FoxO3 expression profile in postnatal and injured hearts
It has been demonstrated that cardiac regeneration can occur in neonatal mouse following an injury produced in the first week of life13,14. This prompts us to ask the expression patterns of FoxO3 in mouse heart after birth. To determine the expression and localization of FoxO3 in cardiomyocytes, primary cardiomyocytes isolated from neonatal mice at postnatal day 1 (p1) were subjected to immunofluorescent staining. Z-stack confocal microscopy analysis revealed that FoxO3 was predominantly expressed in the nuclei of cardiomyocytes (Supplementary Fig. 1a, b). Consistent with the observation in primary cardiomyocytes, FoxO3 was also dominantly detected in the nuclei of cardiomyocytes in hearts from postnatal mice (Supplementary Fig. 1c). Moreover, FoxO3 expression levels significantly increases with the heart growth from neonatal (p1) to adult (p84) stages (Supplementary Fig. 1d). Although FoxO3 dramatically expressed in the nuclei of cardiomyocytes, little FoxO3 expression was also detected in the non-cardiomyocytes as indicated by arrowhead in Supplementary Fig. 1a. To further explore FoxO3 expression patterns in cardiomyocytes (CMs) and non-cardiomyocytes (nCMs) during heart maturation, primary CMs and nCMs were isolated from postnatal mice at p1 and p56, respectively. The qPCR assay revealed that FoxO3 mRNA expression levels were significantly increased in adult CMs and nCMs when compared with neonatal ones, respectively (Supplementary Fig. 1e).
To determine the response of FoxO3 to heart injury, we resected ventricular apex of neonatal heart at p1 and examined the expression of FoxO3 protein at 1 day post-resection (1 dpr) and 5 dpr, respectively. Western blotting analysis showed that heart injury significantly increased the phosphorylation of FoxO3 on Thr 32 and Ser 253 (Fig. 1a-d), indicating the decreased activation of FoxO3. Immunofluorescence staining further revealed that both expression level of FoxO3 and percentage of FoxO3-positive cardiomyocytes in apical and remote zone were significantly decreased at 5 dpr when compared with sham group (Fig. 1e-j). Consistently, western blotting further confirmed the reduction of FoxO3 in the primary cardiomyocytes isolated from the injured hearts at 5 dpr compared with sham group (Fig. 1k). These data suggest that FoxO3 activation decreases in neonatal heart upon injury. It’s well known that Akt is an upstream negative mediator of FoxO3 that can promote the phosphorylation of FoxO3, thereby suppressing its activation29. To further explore the molecular mechanisms regulating FoxO3 activation during neonatal heart injury, we examined the activation of Akt pathway and found that the Akt phosphorylation level was significantly increased in the injured heart at 5 dpr (Fig. 1l). These findings suggest that Akt-mediated inactivation of FoxO3 might play an important role in heart regeneration.
In vitro effects of FoxO3 on cardiomyocyte proliferation
To elucidate the potential role of FoxO3 in cardiomyocyte proliferation in vitro, the siRNA targeting FoxO3 (siFoxO3) and negative control (siNC) were used to treat the primary cardiomyocytes isolated from neonatal mice at p1. The silencing efficiency of siFoxO3 was confirmed at both mRNA and protein levels (Supplementary Fig. 2a, b). Firstly, the cell cycle entry of cardiomyocytes was evaluated by measuring the nuclear incorporation of 5-ethynyl-2’-deoxyuridine (EdU), an efficient marker of DNA synthesis. As shown in Supplementary Fig. 2c, the representative EdU incorporation in cardiomyocytes was captured by confocal microscopy in both siNC- and siFoxO3-treated groups. However, quantification revealed that the percentage of EdU+ cTnT+ cells significantly increased in siFoxO3-treated group compared with control group (Supplementary Fig. 2d). Consistent with these findings, Ki67 and cTnT double staining revealed that FoxO3 knockdown leads to an increase in the percentage of Ki67+ cTnT+ cells, indicating an elevated proliferation level of primary cardiomyocytes (Supplementary Fig. 2e, f). To prove complete cardiomyocyte proliferation, late cell cycle markers including phospho-Histone H3 (pH3) and aurora kinase B (AurkB) were further used in this study. Consistently, the percentage of pH3+ cTnT+ cells was significantly elevated by FoxO3 knockdown (Supplementary Fig. 2g, h), indicating that FoxO3 knockdown really promotes the complete proliferation of cardiomyocytes in vitro. Importantly, increased percentage of AurkB+ cardiomyocytes was also detected in the siFoxO3-treated group compared with control group (Supplementary Fig. 2i, j), implying the increased cytokinesis of cardiomyocytes. Consistent with these results from primary cardiomyocytes, increased proliferation was also detected in the siFoxO3-treated HL-1 cardiomyocytes compared with control cells (Supplementary Fig. 2k–m). These findings from primary cardiomyocytes and cell line strongly demonstrated that FoxO3 might negatively control cardiomyocyte proliferation.
FoxO3 knockout promotes cardiomyocyte proliferation in postnatal mice
To elucidate the potential role of FoxO3 in cardiomyocyte proliferation in postnatal heart, a cardiomyocyte-specific knockout mouse model (CKO) was generated by intercrossing FoxO3-floxed (FoxO3fl/fl) mice21 with Myh6-Cre transgenic mice (Supplementary Fig. 3a). Cardiac-specific deletion of FoxO3 in CKO mice were determined in protein and mRNA levels (Supplementary Fig. 3b–d). To determine whether FoxO3 knockout influences heart growth in postnatal mice, heart weight (HW) to tibial length (TL) ratio (HW/TL) was analyzed at p1 and p14. The increased HW/TL ratio were detected in CKO mice at p14 (Fig. 2a), indicating that FoxO3 knockout might promote postnatal heart growth. This idea was further confirmed by the morphological (Fig. 2b) and histological (Fig. 2c) analysis of hearts from CKO and Con mice at p1 and p14. To explore whether the FoxO3 knockout-induced heart growth results from myocardial hypertrophy, we further assessed cardiomyocyte size at p14 with wheat germ agglutinin (WGA) staining. Surprisingly, cardiomyocyte size was smaller in CKO hearts at p14 compared to controls (Fig. 2d). Moreover, there were not significant increases in the expression of myocardial hypertrophy markers including ANP, BNP, and Myh7 in the CKO hearts at p1 and p14 compared with controls (Supplementary Fig. 4). These results imply that cardiomyocyte number might be increased in the FoxO3-deficient hearts. To confirm this idea, we quantified the cardiomyocytes isolated from CKO and Con hearts at p14, and revealed a significant increase in the total number of cardiomyocytes in CKO hearts compared with controls (Fig. 2e). Consistent with this result, an increase in mononucleated cardiomyocytes and a decrease in binucleated cardiomyocytes were observed in CKO hearts compared with controls (Fig. 2f), which reflecting the proliferation or mitosis of cardiomyocytes as previously demonstrated15,30. These findings suggest that cardiomyocyte-specific knockout of FoxO3 might promote postnatal heart growth through increasing cardiomyocyte proliferation.
a Heart weight to tibial length (HW/TL) ratio in control and CKO mice at p1 and p14, respectively (n = 9 mice for p1 and 10 mice for p14). b Representative whole images of control and CKO postnatal hearts at p1 and p14. c Representative H&E staining images of control and CKO postnatal hearts at p1 and p14. d Representative WGA staining images (upper panel) and quantification (lower panel) of cardiomyocyte size in ventricles at p14 (total ~500 cells in 6 hearts per group). e Representative images of cardiomyocytes completely dissociated from whole heart (left panel) and quantification of total rod-shaped cardiomyocyte numbers (right panel) in control and CKO mice at p14 (n = 6 hearts). f Representative images of cardiomyocyte nucleation (left panel) and quantification of mononuclear (Mono), binuclear (Bi), and multinuclear (Multi) cardiomyocytes (right panel) in control and CKO hearts at p14 (n = 6 hearts per group). g, h Representative confocal images (left) and quantification (right) of EdU+ (g) and pH3+ (h) cardiomyocytes in control and CKO apical ventricles at p1 and p14 (n = 10 hearts). i Representative confocal images at low (left) and high (right) magnification of PCNA+ cardiomyocytes in CKO apical ventricles. j Quantification of PCNA+ cardiomyocytes in control and CKO apical ventricles at p7 (n = 10 hearts). k, l Representative confocal images (left) and quantification (right) of symmetrical (k) and asymmetrical (l) AurkB+ cardiomyocytes in control and CKO apical ventricles at p7 (n = 3 hearts). All data are presented as the mean ± SEM. P values are from two-tailed t test (a, d, e, j–l) or two-way ANOVA followed by Sidak’s multiple comparisons test (f–h). ns, no significant difference. Source data are provided as a Source data file.
To further confirm this conjecture, hearts from CKO and Con postnatal mice at p1 and p14 were then subjected to immunofluorescent staining to evaluate cardiomyocyte proliferation. We firstly examined EdU incorporation and found that the percentage of EdU+ cTnT+ cells was significantly higher in CKO mice at p1 and p14 than that in Con mice, respectively (Fig. 2g). We subsequently quantified the number of cardiomyocytes that were positive for the mitosis marker pH3, and found that there was a significant increase in pH3+ cardiomyocytes in CKO mice at p1 and p14 compared with controls (Fig. 2h), suggesting that FoxO3 knockout really promotes the complete proliferation of cardiomyocytes in postnatal mice. In addition, proliferating cardiomyocytes at p7, a time point just beyond the regenerative windows of neonatal heart, were confirmed by proliferating cell nuclear antigen (PCNA) and myocyte enhancer factor 2 C (Mef2C) double staining (Fig. 2i, j). To further determine whether FoxO3 knockout can promote the cytokinesis of cardiomyocytes at p7, Aurora Kinase B (AurkB) staining were performed. AurkB analysis can distinguish cardiomyocytes that were undergoing karyokinesis with subsequent cytokinesis (symmetric AurkB) from ones without cytokinesis (asymmetric AurkB)31. Our data showed that FoxO3 knockout significantly elevated the percentage of symmetric AurkB-positive cardiomyocytes, implying the increased cytokinesis of cardiomyocytes in CKO heart at p7 (Fig. 2k). Moreover, increased multinucleation (asymmetric AurkB) events were also observed in CKO heart (Fig. 2l). Taken together, these in vivo findings demonstrated that FoxO3 might negatively regulate cardiomyocyte proliferation in early postnatal mice.
FoxO3 knockout improve cardiac function in homeostatic adult mice
Above observation in early postnatal mice prompts us to explore whether FoxO3 knockout influences cardiac function in homeostatic adult mice. For 6-week-old adult mice, we found that heart weight indices (HW/TL) and overall heart size increased in CKO mice compared with controls (Fig. 3a, b). However, there were not significant differences in the expression of myocardial hypertrophy markers including ANP, BNP, and Myh7 between CKO and Con mice (Fig. 3c). WGA staining (Fig. 3d) and cardiomyocyte morphology (Fig. 3e) revealed that cardiomyocyte size was smaller in CKO hearts than that in controls. These data imply that FoxO3 deficiency-increased proliferation of cardiomyocytes in early postnatal mice might lead to increases in cardiomyocyte number and heart size at adult stage. However, cardiac functions were unaffected by FoxO3 knockout in 6-week-old mice (Fig. 3f), indicating that FoxO3 knockout-induced cardiomyocyte proliferation in neonatal mice might need longer time to affect cardiac function at adult stage. We therefore further analyzed the cardiac functions in 12-week-old mice. Histological and morphological analysis revealed that the overall heart size and heart weight indices (HW/TL) were increased in CKO mice compared with controls (Fig. 3g, h). To determine whether FoxO3 knockout increases the total number of cardiomyocytes, cardiomyocytes were isolated from CKO and Con mice (12-week-old) using collagenase digestion. Cell counting revealed a significant increase in the total number of cardiomyocytes in 12-week-old CKO hearts compared with controls (Fig. 3i, j). Moreover, significant increased cardiomyocytes numbers were detected in 12-week-old CKO mice compared with 2-week-old CKO mice, although there were no significant differences between 6- and 2-week-old CKO mice (Fig. 3j). In contrast with CKO mice, no significant differences in cardiomyocyte numbers were detected in adult (6- and 12-week-old) control mice when compared with 2-week-old mice (Fig. 3j). These data imply that the cardiomyocyte number continues to increase from 2- to 12-week-old CKO mice in a lower speed. This was accompanied by the increased percentage of mononucleated cardiomyocytes and the decreased percentage of binucleated cardiomyocytes in CKO hearts (Fig. 3k). Importantly, increased cardiac function was also detected in CKO mice compared with controls (Fig. 3l). Collectively, these results demonstrated that FoxO3 knockout promotes the proliferation of cardiomyocytes at neonatal stage, thereby leading to an increased cardiomyocyte number and cardiac function at adult stage.
a HW/TL ratio in control and CKO mice at postnatal week 6 (n = 8 mice). b Representative images of H&E staining of sections from control and CKO hearts at postnatal week 6. c The qPCR validation of hypertrophic markers including Anp, Bnp, and Myh7 in control and CKO ventricles at postnatal week 6 (n = 3 hearts). d Representative WGA staining images (left) and quantification (right) of the size of cardiomyocytes located in ventricles at postnatal week 6 (total ~700 cells in 6 hearts per group). e Representative cross section of cTnT staining cardiomyocytes (left) and the quantification of cardiomyocyte size and density (right) in ventricles at postnatal week 6 (n = 6 hearts for control and 10 hearts for CKO). f Representative images of M-model echocardiography (left) and quantification (right) of LVEF and LVFS levels at postnatal week 6 (n = 10 hearts). g HW/TL ratio in control and CKO mice at postnatal week 12 (n = 10 mice). h Representative whole images of adult hearts at postnatal week 12. i Representative images of cardiomyocytes completely dissociated from whole heart in control and CKO mice at postnatal week 12. j Quantification of total cardiomyocytes in control and CKO mice from 2 to 12 weeks (n = 5 hearts). k Representative images of cardiomyocyte nucleation (left) and quantification of mononuclear (Mono), binuclear (Bi), and multinuclear (Multi) cardiomyocytes (right) in control and CKO hearts at postnatal week 12 (n = 3 hearts). l Representative images of M-model echocardiography (left) and quantification of LVEF and LVFS levels (right) at postnatal week 12 (n = 10 hearts). All data are presented as the mean ± SEM. P values are from two-tailed t test (a, d–g, l) or two-way ANOVA followed by Sidak’s multiple comparisons test (c, j, k). ns, no significant difference. Source data are provided as a Source data file.
FoxO3 knockout accelerates heart regeneration at regenerative stage
Increased cardiomyocyte proliferation in CKO neonatal mice prompts us to ask whether FoxO3 knockout improves heart regeneration. Firstly, ventricular apex was resected at p1, followed by histological analysis, cardiomyocyte proliferation, and cardiac function evaluation at 5 dpr, 14 dpr, and 28 dpr, respectively. We found that FoxO3 knockout significantly increased the HW/TL ratio at 5 dpr (Fig. 4a). However, the expression of myocardial hypertrophy markers and proinflammatory factors was not significantly changed in CKO hearts compared with controls (Supplementary Fig. 5a, b). EdU incorporation assay revealed that FoxO3 knockout increased the percentage of EdU-positive cardiomyocytes in apical areas at 5 dpr (Fig. 4b). This was consistent with an increase in the percentage of Ki67-positive cardiomyocytes in apical areas in CKO heart compared with controls (Fig. 4c). Consistent with these results at 5 dpr, histological and morphological analysis revealed that scar size was significantly decreased in CKO hearts at 14 dpr compared with control hearts (Fig. 4d–f). Moreover, increased HW/TL ratio was also detected in CKO mice at 14 dpr (Fig. 4g). We also analyzed cardiomyocyte proliferation by pH3 staining and found that FoxO3 knockout increased the numbers of pH3-positive cardiomyocytes in cardiac apical and remote areas (Fig. 4h–k), which indicates that FoxO3 knockout promotes cardiomyocyte proliferation at 14 dpr. However, no significant difference in coronary density was detected in CKO heart at 14 dpr compared with controls (Supplementary Fig. 5c, d). We further extended the repairing period to 28 dpr, and found that both Con and CKO mice greatly regenerated the injured heart without fibrosis (Fig. 4l). Moreover, the increased HW/TL ratio was detected in CKO mice (Fig. 4m), which may imply the improved cardiac function. As expected, left ventricular systolic function was significantly increased in CKO mice, as evaluated by significant increases in left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) levels, although there were no significant differences in cardiac function between the sham-operated CKO and control mice (Fig. 4n, o). Taken together, these findings indicate that the increased cardiomyocyte proliferation and decreased scar size caused by FoxO3 knockout improves cardiac function, thereby accelerating heart regeneration in neonatal mice.
a HW/TL ratio in control and CKO mice at 5 dpr (n = 9 mice). b Representative images (left) and quantification (right) of EdU+ cardiomyocytes in control and CKO apical ventricle at 5 dpr (n = 7 hearts). c Representative images (left) and quantification (right) of Ki67+ cardiomyocytes in control and CKO apical ventricle at 5 dpr (n = 7 hearts). d, e Representative Mason’s trichrome staining images of cardiac apex (d) and quantification of scar size (e) in control and CKO hearts at 14 dpr (n = 9 hearts for control and 7 hearts for CKO). f, g Representative whole images of postnatal hearts (f, arrows denote scar) and quantification of HW/TL ratio (g) at 14 dpr (n = 7 mice). h–k Representative images (h, j) and quantification (i, k) of pH3+ cardiomyocytes in the apical (h, i) and remote (j, k) zone of control and CKO ventricles at 14 dpr (n = 9 hearts for control and 6 hearts for CKO). l, m Representative Masson’s trichrome staining images of cardiac apex (l) and quantification of HW/TL ratio (m) in control and CKO hearts at 28 dpr (n = 8 mice). n, o Representative images of M-model echocardiography (n) and quantification (o) of LVEF and LVFS levels in control and CKO mice at 28 dpr (n = 5 mice for sham and 7 mice for CKO). All data are presented as the mean ± SEM. P values are from two-tailed t test (a–c, e, g, i, k, m) or two-way ANOVA followed by Sidak’s multiple comparisons test (o). ns, no significant difference. Source data are provided as a Source data file.
FoxO3 knockout promotes heart regeneration at non-regenerative stage
To further determine the involvement of FoxO3 in heart regeneration at the non-regenerative stage, ventricular apex resection was performed in postnatal mice at p8, the time point beyond the regenerative windows of neonatal heart. Samples were then collected and analyzed at 5 to 49 dpr (Supplementary Fig. 6a). Significant increase in HW/TL ratio was detected in CKO mice at 5 dpr compared with controls (Supplementary Fig. 6b). EdU incorporation assay revealed that FoxO3 knockout significantly increased the number of EdU-positive cardiomyocytes in both apical and remote areas at 5 dpr (Supplementary Fig. 6c, d), implying increased proliferation of cardiomyocytes in CKO mice. At 21 dpr, the increased HW/TL ratio was detected in CKO mice (Supplementary Fig. 6e). Moreover, increased LVEF and LVFS were detected in CKO mice at 21 dpr compared with controls (Supplementary Fig. 6f), indicating that FoxO3 knockout promotes left ventricular systolic function. Histological analysis revealed that scar size in the cardiac apex at 21 dpr was significantly decreased in FoxO3-deficient hearts compared with controls (Supplementary Fig. 6g), implying the accelerated heart regeneration. Consistent with these results, pH3/cTnT double staining revealed that the percentage of pH3-positive cardiomyocytes was increased in both apical and remote areas in CKO hearts compared with controls (Supplementary Fig. 6h, i). We further extended repairing time to 49 dpr and analyzed cardiac function and regeneration. As expected, apex resection-induced decreases in cardiac function in control mice were significantly restored by FoxO3 knockout, as evidenced by increases in both LVEF and LVFS levels in CKO mice (Supplementary Fig. 6j). In agreement with this result, a significant decrease in scar size was detected in CKO hearts at 49 dpr compared with controls (Supplementary Fig. 6k), indicating a promoted regeneration in CKO heart. In addition, cardiomyocyte sizes were not increased in CKO hearts at 49 dpr compared with controls (Supplementary Fig. 7), indicating that FoxO3 knockout did not lead to cardiac hypertrophy.
To confirm above conclusions in apical resection model, myocardial infarction (MI) model was further performed in p8 mice, followed by sample collection and analysis at the indicated time points (Fig. 5a). Ki67 staining revealed that FoxO3 knockout significantly increased the percentage of Ki67-positive cardiomyocytes in apical areas at 7 days post-MI (dpM), implying the increased cardiomyocyte proliferation in CKO mice (Fig. 5b, d). To prove complete cardiomyocyte proliferation, late cell cycle marker pH3 and cytokinesis marker AurkB were further used in this study. Consistently, the percentage of pH3-positive cardiomyocytes in apical areas was significantly elevated by FoxO3 knockout (Fig. 5c, e), indicating that FoxO3 knockout really promotes the complete proliferation of cardiomyocytes during MI of p8 mice. AurkB staining showed that the percentage of symmetric AurkB-positive cardiomyocytes was significantly increased in cardiac apex in the CKO mice compared with controls, implying the elevated cytokinesis of cardiomyocytes (Fig. 5f). The increased asymmetric AurkB-positive cardiomyocytes was also observed in CKO hearts (Fig. 5g). On the contrary, neither symmetric nor asymmetric AurkB-positive cardiomyocytes were detected in control hearts at 7 dpM (Fig. 5f, g). We also revealed that the expression of proinflammatory factors was not significantly changed in CKO hearts at 7 dpM compared with controls (Supplementary Fig. 8a). Cardiac function analysis showed a modest increase in LVEF and LVFS levels in CKO mice compared with controls at 14 dpM (Fig. 5h). However, significantly increased LVEF and LVFS levels were observed in CKO mice compared with controls at 28 dpM (Fig. 5i), indicating the elevated cardiac function in CKO mice. In agreement with the elevated cardiac function, decreased scar sizes were also detected in CKO hearts at 28 dpM (Fig. 5j), indicating a promoted regeneration in CKO heart. On the contrary, αSMA immunostaining revealed that the angiogenesis capacity was comparable between CKO and control hearts at 28 dpM (Supplementary Fig. 8b). These data from apex resection and MI injury models of p8 mice revealed that FoxO3 knockout can really promote heart regeneration at non-regenerative stage.
a Schematic of sample collection and analysis at indicated time points after MI surgery at p8. b, d Representative images (b) and quantification (d) of Ki67+ cardiomyocytes in the border zone of infarcted hearts at 7 dpM (n = 3 hearts). c, e Representative images (c) and quantification (e) of pH3+ cardiomyocytes in the border zone of infarcted hearts at 7 dpM (n = 3 hearts). f, g Representative images (left) and quantification (right) of symmetrical (f) and asymmetrical (g) AurkB+ cardiomyocytes in the border zone of infarcted hearts at 7 dpM (n = 5 hearts). h, i Representative images of M-model echocardiography (left) and quantification (right) of LVEF and LVFS levels in control and CKO mice at 14 (h) and 28 (i) dpM (n = 6 mice for Con and 7 mice for CKO). j Representative Masson’s trichrome staining images (left) and quantification of scar size (right) at 28 dpM (n = 6 mice). k Schematic of sample collection and analysis at indicated time points after MI surgery in adult iCKO mice. l Validation of the inducible knockout of FoxO3 in heart tissues. m, n Representative images (left) and quantification (right) of Ki67+ (m) and pH3+ (n) cardiomyocytes in the border zone of infarcted hearts in iCKO mice at 7 dpM (n = 5 hearts). o Representative WGA staining images (left) and quantification (right) of cardiomyocyte size in the border zone of infarcted hearts at 7 dpM (n = 10 mice). p Quantification of LVEF and LVFS levels in control and iCKO mice from 0 to 28 dpM (n = 10 mice). Control mice without MI injury were used as sham group (n = 6 mice). q Representative images of M-model echocardiography in control and iCKO mice at 28 (P) dpM. r Representative Masson’s trichrome staining images (left) and quantification of scar size (right) in control and iCKO mice at 28 dpM (n = 10 mice). All data are presented as the mean ± SEM. P values are from two-tailed t test (d–j, m, n, r), one-way ANOVA followed by Tukey’s multiple comparisons test (o), or two-way ANOVA followed by Sidak’s multiple comparisons test (p). Source data are provided as a Source Data file.
To access the pro-regeneration effects of FoxO3 deficiency in adult hearts, FoxO3fl/fl mice were crossbred with inducible Myh6-MerCreMer (Myh6MCM) mice to generate FoxO3fl/fl::Myh6MCM mice (iCKO), of which the cardiomyocyte-specific knockout of FoxO3 could be induced by tamoxifen administration at the indicated time points. The Myh6MCM mice were used as control (Con). Adult (8-week-old) iCKO and Con mice were injected with tamoxifen (50 mg/kg, i.p.) for 5 days to induce the cardiomyocyte-specific knockout of FoxO3, followed by the permanent ligation of the left anterior descending artery (LAD) to induce myocardium infarction (MI) injury (Fig. 5k). Con mice without MI injury were used as sham group. The inducible knockout of FoxO3 (iCKO) in the injured heart was determined by WB at 7 dpM (Fig. 5l). Ki67 staining showed that FoxO3 knockout greatly increased the proliferation of cardiomyocytes in border zone of infarction at 7 dpM, as indicated by the increased percentage of Ki67-positive cardiomyocytes (Fig. 5m). To confirm this conclusion, cardiomyocyte proliferation was further determined using immunofluorescent staining for pH3, a late cell cycle marker. Almost no pH3-positive cardiomyocytes were detected in control hearts at 7 dpM. However, FoxO3 knockout increased the percentage of pH3-positive cardiomyocytes which indicating the elevated cardiomyocyte proliferation (Fig. 5n). Moreover, MI-induced cardiomyocyte hypertrophy was suppressed by FoxO3 knockout at 7 dpM as indicated by the decreased cardiomyocyte size in iCKO hearts (Fig. 5o). We further analyzed the inflammatory response and revealed that the expression of proinflammatory factors was not significantly changed in iCKO hearts at 7 dpM compared with controls (Supplementary Fig. 9a). To further evaluate the effects of FoxO3 knockout on cardiac function, sham-operated and injured mice were subjected to echocardiography analysis from 0 to 28 dpM. Our data showed that FoxO3 knockout leads to significant increases in LVEF and LVFS levels at 14 and 28 dpM when compared with control hearts (Fig. 5p, q). In line with these results, decreased scar size was also detected in iCKO hearts at 28 dpM when compared with control hearts (Fig. 5r and Supplementary Fig. 10). In contract, the angiogenesis capacity in iCKO heart was comparable with that in control hearts at 28 dpM (Supplementary Fig. 9b). These findings further validate the pro-regeneration effect of FoxO3 inactivation in adult heart.
Variations in FoxO3-regulated genes in postnatal hearts
To investigate target genes involved in FoxO3-mediated heart regeneration in postnatal mice, ventricles isolated from Con and CKO mice at p1 and p14 were subjected to RNA-seq analysis. We identified 646 upregulated and 1871 downregulated genes in Con mice at p14 compared with p1 (Supplementary Fig. 11a). Gene ontology (GO) and KEGG pathway enrichment analysis revealed that heart growth-induced differentially expressed (DE) genes in Con postnatal mice were significantly enriched for cell cycle-related gene sets where 80 genes were specifically identified in postnatal heart at p14 (Supplementary Fig. 11b, c). At p1, 989 upregulated and 1259 downregulated genes were identified in CKO hearts compared with controls (Supplementary Fig. 11d). GO and KEGG analysis revealed that FoxO3 knockout-induced DE genes in neonatal hearts at p1 were significantly enriched for cell growth- and cell cycle-related gene sets where 59 genes were specifically identified in CKO hearts at p1 (Supplementary Fig. 11e, f). At p14, 685 upregulated and 1228 downregulated genes were identified in CKO hearts compared with controls (Supplementary Fig. 11g). In agreement with GO and KEGG data from p1, FoxO3 knockout-induced DE genes were significantly enriched for cell cycle-related gene sets where 36 genes were specifically identified in CKO hearts at p14 (Supplementary Fig. 11h, i). These data suggest that cell cycle-associated genes may play important roles in the regulation of heart regeneration in a FoxO3-dependent manner. As expected, gene-set enrichment analysis (GSEA) further revealed that cell cycle and proliferation gene sets are associated with FoxO3-mediated postnatal heart growth which spanning the regenerative window (Supplementary Fig. 12). Using qPCR assay, we further confirmed the downregulation of several cyclin-dependent kinase inhibitors (CDKN) including Cdkn2 and Cdkn3 in CKO hearts at p1 and/or p14 compared with controls (Supplementary Fig. 11j, k). To further determine this idea in adult heart, the expression of cell cycle-related genes was examined in 2-month-old CKO mice. The qPCR analysis revealed that the expression of negative regulators of cell cycle including Cdkn2a, Cdkn2b, and Cdkn3 in adult heart were significantly reduced by FoxO3 knockout compared with controls, implying the increased proliferation levels in CKO hearts (Supplementary Fig. 13). These findings indicate that specific knockout of FoxO3 in cardiomyocytes can reduce the expression of negative regulators of cell cycle in postnatal hearts. However, FoxO3 knockout did not influence the expression of Meis1 in postnatal heart (Supplementary Fig. 11j, k), which has been demonstrated to be important for the regulation of postnatal cardiomyocyte cell cycle arrest15.
It has been demonstrated that sarcomere genes (TNNI3, MYL2, TNNT2, MYOZ2, TPM1, and ACTC1) and fatty acid oxidation (FAO) genes (CPT1A, CPT1B, and SLC27A) are crucial for cardiomyocyte maturation and contraction32,33. We thus analyzed the expression of these genes using primary cardiomyocytes isolated from control and CKO mice. We found that two sarcomere genes (TNNT2 and MYOZ2) and two FAO genes (CPT1A and SLC27A) were upregulated in FoxO3-deficient cardiomyocytes within postnatal day 14, whereas there were no significant differences in the expression levels of these genes between adult wild-type and mutant cardiomyocytes (Supplementary Fig. 14). These findings imply that certain sarcomere and FAO genes might be influenced by FoxO3 in postnatal rather than in adult hearts.
Previous study has reported the antagonistic functions between FoxO1 and FoxM1 in neonatal cardiomyocyte cell cycle withdrawal, and demonstrated the requirement of FoxM1 for cardiomyocyte proliferation34. Moreover, it has been demonstrated that FoxM1 is required for heart regeneration through transcriptional regulation of pro-proliferative genes including G2-phase genes (Ccnf and G2e3) and M-phase genes (Cenpf and Prc1)35. These previous findings imply the potential mutual antagonism between FoxO3 and FoxM1. This prompts us to ask whether FoxO3-deficiency-mediated cardiomyocyte proliferation is contributed by the potential upregulation of FoxM1 under our experimental conditions. To determine the expression of FoxM1 in cardiomyocytes with or without FoxO3 knockout, primary cardiomyocytes isolated from control and CKO neonatal mice at p1 were subjected to qPCR assay. We found that FoxO3 deficiency has no significant effects on the expression of FoxM1 in neonatal cardiomyocytes. Consistently, the expression levels of FoxM1-dependent cell cycle genes are comparable between control and FoxO3-deficient neonatal cardiomyocytes (Supplementary Fig. 15a). To further determine the expression of FoxM1 and its downstream targets in cardiomyocytes upon heart injury, we performed apical resection in p1 neonatal mice and isolated primary cardiomyocytes at 5 dpr. Our data showed that both FoxM1 and its targets expression in cardiomyocytes were comparable between control and CKO groups (Supplementary Fig. 15b). Taken together, these results suggest that the increased proliferation of cardiomyocytes and promoted cardiac regeneration in FoxO3-deficient mice may be independent of FoxM1 signaling.
SFRP2 contributes to FoxO3-mediated cardiomyocyte proliferation
To further identify potential target genes involved in FoxO3-mediated heart regeneration in postnatal mice, downregulated genes in CKO ventricles at p1 (1259 genes) and p14 (1228 genes) versus controls were subjected to GO analysis, respectively. Given that FoxO3 deficiency promotes cardiomyocyte proliferation in vitro (Supplementary Fig. 2) and in vivo (Figs. 2 and 4), we speculate that negative regulators of cardiomyocyte proliferation might be downregulated by FoxO3 deficiency. Therefore, we subsequently focused on the gene sets associated with negative regulation of cell proliferation (NRP) and growth (NRG) in GO terms from the downregulated genes in CKO ventricles at p1 and p14 (Supplementary Tables 1 and 2). NRP- and NRG-related genes from CKO versus Con ventricles at p1 and p14 were compared. Four potential target genes including Trp53, Sfrp2, Cdkn2c, and Cdkn2d were screened by Venn Diagram between different gene sets (Fig. 6a). To elucidate whether and how FoxO3 regulates the expression of these potential targets, we analyzed their promoter sequence and identified a series of FoxO3 binding sites (Supplementary Tables 3–6). The binding site with the highest score for each target was then analyzed by ChIP-qPCR to examine the in vivo interaction between FoxO3 and target gene promoters. Primary cardiomyocytes were isolated from sham and injured control mice at 5 dpr for ChIP-qPCR assay. Among these four potential targets, our data revealed the interaction between FoxO3 proteins and the promoters of Trp53, Sfrp2, and Cdkn2c instead of Cdkn2d in primary cardiomyocytes isolated from sham-operated hearts, as verified by increases in the relative signals of FoxO3 compared to IgG (Fig. 6b). Moreover, only Trp53 and Sfrp2 showed decreases in the interaction with FoxO3 protein in primary cardiomyocytes isolated from injured heart at 5 dpr compared with cardiomyocytes from sham group (Fig. 6b), suggesting that FoxO3-mediated expression of Trp53 and Sfrp2 in cardiomyocytes may be regulated by the stress of cardiac injury. Among these four genes, the most enrichment of FoxO3 signal relative to IgG signal in sham group was detected for Sfrp2 promoter (Fig. 6c). In addition, we found a moderate decrease in Trp53 expression and a remarkable decrease in Sfrp2 expression in primary cardiomyocytes isolated from CKO mice compared with controls (Fig. 6d). These data suggest that Trp53 and Sfrp2 may be the downstream targets of FoxO3 in regulating cardiomyocyte proliferation. As expected, GSEA analysis revealed that Trp53 and Sfrp2 pathways were significantly associated with FoxO3-mediated postnatal heart growth which spanning the regenerative window (Fig. 6e, f). To further address the effects of FoxO3 on target gene expression, we constructed luciferase reporter plasmids using target gene promoters which include FoxO3 binding sites. Reporter gene assay revealed that FoxO3 overexpression greatly promoted relative luciferase activity driven by the promoter of Trp53 and Sfrp2 (Fig. 6g). Mutation of the consensus FoxO3 binding site with highest score in the promoter of Sfrp2 attenuated the FoxO3 overexpression-induced increase in luciferase activity (Fig. 6g). However, the mutation of FoxO3 binding site with highest score in Trp53 promoter (Trp53∆1) failed to attenuate the luciferase activity induced by FoxO3 (Fig. 6g). To further determine the effects of other potential binding sites in Trp53 promoter, we further mutated the other binding site with the second higher score and constructed the new reporter plasmid (pTrp53∆2-GR) for further reporter gene assay. We found that FoxO3 overexpression-induced increase in luciferase activity could be reduced by the second mutation (Trp53∆2) (Fig. 6g). These findings suggest that the second FoxO3 binding site in Trp53 promoter may be more critical for Trp53 regulation. In agreement with mouse, FoxO3 binding sites in Trp53 and Sfrp2 promoters could also be predicted in other organisms including human, frog, and zebrafish, implying the evolutionary conservation of Foxo3 binding with the promoters of these two targets (Supplementary Fig. 16, Supplementary Tables 7 and 8).
a RNA-seq identified 4 overlapping genes (Trp53, Sfrp2, Cdkn2c, and Cdkn2d) between the downregulated gene sets for negative regulation of cell proliferation (NRP) and growth (NRG) in CKO ventricles versus controls at p1 and p14. b ChIP-qPCR validation of FoxO3 binding to the promoters of the identified 4 potential targets in primary cardiomyocytes isolated from sham and injured hearts at 5 dpr (n = 3). c Fold enrichment of FoxO3 signal relative to IgG signal for these 4 potential target promoters in cardiomyocytes isolated from sham-operated hearts (n = 3). d The qPCR validation of these 4 potential targets expression in control and CKO cardiomyocytes at 5 dpr (n = 3). e, f GSEA analysis based on the RNA-seq data revealed that Trp53 and Sfrp2 pathways are associated with FoxO3-mediated postnatal heart regeneration. g The interactions of FoxO3 with the promoters of Trp53 and Sfrp2 are evaluated using reporter gene and mutation assay (n = 5). h–j Primary cardiomyocytes isolated from CKO neonatal mice at p1 are transfected with Adv5-NC and Adv5-Sfrp2 adenoviruses for 48 h, followed by immunofluorescent staining for cTnT to verify cardiomyocytes, for proliferation markers Ki67 (h), EdU (i), and pH3 (j) to verify proliferating activity of cardiomyocytes. Representative images (left) and quantification (right) of proliferating cardiomyocytes in Adv5-NC and Adv5-Sfrp2 groups are shown (n = 8). All data are presented as the mean ± SEM. P values are from two-tailed t test (h–j), one-way ANOVA followed by Tukey’s multiple comparisons test (b, c, g) or two-way ANOVA followed by Sidak’s multiple comparisons test (d). ns, no significant difference. Source data are provided as a Source Data file.
Given that the most enrichment of FoxO3 signal in Sfrp2 promoter (Fig. 6c) and the highest inhibition of Sfrp2 expression in FoxO3-deficient cardiomyocytes (Fig. 6d), Sfrp2 was further analyzed in the following experiments. To determine the effect of Sfrp2 on cardiomyocyte proliferation, primary cardiomyocytes isolated from wild-type neonatal mice at p1 were treated with siRNA targeting Sfrp2 (siSfrp2). We found that Sfrp2 knockdown (Supplementary Fig. 17a) significantly promoted cardiomyocyte proliferation as evidenced by the increased percentage of Ki67-positive cardiomyocytes (Supplementary Fig. 17b, c). In agreement with this result, downregulation of Sfrp2 expression (Supplementary Fig. 18) and increase in cardiomyocytes proliferation (Fig. 2) was detected in CKO ventricles when compared with controls. Thus, we supposed that Sfrp2 might be responsible for FoxO3-medicated cardiomyocyte proliferation. To further elucidate whether and how Sfrp2 influences the proliferation of FoxO3-deficient cardiomyocytes, we overexpressed Sfrp2 using adenovirus type 5 (Adv5) in the primary cardiomyocytes isolated from CKO neonatal mice at p1 (Supplementary Fig. 19). Ki67/cTnT double staining revealed that the percentage of Ki67-positive cardiomyocytes was decreased by Sfrp2 overexpression (Fig. 6h), implying the decreased proliferation of Sfrp2-overexpressing cardiomyocytes. Consistent with the result, EdU incorporation assay showed that the percentage of EdU-positive cardiomyocytes was significantly decreased in Adv5-Sfrp2 group compared with Adv5-NC group (Fig. 6i). In addition, pH3/cTnT double staining further confirmed that Sfrp2 overexpression inhibited cardiomyocyte proliferation as evidenced by the decreased percentage of pH3-positive cardiomyocytes (Fig. 6j). These findings suggest that FoxO3 deficiency-induced increases in cardiomyocyte proliferation can be significantly restored by Sfrp2 overexpression.
SFRP2 overexpression suppresses heart regeneration in FoxO3-deficient mice
To further determine the effects of Sfrp2 on heart regeneration in FoxO3-deficient mice, we overexpressed Sfrp2 in CKO neonatal mice at p1 using AAV9-Sfrp2 and analyzed the cardiomyocyte proliferation and heart regeneration at 5 and 28 dpr, respectively (Fig. 7a). EdU incorporation assay revealed that in vivo overexpression of Sfrp2 (Fig. 7b) significantly decreased the percentage of EdU-positive cardiomyocytes in ventricular apex at 5 dpr compared with AAV9-NC group (Fig. 7c, d). Moreover, Ki67/cTnT double staining showed that Sfrp2 overexpression suppressed cardiomyocyte proliferation in ventricular apex at 5 dpr, as evidenced by the decreased percentage of Ki67-positive cardiomyocytes in the AAV9-Sfrp2 group (Fig. 7e, f). In line with these results, histological analysis revealed that scar size was significantly increased by Sfrp2 overexpression at 28 dpr compared with AAV9-NC group (Fig. 7g, h). In addition, depraved cardiac function was detected in Sfrp2-overexpressing hearts at 28 dpr as demonstrated by the decreased LVEF and LVFS values when compared with AAV9-NC groups (Fig. 7i, j). Taken together, these findings suggest that Sfrp2 overexpression suppresses the accelerated heart regeneration in FoxO3-deficient mice.
a Schematic of AAV9 virus injection in CKO postnatal mice at p1, apex resection at p2 (equal to 0 dpr), EdU injection and sample collection at indicated time points. b Representative images of Sfrp2 expression mediated by AAV9-NC and AAV9-Sfrp2 viruses in postnatal hearts at 5 dpr. Right panel, magnified confocal images of Sfrp2 expression in left panel. At least three times each experiment was repeated independently with similar results. c, d Representative images (c) and quantification (d) of EdU+ cardiomyocytes in the injured apical ventricles at 5 dpr (n = 7 hearts). e, f Representative images (e) and quantification (f) of Ki67+ cardiomyocytes in the injured apical ventricles at 5 dpr (n = 7 hearts). g, h Representative Masson’s trichrome staining images of cardiac apex (g) and quantification of scar size (h) in AAV9-NC and AAV9-Sfrp2 hearts at 28 dpr (n = 7 hearts). i, j Representative images of M-model echocardiography (i) and quantification of LVEF (j, left) and LVFS (j, right) in AAV9-NC and AAV9-Sfrp2 hearts at 28 dpr (n = 7 hearts). All data are presented as the mean ± SEM. P values are from two-tailed t test (d, f, h, j). Source data are provided as a Source data file.
FoxO3 deficiency promotes Wnt/β-catenin signaling activation by suppressing Sfrp2 in cardiomyocytes
It is well known that secreted frizzled-related protein 2 (Sfrp2) is the inhibitor of canonical Wnt/β-catenin signaling. As expected, GSEA analysis further revealed that Wnt signaling is significantly associated with FoxO3-mediated postnatal heart growth which spanning the regenerative window (Supplementary Fig. 20). This implies that canonical Wnt/β-catenin pathway may be involved in FoxO3/Sfrp2-mediated regulation of heart regeneration in postnatal heart. To determine the effects of FoxO3 on the expression of Sfrp2 and β-catenin, primary cardiomyocytes isolated from control and CKO neonatal mice at p1 was subjected to western blotting assay. Our data showed that FoxO3 knockout greatly reduced the production of Sfrp2. On the contrary, β-catenin production in cardiomyocytes was increased by FoxO3 knockout (Fig. 8a, b). These data imply that FoxO3 might negatively control β-catenin production in cardiomyocytes by promoting Sfrp2 expression. In consistent with primary cardiomyocytes, decreased Sfrp2 and increased β-catenin production were also detected in the FoxO3-deficient HL-1 cell line compared with control cells (Fig. 8c, d). Moreover, the transcriptional activity of endogenous β-catenin was measured by TOP-Flash reporter (Red Firefly luciferase) assays. Increased relative firefly luciferase activity was detected in the FoxO3-deficient HL-1 cells compared with controls (Fig. 8e), which indicating that FoxO3 knockdown significantly promotes β-catenin transcriptional activity.
a, b Representative images (a) and quantification of western blotting for Sfrp2 (b, left) and β-catenin (b, right) expression in primary cardiomyocytes isolated from neonatal mice at p1 (n = 3 mice). c Representative images (left) and quantification (right) of immunofluorescent staining for Sfrp2 in control and FoxO3-deficient HL-1 cells (n = ~600 cells from 8 experiments). d Representative images (left) and quantification (right) of immunofluorescent staining for β-catenin in control and FoxO3-deficient HL-1 cells (n = 8). e The relative activation of β-catenin is evaluated in control and FoxO3-deficient HL-1 cells using TOP-Flash reporter assay (n = 5). f, g Representative images (f) and quantification of EdU+ HL-1 cells (g, left) and density (g, right) in control and Sfrp2-overexpressing cells (n = 10). h Cell counting assay for control and Sfrp2-overexpressing HL-1 cells (n = 5). i Representative images (left) and quantification (right) of immunofluorescent staining for β-catenin in control and Sfrp2-overexpressing HL-1 cells (n = 6). j The relative activation of β-catenin is evaluated in control and Sfrp2-overexpessing HL-1 cells using TOP-Flash reporter assay (n = 5). All data are presented as the mean ± SEM. P values are from two-tailed t test (b–e, g–j). Source data are provided as a Source data file.
To further determine whether Sfrp2 influences FoxO3-regulated proliferation of HL-1 cells, we overexpressed Sfrp2 using Adv5 adenovirus in the FoxO3-deficient HL-1 cells. In consistent with primary cardiomyocytes from CKO mice (Fig. 6h–j), we found that Sfrp2 overexpression inhibited the proliferation of FoxO3-deficient HL-1 cells as proved by the decreased percentage of EdU-positive cells and total cell density (Fig. 8f, g). Moreover, cell counting assay also revealed the decreased proliferation in Sfrp2-overexpressing cells compared with controls (Fig. 8h). To determine the influence of Sfrp2 on β-catenin activity, the expression and transcriptional activity of β-catenin were further examined in the FoxO3-deficient HL-1 cells with and without overexpression of Sfrp2. As expected, Sfrp2 overexpression significantly suppressed the expression and nuclear localization of β-catenin as indicated by the decreased percentage of β-catenin-positive nuclei (Fig. 8i). In agreement with this result, TOP-Flash reporter assay further revealed the decreased transcriptional activity of β-catenin in Sfrp2-overexpressing cells (Fig. 8j). These findings suggest that FoxO3 deficiency promotes β-catenin signaling activation by suppressing Sfrp2 in cardiomyocytes, implying the involvement of canonical Wnt/β-catenin pathway.
To further determine whether β-catenin-independent non-canonical Wnt signaling is activated in FoxO3-deficient cardiomyocytes, we examined the activation of JNK (the planar cell polarity (PCP)-dependent non-canonical Wnt pathway) and CaMKII (the Ca2+-dependent non-canonical Wnt pathway)36 in primary cardiomyocytes isolated from CKO and control neonatal mice. We found that the phosphorylation of JNK and CaMKII in FoxO3-deficient cardiomyocytes was comparable with that in control cells (Supplementary Fig. 21a, b), implying that non-canonical Wnt signaling may be not involved in the phenotypes mediated by FoxO3-Sfrp2 axis in cardiomyocytes. Previous studies have reported that several Wnt ligands including Wnt137, Wnt2b38, and Wnt10b39 may be involved in cardiac repair and regeneration. To determine whether these Wnt ligands are involved in the FoxO3-Sfrp2 axis-mediated phenotypes under our experimental conditions, we examined the expression of Wnt1, Wnt2b, and Wnt10b in cardiomyocytes with Sfrp2 silencing. Our data showed that Sfrp2 knockdown has no significant impact on the expression of these three Wnt ligands (Supplementary Fig. 21c, d). To analyze the potential Wnt ligands binding to Sfrp2, we predicted the structure of mouse Sfrp2 and 19 Wnt ligands using AlphaFold3 and calculated the binding affinities between Sfrp2 and Wnt ligands using HDOCK server. Among the 19 Wnt ligands, the prediction data showed that Wnt8a and Wnt4 are the top2 ligands with higher binding affinity for Sfrp2 protein (Supplementary Fig. 21e). We thus examined the expression of these two Wnt ligands in primary cardiomyocytes with Sfrp2 silencing, and found that Sfrp2 silencing significantly increased the production of Wnt8a instead of Wnt4 (Supplementary Fig. 21f, g). These data imply that Sfrp2 may negatively regulate Wnt8a in cardiomyocytes.
Disturbance of endogenous Sfrp2 and β-catenin attenuates cardiomyocyte proliferation in CKO mice
To determine whether downregulation of endogenous Sfrp2 in CKO heart contributes to cardiomyocyte proliferation in vivo, we overexpressed Sfrp2 in CKO mice by injecting AAV9-Sfrp2 viruses at p1 and performed MI surgery at p8, the time point beyond the regenerative windows of neonatal heart. AAV9-NC virus was used as negative control. Cardiomyocyte proliferation in infarct border zone was then analyzed by immunofluorescent staining at 7 dpM (Fig. 9a). Successful overexpression of Sfrp2 in heart tissue at p8 was validated by western blotting validation (Fig. 9b). Ki67 staining showed that Sfrp2 overexpression significantly suppressed cardiomyocyte proliferation, as determined by the decreased percentage of Ki67-positive cardiomyocytes in AAV9-Sfrp2 group compared with AAV9-NC group (Fig. 9c). In consistent with this result, the percentage of pH3-positive cardiomyocytes was significantly decreased by Sfrp2 overexpression (Fig. 9d), indicating that Sfrp2 overexpression really blocks the complete proliferation of cardiomyocytes induced by FoxO3 knockout. These findings suggest that the endogenous downregulation of Sfrp2 indeed contributes to the increased proliferation of cardiomyocytes during heart regeneration of CKO mice at non-regenerative stage.
a Schematic of Sfrp2 overexpression, MI induction, and histological analysis in CKO hearts. b Western blotting validation of Sfrp2 overexpression in CKO hearts at p8 (n = 3 hearts). c, d Representative images (left) and quantification (right) of Ki67+ (c) and pH3+ (d) cardiomyocytes in the border zone of infarcted hearts at 7 dpM (n = 3 hearts). e Schematic of β-catenin knockdown, MI induction, and histological analysis in CKO hearts. f Western blotting validation of β-catenin knockdown in CKO hearts at p8 (n = 3 hearts). g, h Representative images (left) and quantification (right) of Ki67+ (g) and pH3+ (h) cardiomyocytes in the border zone of infarcted hearts at 7 dpM (n = 3 hearts). i Representative Masson’s trichrome staining images (left) and quantification of scar size (right) at 28 dpM (n = 8 mice for shNC and 9 mice for shβCat). j Representative images of M-model echocardiography (left) and quantification (right) of LVEF and LVFS levels at 28 dpM (n = 9 mice). k The general view of how FoxO3 controls cardiomyocyte proliferation and heart regeneration through regulating Sfrp2 expression and β-catenin activation. The graphic was created using BioRender.com. All data are presented as the mean ± SEM. P values are from two-tailed t test (b–d, f–j). Source data are provided as a Source Data file.
To confirm the effect of increased endogenous β-catenin on heart regeneration of CKO mice at non-regenerative stage, we inhibited β-catenin expression in CKO mice by injecting AAV9-shRNA targeting β-catenin (AAV9-shβCat) at p1. AAV9-shNC virus was used as negative control. The MI surgery was performed at p8, followed by analysis at 7 and 28 dpM (Fig. 9e). Our data showed that β-catenin knockdown (Fig. 9f) significantly decreased the percentage of Ki67-positive cardiomyocytes at 7 dpM compared with AAV9-shNC group (Fig. 9g). In agreement with Ki67 staining, pH3 staining revealed that β-catenin knockdown greatly suppressed cardiomyocyte proliferation in CKO heart at 7 dpM, as demonstrated by the decreased percentage of pH3-positive cardiomyocytes in AAV9-shβCat group compared with AAV9-shNC group (Fig. 9h). In consistent with these results, Masson’s staining showed that β-catenin knockdown increased the scar sizes in CKO hearts at 28 dpM (Fig. 9i and Supplementary Fig. 22), indicating a reduced regeneration in CKO heart. Consistently, significantly decreased LVEF and LVFS levels were observed in β-catenin-deficient CKO mice compared with controls at 28 dpM (Fig. 9j), indicating the deteriorated cardiac dysfunction. These findings suggest that endogenous β-catenin is a mediator of FoxO3 in regulating heart regeneration at non-regenerative stage. To further determine whether β-catenin knockdown counteract the pro-proliferation effect of FoxO3 inactivation in adult hearts, AAV9-shβCat and control viruses were injected into adult CKO mice for 2 weeks, followed by MI surgery and cardiomyocyte proliferation determination at 7 dpM (Supplementary Fig. 23a). Our data showed that β-catenin knockdown (Supplementary Fig. 23b, c) significantly suppressed cardiomyocyte proliferation in adult CKO heart, as determined by the decreased percentage of Ki67- and pH3-positive cardiomyocytes in AAV9-shβCat group compared with that in AAV9-NC group (Supplementary Fig. 23d, e). These findings suggest that the endogenous β-catenin contributes to cardiomyocyte proliferation in FoxO3-deficient adult heart. Taken together, our results indicate that downregulation of Sfrp2 caused by FoxO3 inactivation activates canonical Wnt/β-catenin pathway, which upregulates the expression of genes required for cardiomyocyte proliferation, thereby promoting heart regeneration in mice (Fig. 9k).
