Study On Mechanism Of Metformin Regulates Cardiomyocyte Proliferation And Promotes Cardiac Regeneration

Objective: To explore whether metformin,(MET)can promote the proliferation of mammalian cardiomyocytes,whether MET plays a cardiovascular protective role by promoting the proliferation of cardiomyocytes,and the mechanism of MET promoting cardiomyocyte proliferation.Methods: H9C2 cells were treated with MET and cell viability was measured using CCK-8 assay kit.Neonatal rat cardiomyocytes were treated with MET and cell proliferation was detected by immunofluorescence staining.MET was injected subcutaneously into the head and neck of P1-P7 neonatal CD1 mice,and samples were collected from the P8 and P14 hearts.Myocardial cell proliferation was measured by immunofluorescence staining,and the size of myocardial cells was compared with that of WGA staining.Quantification of cardiomyocyte nuclear ploidy from dissociated mouse heart.The 8-day-old CD1 mouse model of myocardial infarction was constructed.MET was injected subcutaneously into the posterior neck and back of the myocardial infarction.Cardiac samples were taken 4 days after myocardial infarction.Ultrasound was used to detect cardiac function at 21,42 days after myocardial infarction.Masson staining was used to detect cardiac fibrosis 42 days after myocardial infarction.Adult mice myocardial infarction model was established,MET was treated after myocardial infarction,and cardiac function was detected by ultrasound before myocardial infarction,before administration,7,14,28,42,and 56 days after myocardial infarction.Masson staining was used to detect cardiac fibrosis 56 days after myocardial infarction.Neonatal rat cardiomyocytes were cultured for 48 h after metascularization and RNA-seq was performed to detect differentially expressed genes.The changes of reactive oxygen species and DNA damage response in cardiomyocytes were detected after MET administration.RNA-seq,immunofluorescence staining,q PCR and other techniques were used to elucidate the regulatory mechanism of MET’s cardiovascular protective effect.Results:The survival rate of H9C2 cells in in MET treated group was higher than that in saline group(NC).Compared with the negative control group,the ratio of p H3 and Ki67 positive cardiac myocytes afte r MET was higher in vitro and in vivo,And WGA staining indicated that the volume of myocardial cells in the MET group was much smaller than that in the negative control group.The quantitive results of myocardial cell nucleation indicated that the percentage of mononuclear myocardial cells in the MET group was higher than that in the negative control group.,and the proportion of binuclear cardiomyocytes was lower than that in negative control group.The p H3 and Ki67 positive cardiomyocytes of8-day-old mice at 4 days after myocardial infarction were significantly higher in MET group than in negative control group,and WGA staining indicated that the cardiac myocytes in MET group were significantly smaller than those in the negative control group.Ultrasound results of 8-day-old mice at 21,42 days after myocardial infarction showed that Left ventricular ejection fraction(LVEF)and LVFS(LV)were significantly increased in MET group..Masson staining showed that the MET group had less fibrosis than the negative control group.Ultrasonic results of adult mice showed that the values of LVEF and LVFS in the MET treatment group were significantly higher than those in the negative control group at days 42 and 56 after myocardial infarction.Masson staining at56 days after adult myocardial infarction showed that the fibrosis area in the MET treatment group was significantly less than that in the negative control group.After MET treatment,the production of reactive oxygen species(ROS)in cardiomyocytes decreased and DNA damage decreased.RNA-seq results showed that the expression of cell cycle related genes in neonatal rat cardiomyocytes was up-regulated after MET administration.Conclusion:MET promotes the viability of H9C2 cells.In vitro and in vivo,MET promotes cardiomyocyte proliferationand prolongs cardiomyocyte proliferation window in vivo.MET promotes myocardial proliferation after myocardial infarction in 8-day-old mice.MET improved cardiac function after myocardial infarction and reduced the area of cardiac fibrosis after myocardial infarction in 8-day-old and adult mice.MET promotes the expression of cell cycle related genes in cardiomyocytes and motivate the proliferation of cardiomyocytes.MET can promote myocardial cell proliferation through decreasing ROS,DNA damage,and promoting cell cycle related gene expression.