Epicardial FSTL1 reconstitution regenerates the adult mammalian heart

Abstract

The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.

Extended Data Figure 1. Characterization of mCMs^ESC cells used in this study

a, Schematic time-line of cell preparation and treatment. b–d, Immunostaining of α-actinin of mCMs^ESC, showing that the majority of the cells are α-actinin⁺ (b), and the α-actinin lacks striation structures (c). d, Immunostaining of α-smooth muscle actin (aSMA) of mCMs^ESC, showing the majority of the cells are αSMA⁺, unlike mature cardiomyocytes with no SMA expression. e, f, Automatic detection of EdU incorporation in mCMs^ESC. Captured image of mCMs^ESC treated with 10 μg ml⁻¹ EdU for 24 h, stained with EdU, α-actinin and DAPI using InCell 1000 (General Electric) (e). Overlay of masks of EdU, α -actinin and DAPI channels with automatic detection software (f). g, EdU incorporation profile of mCMs^ESC over time. mCMs^ESC are treated with 10 μg ml⁻¹ EdU for 24 h at time 0 h, 24 h, 48 h, and 144 h. The percentage of EdU⁺/α-actinin⁺ cardiomyocytes of all α-actinin⁺ cardiomyocytes is calculated for each time period. Note the decrease of EdU incorporation rate over time. h, i, Fluo 4 calcium images of mCMs^ESC, with baseline background image (h) and peak image (i). j, Comparison of representative calcium transients of mCMs^ESC (red) and neonatal rat ventricular cardiomyocytes (NRVC, blue). Note the reduced amplitude, slower rate of up and down strokes, and elongated duration of the calcium transient in mCMs^ESC compared to NRVC, suggesting immature calcium handling in mCMs^ESC. In all experiments, FSTL1 was added one day after plating of the mCMs^ESC (time 0–24 in this figure).

Extended Data Figure 2. Myocardial overexpression of Fstl1 (Fstl1-TG) mice after permanent LAD ligation

a–d, Fstl1 protein expression kinetics after myocardial infarction. Fstl1-TG mice (C57/Bl6 background) and littermate wild-type (WT) mice underwent LAD ligation. Heart tissue and serum were collected at baseline, day 1, day 3, day 7 and day 28 after surgery. Fstl1 protein levels in ischaemic area (IA) and remote area (RM) of heart were analysed by western blotting (a). Fstl1 expression expressed relative to tubulin levels is reported (b). Fstl1 serum levels were analysed by western blotting (c). Also shown in Ponceau-S staining to indicate equal loading of serum. Quantification of serum Fstl1 level 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 response of Fstl1-TG mice to permanent LAD ligation at long-term. Representative Masson’s trichrome staining of WT (e) and Fstl1-TG (f) 4 weeks after MI. Quantification of content in fibrotic tissue at week 4 after MI (g). Echocardiographic measurement of left ventricular internal dimension in systole (LVIDs) (h), and left ventricular internal diameter in diastole (LIVDd) (i) at weeks 2 and 4 after MI. Echocardiographic determination of fractional shortening (FS%) in the indicated genotypes at 2 and 4 weeks after MI (j). k–n, Double immunofluorescent staining of α-actinin (cardiomyocytes) and pH3 (mitosis) (k) and α-actinin (cardiomyocytes) and von Willebrand factor (vascular endothelial cells) (m) in the Fstl1-TG and WT mice, quantified in (i, n). n = 5, *P < 0.05 indicates significantly different from WT.

Extended Data Figure 3. Patch with FSTL1 attenuated fibrosis after MI

a–f, FSTL1 retention in the patch in vitro and in vivo. a, b, Enzyme-linked immunosorbent assay used to measure the amount of FSTL1 retained within collagen scaffolds exposed to PBS in vitro for different time intervals (0–21 days) (a). The Table lists the initial and final FSTL1 concentration, as well as the release values within the first 24 h (b). c–f, FSTL1 retention in the patch in vivo. Representative images of Fstl1 immunostaining in the indicated animal treatment groups, week 4 after surgery. Note that, while Fstl1 is expressed in the uninjured epicardium (arrow in the inset in c), its expression became undetectable within the infarct area after MI (d). Similarly, no FSTL1 was detected in the MI plus patch animals (e), while it still persists (red staining) in the patch area of the MI plus patch with FSTL1 group (f). g, Representative Masson’s Trichrome staining on serial cross sections of hearts under 4 conditions (sham, MI only, MI with patch and MI plus patch with FSTL1) 4 weeks after MI. Note the severe fibrosis in MI only condition, and reduced fibrosis in MI plus patch condition, and further reduction in MI plus patch with FSTL1 condition, quantified in Fig. 3d. h–j, Representative MRI images from the mouse MI only, MI plus patch and MI plus patch with FSTL1 treatment groups showing the 3D-FSPGR (fast spoiled gradient-echo) images and the delayed enhancement images using gadolinium contrasting agents, confirming a reduction in infarct area (demarcated in green) and preserved contractility (Supplementary Videos 3–5). k, Trichrome staining of infarct and border zone of the indicated treatments demonstrates the integration of the patch with the host tissue and massive patch cellularization by the native cardiac cells. Observe the abundant muscle (red) inside the patch and in the border zone of the patch with FSTL1 treated animals (three right panels, green arrowheads).

Extended Data Figure 4. Analysis of patch with FSTL1 function in the mouse model of ischaemia/reperfusion (I/R) with delayed patch grafting

a–c, Heart function evaluation for sham, I/R, and I/R treated with patch with FSTL1, at end- diastolic and systolic, pre-grafting (a, 1 week post-injury), 2 weeks post patch implantation (b), and 4 weeks post grafting (c). Values were normalized by dividing to pre-surgery baseline values for each individual animal. d, Absolute values of fractional shortening (FS, %) at different times pre and post I/R as evaluated by echocardiography of mice from a–c. Abbreviations same as in Fig. 3. *P < 0.05 compared to sham and black circle P < 0.05 compared to I/R. e, Co-immunofluorescence staining of DNA duplication marker phospho-Histone3 Ser10 (pH3, green) and α-actinin (red) in the border zone of patch with FSTL1 treated heart 4 weeks after MI. f, Quantification of incidence of pH3⁺, α-actinin⁺ double positive cells in the 3 experimental groups. Data collected from 3 hearts in each group with 3 different cross sections counted for total pH3⁺, α-actinin⁺ cells in each heart. *P < 0.05 indicates statistically different from all other groups.

Extended Data Figure 5. Representative images and quantification of cardiomyocyte proliferation in vivo after patch with FSTL1 treatment

a–h, Immunostaining of the cardiomyocyte marker α-actinin (red) in the infarct area (b–d) and co-immunofluorescence staining of DNA duplication marker phospho-Histone3 Ser10 (pH3, green) and α-actinin (red) in the border zone (f–h), in the 4 treatment groups analysed 4 weeks post-MI, compared to sham-operated animals (a, e). Insets in (a–d) show lower magnification images with broken lines demarcating the border between the patch and host tissues. Arrowheads in g, h, indicate α-actinin⁺ cardiomyocytes with pH3⁺ nuclei. i–k, Representative images of pH3⁺ cardiomyocytes in a patch with FSTL1 treated heart. Masson’s Trichrome staining of a heart after MI 4 weeks treated with patch with FSTL1 (i). The adjacent slide was stained for α-actinin in j, corresponding to the black box area with infarction and the patch in i. The spotted line in j indicates the boundary 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 were shown in k (white arrowhead), with each image corresponding to the area in numbered white boxes in j. l–n, Quantification of cardiomyocyte proliferation measured in 3 cross sections covering the infarct, patch, and separated by 250 μm, between 1–2 mm from the apex in each heart (Fig. 3j). Data collected from 5–7 hearts in each group with the 3 cross-sections counted exhaustively for incidence of α-actinin⁺ cells positive for pH3 (l), midbody-localized aurora B kinase between α-actinin⁺ cells (m), and double-positive cells for pH3 and the nuclear cardiomyocyte maker PCM1 (n), and normalized to myocardium area quantified by trichrome staining of immediate adjacent section. *P < 0.05 statistically different from sham. **P < 0.05, statistically different from all other groups. o, Quantification of hypertrophy in all experimental groups, measured by counting cardiomyocytes in areas of intraventricular wall with perpendicular cross-sections of cardiomyocytes in all hearts analysed for cardiomyocyte proliferation. No significance were found between samples. p–r, Quantification of incidence of α-actinin⁺ cells positive for pH3 (p), midbody-localized aurora B kinase between α-actinin⁺ cells (q), and double-positive cells for pH3 and the nuclear cardiomyocyte maker PCM1 (r) measured in l, m, to total number of cardiomyocytes, calculated using hypertrophic analysis results in o. *P < 0.05, statistically different from sham, **P < 0.05,: statistically different from all other groups. s, t, Quantification of incidence of α-actinin⁺ cells positive for pH3 (s) and midbody-localized aurora B kinase between α-actinin⁺ cells (t), separated by their localization in the border zone or infarcted area. Note the majority of proliferation quantified by both methods are located in the border zone, *P < 0.05, statistically different from all other groups.

Extended Data Figure 6. Effect of implantation of patch with FSTL1 on apoptosis and inflammation

a, Representative TTC staining of day 2 post MI/patch treatment of all four groups (sham, MI, MI plus patch, MI plus patch with FSTL1). b, Quantification of area at risk comparing all 4 groups. Data collected from 4 hearts in each group, with 4 cross-sections, approximately 2 mm thick each, encompassing each heart. *P < 0.05, statistically different from the sham. c, d, Representative image of TUNEL assays (TUNEL, green, α-actinin, red) comparing hearts 2 days after MI with patch alone and patch with FSTL1. e, Quantification of TUNEL⁺, α-actinin⁺ in infarcted area, as percentage of total number of cardiomyocyte. No difference is observed between MI plus patch and MI plus patch with FSTL1 conditions. Data collected from 3 hearts in each group with 3 different cross-sections (same as in Fig. 3j) Ten 0.09 mm² images were taken from infarcted area of each section and counted for TUNEL⁺, α-actinin⁺ and total α-actinin⁺ cells. f–j, TUNEL staining for cell death and α-actinin staining for cardiomyocytes were performed on hearts treated with patch-only and patch with FSTL1 at day 4 and day 8 after MI (f–i). Minimal TUNEL⁺, α-actinin⁺ cells are detected while there are signification amount of TUNEL⁺, α-actinin⁻ cells. Quantification of all TUNEL⁺ nuclei showed no significant differences between patch and patch with FSTL1 treated hearts at both time points (j). k–o, Immunostaining of F4/80 for macrophages and α-actinin for cardiomyocyte were performed on the same hearts as in panels a–d (k–n). Quantification of F4/80⁺ cells showed no significant differences between patch and patch with FSTL1 treated hearts at both time points (o).

Extended Data Figure 7. FSTL1 does not induce proliferation in adult and neonatal cardiomyocytes, or cardiac progenitor cells

a–f, Adult cardiomyocytes derived from mouse primary isolation. a, Visualization of GFP⁺ cardiomyocytes isolated from Myh6^mERcremER:Rosa26^Z/EG mice treated with 4-OH-tamoxifen (OH-Tam) in 3D-collagen patches. b–d) Gene expression changes in adult cardiomyocyte treated with FSTL1, including proliferation (b), cardiac-specific (c), and hypertrophy (d) markers. Note no changes in expression of cardiac specific genes, no increase in cell cycle markers (consistent with undetectable Ki67 immunostaining), and decreased hypertrophy markers (n = 3). Cardiomyocytes were embedded within 3D patch were treated with FSTL1 (10 ng ml⁻¹) for duration of 7 days with media change every 2 days. e, f, FUCCI assay in 3D-cultured adult cardiomyocytes, conducted 1 week after the 3D culture. e, Treatment with FSTL1 was performed for 7 days with media change every 2 days. f, Adult cardiomyocytes 3D-cultured control in absence of FSTL1. Note no detectable sign of cardiomyocytes in S/G2/M phases (GFP⁺) in either condition. Purple arrows point to purple-colored nuclei resulting from co-localization of Hoechst (blue) and G1 phase FUCCI (red) labelling. g–j, Primary neonatal rat ventricular cardiomyocytes (NRVC). g, h, Freshly isolated NRVCs stimulated with FSTL1 for 48 h with 10 μg ml⁻¹ EdU, and stained for α-actinin (red) and EdU (green). Percentages of EdU⁺/α-actinin⁺ cardiomyocytes of all α-actinin⁺ cardiomyocytes are quantified (h). i, j, NRVCs stimulated with FSTL1 for 48 h, and stained for α-actinin (red) and pH3 (green). Percentages of pH3⁺/α-actinin⁺ cardiomyocytes of all α-actinin⁺ cardiomyocytes are quantified (j). No increase of proliferation is found upon FSTL1 treatment. (n = 4) *P < 0.05, statistically different from control,. k–m, Sca1⁺ progenitor cells were starvation-synchronized for 48 h and stimulated with FSTL1 or control growth medium for 72 h in presence of EdU. Clone 3 was obtained by clonal growth from the Lin-Sca1+SP fraction. Sca1 pool was obtained from lin-Sca1⁺ without clonal growth. k, EdU and DAPI staining of Sca1⁺ cells after 72 h treatment. l, Percentage of EdU⁺ Sca1⁺ cells after 72 h treatment. FSTL1 concentration: 0, 1, 10, 100 ng ml⁻¹. Abbreviation s: SS, serum starvation; CGM, control growth medium. m, Number of Sca1⁺ cells after 72 h FSTL1 treatment (n = 5). No significant change is found upon FSTL1 treatment.

Figure 1. Epicardial secretome has cardiogenic activity, and improves cardiac function after MI via embryonic epicardium-like patches

a–d, Co-culture of mCMs^ESC cardiomyocytes with epicardial EMC cells. a, b, Representative micrographs. c, d, Quantification of myocyte number (c) and cardiac gene expression (d). *P < 0.05 compared to acellular (EMC⁻) control; †P < 0.05 compared to 1 × 10⁵ cells condition. e–i, Culture of mCMs^ESC cardiomyocytes with EMC-conditioned media. Representative micrographs (e, f). Quantification of myocyte number (g), cardiac gene expression (h), and cardiomyocytes with rhythmic calcium transients (i). *P < 0.05 compared to control. j–m, Effect of adult epicardial media on embryonic cardiomyocytes from E12.5 GFP⁺ cells (Tnnt2-cre;Rosa26^mTmG/+) (j). Conditioned media obtained from adult epicardial-derived cells (EPDCs) promotes cardiomyocyte proliferation that can be heat-inactivated (k) and cytokinesis analysed by double immunostaining for aurora B and Tnnt2 (cardiomyocytes) (l, m). *P < 0.05. n, Schematic of collagen patch generation (reconstructed from ref. 26). o, Evaluation of mechanical properties of engineered patch, measured by atomic force microscopy. p, q, Suture procedure of patch over ischaemic myocardium. r, Echocardiography analysis normalized to individual pre-surgery baseline values. s, Absolute values of fractional shortening (FS%). t, Masson’s trichrome staining of the animal cohorts: sham (control, n = 10), infarcted mice without treatment (MI only, n = 8), MI treated with patch only (MI plus patch, n = 8), and infarcted animals treated with patch laden with epicardial conditioned media (MI plus patch plus CM, n = 8), 2 weeks after MI. *P < 0.05 compared to Sham control, ‡P < 0.05 compared to MI-only, and †P < 0.05 compared to MI plus patch (see Methods for details.)

Figure 2. Fstl1 is an epicardial cardiogenic factor with dynamic expression after ischaemic injury

a, MS/MS spectrum of Fstl1. b–g, Fstl1 treatment of mCMs^ESC cardiomyocytes measured by immunostaining (α-actinin, green) (b, c), quantification of myocyte number (d), expression of cardiac-specific markers (e), cardiomyocytes with rhythmic calcium transient (f), and individual cardiomyocyte cell size (g). *P < 0.05 indicates statistically significantly different from control. h, Fstl1 immunostaining in the mouse embryonic heart (days E12.5, E15.5 and E17.5). Fstl1 (red), Wt1 (epicardial marker), α-actinin (myocardial marker), DAPI (nuclei). Fstl1 is expressed in epicardium (white arrowheads), no myocardium (yellow arrowhead). i) Expression shift of Fstl1 in the mouse heart after MI. Trichrome staining (upper), labels fibrosis (blue) Fstl1 immunohistochemistry (lower panels, brown). In injured hearts Fstl1 expression is depleted from the epicardium (brown) and upregulated in the myocardium. j–l, High resolution images of Fstl1 expression-shift after MI (see Methods for details).

Figure 3. FSTL1 recapitulates the in vivo restorative effect of epicardial conditioned media in the engineered epicardial patch, and promotes cardiomyocyte proliferation

a, b, Survival (a) and kinetics of FS(%) (b) analyses after MI in the indicated treatments. c, Effect of epicardial hFSTL1 patches on FS% in Fstl1-TG mice. d–i, Masson’s trichrome staining (d), morphometric analysis by echocardiography (e), and vascularization analysis (f–i) 4 weeks after MI. *P < 0.05 compared to sham, ‡P < 0.05 vs MI only, and †P < 0.05 vs MI plus patch. j, Cross-sections covering infarct/patch area separated 250 μm, 1–2 mm from apex used for cardiomyocytes proliferation analysis (k–p), 4 weeks after MI. k, m, o, Co-staining of pH3 and α-actinin (k), midbody-localized aurora B kinase between α-actinin⁺ cells (m), and double-positive cells for pH3 and PCM1 (cardiomyocyte nuclei) (o) 4 weeks post-MI, quantified in l, n, p, normalized to myocardium area quantified by trichrome staining of immediately adjacent section. *P < 0.05 from sham. **P < 0.05 from all other groups. q–v, Lineage tracing of FSTL1-responsive cells in 4-OH-tamoxifen treated Myh6^mERcremER:Rosa26^Z/EG mice; patch with FSTL1 applied simultaneously to MI, and hearts were collected 4 weeks post-MI (q) with efficient labelling of cardiomyocytes (r). Infarcted hearts showing eGFP⁺ (pre-existing, green) cardiomyocytes positive for pH3 (yellow arrowheads) (white arrowheads: pH3⁺ eGFP⁻ cells) (s–v). (length of treatment (a–c) and n for each experiment indicated in graph, see Methods for details).

Figure 4. FSTL1 proliferative activity on early cardiomyocytes depends on the cells’ selective post-transcriptional FSTL1 modifications

a–f, FSTL1 promotes proliferation of mCMs^ESC, measured by EdU incorporation (a), pH3 (b), and aurora B immunostaining (c), and quantified in d–f. g–i, Western blot analysis of Fstl1 secreted in cultured cardiomyocytes (myoFSTL1 CM) infected with Adeno-Fstl1 and in EMC (EMC CM) in the presence of tunicamycin (glycosylation inhibitor) (g), hFSTL1-V5 tagged expressed in AD-293 cells (h), and mammalian and bacterial-produced FSTL1 (i). Red arrows, glycosylated; black arrows, hypoglycosylated. j, Mammalian-produced FSTL1 attenuates H₂O₂ induced apoptosis, while bacterial-produced FSTL1 cannot. k, l, Bacterially-produced FSTL1 promotes mCMs^ESC EdU incorporation and aurora B positivity whereas mammalian-produced FSTL1 does not. m, n, Quantification of EdU incorporation in mCMs^ESC treated with conditioned media of EMC and Fstl1-overexpressing NRVC (concentration normalized to Fstl1 content). *P < 0.05 indicates statistically different from control (see Methods for details). o, Working model of FSTL1 in distinct cardiac compartments.

Figure 5. Epicardial FSTL1 delivery activates cardiac regeneration in preclinical model of ischaemic heart injury

a–d, Time course MRI analysis of cardiac function in pigs. Functional analysis by measurement of ejection fraction (EF%) (a, b). Scar size at week 4 post-grafting (c, d). Green lines highlight scar perimeter. e–n, Analysis at week 4 post-grafting. Masson’s trichrome staining (e). EdU incorporation (newly synthesized DNA) in the vascular smooth muscle cells (f–h). White line demarcates patch and host tissue. i–n, EdU (i–m) incorporation and aurora B kinase positivity (n) in cardiomyocytes at week-4 post-grafting (see Methods for details).