PDGFR-? Signaling Regulates Cardiomyocyte Proliferation And Myocardial Regeneration

Background The mammalian neonatal myocardium can regenerate after kinds of injury with cardiomyocyte(CM)expansion,and this capacity is lost in 7 days after birth.Understanding the mechanism of CM proliferation and quiescence is crucial for the development of cardiac regeneration medicine.PDGF signaling is involved in proliferation,survival and migration in a wide array of cell types and vital for heart development.Nonetheless,little is known about the role of PDGFR-? signaling in mammalian myocyte proliferation and myocardial regeneration.Method and Results Part1: Sustained activation of PDGFR-? signaling in CMs promotes proliferation and heart regeneration.Objective: To investigate the role of PDGFR-? in cardiomyocyte proliferation and heart regeneration.Methods: We conditionally activated PDGFR-? signaling in CMs by intercrossing the Myh6-Mer Cre Mer strain with mice harboring a floxed-stop cassette upstream of PdgfrbD849 V allele encoding a ligand-independently activated PDGFR-?(D849V mutation in the kinase domain).Following tamoxifen administration,the mice with Myh6-Mer Cre Mer;PdgfrbD849V genotype(PdgfrbMyh6D849V)expressed the constitutively active PDGFR-? mutant isoform at m RNA and protein levels and kept PDGF signaling activation in CMs.We created myocardial infarction(MI)by permanently ligating the left anterior descending coronary artery at P7,a time-point associated with the absence of CM proliferation ability.Results: Histological analysis and echocardiography revealed a significantly smaller myocardial infarct scar and better heart function in PdgfrbMyh6D849 V mice 21 days after MI at P7,compared with littermate controls.Wheat germ agglutinin(WGA)staining exhibited that PDGFR-?-activated CMs were smaller than control group at day 21 after MI,with no change found in ratio of heart to body weight,which might imply that the CM number is augmented in PdgfrbMyh6D849 V mice after injury.We detected the mitosis marker p H3(phosphorylated histone H3)and the cell proliferation marker Ki67 by immunostaining at day 7 after MI.PDGFR-? activation induced an increase in the number of p H3+?-actinin+ and Ki67+ ?-actinin+ double-positive cells.In addition,we found a remarkable increase in the number of Ed U(5-ethynyl-2'-deoxyuridine)-positive CMs in PdgfrbMyh6D849 V hearts,indicating that DNA synthesis was increased.Taken together,these data demonstrated that activation of PDGFR-? signaling could enhance CM proliferation,thereby preserving cardiac function and promoting cardiac regeneration.Part2: Ezh2 is required in the PDGFR-? signaling-induced CMs proliferation and regeneration.Objective: To investigate the molecular mechanisms of PDGFR-? signaling in CM proliferation and cardiac regeneration.Methods: We harvested the RNA of the Ad-treated(Ad-Cre or Ad-Null)CMs,which were isolated from the neonatal PdgfrbD849V/fl mice and performed RNA-Seq.And then conditionally knocked out Ezh2 in mice with sustained activation of PDGFR-? signaling in CMs.We made an MI model at P7 and echocardiographically measured the heart function in Ezh2-c KO;PdgfrbMyh6D849V,PdgfrbMyh6D849 Vand PdgfrbD849V/fl littermate control mice.We generated Myh6-Mer Cre Mer;Ezh2fl/fl(Ezh2-c KO)that allowed conditional knockout of Ezh2 in CMs and performed AR at P1,samples were harvested in 21 days post resection.Results:The EF% and FS% were significantly higher in PdgfrbMyh6D849 V mice than in the littermate controls and Ezh2-c KO,PdgfrbMyh6D849 V mice at day 21 after MI at P7.Masson trichrome staining showed that,compared with the PdgfrbMyh6D849 V heart at day 21 after MI at P7,fibrosis was more serious in the infarct area of Ezh2-c KO,PdgfrbMyh6D849 V and littermate control mice.Then we explored whether Ezh2 knockout influenced the PDGFR-?-induced CM proliferation and found a striking decrease in Ed U incorporation in Ezh2-c KO,PdgfrbMyh6D849 V group 7 days after MI at P7.Collectively,these data suggested that EZH2 was indispensable in the PDGFR-?-induced heart regeneration.IF staining revealed that,compared with sham-treated group,EZH2 expression increased in CMs at day 7 after AR at P1,while EZH2 knock out in CMs eliminated this increase.Masson staining showed that the Ezh2-c KO mice failed to regenerate the lost myocardium but with fibrosis at resected region,whereas the littermate control hearts completely recovered 21 days after AR at P1.Conclusion:In this study,we demonstrated that connective activation of PDGFR-? signaling in CMs could promote juvenile mouse heart regeneration with increased proliferating CMs following myocardial infraction.Relying on RNA sequencing analysis,we identified EZH2 is the inevitable course when PDGFR-? signaling inducing CM proliferation.Conditional knock out Ezh2 in PDGFR-? activation CMs could eliminate the regeneration promotion of PDGFR-? signaling,suggesting that PDGFR-? signaling induced myocardial regeneration requires EZH2.EZH2 is also required in neonatal myocardial regeneration and myocyte proliferation by regulating H3K27me3 level and Ink4a/Arf expression.