle entry of the cardiomyocytes is assessed by expression of phospho-histone H3. (Item 25) The method of claim 23 or 24, which results in at least a two-fold increase in cardiomyocyte cell cycle entry compared to the amount of cardiomyocyte cell cycle entry in cardiac tissue not contacted by epicardium-derived paracrine factors after injury. (Item 26) The method of item 1, wherein the underglycosylated FSTL1 polypeptide is synthesized in a prokaryotic cell. (Item 27) The method of item 1, wherein the underglycosylated FSTL1 polypeptide is synthesized in a eukaryotic cell treated with a glycosylation inhibitor. (Item 28) The method of item 1, wherein the underglycosylated FSTL1 polypeptide is produced by replacing one or more glycosylated amino acids with one or more amino acids that are ineligible for glycosylation. (Item 29) 2. The method of claim 1, wherein the one or more glycosylated amino acids are selected from the group consisting of N144, N175, N180, and N223. (Item 30) The method of any one of items 26 to 28, wherein the underglycosylated FSTL1 polypeptide repairs cardiac tissue. (Item 31) The method of any one of items 26 to 28, wherein the underglycosylated FSTL1 protects cardiomyocytes from apoptosis after injury. (Item 32) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is delivered to the injured myocardial tissue by a collagen patch. (Item 33) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is directly injected into damaged myocardial tissue. (Item 34) The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is delivered systemically. (Item 35) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is delivered to the endocardium. (Item 36) 36. The method of claim 35, wherein the endocardial delivery is by catheter. (Item 37) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is delivered to the epicardium by a catheter. (Item 38) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is delivered by a catheter using a drug-eluting stent. (Item 39) 39. The method according to any one of items 1 to 38, wherein the underglycosylated Fstl1 polypeptide is embedded or seeded in a three-dimensional collagen patch. (Item 40) 40. The method of any one of the preceding claims, 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. (Item 41) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is expressed in the heart by the use of modified RNA (modRNA). (Item 42) 32. The method of any one of items 1 to 31, wherein the underglycosylated FSTL1 polypeptide is expressed by genome editing. (Item 43) 1. A method for repairing cardiac tissue after injury in a subject in need thereof, the method comprising contacting said cardiac tissue with an epicardium-derived paracrine factor. (Item 44) A sterile pharmaceutical composition comprising an underglycosylated follistatin-like 1 (FSTL1) polypeptide and one or more pharma- ceutically acceptable excipients. (Item 45) The sterile pharmaceutical composition according to item 44, further comprising an inhibitor of glycosylation of FSTL1. 46. ​​The sterile pharmaceutical composition of item 45, wherein the glycosylation inhibitor of FSTL1 comprises tunicamycin. (Item 47) 45. The sterile pharmaceutical composition of item 44, wherein the underglycosylated FSTL1 polypeptide is synthesized in a prokaryotic cell. (Item 48) 48. The sterile pharmaceutical composition according to item 47, wherein the prokaryotic cell is a bacterial cell. (Item 49) 45. The sterile pharmaceutical composition according to item 44, wherein the underglycosylated FSTL1 polypeptide is synthesized in eukaryotic cells treated with a glycosylation inhibitor. (Item 50) 50. The sterile pharmaceutical composition of item 49, wherein the glycosylation inhibitor is tunicamycin. (Item 51) 51. A sterile pharmaceutical composition according to any one of items 44 to 50, formulated for direct injection into injured cardiac tissue. (Item 52) 51. The sterile pharmaceutical composition according to any one of items 44 to 50, which is formulated for systemic administration. (Item 53) 45. The sterile pharmaceutical composition of item 44, wherein the underglycosylated FSTL1 polypeptide is synthesized in cells containing modified RNA (modRNA). (Item 54) 45. The sterile pharmaceutical composition of item 44, wherein the underglycosylated FSTL1 polypeptide is expressed by genome editing. (Item 55) 55. The sterile pharmaceutical composition according to any one of items 44 to 54, wherein the underglycosylated FSTL1 polypeptide is embedded or seeded in a three-dimensional (3D) collagen patch. (Item 56) 56. The sterile pharmaceutical composition according to item 55, wherein said 3D collagen patch has an elastic modulus of 12±4 kPa. (Item 57) A kit comprising: (i) an underglycosylated follistatin-like 1 (FSTL1) polypeptide; and (ii) one or more pharma- ceutically acceptable excipients. (Item 58) (iii) The kit according to Item 57, further comprising a three-dimensional (3D) collagen patch. (Item 59) 59. The kit of item 57 or 58, wherein the underglycosylated FSTL1 polypeptide is embedded or seeded in a three-dimensional (3D) collagen patch. (Item 60) 59. The kit according to item 57 or 58, wherein the 3D collagen patch has an elastic modulus of 12±4 kPa. (Item 61) (iv) The kit according to any one of items 57 to 60, further comprising an adhesion means for adhering said 3D collagen patch to the epicardium or myocardium of the injured heart. (Item 62) Item 62. The kit according to item 61, wherein the attachment means is a suture. (Item 63) A method for repairing cardiac tissue after injury in a subject in need of such repair, comprising contacting the cardiac tissue or epicardium with a three-dimensional (3D) collagen patch seeded or infused with recombinant underglycosylated follistatin-like 1 (FSTL1) polypeptide. (Item 64) 64. The method of claim 63, wherein the injury is ischemia-reperfusion injury. (Item 65) 64. The method of claim 63, wherein the injury is a myocardial infarction. (Item 66) 66. The method according to any one of items 63 to 65, wherein the 3D collagen patch is sutured to the cardiac tissue or epicardium. (Item 67) Three-dimensional (3D) collagen patches injected or seeded with recombinant underglycosylated follistatin-like 1 (FSTL1) polypeptide. (Item 68) 68. The collagen patch according to item 67, wherein the recombinant underglycosylated FSTL1 polypeptide is synthesized in prokaryotic cells. (Item 69) Item 69. The collagen patch according to item 68, wherein the prokaryotic cells are bacterial cells. (Item 70) 68. The collagen patch according to item 67, wherein the recombinant underglycosylated FSTL1 polypeptide is synthesized in eukaryotic cells treated with a glycosylation inhibitor. (Item 71) 71. The collagen patch of item 70, wherein the glycosylation inhibitor is tunicamycin. (Item 72) 72. The collagen patch according to any one of items 67 to 71, having an elastic modulus of 12±4 kDa. [Brief description of the drawings]

[0013] [Figure 1]Figure 1 shows cardiogenic activity in the epicardial secretome. a-d) Cardiogenic effect of co-culture of ESC-derived cardiomyocytes (mCMsESCs) with epicardial (EMC) cell lines. a, b) mCMsESCs only (a) and mCMsESCs co-cultured with EMCs for 4 days (b). Cardiomyocytes are visualized by α-actinin immunofluorescence (green) and EMCs by H2B-mCherry fluorescence (red). c) Quantification of cardiomyocyte numbers (as in a and b) expressed by fold change (n=3). d) Cardiogenic response of mCMsESCs to the number of EMCs added to the co-culture (+: 1x105EMC per well, ++: 5x105EMC per well), quantified using myocyte markers Myh6, Myh7, Mlc2a and Mlc2v (n=3), normalized to Gapdh gene expression. *Statistically significant difference (p<0.05) compared to control, filled squares: p<0.05 compared to "+" condition. e-i) Effect of epicardial EMC-conditioned medium on cardiac development. e, f) α-actinin staining (green) of mCMsESCs after 8 days of treatment with control (e) or EMC-conditioned medium (f). g) Quantification of the number of cardiomyocytes. h) Quantification of cardiac specific markers in mCMsESCs, normalized to Gapdh expression. i) Quantification of the number of cardiomyocytes with rhythmic calcium transients measured automatically using a Kinetic Imaging Cytometer (Vala Sciences). Quantification in g-i is expressed by fold change. n=3 in all experiments. *Statistically significant difference (p<0.05) compared to control. j-m) Conditioned medium from adult epicardium-derived cells (EPDCs) promotes cytokinesis in embryonic cardiomyocytes. j) Isolation of embryonic cardiomyocytes from E12.5 GFP-positive hearts (Tnnt2-Cre;Rosa26mTmG / +). k) EPDC-conditioned medium promoted proliferation of cardiomyocytes, and boiling of conditioned medium abolished the growth-promoting effect. l, m) Analysis of cytokinesis by immunostaining for Aurora B and cardiac marker Tnnt2 showed increased cytokinesis of cardiomyocytes after treatment with adult EPDC medium (vehicle group: 38 positive cells out of 19668, conditioned medium group: 74 out of 22143). *P<0.05; n=5.

[0014] [Diagram 2] Figure 2 shows that an embryonic epicardial-mimetic patch improves cardiac function after permanent LAD ligation. a) Schematic of collagen patch production (plastic compression procedure reconstructed from 30). b) Evaluation of the mechanical properties of the fabricated patch measured by atomic force microscopy. The distribution histogram of the measured patch microstiffness is shown in red. These values ​​are plotted against the reported range of elasticity for common scaffold biomaterials31. The grey area indicates the optimal range of elasticity to maximize myocyte contractility, as previously described32. c, d) Epicardial-mimetic patch implantation. After permanent LAD ligation to induce myocardial infarction (c), the epicardial patch was sutured at two points to the surface of the ischemic myocardium (d). The inset in the panel shows the prepared patch, soaked in culture medium before implantation. e-g) Physiological effects of the epicardial-conditioned medium-loaded patch after myocardial infarction (MI). All samples were taken 2 weeks after patch implantation. e) Summary of echocardiographic analysis 2 weeks after infarction, including sham (sham control), infarcted mice without treatment (MI only), MI treated with patch only (MI+patch), and infarcted animals treated with patch loaded with epicardial conditioned medium (MI+patch+CM). All data are normalized to individual pre-surgery baseline values. f) Absolute values ​​of fractional shortening (FS%) from e. g) Gross histological analysis of Masson's trichrome stained hearts. Samples are as indicated. A minimum number (n) of 8 mice per experimental group was used. *: p<0.05 compared to sham control, filled circle: p<0.05 compared to MI only, and filled square: p<0.05 compared to MI+patch.

[0015] [Figure 3-1]Figure 3 shows that FSTL1 is an epicardial cardiogenic factor with dynamic expression after injury. a) MS / MS spectrum of R.GLCVDALIELSDENADWK.L identified as FSTL1. Peptide probability = 1.0, Xcorr = 6.276, delta Cn = 0.471. b-f) Effect of recombinant FSTL1 on mouse embryonic stem cell-derived cardiomyocytes (mCMsESCs) after treatment for 8 days with control medium only (control) or medium containing human FSTL1 (FSTL1, 10 ng / ml). Medium was changed every 2 days in all experiments and conditions. b, c) Cardiomyocytes are identified by α-actinin immunostaining (green). d) Quantification of the number of cardiomyocytes in b, c expressed by fold change (n = 8). e) Expression of cardiac specific markers in b, c, normalized to Gapdh expression as fold change (n = 3). f) Quantification of the number of cardiomyocytes with rhythmic calcium transients (fold change vs. control) with and without FSTL1 treatment using Kinetic Image Cytometer (KIC) analysis (n=6). g) Measurement of individual cardiomyocyte size (pixels) after 2 days of culture at the indicated FSTL1 concentrations (n=5). *: statistically significant difference from control (p<0.05). h) Epicardial expression of FSTL1 from mid-gestation onwards. Direct protein visualization using FSTL1 antibody (red) shows that FSTL1 is expressed in the mouse epicardium at embryonic stages E12.5, E15.5 and E17.5. Some interstitial expression is also detected. The E12.5 image shows co-localization of FSTL1 with the epicardial transcription factor Wilms' tumor 1 (Wt1, green nuclear stain, white arrows). Images at E15.5 and E17.5 co-stained with myocyte marker α-actinin (green) show no overlap of FSTL1 (white arrows) with myocyte markers (yellow arrows). i-l) Dynamic expression of FSTL1 in injured adult epicardium. i) Upper panel: histological (Masson's Trichrome staining) evaluation of induced fibrotic tissue at sequential times after myocardial infarction (MI). Lower panel: immunohistochemistry of FSTL1 (brown) at sequential times after MI. Insets show expression of FSTL1 in the epicardium of sham-operated hearts and depletion of FSTL1 in the epicardium of injured hearts.FSTL1 is also undetectable in fibrotic tissue, whereas it is upregulated in post-MI myocardium (observe brown FSTL1 immunostaining in post-MI myocardium). j-l) High-resolution immunofluorescence images: Colocalization of FSTL1 (red) with epicardial marker Wt1 (green) in uninjured (sham) adult hearts (j). Selective epicardial localization of FSTL1 (red) in adult sham hearts (k). FSTL1 is absent in epicardial cells and their derivatives after MI (l). Epicardial lineage labeling (green) after oral delivery of tamoxifen in Wt1-CreER;Rosa26RFP / + mice (delivered six times over 3 weeks and stopped 1 week before MI). Hearts were harvested 2 weeks after MI. Immunostaining for RFP for Wt1 lineage cells (gray), FSTL1 (red) and Tnni3 (green) shows that FSTL1 is absent from epicardial cells and their derivatives (gray) but abundant in the post-MI myocardium (green). [Figure 3-2] Same as above

[0016] [Figure 4-1]FIG. 4 shows that FSTL1 recapitulates the in vivo restorative effect of epicardial conditioned medium in engineered epicardial patches. a-c) Physiological analysis. a) Time course of survival in each condition was analyzed using the Kaplan-Meier method. b) Kinetics of fractional shortening [FS (%)] measured by echocardiography during the first 3 months of treatment as indicated. Data presented as absolute FS. Sham (sham control), infarcted mice without treatment (MI only), MI treated with patch only (MI+patch), and infarcted animals treated with patch loaded with FSTL1 (MI+patch+FSTL1). c) FS% at 2 and 4 weeks post-MI in FSTL1-TG mice compared to MI+patch and MI+patch+FSTL1. *: p<0.05 compared to sham control, filled circle: p<0.05 compared to MI only, and filled square: p<0.05 compared to MI+patch. d-e) Morphometric analysis. d) Representative Masson's trichrome staining of hearts at 4 weeks post-MI and quantification of fibrosis area as a percentage of total LV wall (n>4). e) Echocardiographic assessment of left ventricular morphology. Abbreviations indicate: LVIDd (left ventricular internal diameter at end diastole); LVIDs (left ventricular internal diameter at end systole); LVPWd (left ventricular posterior wall dimension at end diastole); LVPWs (left ventricular posterior wall thickness at systole). *: p<0.05 compared to sham, filled circle: p<0.05 compared to MI only, and filled square: p<0.05 compared to MI+patch. f-i) Analysis of vasculature at 4 weeks post-MI. f) Immunostaining for endothelial marker (vWF). g) Vascular area quantified by measurement of the mean luminal area of ​​individual vessels relative to the total area of ​​tissue sections. h) Immunostaining for smooth muscle marker (αSMA). i) Quantification of number of vessels per area unit. *: p<0.05 compared to sham controls, filled circles: p<0.05 compared to MI only, and filled squares: p<0.05 compared to MI+patch. j) Visualization of patch border zone at 4 weeks post-MI. Trichrome staining of the infarct and border zone of the indicated treatments shows integration of the patch with host tissue and extensive cellularization of the patch with native cardiac cells. Observe abundant muscle (red) within the interior of the patch and border zone of animals treated with patch+FSTL1 (right three panels, green arrows). [Figure 4-2] Same as above

[0017] [Figure 5-1]Figure 5 shows that restored epicardial expression of FSTL1 promotes cardiomyocyte proliferation. All experiments were performed after permanent LAD ligation. Samples were analyzed 4 weeks after treatment unless otherwise indicated. a-h) Immunostaining. Immunostaining for cardiomyocyte marker α-actinin (red) in the infarct area (b-d) and co-immunofluorescent staining for DNA replication marker phospho-histone 3 Ser10 (pH3, green) and α-actinin (red) in the border zone (f-h) were compared to sham-operated animals (a, e) in the four treatment groups analyzed 4 weeks after MI. Insets in (a-d) show lower magnification images with dashed lines demarcating the border between the patch and the host tissue. Arrows in (g, h) indicate α-actinin+ cardiomyocytes with pH3+ nuclei. i-o) Quantification of cardiomyocyte proliferation. i) Cross sections used for quantitative analysis. Each section encompasses the infarct, patch, and is separated by 250 μm, between 1-2 mm from the apex. j) 3D rendering of high magnification images of pH3 (green) and α-actinin (red) showing colocalization of cardiomyocyte nuclei and pH3 staining. k) Quantification of the frequency of pH3+, α-actinin+ double positive cells in the four experimental groups. Data were collected at three different sections from five to seven hearts in each group, and total pH3+, α-actinin+ cells were counted in each heart. l-m) Determination of cytokinesis. l) Co-immunofluorescence of cardiomyocytes (α-actinin, green) and cytokinesis marker Aurora B kinase (red) in the patch+FSTL1 cohort. 3D rendering shows the formation of grooves between α-actinin+ cardiomyocytes in the Z-axis by Aurora B kinase+ cleavage. m) Quantification of the frequency of Aurora B+ / α-actinin+ cells in the four experimental groups. Data were collected at three different sections from 5-7 hearts in each group and total Aurora B+ / α-actinin+ cells were counted in each heart. n-o) Proliferation determination using cardiomyocyte nuclear markers. n) Co-immunofluorescence of cardiomyocyte nuclear markers PCM1 (red) and pH3 (green) in Patch+FSTL1 cohort. 3D rendering shows co-localization of cardiomyocyte nuclei and pH3 staining. o) Quantification of the frequency of PCM1+ / pH3+ cells in the four experimental groups.Data were collected from 5–7 hearts in each group at three different sections, and total PCM1+ / pH3+ cells in each heart were counted. *: statistically different from sham, P<0.05. **: statistically different from all other groups, P<0.05. p~v) Lineage tracing of newly generated myocytes. Treatment of αMHC-mERCremER;Rosa26Z / EG / +(MCM+ / ZEG+) mice with 4-OH-Tamoxifen (OH-Tam) induced eGFP expression in pre-existing cardiomyocytes (schematically illustrated in p). FSTL1-loaded collagen patches were applied simultaneously with coronary artery ligation (MI). Hearts were dissected, fixed, and stained 4 weeks after MI. q) LV area of ​​sham-operated hearts shows efficient labeling of cardiomyocytes (α-actinin: white, eGFP: green). r-t) LV areas of infarcted hearts show eGFP+ (pre-existing, green) cardiomyocytes in the intact area (r), infarcted area (s), and border zone (t, u) 4 weeks post-surgery. White arrows indicate pH3+ non-cardiomyocytes. Yellow arrows indicate pH3+, eGFP+ double positive cells, indicating pre-existing cardiomyocytes in the middle of the cell cycle. Observe clusters of pH3+, eGFP+ double positive cells in the border zone and infarcted area. [Figure 5-2] Same as above

[0018] [Figure 6]Figure 6 shows that the proliferation activity of FSTL1 on early cardiomyocytes is due to cell-selective post-transcriptional FSTL1 modification. a-f) FSTL1 promotes proliferation of mESC-derived immature cardiomyocytes. a, d) mCMsESCs were stimulated with the indicated concentrations of FSTL1 together with 10 μg / ml EdU for 24 h and stained for α-actinin (red) and EdU (green). The percentage of EdU+, α-actinin+ cardiomyocytes among total α-actinin+ cardiomyocytes is quantified (d). b, e) mCMsESCs were stimulated with 10 ng / ml FSTL1 for 48 h and stained for α-actinin (red) and phospho-histone 3 (green). The percentage of pH3+, α-actinin+ cardiomyocytes among total α-actinin+ cardiomyocytes is quantified (e). c, f) mCMsESCs are stimulated with the indicated concentrations of FSTL1 for 48 hours and stained for α-actinin (red) and cytokinesis marker Aurora B (green). The percentage of Aurora B+, α-actinin+ cardiomyocytes among total α-actinin+ cardiomyocytes is quantified (f). g, h) FSTL1 expressed in mammalian cells is glycosylated. g) Western blot of FSTL1 from conditioned medium of HEK293 cells with and without FSTL1 overexpression and with and without tunicamycin (Tuni.), which blocks protein glycosylation, shows that FSTL1 is glycosylated. h) Western blot of recombinant human FSTL1 expressed in mammalian cells or in bacteria shows differential glycosylation (red arrow: glycosylated form, black arrow: non-glycosylated form). i) mCMsESCs are stimulated with 10 nM H2O2 and 10 ng / ml bacterial and mammalian produced FSTL1 for 24 hours and stained with α-actinin and TUNEL for cell death. The percentage of TUNEL+, α-actinin+ cardiomyocytes among total α-actinin+ cardiomyocytes is quantified (i), showing that mammalian produced FSTL1 can attenuate apoptosis induced by H2O2, whereas bacterial produced FSTL1 cannot, while both proteins have no effect on apoptosis in the absence of H2O2 stimulation.j, k) Comparison of bacterial and mammalian produced FSTL1 in EdU incorporation and Aurora B quantification (same assays as a, c, d, f), showing that bacterial produced FSTL1 promotes mCMsESC proliferation, whereas mammalian produced FSTL1 does not. l) FSTL1 is differentially glycosylated in cardiomyocytes and epicardial cells. Western blots of FSTL1 in conditioned medium from NRVC infected with adeno-FSTL1 and conditioned medium from EMC treated or not with tunicamycin showed that the non-glycosylated protein has the same size, whereas glycosylated FSTL1 has a different size, suggesting that FSTL1 is modified differently by cardiomyocytes and epicardial cells. m, n) Comparison of the effect of conditioned medium from NRVC infected with adeno-FSTL1 and conditioned medium from EMC by EdU incorporation assay. Normalized to the concentration of the same amount of FSTL1 using Western blot, EMC-conditioned medium induced proliferation of mCMsESCs to the same extent as bacterially produced FSTL1, whereas conditioned medium from NRVCs infected with adeno-FSTL1 failed to do so, suggesting that the glycosylation status determines the function of FSTL1. n=5 for all experiments. *: statistically different from control, P<0.05. o) Action of FSTL1 during cardiac injury, working model. FSTL1 becomes very highly expressed in the myocardium during ischemic heart disease. FSTL1 secreted into the myocardium is highly glycosylated (glyco-FSTL1), protects against apoptosis, and does not show proliferative activity. FSTL1 is not expressed in the epicardium of injured hearts. The hypoglycosylated form, whether secreted by the intact epicardium or delivered into an epicardial patch, activates proliferation of replicative-competent cardiomyocyte precursor cells located in the subepicardial space.

[0019] [Figure 7-1]FIG. 7 shows that epicardial FSTL1 delivery activates cardiac regeneration in a preclinical model of ischemic heart injury. a-d) Physiological effects of epicardial FSTL1 patch delivery in a porcine experimental model of ischemia-reperfusion (I / R). Baseline ejection fraction (EF) of about 50% was measured by MRI, which dropped to about 30% after 1 week (a-b). Pigs treated with patch+FSTL1 after 1 week of I / R regained contractility by 2 weeks after treatment, with an EF of about 40%. EF remained stable until the following 2 weeks, the longest period analyzed (a, b). This contrasted with the steady decline in cardiac function in untreated animals (I / R without treatment) or animals treated with patch alone (I / R+patch) (b). c-d) Pigs treated with patch+FSTL1 (c) showed the smallest scar size (area) among all study groups, including I / R+patch animals (d). The green line and arrows highlight the scar perimeter. e-o) Evaluation of patch integration with host cardiac tissue, vascularization, and cellularization and regeneration 4 weeks after implantation. e) Masson's trichrome staining of pig hearts shows patch+FSTL1 attachment to ischemic tissue and limited fibrosis. f-h) Immunostaining for smooth muscle markers (αSMA, red) and EdU (green) shows newly formed arterial smooth muscle. Patch+FSTL1 treated pig hearts showed evidence of de novo DNA formation of vascular smooth muscle cells in both the ischemic area (f, g) and the border zone with the FSTL1 loaded patch (h). The white line and arrow in panel h delineate the approximate border between the patch and the host tissue. i-m) EdU incorporation analysis of cardiomyocytes present in the infarct and border zone in Patch+FSTL1 treated porcine hearts shows linear cardiomyocytes (α-actinin+, red) some of which (arrow in i) also stain positive for DNA synthesis (EdU, green; higher magnification examples in j-m). n) Co-immunofluorescence of cardiomyocytes (α-actinin, green) and cytokinesis marker Aurora B kinase (red) in Patch+FSTL1 hearts. 3D rendering shows Aurora B kinase+ centrosomes between α-actinin+ cardiomyocytes in the Z axis. [Figure 7-2] Same as above

[0020] [Figure 8] Figure 8 shows the characterization of mCMsESC cells used in this study. a) Schematic timeline of cell preparation and treatment. b-d) Immunostaining of α-actinin of mCMsESCs showing that the majority of cells are α-actinin+ (b) and that α-actinin lacks striated structure (c). d) Immunostaining of α-smooth muscle actin (αSMA) of mCMsESCs showing that the majority of cells are α-SMA+, unlike mature cardiomyocytes42, which lack expression of SMA. e-f) Automated detection of EdU incorporation in mCMsESCs. Captured image of mCMsESCs treated with 10 μg / ml EdU for 24 h and stained with EdU, α-actinin and DAPI using InCell 1000 (General Electric) (e). Overlay of masks of EdU, α-actinin and DAPI channels using the automated detection software (f). g) EdU incorporation profile of mCMsESCs over time. mCMsESCs are treated with 10 μg / ml EdU for 24 hours at 0, 24, 48, and 144 hours. The percentage of EdU+ / α-actinin+ cardiomyocytes among all α-actinin+ cardiomyocytes is calculated for each time point. Note the decrease in EdU incorporation rate over time. h, i) Fluo4 calcium images of mCMsESCs with baseline background image (h) and peak image (i). j) Representative calcium transients of mCMsESCs (red) compared to neonatal rat ventricular cardiomyocytes (NRVC, blue). Note the reduced amplitude, slower rate of up- and down-pulsation, and extended duration of calcium transients of mCMsESCs compared to NRVCs, suggesting immature calcium handling in mCMsESCs. In all experiments, FSTL1 was added 1 day after plating of mCMsESCs (times 0-24 in this figure).

[0021] [Figure 9]Figure 9 shows atomic force microscopy (AFM) analysis of the fabricated epicardial patch. A custom flat tip fabricated using electron beam deposition in the AFM of the patch (a) was used to investigate the stiffness of the gel with a scan area of ​​90 μm × 90 μm (b, c).

[0022] [Figure 10-1]FIG. 10 shows myocardial overexpression of FSTL1 (FSTL1-TG) mice after permanent LAD ligation. a-d) Kinetics of FSTL1 protein expression after myocardial infarction. FSTL1-TG mice (C57 / Bl6 background) and littermate wild-type (WT) mice were subjected to LAD ligation. Heart tissue and serum were collected at baseline, 1, 3, 7 and 28 days after surgery. FSTL1 protein levels in ischemic (IA) and remote (RM) areas of the heart were analyzed by Western blotting (a). FSTL1 expression relative to tubulin levels is reported (b). Serum levels of FSTL1 were analyzed by Western blotting (c). Ponceau-S staining showing equal serum loading is also shown. Quantification of serum FSTL1 levels is shown in (d). n>3 in all groups. *: P<0.05 compared to WT baseline, #: P<0.05 compared to FSTL1-TG baseline. ANOVA was used for statistical significance (P<0.05). e–j) Morphometric and functional e–j) Morphometric and functional response of FSTL1-TG mice to long-term permanent LAD ligation. Representative Masson's Trichrome staining of WT (e) and FSTL1-TG (f) at 4 weeks post-MI. Quantification of fibrotic tissue content at 4 weeks post-MI (g). Echocardiographic measurements of systolic left ventricular internal dimensions (LVIDs) (h) and diastolic left ventricular internal diameter (LIVDd) (i) at 2 and 4 weeks post-MI. Echocardiographic determination of fractional shortening (FS%) in the indicated genotypes at 2 and 4 weeks post-MI (j). (k-n) Double immunofluorescence staining of α-actinin (cardiomyocytes) and pH3 (mitosis) (k), and α-actinin (cardiomyocytes) and von Willebrand factor (vascular endothelial cells) (m) in FSTL1-TG and WT mice quantified in (l, n). n = 5, *: significantly different from WT (P < 0.05). [Figure 10-2] Same as above

[0023] [Figure 11]FIG. 11 shows that FSTL1 is absent from the epicardium after myocardial infarction and is expressed in the myocardium. a-c) Detection of FSTL1 after MI in lineage-labeled Wt1-CreER;Rosa26RFP / + mice. Epicardial lineage labeling (green) after oral delivery of tamoxifen to Wt1-CreER;Rosa26RFP / + mice (delivered six times over 3 weeks and stopped 1 week before MI). Hearts were harvested 2 weeks after MI. Immunostaining of RFP for Wt1 lineage cells (green), FSTL1 (red) and Tnni3 (white) shows that FSTL1 is absent from epicardial cells and their derivatives (green) but abundant in the myocardium (gray) after MI (higher magnification images in a, b are shown in c).

[0024] [Figure 12] Figure 12 shows retention of FSTL1 in patches in vitro and in vivo. a, b) Enzyme-linked immunosorbent assay was used to measure the amount of FSTL1 retained in collagen scaffolds exposed to PBS in vitro at various time intervals (0-21 days) (a). The initial and final FSTL1 concentrations, as well as the release values ​​within the first 24 hours, are listed in the table (b). c-f) Retention of FSTL1 in patches in vivo. Representative images of immunostaining of FSTL1 in the indicated animal treatment groups at 4 weeks after surgery. Note that FSTL1 is expressed in the uninjured epicardium (arrow in inset of c), but its expression becomes undetectable within the infarct area after MI (d). Similarly, FSTL1 was not detectable in MI+patch animals (e), but still persists in the patch area (red staining) in the MI+patch+FSTL1 group (f).

[0025] [Figure 13]Figure 13 shows that patch + FSTL1 attenuated fibrosis after MI. Representative Masson's trichrome staining of serial sections of hearts under four conditions (sham, MI only, MI + patch, and MI + patch + FSTL1) at 4 weeks after MI. Note the severe fibrosis in the MI only condition and the reduced fibrosis in the MI + patch condition, as well as the further reduction in the MI + patch + FSTL1 condition, as quantified in Figure 4d.

[0026] [Figure 14] Figure 14 shows MRI imaging after treatment. Representative MRI images from mice treated with MI only, MI+patch, and MI+patch+FSTL1 show 3D-FSPGR (fast spoiled gradient-echo) images and delayed-weighted images using gadolinium contrast, confirming the reduction of infarct area (delineated in green) and the maintenance of contractility.

[0027] [Figure 15]FIG. 15 shows the analysis of the function of Patch+FSTL1 in a mouse model of ischemia / reperfusion (I / R) with delayed patch grafting. a-c) Cardiac function assessment at end diastole and end systole for sham, I / R, and I / R treated with Patch+FSTL1 before grafting (a, 1 week after injury), 2 weeks after patch implantation (b), and 4 weeks after grafting (c). Values ​​were normalized by dividing by the preoperative baseline value for each individual animal. d) Absolute values ​​of fractional shortening (FS, %) at various times before and after I / R assessed by echocardiography in mice in a-c. Abbreviations are the same as in FIG. 4. *: p<0.05 compared to sham, and filled circle: p<0.05 compared to I / R. e) Co-immunofluorescence staining of DNA replication markers phospho-histone 3 Ser10 (pH3, green) and α-actinin (red) in the border zone 4 weeks after MI in hearts treated with Patch+FSTL1. f) Quantification of the occurrence of pH3+, α-actinin+ double positive cells in the three experimental groups. Data were collected from three hearts at three different sections in each group, and the total pH3+, α-actinin+ cells were counted in each heart. *: statistically different from all other groups, P<0.05.

[0028] [Figure 16] FIG. 16 shows a representative example of all pH3+ cardiomyocytes detected in one section of a patch+FSTL1 treated heart. Masson's trichrome staining of a patch+FSTL1 treated heart 4 weeks after MI (a). The adjacent slide was stained for α-actinin in (b) and corresponds to the black boxed areas of the infarct and patch in (a). The dotted line in (b) indicates the border between the heart and the patch. The adjacent slide was stained for α-actinin and pH3 and all α-actinin+, pH3+ double positive cardiomyocytes found are shown in (c) (white arrow). Each image corresponds to the numbered white boxed areas in (b).

[0029] [Figure 17-1]FIG. 17 shows the effect of implantation of patch+FSTL1 on apoptosis and inflammation. a) Representative TTC staining 2 days after MI / patch treatment for all four groups (sham, MI, MI+patch, MI+patch+FSTL1). b) Quantification of the area at risk comparing all four groups. Data was taken from four hearts in each group at four cross sections of approximately 2 mm thickness each encompassing each heart. *: statistically different from sham, P<0.05. c, d) Representative images of TUNEL assay (TUNEL, green; α-actinin, red) comparing hearts 2 days after MI with patch alone and patch+FSTL1. e) Quantification of TUNEL+, α-actinin+ in the infarcted area as a percentage of the total number of cardiomyocytes. No differences are observed between MI+patch and MI+patch+FSTL1 conditions. Data was taken from three hearts in each group at three different cross sections (same as in FIG. 5i). Ten images of 0.09 mm2 were taken from the infarcted area of ​​each section and TUNEL+, α-actin+, and total α-actin+ cells were counted. a-e) Patch-only and patch+FSTL1 treated hearts were stained for TUNEL for cell death and α-actin for cardiomyocytes at 4 and 8 days post-MI (a-d). Minimal TUNEL+, α-actin+ cells are detected, but there are significant amounts of TUNEL+, α-actin cells. Quantification of total TUNEL+ nuclei showed no significant differences between patch and patch+FSTL1 treated hearts at both time points (e). f-j) The same hearts as in panels a-d were immunostained for F4 / 80 for macrophages and α-actin for cardiomyocytes (f-i). Quantification of F4 / 80+ cells showed no significant differences between patch and patch+FSTL1 treated hearts at both time points (j). [Figure 17-2] Same as above

[0030] [Figure 18]FIG. 18 shows that FSTL1 does not induce proliferation of adult and neonatal cardiomyocytes or cardiac progenitor cells. a-f) Adult cardiomyocytes derived from primary isolation of mice. a) Visualization of GFP+ cardiomyocytes isolated from MCM+ / ZEG+ mice treated with 4-OH-Tamoxifen (OH-Tam) in 3D collagen patches. b-d) Changes in gene expression including proliferation (b), cardiac specific (c), and hypertrophy (d) markers in adult cardiomyocytes treated with FSTL1. Note the absence of changes in expression of cardiac specific genes, the absence of increase in cell cycle markers (consistent with undetectable Ki67 immunostaining), and the decrease in hypertrophy markers. Cardiomyocytes embedded in 3D patches were treated with FSTL1 (10 ng / ml) for 7 days with medium changes every 2 days. e, f) FUCCI assay of 3D cultured adult cardiomyocytes was performed after 1 week of 3D culture. e) Treatment with FSTL1 was performed for 7 days with medium changes every 2 days. f) 3D culture control of adult cardiomyocytes without FSTL1. Note that there were no detectable signs of cardiomyocytes in S / G2 / M phase (GFP+) in either condition. Purple arrows indicate purple stained nuclei resulting from colocalization of Hoechst (blue) and G1 phase FUCCI (red) labeling. (g-j) Primary neonatal rat ventricular cardiomyocytes (NRVCs). g, h) Freshly isolated NRVCs were stimulated with FSTL1 for 48 h together with 10 μg / ml EdU and stained for α-actinin (red) and EdU (green). The percentage of EdU+ / α-actinin+ cardiomyocytes among total α-actinin+ cardiomyocytes is quantified (h). i, j) NRVCs stimulated with FSTL1 for 48 h and stained for α-actinin (red) and pH3 (green). The percentage of pH3+ / α-actinin+ cardiomyocytes among total α-actinin+ cardiomyocytes is quantified (j). No increased proliferation is observed upon FSTL1 treatment. (n=4) *: statistically different from control, P<0.05. k~m) Sca1+ progenitor cells 19 were starvation synchronized for 48 hours and stimulated with FSTL1 or control growth medium in the presence of EdU for 72 hours. Clone 3 was obtained from the Lin-Sca1+ SP fraction by clonal outgrowth. The Sca1 pool was obtained from lin-Sca1+ without clonal outgrowth.k) EdU and DAPI staining of Sca1+ cells after 72 hours of treatment. l) Percentage of EdU+Sca1+ cells after 72 hours of treatment. FSTL1 concentrations: 0, 1, 10, 100ng / ml. Abbreviations: SS, serum starvation; CGM, control growth medium. m) Number of Sca1+ cells after 72 hours of FSTL1 treatment (n=5). No significant changes are observed upon FSTL1 treatment.

[0031] [Figure 19] Figure 19 shows detection of FSTL1 in NRVC and EMC conditioned medium. Western blots of NRVC conditioned medium and EMC conditioned medium for FSTL1 show secretion of glycosylated FSTL1 into EMC, but not in NRVC, with or without tunicamycin treatment.

[0032] [Figure 20] Figure 20 shows detection of phospho-Akt and PCNA in mCMsESCs after FSTL1 treatment. Western blots for phospho-Akt (Ser473 and Thr308, both of which are shown in cardiomyocyte survival responses) and PCNA (proliferation marker) after 1 hour and 24 hours of FSTL1 treatment at 10 ng / ml and 50 ng / ml show no change in phospho-Akt and PCNA upon FSTL1 treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The invention disclosed herein is based, in part, on the inventors' observation that conditioned medium from epicardial-like cell cultures enhances cardiomyogenesis in vitro and in the injured adult heart. The cardiac epicardium is a cell-derived cell that is composed of progenitor cells during development. 1、2 and FGF, IGF2, and PDGF 3~5 The epicardium is an outer epithelial layer that contributes to myocardial growth by providing mitogens, including mitogens for myocardium. Recent studies have suggested that the epicardium may maintain the function of adult myocardium after injury, possibly as a source of myogenic precursors. 6、7However, to date, no epicardium-derived paracrine factors have been shown to support myocardial regeneration in mammals after injury, although identification of such factors as well as their mechanisms of action would provide insight into this poorly understood and inherently inefficient process. 8 .

[0034] As described in more detail below, after subjecting the conditioned medium to mass spectrometry and subsequent analysis, follistatin-like 1 (FSTL1) was identified as a component of the observed cardiomyotrophic activity. FSTL1 was found to be expressed in the adult epicardium, but rapidly decreased after myocardial infarction (MI), and then replaced by expression in the myocardium. As illustrated in the non-limiting examples below, endogenous myocardial or transgenic overexpression in the myocardium has no regenerative effect, but application of FSTL1 to the epicardial surface of the heart by a compressed collagen patch recapitulated the activity of epicardial conditioned medium. In some embodiments, after MI, the generated FSTL1 epicardial treatment abolished pathological remodeling, restored angiogenesis, and upregulated pre-existing αMHC + Cell cycle entry of cells is induced after MI, resulting in improved cardiac function. As further illustrated in the non-limiting examples below, in vitro studies have shown that FSTL1 stimulates the proliferation of immature myocytes, but not progenitor cells. In some embodiments, the proliferation-promoting properties of FSTL1 correlate with tissue-specific post-transcriptional modifications, such as the glycosylation status of the protein. In other embodiments of the invention, administration of hypoglycosylated FSTL1 does not activate Akt-1 signaling activity. In further non-limiting examples below, epicardial patch delivery of hypoglycosylated FSTL1 is also effective in a preclinical porcine model of myocardial infarction, highlighting the evolutionary conservation of this regenerative mechanism in mammals. Thus, without being bound by theory, engineered epicardial delivery of FSTL1 may be an attractive option for therapeutically achieving regeneration of cardiomyocytes after ischemic injury.

[0035] I. Definition 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 of the cell types found within the heart.

[0036] As used herein, the phrase "epicardium-derived paracrine factors" refers to any protein, polypeptide, or fragment thereof produced by cells of the outer epithelial layer of the heart that can induce one or more physiological, protective, proliferative, and / or reparative responses in cardiac tissue (e.g., myocardial tissue) following injury from cardiovascular disease, myocardial infarction, or other ischemic event. In one embodiment, the epicardium-derived paracrine factors are components of conditioned medium obtained from epicardial cell cultures.

[0037] The term "hypoglycosylated" as used in the context of the present invention refers to a protein that is post-translationally modified with a minimal number of carbohydrate moieties or that is completely devoid of carbohydrate moieties. In some embodiments, hypoglycosylation refers to a protein that is completely devoid of any carbohydrate modification (e.g., N-linked glycans, O-linked glycans, or phospho-glycans, etc.). In another embodiment, the term refers to a protein that has reduced carbohydrate modification (e.g., at least about any of 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%) compared to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells. In another embodiment, the term refers to a protein that has reduced carbohydrate modifications (e.g., at most about any of 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%) compared to the amount of glycosylation that occurs in vivo under normal physiological conditions in a mammalian cell. In another embodiment, the term refers to a protein that has reduced carbohydrate modification (e.g., at least about 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% reduced carbohydrate modification) compared to the amount of glycosylation that occurs in vivo under normal physiological conditions in a mammalian cell.In another embodiment, the term refers to a protein having reduced carbohydrate modification (e.g., at most about 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% reduced carbohydrate modification) compared to the amount of glycosylation that occurs in vivo under normal physiological conditions in mammalian cells. In yet another embodiment, a hypoglycosylated protein is engineered such that all amino acid residues eligible for glycosylation (amino acid residues eligible for N-linked, O-linked, or phospho-glycans) are replaced with amino acid residues that are ineligible for glycosylation.

[0038] As used herein, the phrase "repairing cardiac tissue after an injury" or "repairing cardiac tissue after an injury" refers to any type of action or treatment that reduces, minimizes, or even maintains the level of injury due to cardiovascular disease, myocardial infarction, or other ischemic events. Thus, repairing injury indicates that the condition of a subject with respect to the injury in question does not worsen, and may be improved, compared to the level of injury in the absence of the treatment or action described herein to reduce injury.

[0039] As used herein, "cardiovascular disease" or "heart disease" is a term used to describe a series of diseases or events that affect the heart and / or vascular system. Types of heart disease include, but are not limited to, coronary heart disease, cardiomyopathy, ischemic heart disease, heart failure, inflammatory heart disease, valvular heart disease, and aneurysm. Heart disease can be evaluated using clinical parameters and / or assessments known to those skilled in the art of diagnosing and / or treating heart disease, such as physical examination, detection of signs and symptoms of cardiovascular disease, electrocardiogram, echocardiogram, chest x-ray, blood tests to detect cardiac biomarkers, etc. Biomarkers typically used in clinical situations include, but are not limited to, cardiac troponin (C, T, and I), CK and CK-MB, and myoglobin.

[0040] As used herein, "myocardial infarction" or "MI" refers to the occurrence of myocardial necrosis, which occurs due to interruption of blood supply to the heart, and can result in a fatal imbalance between oxygen supply and myocardial demand. It is caused by plaque rupture with thrombus formation in the coronary vessels, which can lead to an abrupt reduction in blood supply to a portion of the myocardium. That is, the rupture of susceptible atherosclerotic plaque is followed by occlusion or blockage of the coronary artery. If not treated in sufficient time, the resulting ischemia or restriction of blood supply and oxygen deficiency can cause injury or death, i.e., infarction of the heart. In general, this injury is largely irreversible, and clinical treatments to date have been primarily aimed at slowing the progression of heart failure and prolonging life. Myocardial infarction can be assessed using clinical parameters and / or assessments known to those of skill in the art of diagnosing and / or treating myocardial infarction, such as a physical examination, detection of signs and symptoms of myocardial infarction, electrocardiogram, echocardiogram, chest x-ray, blood tests to detect cardiac biomarkers including troponin, CK, and CK-MB, etc.

[0041] As used herein, "reperfusion" refers to the restoration of blood flow or blood supply to a heart or cardiac tissue (e.g., myocardial tissue) that has become ischemic or hypoxic. Modalities of reperfusion include, but are not limited to, chemical dissolution of an obstructing thrombus, i.e., thrombolysis, administration of vasodilators, angioplasty, percutaneous coronary intervention (PCI), catheterization, and coronary artery bypass graft (CABG) surgery.

[0042] A "subject" or "individual" may be a vertebrate, mammal, or human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. In one aspect, the subject is a human.

[0043] Unless otherwise defined herein, 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 invention pertains.

[0044] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0045] II. Compositions of the Invention Provided herein are pharmaceutical compositions containing an epicardium-derived paracrine factor (eg, FSTL1, such as underglycosylated FSTL1) and one or more pharma- ceutically acceptable excipients or carriers.

[0046] In some embodiments, the epicardium-derived paracrine factor is follistatin-like protein 1 (FSTL1, also known as follistatin-related protein 1). FSTL1 is a protein encoded by the FSTL1 gene in humans. This gene encodes a protein with similarity to the activin-binding protein follistatin. 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 type C domain ("Entrez gene", "FSTL1 follistatin-like 1"). In other embodiments, FSTL1 has the amino acid sequence of SEQ ID NO: 1 (NCBI Reference Sequence: NP_009016.1): [ka] Includes.

[0047] Nucleic acids encoding FSTL1 are provided and contemplated within the scope of the present invention. In various embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, FSTL1 is encoded by the nucleic acid of SEQ ID NO:2 (NCBI Reference Sequence: NM_007085.4): [ka] [ka] is coded by

[0048] FSTL1 nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques known to those skilled in the art. The methods used to ligate DNA constructs containing the nucleic acid of interest, such as FSTL1, promoters, terminators and other sequences, and insert them into a suitable vector are well known in the art. Furthermore, vectors can be constructed using known recombinant techniques (e.g., Invitrogen Life Technologies, Gateway technology).

[0049] In some embodiments, it may be desirable to overexpress FSTL1 nucleic acids at levels much higher than currently found in naturally occurring cells. This result can be achieved by selectively cloning the nucleic acids encoding these polypeptides into multicopy plasmids or by placing these nucleic acids under a strong inducible or constitutive promoter. Methods for overexpressing desired polypeptides are common and well known in the art of molecular biology, and examples can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, 2001.

[0050] Various host cells can be used to generate recombinant host cells capable of expressing 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 epicardial derived cell cultures can be used to produce FSTL1. However, in other embodiments, cells from organisms that do not post-translationally glycosylate proteins (i.e., cells that do not post-translationally modify proteins with one or more carbohydrate moieties) are used to produce recombinant Fstl.

[0051] Non-limiting examples of cells that do not post-translationally glycosylate proteins include bacterial cells. Thus, in one embodiment, the host cell is a bacterial cell. In another embodiment, the bacterial cell is a gram-positive bacterial cell or a gram-negative bacterial cell. In another embodiment, the bacterial cell is selected from the group consisting of E. coli, L. acidophilus, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., P. alcaligenes, Clostridium sp., Corynebacterium sp., and C. glutamicum cells.

[0052] Nucleic acids encoding FSTL1 or vectors containing them can be inserted into host cells (e.g., bacterial cells) using standard techniques to express the encoded FSTL1 polypeptide. Introduction of DNA constructs or vectors into host cells can be accomplished using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection- or DEAE-dextrin-mediated transfection or transfection using recombinant phage viruses), incubation with calcium phosphate DNA precipitates, high-velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are well known in the art (e.g., Current Protocols in Molecular Biology (see 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, vol. 16, pp. 53-56, 1989), each of which is incorporated herein by reference in its entirety, particularly with respect to transformation methods). The introduced nucleic acid can be integrated into chromosomal DNA of the host cell or maintained as an extrachromosomal replicating sequence. In yet another embodiment, FSTL1 polypeptides can be produced in host cells by delivery of chemically modified mRNAs encoding mutant Fstl1 glycosylation-deficient polypeptides (see Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Zangi L et al., Nat Biotechnol. October 2013, 31(10):898-907, which is incorporated herein by reference in its entirety). Chemically modified RNAs, also referred to herein as modRNAs, can include, for example, modifications of phosphates to phosphorothioate internucleotide linkages, modifications of the 2'-hydroxyl group of ribose, or other modifications to the phosphate backbone or sugar moieties of mRNA.

[0053] In some embodiments, FSTL1 is hypoglycosylated FSTL1. Hypoglycosylated FSTL1 can be obtained by producing recombinant FSTL1 in a host cell (such as bacteria, e.g., E. coli) that does not naturally post-translationally modify proteins with carbohydrate moieties, or a host cell engineered to be unable to post-translationally modify proteins with carbohydrate moieties. Alternatively, hypoglycosylated FSTL1 can be produced in a mammalian or other eukaryotic cell that normally post-translationally modifies proteins with carbohydrate moieties, but that has 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), streptovirudin, mycospocidin, amphomycin, tsushimycin, antibiotic 24010, antibiotic MM19290, bacitracin, corynetoxin, showdomycin, duimycin, 1-deoxyglucose ... Deoxymannonojirimycin, N-methyl-1-deoxymannojirimycin, brefeldin A, glucose analogue, mannose analogue, 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-CoA reductase inhibitor, 25-hydroxycholesterol, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin, m-chlorocarbonyl-cyanide phenylhydrazone (CCCP), compactin, dolisyl-phosphoryl-2-deoxyglucose, N-acetyl-D-glucosamine, hygoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromoconduritol, conduritol epoxide epoxide), conduritol derivatives, aglycosylmethyl-p-nitrophenyltriazenes, β-hydroxynorvaline, threo-β-fluoroasparagine, D-(+)-gluconic acid δ-lactone, di(2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl ester of (diphenylmethyl)-phosphate, [2-(diphenylphosphinyloxy)ethyl]trimethylammonium iodide, iodoacetate, 2-deoxy-D-glucose, and fluoroacetate.

[0054] Alternatively, in other embodiments, recombinant FSTL1 is engineered to be incapable of glycosylation when produced using eukaryotic cells or other glycosylation-competent host cells. In most biological contexts, glycosylation is either N-linked or O-linked. The N-linked glycosylation process occurs widely in eukaryotic cells and in archaea, but is very rare in eubacteria. In N-linked glycosylation, glycans (i.e., carbohydrate-containing moieties) are attached to the nitrogen atom of asparagine or arginine amino acid side chains. N-linked glycans are almost always attached to the nitrogen atom of asparagine (Asn) side chains that are present as part of the Asn-X-Ser / Thr consensus sequence, where X is any amino acid other than proline (Pro), serine (Ser) and threonine (Thr). O-linked glycosylation is the form of glycosylation that occurs in the Golgi apparatus of eukaryotic cells. In O-linked glycosylation, the glycan is attached to the hydroxyl oxygen of a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline amino acid side chain.

[0055] Thus, in some embodiments, recombinant FSTL1 is engineered to be incapable of N-linked glycosylation. In this case, some or all of the arginine or asparagine amino acids in the polypeptide sequence that are competent for glycosylation can be replaced with amino acids that are ineligible for glycosylation (e.g., glutamine). In other embodiments, recombinant FSTL1 is engineered to be incapable of O-linked glycosylation. In this case, all of the serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residues in the polypeptide sequence that are competent for glycosylation can be replaced with amino acids that are ineligible for glycosylation (e.g., alanine). In yet further embodiments, recombinant FSTL1 is engineered to be incapable of both O-linked and N-linked glycosylation by replacing all of the amino acids that are competent for glycosylation with amino acids that are ineligible for glycosylation. In further embodiments, one or more asparagine (N) residues at positions X144, X180, X175, and / or X223 in the FSTL1 amino acid sequence are replaced with glycosylation-incompetent amino acids (for example, but not limited to, glutamine (Q)). The engineered glycosylation-incompetent FSTL1 can be produced in a host cell by transfection of a plasmid, a viral vector carrying a gene encoding glycosylation-incompetent FSTL1, or a chemically synthesized mRNA or mRNA mimic. Alternatively, the gene encoding glycosylation-incompetent FSTL1 can be integrated into the host cell chromosome under the control of an inducible or constitutive expression promoter. In yet another embodiment, the glycosylation-incompetent FSTL1 polypeptide can be produced in a host cell by delivery of a modified mRNA encoding a glycosylation-incompetent FSTL1 polypeptide.

[0056] The invention described herein contemplates FSTL1 incorporated into a pharmaceutical composition (e.g., a sterile pharmaceutical composition) containing one or more pharma- ceutically acceptable carriers. As used herein, a "pharma-ceutically acceptable carrier" or "pharma-ceutically acceptable excipient" according to the present invention is a component of the composition, such as a carrier, diluent, or excipient, that can be combined with the agent and / or composition of the present invention without losing the biological activity of the agent or composition (e.g., FSTL1, such as underglycosylated FSTL1), is compatible with the other components of the composition in that it is suitable for use in subjects provided herein without excessive adverse side effects (e.g., toxicity, irritation, allergic response, and death). A side effect is "excessive" when the risk outweighs the benefit provided by the pharmaceutical composition. Non-limiting examples of pharma- ceutically acceptable components include any of the standard pharmaceutical carriers, such as, but not limited to, phosphate buffered saline solution, water, sterile water, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil / water or water / oil emulsions, microemulsions, nanocarriers, and various types of wetting agents. Water, alcohol, oil, glycols, preservatives, flavorings, colorants, suspending agents, other additives, and the like, may also be included in the composition along with carriers, diluents, or excipients. In one embodiment, pharma- ceutically acceptable carriers suitable for use in the compositions disclosed herein are sterile, pathogen-free, and / or otherwise safe for administration to a subject without the associated risk of infection and other undue harmful side effects.

[0057] Any of the FSTL1-containing (e.g., underglycosylated FSTL1-containing) pharmaceutical compositions disclosed herein can be formulated for administration using various administration methods available in the art. Administration can be by various routes, including patch, catheter, stent, oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, intranasal, and others. In some embodiments, the above administration methods can be used to deliver a suspension containing FSTL1 (e.g., underglycosylated FSTL1 mixed with gelfoam particles). These compositions are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical arts and include at least one active compound. When used as an oral composition, the polypeptide composition is protected from acid digestion in the stomach by a pharma-ceutically acceptable protectant.

[0058] In some embodiments, any of the FSTL1-containing (e.g., hypoglycosylated FSTL1-containing) pharmaceutical compositions disclosed herein can be incorporated into a patch designed for direct administration to epicardial or myocardial damaged tissue. In one embodiment, a compressed collagen gel is used to fabricate a three-dimensional (3D) collagen patch for delivering hypoglycosylated FSTL1 directly to the epicardium. In some embodiments, a highly hydrated collagen gel can be compressed to remove excess water and fabricate a dense biomaterial with improved biological and mechanical properties.

[0059] As described in Example 2 below, the highly moistened collagen gel was subjected to unrestrained compression by applying a static compressive stress of about 1,400 Pa for 5 minutes, resulting in a volume reduction of about 98-99%. The elastic modulus of the compressed collagen approximates that of embryonic epicardium, which is optimal for immature cardiomyocyte contractility. The elasticity of the compressed collagen patch can be assessed by atomic force microscopy (AFM) in nano-indentation mode using a force trigger with an indentation of about 100 nm to minimize the effect of substrate-related artifacts, resulting in a minimum local strain of less than about 10% (e.g., less than about any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%, including all values ​​between these percentages). The 3D collagen patch can be seeded with recombinantly produced FSTL1 (e.g., hypoglycosylated FSTL1) and then administered directly to the damaged or injured area of ​​the epicardium or myocardium, e.g., by suturing. In some embodiments, the 3D collagen patch has an elastic modulus similar to that reported for embryonic epicardium (E=about 12±4 kPa, e.g., about any of 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, or 16 kDa). In other embodiments, the 3D collagen patch has an elastic modulus lower than that of mature epicardium (E>30-40 kPa). In other embodiments, the 3D collagen patch has an elastic modulus lower than that of fibrotic cardiac tissue (E>100 kPa), but higher than that of the majority of currently used scaffold biomaterials (E≦1 kPa). In another embodiment, the 3D collagen patch has an elastic modulus of about any of 1 kPa, 2 kPa, 3 kPa, 4 kPa, 5 kPa, 6 kPa, 7 kPa, 8 kPa, 9 kPa, 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, 15 kPa, 16 kPa, 17 kPa, 18 kPa, 19 kPa, 20 kPa, 21 kPa, 22 kPa, 23 kPa, 24 kPa, 25 kPa, 26 kPa, 27 kPa, 28 kPa, or 29 kPa.

[0060] Further information regarding the construction and use of 3D collagen-based patches to deliver substances directly to the heart can be found in Serpooshan, V. et al., Acta Biomater, 2010, 6, 3978-3987; Serpooshan, V. et al., J Biomed Mater Res A, 2011, 96, 609-620; and Abou Neel et al., Soft Matter, 2006, vol. 2, pp. 986-992, the disclosure of which is incorporated herein by reference.

[0061] Another option for delivery of low glycosylated FSTL1 polypeptide to cardiac tissue is as a component of a self-polymerizing hydrogel delivered by catheter technology. Further information on this type of delivery can be found in Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014), vol. 7, pp. 232-241, the disclosure of which is incorporated herein by reference. Catheter delivery can also be used for suspensions containing FSTL1 (e.g., low glycosylated FSTL1 mixed with gelfoam particles).

[0062] III. Methods of the Invention Provided herein is a method for repairing cardiac tissue (e.g., myocardial tissue) after injury in a subject in need thereof, comprising contacting the cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor. In some embodiments, the epicardium-derived paracrine factor is a hypoglycosylated follistatin-like 1 (FSTL1) polypeptide. In other embodiments of the invention, administration of hypoglycosylated FSTL1 does not activate Akt-1 signaling activity (see FIG. 20). In other embodiments of the invention, administration of hypoglycosylated FSTL1 does not result in reduced apoptosis of cardiomyocytes (see FIG. 6).

[0063] Injury to cardiac tissue (e.g., myocardial tissue) may be associated with a variety of diseases or conditions known to affect the heart or circulatory system, including, but not limited to, coronary heart disease, cardiomyopathy, ischemic heart disease, heart failure, inflammatory heart disease, valvular heart disease, and aneurysms. In one embodiment, the injury is caused by myocardial infarction (MI, e.g., acute myocardial infarction (AMI)). In another embodiment, the injury is caused by an ischemic event followed by reperfusion.

[0064] Repair of damaged cardiac tissue (e.g., myocardial tissue) includes increasing the number of cardiomyocytes, which can be an indirect measure in living subjects by some imaging methods (e.g., delayed-enhancement MRI, DE-MRI) as a reduction in the size of myocardial infarction. 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 its entirety). In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in recovery of lost muscle and reduction in infarct size by about any of about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100% or about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%. In other embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in recovery of lost muscle and reduction in infarct size by at least about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 90%, 100%, or any of about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%. In other embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in recovery of lost muscle and reduction in infarct size by at most about 2%, 5%, 10%, 15%, 20%, 30%, 40% 50%, 60%, 90%, 100%, or any of about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%.

[0065] In some embodiments of any of the methods disclosed herein, epicardium-derived paracrine factors (e.g., hypoglycosylated FSTL1) are injected, seeded, or embedded into a 3D collagen-based patch (e.g., any of those described herein). The collagen-based patch can then be directly contacted to an injured area of ​​the epicardium or myocardium (e.g., an area of ​​myocardium exposed to an ischemic event such as a myocardial infarction). The 3D collagen patch can be applied to the epicardium or myocardium by suturing or any other means known in the art for contacting the patch to the injured tissue.

[0066] In yet other embodiments, the epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) is a component of a hydrogel that is delivered (e.g., by catheter techniques; Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014) 7:232-241, incorporated herein by reference in its entirety) to injured areas of the epicardium, endocardium, or myocardium.

[0067] In some embodiments of any of the methods disclosed herein, an increase in the number of cardiomyocytes of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more is achieved compared to the number of cardiomyocytes in cardiac tissue (e.g., myocardial tissue) that was not contacted by epicardium-derived paracrine factors after injury. Assessment of cardiomyocyte replication is routine in the art and can be measured, for example, by determining the number of α-actinin positive cells in a cardiac tissue (e.g., myocardial tissue) sample from a subject. In some other embodiments, the effect on cardiac tissue (e.g., myocardial tissue) can be achieved by placing underglycosylated FSTL1 in the vicinity of the endocardial compartment (i.e., endocardium), for example, by delivery via catheter technology. In some embodiments, a suitable catheter can be a NOGA catheter (Johnson&Johnson).

[0068] In some embodiments, an effect on cardiac tissue (e.g., myocardial tissue) can be achieved by placing underglycosylated FSTL1 into the heart through the endocardium via a subcutaneous catheter delivery system, for example an available system developed by BioCardia (www.biocardia.com).

[0069] In some embodiments, effects on cardiac tissue (e.g., myocardial tissue) can be achieved by placing underglycosylated FSTL1 through the epicardium into the heart using a catheter device similar to those used for other applications (e.g., Epicardial Catheter System™, St. Jude Medical).

[0070] In some embodiments, the effect on cardiac tissue (e.g., myocardial tissue) can be measured using a drug-diluted stent (e.g., available from Abbott Laboratories or Biosensors, among others). This can be achieved by placing underglycosylated FSTL1 via impregnation with GFP (available from Sigma-Aldrich International).

[0071] In some embodiments, the effect on cardiac tissue (eg, myocardial tissue) can be achieved by systemic placement of underglycosylated FSTL1 using approved formulations.

[0072] In some embodiments, the effect on cardiac tissue (e.g., myocardial tissue) can be achieved by placing underglycosylated FSTL1 using compounds or drugs that inhibit glycosylation of endogenous glycosylated FSTL1 protein that are readily available and known to those of skill in the art.

[0073] In some embodiments, the effect on cardiac tissue (e.g., myocardial tissue) can be achieved by introducing a modRNA encoding a specific mutagenesis targeting an N-glycosylation site in the FSTL1 mRNA sequence to place an underglycosylated FSTL1.

[0074] In some embodiments, the effect on cardiac tissue (e.g., myocardial tissue) can be achieved by placing hypoglycosylated FSTL1 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).

[0075] In some embodiments, the effect on cardiac tissue (eg, myocardial tissue) is achieved by delivery of a small molecule that mimics underglycosylated FSTL1.

[0076] In other embodiments, repairing the injured cardiac tissue (e.g., myocardial tissue) includes improving the fractional shortening of the cardiac tissue (e.g., myocardial tissue) after injury compared to the amount of fractional shortening in the cardiac tissue (e.g., myocardial tissue) not contacted by the epicardium-derived paracrine factor. In some embodiments, contacting the cardiac tissue (e.g., myocardial tissue) with the epicardium-derived paracrine factor (e.g., underglycosylated Fstl1) results in an improvement in the fractional shortening of the cardiac tissue (e.g., myocardial tissue) 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%, 75%, 80%, 85%, 90%, 95%, or 100% or more, including all values ​​between these percentages, compared to the same subject before treatment. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated Fstl1) results in an improvement in the shortening fraction of the cardiac tissue (e.g., myocardial tissue) compared to the same subject prior to treatment of at most about any of 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 ​​between these percentages. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated Fstl1) results in an improvement in the shortening fraction of the cardiac tissue (e.g., myocardial tissue) compared to the same subject prior to treatment of at least 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%, 90-100%, including all values ​​between these percentages.In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., hypoglycosylated Fstl1) results in at most about 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%, including all values ​​between these percentages, in the shortening fraction of the cardiac tissue (e.g., myocardial tissue) compared to the same subject before treatment. Repair of injured cardiac tissue (e.g., myocardial tissue) may also include an increase in the amount of myocardial cell cytokinesis compared to the amount of myocardial cell cytokinesis in cardiac tissue (e.g., myocardial tissue) not contacted with epicardium-derived paracrine factors after injury. In some embodiments, in some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an improvement in the amount of cytokinesis in cardiomyocytes of at least about any of 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% or more. In some embodiments, in some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an improvement in the amount of cytokinesis in cardiomyocytes of at most about any of 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% or more.In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an improvement in the amount of cytokinesis in cardiomyocytes of at least 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%. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an improvement in the amount of cardiomyocyte cytokinesis of at most about 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%. Assessment of cardiomyocyte cytokinesis is routine in the art and can be measured, for example, by determining the expression level of Aurora B kinase in a cardiac tissue (e.g., myocardial tissue) sample from a subject. With current methods, these studies can only be performed post-mortem or post-biopsy or post-transplant.

[0077] In some embodiments, repair of injured cardiac tissue (e.g., myocardial tissue) may include a reduction in cardiomyocyte apoptosis compared to the amount of cardiomyocyte apoptosis in cardiac tissue (e.g., myocardial tissue) not contacted with epicardium-derived paracrine factors after injury. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in a reduction in cardiomyocyte apoptosis in cardiac tissue (e.g., myocardial tissue) 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%, 75%, 80%, 85%, 90%, 95%, 100% or more, including all values ​​between these percentages. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) results in a reduction in apoptosis of cardiomyocytes in the cardiac tissue (e.g., myocardial tissue) of at most about any of 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 ​​between these percentages. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) results in a reduction in apoptosis of cardiomyocytes in the cardiac tissue (e.g., myocardial tissue) of at least about 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% or more, including all values ​​between these percentages.In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in at most about 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% reduction in cardiomyocyte apoptosis in cardiac tissue (e.g., myocardial tissue), including all values ​​between these percentages. Assessment of cardiomyocyte apoptosis is routine in the art (post-mortem or ex vivo, after heart isolation) and can be measured, for example, by TUNEL staining of a cardiac tissue (e.g., myocardial tissue) sample from a subject.

[0078] Repair of injured cardiac tissue (e.g., myocardial tissue) may also include an increase in the level of one or more transcripts encoding cardiac-specific contractile proteins in cardiomyocytes compared to the transcription level of the cardiac-specific contractile proteins in cardiac tissue (e.g., myocardial tissue) not contacted with epicardium-derived paracrine factors after injury. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in at most 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%, 100% or more increase in the level of one or more transcripts encoding cardiac-specific contractile proteins. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding cardiac-specific contractile proteins of at least about any of 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%. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding cardiac-specific contractile proteins of at least 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%.In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) results in an increase in the level of one or more transcripts encoding cardiac-specific contractile proteins of at most about 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%. In some embodiments, the cardiac-specific contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a. Assessment of cardiac-specific contractile protein transcripts is routine in the art and can be measured, for example, by Northern blot, Western blot, reverse transcriptase (RT) PCR, FACS analysis, immunohistochemistry, or in situ hybridization.

[0079] Repair of damaged cardiac tissue (e.g., myocardial tissue) requires rhythmic Ca release in cardiac myocytes. 2+ Actinin with + In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated Fstl1) can increase rhythmic contractile Ca2+ levels in cardiac tissue (e.g., myocardial tissue) that was not contacted by the epicardium-derived paracrine factor after injury. 2+ Actinin with + Rhythmic contractile Ca in cardiomyocytes compared to the number of cells 2+ Actinin with + Any increase in cell mass of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold or more is obtained. 2+ Actinin with + Cellular evaluation is routine in the art (see Example 1 below).

[0080] Injured cardiac tissue (e.g., myocardial tissue) can be contacted with any of the epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) compositions (e.g., pharmaceutical compositions) disclosed herein before, during, or after injury to the cardiac tissue (e.g., myocardial tissue). In some embodiments, cardiac tissue (e.g., myocardial tissue) is contacted with an epicardium-derived paracrine factor composition to reduce or prevent myocardial injury from an event in a subject considered at risk for cardiovascular disease, MI, or another myocardial ischemic event. In other embodiments, cardiac tissue (e.g., myocardial tissue) is administered immediately, e.g., at 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, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, 75 minutes, 76 minutes, 77 minutes, 78 minutes, 79 minutes, 80 minutes, 81 minutes, 82 minutes, 83 minutes, 84 minutes, 85 minutes, 86 minutes, 87 minutes, 88 minutes, 89 minutes, 90 minutes, 91 minutes, 92 minutes, 93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 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 periods between these values), contacted with the epicardium-derived paracrine factor composition. In some embodiments, the composition is administered less than 1 minute after the cardiac injury. Alternatively, in other embodiments, cardiac tissue (e.g., myocardial tissue) is contacted with the epicardium-derived paracrine factor composition subsequent to injury, for example, 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, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more (including all periods in between these values) after the onset of the ischemic event caused by cardiovascular disease or MI.

[0081] Any of the methods disclosed herein for treating injury to cardiac tissue (e.g., myocardial tissue) can increase survival in a subject after injury. As used herein, increased survival includes, for example, 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 200% or more) increase in subject survival compared to the relative survival in a subject not subjected to the methods described herein. Survival can be measured, for example, in days, weeks, months, or years. In some embodiments, contacting injured cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors by any of the methods described herein can extend the survival of a subject by at least 6 months, 7 months, 8 months, 9 months, 10 months, 12 months, 18 months, 24 months, 36 months, or more.

[0082] In some embodiments, repair of injured cardiac tissue (e.g., myocardial tissue) may include attenuating or reducing fibrosis in cardiac tissue (e.g., myocardial tissue) compared to the amount of fibrosis in cardiac tissue (e.g., myocardial tissue) not contacted with epicardium-derived paracrine factors after injury. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in a reduction in fibrosis in cardiac tissue (e.g., myocardial tissue) 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%, 75%, 80%, 85%, 90%, 95%, 100% or more, including all values ​​between these percentages. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) results in a reduction in fibrosis of the cardiac tissue (e.g., myocardial tissue) of at most about any of 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 ​​between these percentages. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in a reduction in fibrosis in cardiac tissue (e.g., myocardial tissue) of at least about 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 ​​between these percentages. Assessment of cardiomyocyte fibrosis is routine in the art and can be measured by DE-MRI or by histological examination (postmortem or biopsy) of cardiac tissue (e.g., myocardial tissue).In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in a reduction in fibrosis in cardiac tissue (e.g., myocardial tissue) of at most about 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 ​​between these percentages. Assessment of cardiomyocyte fibrosis is routine in the art and can be measured by DE-MRI or by histological examination (postmortem or biopsy) of cardiac tissue (e.g., myocardial tissue).

[0083] Repair of injured cardiac tissue (e.g., myocardial tissue) may further include increasing vascularization of the injured region of the cardiac tissue (e.g., myocardial tissue). In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factors (e.g., underglycosylated FSTL1) results in 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%, 75%, 80%, 85%, 90%, 95%, 100% or more of recovery in the amount of vascularization in cardiac tissue (e.g., myocardial tissue) compared to the relative amount of vascularization in cardiac tissue (e.g., myocardial tissue) not contacted with epicardium-derived paracrine factors after injury or increased blood perfusion. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) results in at most about any 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% restoration or increase in blood perfusion in the cardiac tissue (e.g., myocardial tissue) compared to the relative amount of angiogenesis in cardiac tissue (e.g., myocardial tissue) not contacted by the epicardium-derived paracrine factor after injury. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) results in a recovery or increase in blood perfusion of at least 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% in the amount of angiogenesis in cardiac tissue (e.g., myocardial tissue) following injury, compared to the relative amount of angiogenesis in cardiac tissue (e.g., myocardial tissue) not contacted with the epicardium-derived paracrine factor.In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with an epicardium-derived paracrine factor (e.g., underglycosylated FSTL1) results in at most about 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% recovery or increase in blood perfusion in the cardiac tissue (e.g., myocardial tissue) compared to the relative amount of angiogenesis in cardiac tissue (e.g., myocardial tissue) not contacted by the epicardium-derived paracrine factor following injury. Assessment of angiogenesis in cardiac tissue (e.g., myocardial tissue) is routine in the art and can be assessed by measuring the expression of proteins such as von Willebrand factor (vWF) or smooth muscle actin in vascular cells (see Example 4 below).

[0084] In further embodiments, repair of damaged cardiac tissue (e.g., myocardial tissue) includes increasing the cell cycle entry of cardiomyocytes. In some embodiments, contacting cardiac tissue (e.g., myocardial tissue) with epicardium-derived paracrine factor (e.g., hypoglycosylated Fstl1) results in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or more increase in the amount of cell cycle entry of cardiomyocytes in cardiac tissue (e.g., myocardial tissue) compared to the amount of cell cycle entry of cardiomyocytes in cardiac tissue (e.g., myocardial tissue) not contacted with epicardium-derived paracrine factor after injury. Assessment of cell cycle entry of cardiomyocytes in cardiac tissue (e.g., myocardial tissue) is routine in the art and can be assessed, for example, by measuring expression of phospho-histone H3 (see Example 5 below).

[0085] In some embodiments of any of the methods disclosed herein, epicardium-derived paracrine factors (e.g., hypoglycosylated Fstl1) are injected, seeded, or embedded into a 3D collagen-based patch (e.g., any of those described herein). The collagen-based patch can then be directly contacted to an injured area of ​​the epicardium or myocardium (e.g., an area of ​​myocardium exposed to an ischemic event such as a myocardial infarction). The 3D collagen patch can be applied to the epicardium or myocardium by suturing or any other means known in the art for contacting the patch to the injured tissue.

[0086] In yet other embodiments, epicardium-derived paracrine factors (e.g., underglycosylated Fstl1) are components of a hydrogel that is delivered to injured areas of the epicardium, endocardium, or myocardium (e.g., by catheter techniques; Koudstaal et al., J. of Cardiovasc. Trans. Res. (2014) 7, 232-241).

[0087] IV. Kit Also provided herein is a kit comprising (i) an epicardium-derived paracrine factor (e.g., an underglycosylated FSTL1 polypeptide); and (ii) one or more pharma-ceutically acceptable excipients. Either or both of these kit components can be sterile so that they can be administered to an individual in need (e.g., an individual having a cardiac injury such as an MI). The kit may optionally contain a 3D collagen patch (e.g., any of those disclosed herein) that can be seeded or injected with the epicardium-derived paracrine factor prior to administration to the subject. Alternatively, the kit may include a pre-seeded or pre-injected 3D collagen patch, along with instructions for use and application to the injured cardiac tissue (e.g., myocardial tissue) or epicardium of a subject in need thereof. The kit may further comprise a means for attaching the 3D collagen patch to the epicardium or injured cardiac tissue (e.g., myocardial tissue), such as, but not limited to, a suture material.

[0088] Any of the kits disclosed herein may include a hydrogel (e.g., a self-polymerizing hydrogel) as a carrier for the epicardium-derived paracrine factors (e.g., underglycosylated FSTL1 polypeptide). In one embodiment, the kit may also include one or more catheters for delivering the hydrogel (e.g., the underglycosylated FSTL1 polypeptide-infused hydrogel) to one or more damaged areas of the endocardium, epicardium, and / or myocardium.

[0089] The kit may also include instructions for use of the kit, such as instructions for injecting the epicardium-derived paracrine factors into the 3D collagen patch, suturing the patch to the myocardium or epicardium, injecting the epicardium-derived paracrine factors into a hydrogel (e.g., a self-polymerizing hydrogel), and delivering the hydrogel to one or more damaged areas of the epicardium or myocardium by catheter techniques.

[0090] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that is subsumed within the broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0091] The present invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting. EXAMPLES

[0092] Example 1: Epicardial paracrine signaling activates proliferation of cardiomyocytes The cardiac epicardium is a precursor cell 1、2 and mitogens including FGF, IGF2, and PDGF 3~5The epicardium is an outer epithelial layer that contributes to the growth of the developing myocardium by providing a source of myogenic precursors. Recent studies suggest that the epicardium may also be able to maintain the function of the adult myocardium after injury, possibly as a source of myogenic precursors. 6、7 However, epicardium-derived paracrine factors have not been shown to support myocardial regeneration in mammals, although their identity and mechanism of action may provide insight into this poorly understood and inherently inefficient process. 8 This example describes the identification of such epicardium-derived paracrine factors.

[0093] material and method Progenitor cell Sca1 + ,Myh6 - Cardiomyocyte precursors were cultured as described 19 , obtained from the Schneider laboratory.

[0094] Epicardial mesothelial cells (EMCs) were cultured as described 33 EMCs were stably transduced with H2B-mCherry lentivirus for nuclear labeling.

[0095] Mouse embryonic stem cell-derived cardiomyocytes (mCMs) ESC ): A stable mouse ESC line (Myh6-Puror; Rex-Blastr) for drug resistance selection of cardiomyocytes was created using the human line previously reported by the inventors. 34 were produced by lentiviral transduction and blasticidin selection, similar to

[0096] mCMs ESC 10% FBS, 2 mM glutamine, 4.5 × 10 -4Myh6-Puror, Rex-Blastr mESCs were obtained by differentiation as embryoid bodies (EBs) by day 4 in differentiation medium containing 0.5 mM monothioglycerol, 0.5 mM ascorbic acid, 200 μg / mL transferrin (Roche), 5% protein-free hybridoma medium (PFHM-II, Invitrogen) and Iscove's modified Dulbecco's medium (IMDM) supplemented with antibiotics / antimycotics, and plated on adherent cell culture plates until day 9, i.e., 1 day after the onset of spontaneous beating. Myh6 + To purify cardiomyocytes, puromycin was added on day 9 of differentiation for 24 hours. Cells were then trypsinized and plated as monolayers of cardiomyocytes. Conditioned medium and FSTL1 treatments were typically performed 24 hours after plating of the monolayer. The length of treatment is indicated in each figure legend.

[0097] Embryonic cardiomyocytes. Tnt-Cre;Rosa26 cells derived from e12.5 embryos were isolated using fluorescence-activated cell sorting (FACS). mTmG / + Cardiomyocytes were purified from the hearts. Hearts were dissociated by collagenase IV digestion and then incubated for 1 h at 37 °C for 1 h. + Cells were FACS purified. GFP + The cells were cultured and confirmed to be cardiomyocytes by expression of the cardiomyocyte-specific markers alpha-actinin (ACTN2) and cardiac troponin T (TNNT2), and they beat rhythmically when cultured in vitro.

[0098] Rat epicardial mesothelial cell (EMC) conditioned medium. EMC33 cells were cultured to confluency (approximately 1 × 10 6 / cm 2), then washed three times with PBS, and the medium was replaced with serum-free DMEM without phenol red and containing penicillin / streptomycin for another 2 days of culture, after which the medium was harvested as conditioned medium (20 ml of medium was added for conditioning, and 18 ml was harvested after 2 days). The harvested medium was filtered through a 0.22 μm pore membrane (Millipore). A control conditioned medium was prepared in the same manner but without EMC cells.

[0099] Adult mouse EPDC-conditioned medium was produced in the Zhou laboratory. 9 Briefly, 8-week-old adult Wt1CreERT2 / +;Rosa26mTmG / + cardiac mice were orally gavaged with 4 mg tamoxifen, administered 4 to 5 times by oral infusion over a 2-week period. Myocardial infarction was then induced in adult mice (aged 11 weeks) by ligation of the left anterior descending coronary artery. One week after injury, Wt1 CreERT2 / + ;Rosa26 mTmG / + The hearts were harvested and then digested with collagenase IV to single cells. The digestion solution was made by adding 4 ml of 1% collagenase IV and 1 ml of 2.5% trypsin to 44.5 ml of Hank's balanced salt solution and supplemented with 0.5 ml of chicken serum and 0.5 ml of horse serum. The cells were resuspended in Hank's balanced salt solution and 4 ml of digestion solution was added to each tube and gently rocked in a shaker at 37°C for 6 minutes. After removing the supernatant containing the dissociated cells, another 4 ml of digestion solution was added and the digestion was repeated six times. After the final digestion, the cells were filtered through a 70 μm filter and the cells were pelleted by centrifuging at 200g for 5 minutes at 4°C. The cells were then resuspended with Hank's balanced salt solution for FACS isolation. The dissociated cells from the GFP hearts were used as a control for gating in the FACS. GFP+ cells (epicardium-derived cells, EPDCs) were collected from the GFP hearts and analyzed by the FACS gating control. + Wt1 CreERT2 / + ;Rosa26 mTmG / + These GFP-GFP antibodies were isolated from the heart by FACS. + A purified population of GFP was detected under a fluorescent microscope. +The cells were confirmed to be GFP+ EDDCs. Expression of FSTL1 (determined by PCR) was restored in cultured GFP+ EDDCs. Complete conditioned medium from EPDCs was then added to the myocyte assay. Dilutions are as indicated in the figure legends.

[0100] The proliferation of cardiomyocytes treated with conditioned medium was determined as previously described. 9 , measured by MTT assay using Celltiter 96 Aqueous One solution (Promega). After adding Celltiter 96 Aqueous One reagent into the cell culture medium, the plates were incubated at 37 °C for 3-4 h, and then the absorbance was recorded at 490 nm using a 96-well plate reader. The absorbance at 490 nm correlates closely with the cell number. The MTT readout on the y-axis, labeled MTT assay (A490), therefore reflects the relative number of cells from each well between treatment groups.

[0101] Calcium imaging: Contractile calcium transients were recorded using a Kinetic Image Cytometer (KIC, Vala Sciences) using Fluo4 NW calcium indicator (Life Science). Data were analyzed using Cyteseer software containing the KIC analysis package (Vala Sciences) as described. 38 Processed.

[0102] RNA extraction and Q-RT-PCR: Total RNA was extracted with TRIzol (Invitrogen) and reverse transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. cDNA samples synthesized from 100 ng of total RNA were subjected to RT-QPCR using LightCycler 480 SYBR Green I Master Kit (Roche) performed using LightCycler 480 real-time PCR system (Roche). The sequences of the primers used in this example as well as other examples disclosed herein are listed below. [Table 1-1]

[0103] result To explore epicardial signals that promote cardiogenesis, epicardial mesothelial cell (EMC) lines were transformed with Myh6 + Mouse embryonic stem cell (ESC)-derived cardiomyocytes (mCMs) ESC The mice were co-cultured with mCMs. ESC differentiated Myh6-Puro r Mouse ESCs were prepared from puromycin selection (see Figure 8 and Materials and Methods for details and cell phenotype). Co-culture with ESCs increased the expression of α-actinin + Undiluted EMC conditioned medium consistently increased myocyte numbers (Figure 1a-c) and expression of cardiomyocyte markers including Myh6, Myh7, Mlc2a and Mlc2v (Figure 1d). + EMC-conditioned medium reproduced the effects of co-culture by increasing the expression of Myh6 (1.8-fold), Mlc2v (1.9-fold), and Mlc2a (1.3-fold) (Figure 1h) as well as the expression of Myh6 (1.8-fold), Mlc2v (1.9-fold), and Mlc2a (1.3-fold) (Figure 1h). Furthermore, EMC-conditioned medium suppressed rhythmic contractile Ca 2+ α-actinin showed a transient of + The number of cells was increased (8.6-fold) compared to standard medium (Figure 1i). Thus, the factor(s) secreted from the epicardium-like cultures increased the number of contractile cells in ESC-derived cardiomyocyte cultures. Co-culture did not promote cardiogenesis of undifferentiated (Myh6-) ESCs (not shown).

[0104] To assess whether adult epicardium also contains such activity, we used WT1 cells from 3- to 4-month-old mice. CreERT2 / + ;Rosa26m TmG / + mouse 9 We prepared conditioned medium from epicardium-derived cells (EPDCs) isolated by FACS from (Figure 1j and Materials and Methods). E12.5 embryonic cardiomyocytes (again, previously identified as TNT-Cre; Rosa 26mTmG / +When added to adult EPDC-conditioned medium (FACS-isolated from mice), adult EPDC-conditioned medium significantly enhanced proliferation of cardiomyocytes in serum-free medium (p<0.05, Fig. 1k). Boiling the EPDC medium prior to incubation abolished the effect, consistent with the constitutive activity being protein-based. EPDC-conditioned medium inhibited the proliferation of the adjacent Tnnt2 + It nearly doubled the occurrence of Aurora B kinase in the cleavage furrow connecting cells (0.19% to 0.33%, P < 0.05, Fig. 1l,m), demonstrating activity in the adult epicardium that promotes cytokinesis in embryonic cardiomyocytes.

[0105] Example 2: Engineered epicardium attenuates remodeling and improves cardiac function This example illustrates the effects of epicardially secreted factors in the adult heart.

[0106] material and method Adult ventricular myocytes were cultured from 3-month-old FVB mice as previously published. 35 Briefly, mice were anesthetized with sodium pentobarbital (100 mg / kg, i.p.). The hearts were removed and Ca 2+ The ventricles were perfused retrogradely at 37 °C with Ca-free solution (in mM: 120 NaCl, 14.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4, 1.2 MgSO4-7H2O, 4.6 NaHCO3, 10 Na-HEPES, 30 taurine, 10 BDM, 5.5 glucose) and subsequently enzymatically digested with collagenase. The ventricles were cut into small pieces and further digested with stop buffer (Ca 2+A solution containing 100% CaCl2 (12.5 μM + 10% calf serum) was added and the cell suspension was centrifuged at 40 g for 3 min. The muscle cells were resuspended in stop buffer with increasing CaCl2 concentration to 1 mM. The cells were then resuspended in MEM + 5% calf serum + 10 mM BDM + 2 mM L-glutamine and added to the collagen solution and prepolymerized (250,000 cells per ml or per patch). Following collagen gelation and plastic compaction, the cell patches were cultured overnight in the previously mentioned (plating) medium and then transferred into culture medium (MEM + 1 mg / ml bovine serum albumin + 25 μM blebbistatin + 2 mM L-glutamine) in the presence or absence of recombinant FSTL1 (AVISCERA BIOSCIENCE, 10 ng / ml). On day 7, a fluorescent ubiquitination-based cell cycle indicator (FUCCI, Premo™ FUCCI cell cycle sensor, Life Technologies, US) assay was performed as previously described. 36 , performed on 3D culture specimens. In this assay, G1 and S / G2 / M cells emit red and green fluorescence, respectively. The volumes of Premo™ geminin-GFP and Premo™ Cdt1-RFP were calculated using the equation below. [ka] where Number of cells is the estimated total number of cells at the time of cell labeling (equal to the CM seeding density), PPC (particles per cell) is the number of viral particles per cell (=40 in this assay), and 1 × 10 8 is the number of viral particles per mL of reagent. The above calculated volume of reagent was added directly to the cell patch in complete cell medium, mixed gently and incubated overnight (≧16 hours) in a culture incubator. The patch samples were imaged using a conventional fluorescent microscope utilizing GFP and RFP filter sets.

[0107] Compressed collagen gels for use as epicardial patches were created: Highly moistened collagen gels (used as cardiac patches in this study) were produced by adding 1.1 ml of 1×DMEM (Sigma, MO, US) to 0.9 ml of sterile rat tail collagen type I acetate solution (3.84 mg / ml, Millipore, MA, US). The resulting 2 ml collagen-DMEM mixture was mixed thoroughly and neutralized with 0.1 M NaOH (approximately 50 μl). The entire process was carried out on ice to avoid premature gelation of collagen. In the case of patches containing epicardial factors, EMC culture medium was taken as above and 0.6 ml of it was mixed with 0.5 ml of DMEM. The collagen solution (0.9 ml) was then dispensed into wells (15.6 mm diameter) of a 24-well plate and placed at 37° C. for 30 min in a tissue culture incubator for polymerization. Plastic compression was performed as previously described. 39、40 This is carried out to remove excess water and produce a dense biomaterial with improved biological and mechanical properties. Briefly, when cast, a highly moistened collagen gel (with a volume of about 0.9 ml) is subjected to unrestrained compression by applying a static compressive stress of about 1,400 Pa for 5 minutes (see for details). 39、41 ), resulting in a volume reduction of approximately 98-99%. 32 The elastic modulus of the compressed collagen, targeted to approximate that of embryonic epicardium, was assessed by atomic force microscopy (AFM) in nanoindentation mode using a force trigger that produced a minimum local strain of less than 10% (indentation of approximately 100 nm), minimizing the effects of substrate-related artifacts. A custom flat AFM tip was fabricated using focused ion beam milling and utilized to probe the stiffness of the gel by scanning an area of ​​90 μm × 90 μm (Figure 9a-c).

[0108] Permanent LAD occlusion (MI): Male 10-12 week old C57BL / 6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, US). Procedures including animal use and surgery were approved by the Stanford Institutional Animal Care and Use Committee (IACUC). Animal care and interventions were provided in accordance with the Laboratory Animal Welfare Act. Using an isoflurane inhalation chamber, mice were anesthetized and intubated with a 22-gauge angiocatheter (Becton, Dickinson Inc., Sandy, Utah) in the trachea and connected to a small animal volume-controlled ventilator (Harvard Apparatus, Holliston, MA). A left thoracotomy was performed through the fourth intercostal space to deflate the lungs and expose the heart. After opening the pericardium, a 7-0 suture was placed to occlude the left anterior descending artery (LAD), approximately 2 mm below the edge of the left atrium. Ligation was considered successful when the LV wall turned pale. In the case of the experimental group treated with patch, the prepared collagen patch was sutured (at two places) onto the surface of the ischemic myocardium immediately after ligation. The animals were kept on a heating pad until recovery. Another group of mice received sham ligation. They underwent a similar surgical procedure without LAD ligation. A minimum number of n=8 was used in each study group.

[0109] TTC staining: Two days after MI / patch treatment, mouse hearts from all four groups were collected and cut perpendicular to the long axis into four slices (approximately 2 mm thick). The slices were placed in wells of a 12-well cell culture plate and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) solution for 15 min at 37°C. The slices were then washed with PBS and visualized using a stereomicroscope and photographed with a digital camera.

[0110] Echocardiography: In vivo cardiac function was assessed by echocardiography 2 and 4 weeks after LAD ligation. Two-dimensional (2D) analysis was performed in mice using a GE Vivid 7 ultrasound platform with a 13 MHz transducer (GE Health Care, Milwaukee, WI). Mice were sedated with isoflurane (100 mg / kg, inhalation) and the chest was shaved. Mice were placed in supine or left lateral recumbency on a heated platform to facilitate echocardiography. 2D clip and M-mode images were recorded in short-axis view from the mid-left ventricle at the apex of the papillary muscles. LV internal diameter (LVID) and posterior wall thickness (LVPW) were measured at both end-diastole and end-systole. Fractional shortening (FS, %) and ejection fraction (EF, %, by extrapolation of 2D data) were calculated from LV dimensions in the 2D short-axis view. A minimum number (n) of 8 mice per experimental group was used for echo assessment. Measurements were performed in a blinded fashion by two independent groups.

[0111] result Next, the effect of factors secreted by the epicardium in the adult heart was assessed by delivering conditioned medium using a 3D collagen patch of the epicardium. The 3D collagen patch (Figure 2a) has an elastic modulus (E~12±4 kPa) similar to that reported for embryonic epicardium. 10The patch was designed to have an elastic modulus comparable to that of the mature epicardium (E>30-40 kPa) and fibrotic cardiac tissue (E>100 kPa), but higher than that of the majority of currently used scaffold biomaterials (E≦1 kPa) (Figure 2b and Figure 9). The fabricated patch was seeded with EMC-conditioned medium and sutured onto the epicardium of infarcted adult mouse hearts (Figure 2c, d). Patch implantation was performed immediately after permanent ligation of the left anterior descending (LAD) coronary artery (myocardial infarction (MI)). After 2 weeks, hearts treated with epicardium-medium-patch (MI+patch+CM cohort) and empty patch (MI+patch, no conditioned medium) showed significantly better morphometric parameters compared to MI-only animals, including left ventricular internal diameters at end diastole and end systole (LVIDd and LVIDs, respectively), and left ventricular posterior wall dimensions at end diastole and end systole (LVPWd and LVPWs, respectively) (Fig. 2e and Table 1), supporting the model in which the collagen patch provides mechanical support that inhibits pathological remodeling. 10 Notably, MI+patch+CM treatment provided an additional benefit compared to all other conditions, with significantly better parameters of ventricular contractility (Fig. 2e, f and Table 1), thus indicating epicardial secreted activity involved in functional maintenance.

[0112] Example 3: FSTL1 is an epicardial factor that can induce cardiomyocyte proliferation This example provides data suggesting that FSTL1 plays a role in epicardial and myocardial crosstalk that promotes cardiomyogenesis.

[0113] material and method LC-MS / MS analysis of conditioned medium: First, tris(2-carboxyethyl)phosphine (TCEP) was added to 1 mL of conditioned medium to reduce the protein samples for 30 min at 37°C. Iodoacetamide was then added to 20 mM and the solution was alkylated in the dark at 37°C for 40 min. Mass spectrometry grade trypsin (Promega) was then added to the solution at a ratio of 1:100. After overnight digestion at 37°C, the samples were then desalted using a SepPack cartridge, dried using a SpeedVac, and resuspended in 100 μL of 5% formic acid. The resulting peptides were analyzed using a Michrom HPLC, a 15 cm Michrom Magic C18 column, a low-flow ADVANCED Michrom MS source, and an LTQ-Orbitrap XL (Thermo The peptides were analyzed online by an LC-MSMS system consisting of a 120-min gradient of 0–30% B (0.1% formic acid, 100% acetonitrile) with a total LC time of 141 min. The LTQ-Orbitrap XL was set up to scan the precursors in the Orbitrap at a resolution of 60,000, followed by data-dependent MS / MS of the top four precursors. The crude LC-MSMS data containing the sequences of the semi-tryptic peptides were then submitted to Sorcerer Enterprise (Sage-N Research Inc.) for protein identification against the IPI rat protein database, with a tolerance of up to two missed cleavages and a precursor mass tolerance of 50.0 ppm. A molecular weight of 57 Da was added to all cysteines to account for carboxyamidomethylation. The differential search included 16 Da for methionine oxidation. Search results were screened, sorted, filtered, and statistically analyzed using PeptideProphet and ProteinProphet (ISB). The minimum Trans-Proteomic Pipeline (TPP) probability scores for proteins and peptides were set to 0.95 to ensure a TPP error rate of less than 0.01, respectively.

[0114] Recombinant FSTL1 was purchased from AVISCERA BIOSCIENCE (00347-02-100, produced in E. coli) and R&D system (1694-FN-050, produced in mouse myeloma cell line, derived from NS0).

[0115] Histology and Immunohistochemistry: Histological analysis for this and other examples was performed according to standard protocols for paraffin embedding. For immunohistochemistry, embedded embryos were sectioned at 7 μm thickness unless otherwise noted. The antibodies used in this and other examples disclosed herein are as follows: 1:200 α-actinin (Sigma, A7811), 1:300 α-smooth muscle actin (Sigma A2547), 1:100 phospho-histone 3 (rabbit Millipore 06-570), 1:300 phospho-histone 3 (mouse Abcam ab14955), 1:100 WT1 (Abcam, ab15249), 1:250 AuroraB Millipore04-1036 (batch 221196), 1:200 PCM1 (Sigma-Aldrich HPA023370), 1:200 FSTL1 (R&D, MAB17381). At least 5 sections for histological and 3 sections for immunohistochemical studies were used per staining. Inclusion criteria for patch engraftment was that the patch covered >70% of the infarct (controlled by histology). TUNEL assays were performed on frozen sections as indicated (Roche 11684795910).

[0116] result To identify bioactive epicardially secreted protein(s), EMC conditioned media was analyzed by mass spectrometry. Spectra were compared to the IPI rat database to identify 1596 peptide reads corresponding to 311 unique proteins, of which 95 reads were attributable to 16 distinct secreted proteins. Ten proteins with the highest spectral counts were selected for testing in the mCMsESC assay. Of these, cardiogenic activity was only observed in follistatin-like-1 (FSTL1, also known as FRP or TSC36) (Figure 3a), a secreted glycoprotein of the BM-40 / SPARC / osteonectin family that shares a single cysteine-rich domain with follistatin. Unlike follistatin, FSTL1 does not block activin and its biochemical and biological functions have not been fully characterized. 11 Delivery of FSTL1 in the heart produces short-term antiapoptotic effects, but 12、13 However, no myocardial repair function has been ascribed to FSTL1; in fact, FSTL1 levels are increased in the bloodstream after acute MI, and for this reason it has been considered a biomarker for acute coronary syndromes. 14 .

[0117] mCMs ESC Treatment of mice with bacterially synthesized recombinant human FSTL1 (10 ng / ml) for 8 days resulted in a threefold increase in the number of cardiomyocytes (Fig. 3b–d), as well as a twofold increase in the levels of transcripts encoding cardiac-specific contractile proteins (myh6, mlc2v, and mlc2a; Fig. 3e), which enhanced rhythmic contractile Ca 2+ α-actinin with a transient of + Although the number of cells increased sevenfold (Fig. 3f), no hypertrophy was induced. In fact, FSTL1 dose-dependently reduced the size of myocytes (Fig. 3g). Together, these data suggest that FSTL1 plays a role in the crosstalk between the epicardium and myocardium that promotes cardiomyogenesis.

[0118] Direct visualization by immunostaining revealed that FSTL1 is restricted to the epicardium as early as mid-gestation (Fig. 3h), but that it is present earlier in the myocardium of the early heart tube. 15 Although epicardial expression has not been noted previously, it persists throughout adulthood (Figure 3i-k). Remarkably, FSTL1 localization shifts dramatically following ischemic insult, such that it becomes enriched in the myocardium (Figure 3i-l) and, strikingly, is absent from the epicardium and infarcted areas (Figure 3i,l and Figure S10).

[0119] Example 4: Localized FSTL1 delivery improves cardiac function after MI Previous studies have shown that transient overexpression of FSTL1 in cardiomyocytes or direct systemic injection of human recombinant FSTL1 is antiapoptotic after acute ischemia / reperfusion. 12、13 However, this example examines whether it confers any long-term benefits.

[0120] material and method In vivo delayed enhancement magnetic resonance imaging (DEMRI): In preparation for scanning, induction of anesthesia was achieved with 2% isoflurane and maintained at 1.25-1.5% isoflurane by monitoring respiratory rate. ECG leads were inserted subcutaneously to monitor heart rate, while body temperature was maintained at 37°C. Functional parameters were recorded 1 and 4 weeks after the procedure using a 3T GE Signa Excite clinical scanner fitted with a dedicated mouth coil (Rapid MR International, Germany). MRI acquisition was performed in the following order: (1) DEMRI was performed after IP injection of 0.2 mmol / kg gadopentetate dimeglumine (Magnevist, Berlex Laboratories) using a gated fGRE-IR sequence with FOV 3.4 cm, slice thickness 0.9 mm, matrix 128 × 128, TE 5 ms, TI 150-240 ms, and FA 60°; and (2) volumetric cardiac MRI was performed using fSPGR with FOV 7 cm, slice thickness 0.9 mm, matrix 256 × 256, TE 5.5 ms, and FA 30. Coronal and axial scout images were used to position the two-dimensional imaging plane along the short axis of the left ventricular (LV) cavity. A minimum number (n) of 2 mice per experimental group was used for this qualitative study.

[0121] Vascular counting: Vascular density parameters were measured from histological sections of cardiac samples stained for von Willebrand factor (vWF) as a marker of endothelial cells in the vessel wall. Up to 60 sections were analyzed for each treatment group (4 mice per group). The analysis was performed using ImageJ, calculating: 1) the total luminal area of ​​the vessels, and 2) the number of vessels stained positive for vWF. In each case, histograms of the vascular parameters were obtained as a percentage of the total surface area analyzed, and the mid-values ​​were plotted for each treatment group. The statistical significance of the differences from the sham group (p<0.05) was determined by one-way ANOVA.

[0122] Enzyme-linked immunosorbent assay: To evaluate FSTL1 retention in the engineered patch system in vitro, collagen scaffolds loaded with FSTL1 (5 μg / ml) were immersed in PBS and shaken at 37°C for various times (0, 12 h, 1 day, and 21 days), and FSTL1 concentrations were determined using an enzyme-linked immunosorbent assay kit (USCN Life Science, Inc., Houston, USA). The detection limit of this technique was 0.50 ng / ml. The scaffolds were pretreated with 1 mg / ml type I collagenase (Sigma Aldrich, MO, US) and 5 mg / ml hyaluronidase (Sigma Aldrich, MO, US) dissolved in phosphate-buffered saline for 5 min, followed by centrifugation at 5,000×g for 20 min. 100 μl aliquots of the collected samples were added to a 96-well plate and incubated at 37°C for 2 h. Then, 100 μL of prepared detection reagent A was added to the wells, followed by incubation at the same temperature for 1 hour. After aspirating and washing three times, 100 μL of prepared detection reagent B was added to the wells and incubated at 37° C. for 30 minutes. After aspirating and washing five times, 90 μL of substrate solution was added to the wells, followed by incubation at 37° C. for 25 minutes. 50 μL of stop solution was added to the wells, and the absorbance of each well was immediately read at 450 nm. The concentration of FSTL1 was defined using a standard curve of standard solutions. The test was performed in quadruplicate.

[0123] Ischemia-reperfusion (I / R): Male C57 / BL6 mice aged 10-11 weeks were anesthetized and intubated as described above. A left lateral thoracotomy was then performed. The pericardium was gently pulled away and the left anterior descending coronary artery was ligated to the PE10 tubing using 8-0 nylon suture (Ethicon, Inc. Johnson & Johnson Co., USA) and removed after 30 min of occlusion. Successful performance of the coronary artery occlusion was verified by visual inspection (by noting the development of a pale color in the myocardium distal to the ligature). The chest was then closed around the adjacent ribs using 7-0 sutures and the skin was closed with 6-0 sutures. Buprenorphine was administered subcutaneously BID dosing for a minimum of 1 day. For the patch-treated animal group, a second thoracotomy was performed 1 week after the onset of I / R, and the prepared collagen patch was sutured (at two sites) onto the surface of the ischemic myocardium. Sham-operated controls consisted of age-matched mice that underwent the same surgical procedure (two thoracotomies) except for LAD ligation. In ischemia-reperfusion studies, in vivo cardiac function was evaluated at baseline before surgery, 1 week after the onset of I / R, and 2 and 4 weeks after implantation.

[0124] The FSTL1-TG mice used in the MI experiments were female and male mice of C57BL6 background, aged 12–15 weeks. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Boston University.

[0125] result Cardiac function was evaluated in transgenic mice expressing FSTL1 under the control of the MCK promoter restricted to striated muscles (FSTL1-TG16, Fig. 11a, b). FSTL1-TG mice showed a small but significant improvement in contractility after permanent LAD ligation, but did not show long-term favorable morphometric parameters or scar size, despite abundant FSTL1 overexpression (Fig. 11a-j). Thus, myocardial FSTL1 overexpression is insufficient to recapitulate the cardioprotective effect of epicardial-conditioned medium delivered to the epicardial surface. The effect of epicardial hFSTL1 delivery on cardiac function was next evaluated. Collagen patches were prepared as before, but this time loaded with 10 μg / patch of bacterially synthesized recombinant hFSTL1 prior to polymerization and application onto the epicardial surface of infarcted hearts (see Materials and Methods for details). The patch retained immunodetectable hFSTL1 for the longest time tested, up to 21 days in vitro and up to 28 days in vivo (Figure 12). Freshly created hFSTL1 patches (patch+FSTL1) were applied to the epicardial surface of the heart immediately after MI. Patch+FSTL1 resulted in significantly improved survival of animals compared to MI-only and patch-alone animals (Figure 4a).

[0126] Echocardiographic time course measurements of contractility (% fractional shortening, FS%) showed that patch+FSTL1 led to a steady recovery of cardiac function between 2 weeks and 3 months after MI, with FS% approaching that of sham-operated animals (Figure 4b and Table 1). In contrast, untreated animals showed a severe decline in FS% after 4 weeks, with no subsequent improvement. Treatment with patch alone attenuated the decline in cardiac function compared to untreated, but unlike FSTL1, there was no subsequent improvement in cardiac function (Figure 4b and Table 1). [Table 1-2]

[0127] Secondly, FSTL1 is upregulated in the myocardium after MI. 16Given this, we tested whether epicardial delivery of FSTL1 was required to induce beneficial effects by implanting a patch alone or a patch + FSTL1 in infarcted FSTL1-TG mice. 16 Contractility parameters were dramatically and specifically increased in transgenic animals seeded with patch+FSTL1, with notable changes at the second week of treatment, reaching up to 50% improvement by the fourth week compared to patch-only treatment (Figure 4c). Thus, epicardial delivery of recombinant FSTL1 was effective even with myocardial transgenic overexpression of FSTL1, further demonstrating the specific benefit of epicardial delivery of FSTL1 over patch alone or myocardial overexpression.

[0128] Improved cardiac function and survival were accompanied by significantly attenuated fibrosis after implantation of patch+FSTL1 (Fig. 4d, Fig. 13). LV thinning was similar in patch+FSTL1 and patch-only cohorts, with both treatments significantly reducing LV thinning compared to MI-only conditions (Fig. 4d, e and Table 1). In an independent experimental group, delayed enhancement magnetic resonance imaging (DEMRI) analysis 4 weeks after injury confirmed that MI+patch+FSTL1 treatment reduced scar size (Fig. 14).

[0129] We also investigated whether patch+FSTL1 had a similar beneficial effect when applied after cardiac function had declined. To this end, an ischemia-reperfusion (I / R) model was used, with the patch implanted 1 week after injury. All animals showed reduced contractility (FS 37% before injury to 22% 1 week after I / R and before patching). Cardiac function in untreated animals progressively declined (22%, 20% and 16% FS at 1, 3 and 5 weeks after I / R). In contrast, the patch+FSTL1 cohort recovered and stabilized to 34% at 3 weeks after I / R, corresponding to full FS recovery (Figure 15 and Table 2). Similar to the permanent ligation model (Figure 4), functional recovery was accompanied by restoration of morphometric parameters (Figure 15 and Table 2). These data indicate that epicardially delivered FSTL1 produces a reversal of injury after ischemic injury. [Table 2]

[0130] FSTL1 in the patch increased vascularization of both the collagen patch and the underlying myocardium at the border of the infarcted area, as assessed by von Willebrand factor (vWF) and smooth muscle actin (αSMA) immunostaining (Fig. 4f-i). Approximately 1.5% of the patch area and underlying myocardium was occupied by vessels in the MI+patch+FSTL1 group, compared with 0.9% in the MI+patch and 0.4% area in the MI-only group (Fig. 4g). This value represents nearly half the restoration of the vasculature (3.1% area) observed in an area comparable to the distal LV wall in sham-operated animals. The number of vessels (of any size) per unit surface area of ​​the histological sections was also significantly higher in the MI+patch (35 vessels / mm2) than in the MI+patch (35 vessels / mm2). 2 ) and MI only (15 vessels / mm 2 ) treatment group, the MI+patch+FSTL1 group had an increased 2 ) (Figure 4i). In contrast, sham-operated animals had 136 vessels / mm 2 4h), again indicating that patch+FSTL1 restored angiogenesis to approximately half the level of non-infarcted mice. Furthermore, smooth muscle cells surrounded numerous vessels, especially in the MI+patch+FSTL1 group (Fig. 4h). Masson's trichrome staining showed contiguous engraftment of patch+FSTL1 onto the host myocardium, with migration of host cells into the patch, including evidence of striated cells, by 4 weeks after MI and patch placement (green arrows in the last two columns in Fig. 4j).

[0131] Example 5: FSTL1 induces cell cycle entry of cardiomyocytes in vivo This example shows that FSTL1 delivered to the epicardium may have a different function than FSTL1 produced in the myocardium.

[0132] material and method The methods used in Example 5 were as described herein.

[0133] result The patch+FSTL1 cohort showed α-actinin + Evidence of FSTL1 expression, we showed linear myocytes within the patch (Fig. 5a-d). Linear cells were rarely observed in the patch in the absence of FSTL1. Importantly, FSTL1 was also positive for phospho-histone H3 (Ser10) (pH3) and α-actinin. + This resulted in a 6.2-fold increase in the frequency of cardiomyocytes over that seen in MI-only animals (2.5 per section in MI-only (Fig. 5i) to 15.6 per section in the MI+Patch+FSTL1-treated group, p<0.05; Fig. 5e-k and Fig. S16), suggesting that FSTL1 promotes S-phase entry and DNA replication. Localization to centrosomes, the transient bridges connecting dividing cells, was confirmed by the detection of Aurora B kinase immunoreactivity between α-actinin stained cells and no overlap with nuclear DAPI staining in 3D reconstructions of confocal optical sections (Fig. 5l), as well as the significantly increased frequency of cardiomyocytes with centrosome-localized Aurora B kinase in MI+Patch+FSTL1 hearts compared to other conditions (Fig. 5m), which was consistent with the detection of α-actinin Aurora B kinase immunoreactivity in 3D reconstructions of confocal optical sections (Fig. 5m). + This suggests that the cells are induced to undergo cytokinesis. PCM1 is a marker for cardiomyocyte nuclei. 17 Using FSTL1 as a marker, a significant increase in the incidence of PCM1+ nuclei positive for pH3 was observed (Fig. 5n, o). Increased cardiomyocyte proliferation is observed even 4 weeks after engraftment in hearts treated with patch+FSTL1 after I / R injury (Fig. 15). FSTL1 has been shown to prevent apoptosis and may acutely modulate inflammation after ischemic injury. 12、13、16 There was no effect of FSTL1 on cardiomyocyte apoptosis or areas at risk immediately after MI, or on apoptosis and inflammation at days 4 and 8 post-MI (FIG. 17).

[0134] In contrast to patch FSTL1 delivery, pH3 in the border zone myocardium +The number of cardiomyocytes was increased in FSTL1-TG mice due to increased angiogenesis (Figure 10m, n and 16 ), was not increased compared to the wild-type control (FIG. 10k, l), indicating that FSTL1 delivered to the epicardium may have a different function than FSTL1 produced in the myocardium.

[0135] Example 6: Source of proliferative FSTL1-responsive cardiomyocytes This example shows that the glycosylation status of FSTL1 is linked to changes in its functional state.

[0136] material and method Neonatal rat ventricular cardiomyocytes (NRVCs) were isolated using a Neonatal Rat Ventricular Cardiomyocyte Isolation Kit (Cellutron) and cultured at 37 °C with 5% CO2. Briefly, ventricles were dissected from 1-2 day old Hsd:SD rats (Sprague Dawley) and then digested five times for 15 min each at 37 °C with an enzyme cocktail. Cells were pooled and pre-plated on uncoated cell culture dishes for 90 min to remove fibroblasts, and cultured at 3 × 10 in high serum medium (DME / F12 [1:1], 0.2% BSA, 3 mM sodium pyruvate, 0.1 mM ascorbic acid, 4 mg / liter transferrin, 2 mM L-glutamine, and 5 mg / liter ciprofloxacin, supplemented with 10% horse serum and 5% fetal bovine serum (FBS)) on 1% gelatin-coated cell culture plastic dishes. 5 Cells were plated at 200 cells / cm2. After 24 hours, the medium was replaced with low serum medium (same but containing 0.25% FCS) and the cells were cultured until use.

[0137] Automated in vitro cell proliferation and cell death assays: cells (mCMs ESCCardiomyocytes (MCs) and NRVCs (NRVCs) were incubated with EdU (dosage and length of exposure details are specified in figure legends) in a 384-plate format, fixed in 4% PFA for 2 h, washed in PBS, and stained for EdU using the Click-it EdU Assay Kit (Life Technologies). Cells were then washed in PBS and immunostained with α-actinin antibody (Sigma, A7811) to identify cardiomyocytes and stained with DAPI (4',6-diamidino-2-phenylindole) to identify nuclei. Plates were then imaged using an InCell 1000 system (GE Healthcare) and analyzed as described. 37 The percentage of cardiomyocytes that had incorporated EdU into their chromosomal DNA was analyzed automatically using the Developer Toolbox (GE Healthcare). + Nucleus and α-actinin + Similarly, cells in 384-plate format (mCMsESC and NRVC) were fixed in 4% PFA for 2 hours, washed in PBS, and immunostained with pH3 antibody (Millipore06-570) for mitotic nuclei, Aurora B (Millipore04-1036) for cytokinesis, TUNEL (Roche) for cell death, and α-actinin antibody (Sigma, A7811) for cardiomyocytes, and DAPI for nuclei. The same imaging and analysis as for EdU assay were performed. pH3 + , α-actinin + Double positive nuclei of Aurora B + , α-actinin + Double positive cells, and TUNEL + , α-actinin + The percentage of double positive nuclei relative to the total number of α-actinin + cell nuclei was calculated to determine the percentage of cardiomyocytes undergoing mitosis, cytokinesis, and apoptosis, respectively.

[0138] FSTL1 overexpression and Western blot: Hek293 cells were transiently transfected with human FSTL1 plasmid (GE Dharmacon, ID: ccsbBroad304_02639 pLX304-Blast-V5-FSTL1) using Lipofectamine 2000 (mock transfection was performed with Lipofectamine without plasmid). Serum-containing medium 48 hours after transfection was replaced with serum-free DMEM and incubated with cells for 24 hours. Tunicamycin was used at 2 μg / ml. Conditioned medium was collected from the tunicamycin sample during 16 hours (cells appeared healthy). Conditioned medium was centrifuged at 400 g for 7 minutes and then concentrated approximately 20-fold using Microcon-10 kDa cutoff columns (Millipore). Samples were combined in a 1:1 ratio with 2xSDS sample buffer containing protease inhibitors, DTT and 5mM EDTA, boiled at 95°C for 10 minutes, run in a 4-15% acrylamide Mini-Protean TGX gel, transferred to nitrocellulose membrane and incubated with anti-V5 primary antibody MAB15253 (Pierce) at a 1:1,000 dilution and anti-mouse 800nm ​​conjugated secondary antibody at a 1:10,000 dilution (Odyssey). Neonatal rat ventricular cardiomyocytes were infected with adenovirus expressing untagged mouse FSTL1 at an MOI of 50. Culture medium was replaced with serum-free medium 24 hours post-infection. Serum-free DMEM / F12 penicillin / streptomycin medium was conditioned by infected NRVC and EMC cells for 24 hours. Tunicamycin was used at 1ug / ml to condition the medium for 16 hours. Conditioned media was centrifuged at 400g for 7 min and then concentrated using Microcon-10 kDa cut-off columns (Millipore). Samples were combined 1:1 with 2xSDS sample buffer containing protease inhibitors, DTT and 5 mM EDTA, boiled for 10 min at 95°C, run on Any KD Mini-Protean TGX gels, transferred to nitrocellulose membranes and incubated with anti-FSTL1 MAB1694 (R&D) primary antibody at 1:500 dilution and anti-rat 800 nm conjugated secondary antibody at 1:10,000 dilution (Odyssey).Blocking and antibody incubations were performed in Odyssey blocker. Western blots for recombinant FSTL1 (100 ng each) were performed similarly.

[0139] Cardiomyocyte lineage labeling: Cardiomyocyte lineage labeling was performed using 4-OH tamoxifen-treated Myh6 mice on a C57BL6 background at 8 weeks of age. mERcremER :Rosa26 Z / EG mouse 18 This was achieved by injecting 100 mg / kg per day intraperitoneally at a dose of 20 mg / kg per day for 2 weeks and stopping 1 week prior to cardiomyocyte harvest (Figure 5p) or MI surgery and patch grafting. Four weeks after MI, animals were collected for immunostaining (Figure 5q-u).

[0140] result In vivo, cardiomyocytes induced to enter the cell cycle by FSTL1 outgrow pre-existing myocytes (Myh6 + They may arise from existing Myh6 cells or de novo from a precursor population. To distinguish between these possibilities, + Cardiomyocytes were transfected with tamoxifen-inducible Cre expressing the myocyte-specific Myh6 promoter 18 were used under the control of Myh6 to genetically label and follow their fate after MI and engraftment of patch+FSTL1 (Figure 5p). mERCremER :Rosa26 Z / EG 4-OH tamoxifen injected into mice prior to MI efficiently labeled existing cardiomyocytes with eGFP (Figure 5q). Four weeks after patch engraftment, eGFP + , pH 3 + Cells were clearly visible in the infarct area and border zone (FIG. 5r-u), indicating that patch+FSTL1 acts on cells that expressed Myh6 prior to LAD ligation and patch engraftment.

[0141] What is cycling α-actinin? +Adult cardiomyocytes are generally poor at entering the cell cycle, and FSTL1 did not promote DNA replication or cell division in adult or neonatal mouse ventricular cardiomyocytes in vitro (Figure ​(Figure18a–j). 18a–j). Similarly, FSTL1 did not promote DNA replication or cell division in adult mouse ventricular cardiomyocytes that could form cardiomyocytes when reimplanted into the adult heart. 19 Clonally expanded primary cardiomyogenic progenitor cells derived from Lin - , Sca1 + , S.P. + ) did not stimulate proliferation or differentiation of mCMs (Fig. 18k-m). ESC Cells were able to identify FSTL1-dependent pathways that induced increased incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into α-actinin+ mCMsESCs (Fig. 6a, d), at pH 3 + , α-actinin + They responded in a dose-dependent manner with increased numbers of cells (Fig. 6b, e) and Aurora B kinase localized to the cleavage furrow / centrosome (Fig. 6c, f). These results support the conclusion that Myh6 + Combined with the lineage tracing results showing cell proliferation (Fig. 5p-u), we hypothesized that Myh6, located near the epicardium, is proliferative in response to epicardial FSTL1. + / α-actinin + Suggests the presence of cells.

[0142] However, it remained contradictory that neither endogenous myocardial induction of FSTL1 expression nor direct transgenic overexpression of FSTL1 could activate regeneration (Figure 4, Figure 10). Therefore, whether cell-specific modification of FSTL1 might be involved was of particular interest and was tested since all of our previous experiments were performed using bacterially synthesized human FSTL1 (Figures 4, 5, 6a-f). FSTL1 is highly glycosylated in mammalian cells (Figure 6g), whereas bacterially produced recombinant FSTL1 is not glycosylated (Figure 6h). Therefore, we investigated the function of bacterially produced (naked) and mammalian cell produced (glycosylated) recombinant FSTL1 in mCMs. ESCHuman FSTL1 expressed in mammalian cells was tested in apoptosis and proliferation assays in mCMs. ESC FSTL1 protects mCMs from H2O2-induced apoptosis, whereas bacterially expressed FSTL1 does not (Fig. 6i). Conversely, bacterially expressed human FSTL1 protects mCMs ESC FSTL1 promotes proliferation of myocardial and epicardial cells, whereas mammalian expressed human FSTL1 does not (Fig. 6j, k), thus these highly significant functional differences correlate with the glycosylation status of FSTL1. We compared FSTL1 overexpressed in neonatal rat ventricular cardiomyocytes (NRVCs) (NRVCs do not produce detectable amounts of endogenous FSTL1 (Fig. 19)) with endogenous FSTL1 expressed in EMC-epicardial cells. Western blot analysis showed significant size differences between myocardial and epicardial FSTL1 forms, differences that "disappeared" upon tunicamycin treatment (a glycosylation inhibitor) (Fig. 6i), suggesting that FSTL1 is post-transcriptionally modified (glycosylated) in a cell-specific manner. We then compared the forms of Fstl produced in these myocardial and epicardial cells with those produced in mCMs. ESC The myocardial conditioned medium from NRVCs infected with untagged FSTL1 adenovirus showed no effect on mCMs ESC proliferation. In contrast, EMC conditioned medium promoted mCMs ESC proliferation in a manner dependent on underglycosylated FSTL1. ESC significantly promoted the growth of hFSTL1, comparable to that of bacterially synthesized hFSTL1 (Fig. 6m, n), indicating that cell-specific post-transcriptional modifications are linked to changes in the functional state of FSTL1.

[0143] Example 7: Epicardial FSTL1 delivery activates cardiac regeneration in a preclinical porcine model This example shows that the restorative effect of patch+FSTL1 delivery in the epicardium appears to be evolutionarily conserved.

[0144] material and method Application of the patch in a porcine model of ischemia-reperfusion: A study in pigs was performed by inflation of a percutaneous coronary angioplasty dilatation catheter to occlude the LAD in Yorkshire pigs (45 days old). A 90 minute occlusion time was followed by full reperfusion mimicking the clinical MI disease model. One week after MI, a left thoracotomy was performed and a patch was sutured onto the infarct. Animal groups included: sham control, untreated I / R (n=3), I / R treated with patch alone (I / R+patch, n=1), and I / R treated with patch loaded with FSTL1 (I / R+patch+FSTL1, n=2). EdU delivery: 250 mg / week of EdU was infused into the circulation using an osmotic minipump during the 4 week time course of the study (weeks 1-5 after I / R).

[0145] Statistical analysis: The number of samples (n) used in this and all other examples is recorded in the text and shown in the figures. All in vitro experiments were performed at least twice independently. Gene expression experiments were performed three times independently, and EdU proliferation assays and cell size measurements were performed more than 10 times independently. Sample size was not predefined, but retrospective analysis of significantly different results in most in vitro studies using Gpower 3.1 yielded a power of >0.8. Sample size was estimated for animal studies. Animals that did not survive 4 weeks after surgery were excluded from functional and histological studies. No randomization was applied. Blinding to group allocation was performed between the animal surgery and the analysis of the results of the mouse myocardial infarction experiments. Values ​​presented are expressed as mean ± SEM. The rationale for using mean ± SEM instead of SD is that SEM quantifies the uncertainty in the estimate of the mean, whereas SD indicates the variance of the data from the mean. In other words, the SEM provides an estimate of the reported mean, while the SD gives an appreciation of the variability of single observations. One-way ANOVA and Student's T-test were used to test for statistical significance (P<0.05). Survival curves were generated using PRISM (GraphPad) and the Log-rank (Mantel-Cox) test was used to test for significant differences between the survival of mice in different conditions.

[0146] result Delivery of FSTL1 to the engineered epicardium was evaluated in a porcine model of myocardial ischemia-reperfusion injury. Before infarction, ejection fraction (EF) was approximately 50% as determined by magnetic resonance imaging (MRI). One week after I / R, EF% dropped to approximately 30%, after which patch+FSTL1 was applied to the epicardium of the injured tissue. Pigs treated with patch+FSTL1 regained contractility by the second week of treatment (third week of the experiment), achieving an EF of approximately 40% that remained stable for 2 weeks, the longest time analyzed (Fig. 7a, b). This was in contrast to the steady decline in cardiac function in untreated animals and animals treated with patch alone (Fig. 7b). Pigs treated with patch+FSTL1 showed the smallest content of fibrotic tissue formation (scar size) of all treatments, including patch-only animals (see representative MRI images (Fig. 7c, d)). Pig tissue analyzed 4 weeks after patch grafting (5th week of the experiment) showed integration of the patch into the host tissue and limited fibrosis (Figure 7e) as well as EdU labeling of vascular smooth muscle cells (Figure 7f-h) and cardiomyocytes in the border zone of the ischemic area (Figure 7i-m). Cardiomyocytes with Aurora B kinase localized to the centrosome (indicating cytokinesis) were also detected in the border zone of hearts treated with patch+FSTL1 (Figure 7n). Thus, the restorative effect of delivery of patch+FSTL1 in the epicardium appears to be evolutionarily conserved.

[0147] Example 8: Administration of underglycosylated FSTL1 does not activate the signaling activity of Akt-1 This embodiment uses mCMs ESC FSTL1 treatment does not result in activation of Akt-1.

[0148] mCMs with FSTL1 ESC Treatment of and Western blots for phospho-Akt were performed as described above.

[0149] The results are shown in FIG. 20, which shows that mCMs after FSTL1 treatment ESCDetection of phospho-Akt and PCNA in FSTL1 at 10 ng / ml and 50 ng / ml Western blot for phospho-Akt (Ser473 and Thr308 are both involved in survival response in cardiomyocytes) and PCNA (proliferation marker) 1 hour and 24 hours after FSTL1 treatment showed no significant change in either phospho-Akt or PCNA upon FSTL1 treatment. [ka] [ka] [ka]

Claims

1. A composition comprising a modified RNA (modRNA) construct expressing a hypoglycosylated follistatin-like 1 (FSTL1) polypeptide for use in a method for repairing cardiac tissue after injury in a subject in need of such repair, the method comprising the step of contacting the cardiac tissue with the composition.

2. The modRNA, (a) an underglycosylated FSTL1 polypeptide lacking N-linked glycosylation at one or more asparagine residues at positions 144, 175, 180, and 223 of the amino acid sequence of SEQ ID NO:1; or (b) a hypoglycosylated FSTL1 polypeptide comprising one or more substitutions of one or more asparagine residues at positions 144, 175, 180, and 223 of the amino acid sequence of SEQ ID NO:1, wherein the hypoglycosylated FSTL1 polypeptide is substituted with one or more residues ineligible for glycosylation. The composition of claim 1 ,

3. The modRNA, (a) an underglycosylated FSTL1 polypeptide lacking N-linked glycosylation at position 180 of the amino acid sequence of SEQ ID NO:1; or (b) a hypoglycosylated FSTL1 polypeptide comprising an asparagine residue at position 180 of the amino acid sequence of SEQ ID NO:1, wherein the asparagine residue is replaced with a residue that is ineligible for glycosylation. The composition according to any one of claims 1 to 2, which encodes:

4. (a) the injury is an ischemia-reperfusion cardiac injury, ischemic heart disease, and / or is due to a deficiency in the heart, or (b) the injury is a myocardial infarction and / or the heart contains scar tissue; The composition according to any one of claims 1 to 3.

5. Repair of cardiac tissue, (a) increasing the number of cardiomyocytes in the cardiac tissue, optionally wherein the number of cardiomyocytes is increased by at least three-fold compared to the number of cardiomyocytes in cardiac tissue not contacted with the composition following injury; or (b) increasing the recovery of damaged cardiac tissue, including cardiomyocytes and / or the cardiovascular system, optionally increasing the number of cardiomyocytes by at least three-fold compared to the number of cardiomyocytes in cardiac tissue not contacted by the composition following injury; The composition according to any one of claims 1 to 4.

6. The repair of cardiac tissue, (a) improving the fractional shortening of cardiac tissue following injury compared to the amount of fractional shortening in cardiac tissue not contacted by the composition; or (b) a reduction in scar area (fibrosis) of at least 2% compared to the amount of fibrosis in the same heart prior to treatment with the composition after injury; or (c) an increase in vascular perfusion area of ​​at least 2% compared to the amount of perfusion area in the same heart prior to treatment with the composition after injury; or (d) increasing the amount of cytokinesis of cardiomyocytes in the cardiac tissue after injury compared to the amount of cytokinesis of cardiomyocytes in cardiac tissue not contacted by the composition, optionally wherein the increased amount of cytokinesis of cardiomyocytes is determined by expression of Aurora B kinase. The composition according to any one of claims 1 to 5.

7. The method of claim 1, wherein the method results in increased levels of transcripts encoding cardiac specific contractile proteins in cardiomyocytes, and optionally (a) the method results in a two-fold increase in the level of a transcript encoding a cardiac-specific contractile protein in cardiomyocytes; and / or (b) the cardiac-specific contractile protein is selected from the group consisting of myh6, mlc2v, and mlc2a; The composition according to any one of claims 1 to 6. (a) the method results in an increase in actinin+ cells with rhythmic Ca2+ contractions in cardiomyocytes; or (b) the cardiac tissue is contacted with the composition immediately following the injury; or (c) the cardiac tissue is contacted with the composition any time after the injury; or (d) the method reduces cardiac events and hospitalizations; or (e) the method attenuates fibrosis in the cardiac tissue following the injury. The composition according to any one of claims 1 to 7.

9. A composition described in any one of claims 1 to 8, wherein the method results in increased vascularization of the damaged area of ​​the cardiac tissue, and optionally, the increased vascularization is determined by expression of von Willebrand factor (vWF) or smooth muscle actin in vascular cells.

10. The method of claim 1, further comprising inducing cell cycle entry of cardiomyocytes, and optionally comprising: (a) the cell cycle entry of said cardiomyocytes is assessed by expression of phospho-histone H3, and / or (b) the method results in at least a two-fold increase in cell cycle entry of cardiomyocytes following injury, as compared to the amount of cell cycle entry of cardiomyocytes in cardiac tissue not contacted by the composition. The composition according to any one of claims 1 to 9. (a) the composition is injected directly into damaged myocardial tissue; or (b) the composition is delivered systemically; or (c) the composition is delivered to the endocardium, optionally wherein the endocardium delivery is by catheter; or (d) the composition is delivered to the epicardium by a catheter; or (e) the composition is delivered by catheter using a drug eluting stent; or (f) the composition is delivered by catheter via coronary artery injection; or (g) the composition is embedded or seeded in a hydrogel; or (h) the cardiac tissue is contacted from one or more of an epicardial site, an endocardial site, and / or by direct injection into the myocardium; The composition according to any one of claims 1 to 10.

12. A sterile pharmaceutical composition for use in a method for repairing cardiac tissue after injury in a subject in need thereof, comprising: (a) a composition according to any one of claims 1 to 11; and (b) one or more pharma- ceutically acceptable excipients; A sterile pharmaceutical composition comprising: (a) a composition according to any one of claims 1 to 12, or a sterile pharmaceutical composition according to claim 12; and (b) Instructions for use Kit including:

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