The Role And Mechanism Of Mydgf In Heart Regeneration

Background: Heart failure,one of the main causes of death from cardiovascular disease resulting from the massive loss of functional cardiomyocytes,has substantially threatened social development.A number of studies have shown that the ability of adult mammalian heart to regenerate viable myocardial tissue is very limited and not sufficient to restore contractile function after myocardial infarction.However,the mammalian neonatal myocardium can regenerate after different types of injuries owing to cardiomyocyte proliferation,and this capacity is lost within 7 days of birth.Understanding the mechanism underlying cardiomyocyte proliferation is crucial for the development of cardiac regeneration medicine.In recent years,a large number of studies have shown that neonatal mice have powerful myocardial regeneration capabilities,providing an ideal model for heart regeneration.As the basis of cardiovascular disease research,it is essential to obtain cardiomyocytes with high purity and activity,as well as good morphology.Bone marrow-derived growth factor(Myeloid-derived growth factor,Mydgf)is a new type of paracrine protein produced by bone marrow-derived monocytes and plays an important role in myocardial infarction in adult mice.However,whether Mydgf is involved in regulating cardiac regeneration in neonatal mice has not been reported.Objective: In this study,we use neonatal mouse heart regeneration model to explore whether Mydgf could promote myocardial regeneration and its mechanism.Methods:1.We examined the expression of Mydgf by q RT-PCR and Western blot at different ages of WT mice.Apex resection(AR)was performed on neonatal mice.Apical tissue was collected at 1,4,and 7 days post resection(dpr),and Mydgf expression was detected by q RT-PCR and Western blot.2.We preformed Mydgf-KO mice and used q RT-PCR to detect knockout efficiency.Then,we performed AR surgery on neonatal Mydgf-KO mice and detected cardiac function and myocardial fibrosis at 21 dpr by echocardiography and Masson staining.In addition,we constructed myocardial infarction(MI)model on MydgfKO neonatal and adult mice.Masson staining was used to detect cardiac function and myocardial fibrosis at 21 days after surgery.3.We performed AR surgery on neonatal Mydgf-KO mice and detected cardiomyocyte proliferation at 7 dpr by immunofluorescence.Primary neonatal mice cardiomyocytes were isolated and treated with Mydgf recombinant protein.Cardiomyocytes were collected 16 hours later and immunofluorescence was used to detect cardiomyocyte proliferation.4.RNA-sequencing(RNA-Seq)was used to analyze differentially expressed genes and KEGG(Kyoto Encyclopedia of Genes and Genomes)was used to enrich signal pathways.Western blot experiments were performed on RNA-Seq results were verified and the expressions of c-Myc and Fox M1 were detected.5.The expression of cell cycle protein and cardiomyocyte proliferation were detected after treated with Akt,c-Myc,and Fox M1 small interfering RNA.6.We constructed myocardial infarction(MI)model on WT adult mice,and microinjected Mydgf recombinant protein.Masson staining was used to detect cardiac function and myocardial fibrosis at 21 days after surgery.Results: 1.Mydgf expression decreased with age,which might be related to the loss of cardiomyocyte proliferation ability.The expression of Mydgf increased after AR,which might be involved in myocardial regeneration in neonatal mice.2.In Mydgf-KO mice,cardiac function was significantly reduced,myocardium was unable to regenerate.In addition,the survival rate of adult mice after myocardial injury was significantly reduced and affected cardiac function.The cardiac repair ability of adult mouse was significantly enhanced after treated with Mydgf recombinant protein.3.In Mydgf-KO mice,cardiomyocytes of p H3+,Ki67+,and Aurora B+ were significantly reduced.The cardiomyocytes of p H3+,Ki67+,and Aurora B+ were dramatically increased after treating with Mydgf recombinant protein.4.RNA-seq revealed a total of 1005 differential gene expressions,of which 707 genes were up-regulated and 298 genes were down-regulated.KEGG analysis of the upregulated genes revealed significant enrichment in the cell cycle and phosphatidylinositol-3-kinase(PI3K)-Akt signaling pathways.Western blot showed that the expression of p-Akt,c-Myc and Fox M1 increased after Mydgf recombinant treatment.Mydgf deletion decreases the expression of p-Akt,c-Myc,and Fox M1 at 4 dpr and 7 dpr in Mydgf-KO mice.Additionally,the expression of c-Myc and Fox M1 declined over a wide age range and increased after AR compared with sham-operation.5.In cardiomyocytes transfected with si RNA-c-Myc,-Fox M1,or-Akt,cell cycle factors,such as Cyclin B1,Cyclin D1,CDK1 and CDK6,c-Myc and Fox M1 were significantly reduced,and cardiomyocytes of p H3+,Ki67+,and Aurora B+ were significantly decreased.6.The treatment with Mydgf recombinant protein could promote adult cardiac repair after MI.Conclusion: 1.Mydgf promotes neonatal myocardial regeneration by stimulating cardiomyocyte proliferation,Mydgf-KO mice impedes cardiac regeneration in neonatal mice and aggravates cardiac damage in adult mouse after heart injury.2.Mydgf controls cardiomyocyte proliferation through PI3K-Akt-c-Myc-Fox M1 pathway.3.The treatment with Mydgf recombinant protein could promote the adult cardiac repair after myocardial injury,suggesting Mydgf might be a potential intervention target to promote the repair of injured myocardium.