Mydgf promotes Cardiomyocyte proliferation and Neonatal Heart regeneration

Abstract

Myeloid-derived growth factor (Mydgf), a paracrine protein secreted by bone marrow-derived monocytes and macrophages, was found to protect against cardiac injury following myocardial infarction (MI) in adult mice. We speculated that Mydgf might improve heart function via myocardial regeneration, which is essential for discovering the target to reverse heart failure. Methods: Two genetic mouse lines were used: global Mydgf knockout (Mydgf-KO) and Mydgf-EGFP mice. Two models of cardiac injury, apical resection was performed in neonatal and MI was performed in adult mice. Quantitative reverse transcription-polymerase chain reaction, western blot and flow cytometry were performed to study the protein expression. Immunofluorescence was performed to detect the proliferation of cardiomyocytes. Heart regeneration and cardiac function were evaluated by Masson's staining and echocardiography, respectively. RNA sequencing was employed to identify the key involved in Mydgf-induced cardiomyocyte proliferation. Mydgf recombinant protein injection was performed as a therapy for cardiac repair post MI in adult mice. Results: Mydgf expression could be significantly induced in neonatal mouse hearts after cardiac injury. Unexpectedly, we found that Mydgf was predominantly expressed by endothelial cells rather than macrophages in injured neonatal hearts. Mydgf deficiency impeded neonatal heart regeneration and injury-induced cardiomyocyte proliferation. Mydgf recombinant protein promoted primary mouse cardiomyocyte proliferation. Employing RNA sequencing and functional verification, we demonstrated that c-Myc/FoxM1 pathway mediated Mydgf-induced cardiomyocyte expansion. Mydgf recombinant protein improved cardiac function in adult mice after MI injury with inducing cardiomyocyte proliferation. Conclusion: Mydgf promotes cardiomyocyte proliferation by activating c-Myc/FoxM1 pathway and improves heart regeneration both in neonatal and adult mice after cardiac injury, providing a potential target to reverse cardiac remodeling and heart failure.

Keywords: FoxM1; Mydgf; c-Myc; cardiomyocyte proliferation; heart regeneration.

Figure 1

Mydgf is induced during neonatal heart regeneration. (A) Schematic diagram showed the experimental design for B-D. (B-C) Western blot analysis of Mydgf expression in wild-type (WT) mouse heart at different ages. Statistical analysis revealed that the expression of Mydgf decreased with age (n = 3 per group). (D) qRT-PCR analysis of Mydgf expression in WT mouse heart at different ages. Statistical analysis revealed that the expression of Mydgf decreased with age (n = 3 per group). *P < 0.05 and **P < 0.01 compared to postnatal day 1 (P1) by one-way ANOVA with Bonferroni's multiple comparisons test (C, D). (E) Schematic diagram showed the experimental design for F-M. (F-G) Western blot analysis of Mydgf in WT mouse hearts harvested at 1, 4, 7 days after apical resection (AR). Statistical analysis revealed that the expression of Mydgf was upregulated post neonatal heart injury (n = 3 per group). (H) qRT-PCR analysis of Mydgf in WT mouse hearts harvested at 1, 4, 7 days after AR. Statistical analysis revealed that the expression of Mydgf was upregulated post neonatal heart injury (n = 3 per group). (I) qRT-PCR analysis of Mydgf expression in three cell populations sorted by flow cytometry at 1 day after AR. Statistical analysis revealed that the expression of Mydgf was upregulated in endothelial cells post neonatal heart injury (n = 3 per group). *P < 0.05 and **P < 0.01 compared to SH at corresponding time-point by Student's t-test (G, H and I). (J) Mydgf and CDH5 (endothelial cell marker) were co-located by staining for EGFP (green), CDH5 (red), and nuclei (blue) at 7 days after AR in Mydgf-EGFP mice. Scale bars, 20 µm. Values were presented as the mean ± S.E.M.

Figure 2

Mydgf is necessary for heart regeneration and cardiomyocyte proliferation. (A and B) Masson's staining showed heart regeneration in Mydgf-KO mice after apical resection (AR) injury relative to wild-type (WT) at 21 days post resection (dpr). Statistical analysis revealed that heart regeneration was reduced in Mydgf-KO mice (n = 15 for Mydgf-KO mice and n = 10 for WT mice). Scale bars, 500 µm. (C) Representative images of echocardiography analysis in Mydgf-KO and WT mice at 21 dpr. (D) Echocardiography analysis of left ventricular ejection fraction (LVEF) in Mydgf-KO and WT mice at 21 dpr (n = 10 for Mydgf-KO mice and n = 9 for WT mice). *P < 0.05 and **P < 0.01 compared to WT by Student's t-test (B and D). (E) Schematic diagram showed the experimental design for F-K. (F and G) Immunostaining illustrated proliferative (pH3⁺, green, white arrows) cardiomyocytes (CMs) were decreased in Mydgf-KO mice relative to wild-type (WT) at 7 days post resection (dpr) (n = 3 per group). Scale bars, 20 µm. (H and I) Immunostaining illustrated proliferative (Ki67⁺, green, white arrows) CMs were decreased in Mydgf-KO mice relative to WT at 7 dpr (n = 3 per group). (J and K) Immunostaining illustrated proliferative (Aurkb⁺, green, white arrows) CMs were decreased in Mydgf-KO mice relative to WT at 7 dpr (n = 3 per group). *P < 0.05 and **P < 0.01 compared to WT by Student's t-test (G, I and K). (L and M) Example photomicrographs of isolated CMs from Mydgf-KO mice heart and quantification of ploidy at 14 dpr (n = 6 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to WT by two-way ANOVA with Bonferroni's multiple comparisons test (M). Scale bars, 20 µm. Values were presented as the mean ± S.E.M.

Figure 3

Mydgf is sufficient for cardiomyocyte proliferation and heart regeneration. (A) Schematic diagram showed the experimental design for B-G. (B and C) Immunostaining illustrated proliferative (pH3⁺, green, white arrows) primary cardiomyocytes (CMs) were increased in Mydgf-treated group relative to PBS-treated group after 16 hours (n = 3 per group). (D and E) Immunostaining illustrated proliferative (Ki67⁺, green, white arrows) primary CMs were increased in Mydgf-treated group relative to PBS-treated group after 16 hours (n = 3 per group). (F and G) Immunostaining illustrated proliferative (Aurkb⁺, green, white arrows) primary CMs were increased in Mydgf-treated group relative to PBS-treated group after 16 hours (n = 3 per group). *P < 0.05 and **P < 0.01 compared to PBS by Student's t-test (C, E and G). (H) Schematic diagram showed the experimental design for I-R. (I and J) Masson's staining showed heart regeneration in Mydgf-KO mice treated with Mydgf relative to PBS at 21 days post resection (dpr). Statistical analysis revealed that heart regeneration was induced in Mydgf-treated mice (n = 20 for Mydgf-treated mice and n = 15 for PBS-treated mice). Scale bars, 500 µm. (K) Representative images of echocardiography analysis in Mydgf-KO mice treated with Mydgf relative to PBS at 21 dpr. (L) Echocardiography analysis of left ventricular ejection fraction (LVEF) in Mydgf-KO mice treated with Mydgf relative to PBS at 21 dpr (n = 10 for Mydgf-treated mice and n = 9 for PBS-treated mice). *P < 0.05 and ***P < 0.001 compared to PBS-treated mice by Student's t-test (J and L). (M and N) Immunostaining illustrated proliferative (pH3⁺, green, white arrows) CMs were increased in Mydgf-KO neonatal mice treated with Mydgf relative to PBS at 7 dpr (n = 3 per group). (O and P) Immunostaining illustrated proliferative (Ki67⁺, green, white arrows) CMs were increased in Mydgf-KO neonatal mice treated with Mydgf relative to PBS at 7 days post resection (dpr) (n = 3 per group). (Q and R) Immunostaining illustrated proliferative (Aurkb⁺, green, white arrows) CMs were increased in Mydgf-KO neonatal mice treated with Mydgf relative to PBS at 7 dpr (n = 3 per group). Scale bars, 20 µm. *P < 0.05 compared to AR+PBS by Student's t-test (N, P and R). Values were presented as the mean ± S.E.M.

Figure 4

Mydgf controls cardiomyocyte proliferation through c-Myc/FoxM1 pathway. (A) Schematic diagram showed the experimental design for B-F. (B) Western blot of p-Akt and cell cycle relative protein in PBS and Mydgf-treated cardiomyocytes (CMs). (C) Heat map of genes under cell cycle regulated networks. (D) Western blot of c-Myc and FoxM1 in primary CMs treated with PBS and Mydgf. (E) Western blot of c-Myc and FoxM1 in wild-type (WT) mouse hearts harvested at 1, 4, 7 dpr. (F) Western blot of p-Akt, c-Myc and FoxM1 of hearts harvested at 4, 7 days post resection (dpr) in wild-type (WT) or Mydgf-KO mouse hearts. (G and H) Immunostaining illustrated proliferative (pH3⁺, green, white arrows) primary CMs were decreased treated with siRNA-Akt, c-Myc and FoxM1 respectively prior to Mydgf treatment relative to siRNA-NC treatment group after 48 hours (n = 3 per group). (I and L) Immunostaining illustrated proliferative (Ki67⁺, green, white arrows) primary CMs were decreased treated with siRNA-Akt, c-Myc and FoxM1 respectively prior to Mydgf treatment relative to siRNA-NC treatment group after 48 hours (n = 3 per group). (K and L) Immunostaining illustrated proliferative (Aurkb⁺, green, white arrows) primary CMs were decreased treated with siRNA-Akt, c-Myc and FoxM1 respectively prior to Mydgf treatment relative to siRNA-NC treatment group after 48 hours (n = 3 per group). ^#P < 0.05 and ^##P < 0.01 compared to Mydgf treatment by Student's t-test (H, J and L). (M) Western blot of p-Akt, c-Myc, FoxM1, Cyclin B1, Cyclin D1 and CDK1 in primary CMs transfected with different treatment. Values were presented as the mean ± S.E.M.

Figure 5

Mydgf promotes heart regeneration in adult mice. (A) Schematic diagram showed the experimental design for B-L. (B and C) Masson's staining elucidated the infarcted area in wild-type (WT) adult mice treated with PBS/Mydgf at 21 days post infarction (dpi). Statistical analysis of fibrotic area showed the infarcted size was significantly smaller in adult WT mice treated with Mydgf at 21 dpi relative to PBS (n = 25 for Mydgf-treated mice and n = 13 for PBS-treated mice). Scale bars, 500 µm. (D) Representative images of echocardiography analysis in adult WT mice treated with PBS/Mydgf at 21 dpi. (E) Echocardiography analysis of left ventricular ejection fraction (LVEF) in adult WT mice treated with PBS/Mydgf at 21 dpi (n = 19 for Mydgf-treated mice and n = 13 for PBS-treated mice). *P < 0.05 and **P < 0.01 compared to WT by Student's t-test (C and E). (F) Cumulative survival after MI in WT mice treated with 25 PBS and 20 Mydgf. *P < 0.05 compared to PBS-treated group by log-rank test. (G and H) Immunostaining illustrated proliferative (pH3⁺, green, white arrows) cardiomyocytes (CMs) were increased in WT adult mice treated with Mydgf relative to PBS at 7 dpi (n = 3 per group). (I and J) Immunostaining illustrated proliferative (Ki67⁺, green, white arrows) CMs were increased in WT adult mice treated with Mydgf relative to PBS at 7 dpi (n = 3 per group). (K and L) Immunostaining illustrated proliferative (Aurkb⁺, green, white arrows) CMs were increased in WT adult mice treated with Mydgf relative to PBS at 7 dpi (n = 3 per group). Scale bars, 20 µm. *P < 0.05 and **P < 0.01 compared to PBS-treated group by Student's t-test (H, J and L). Values were presented as the mean ± S.E.M.