The Role Of G Protein Coupled Receptor Kinase 4 And YY1 In Cardiomyocyte Injury After Myocardial Infarction

Background: G protein coupled receptor kinase 4(GRK4)plays an important role in the development of hypertension,but its effect on cardiac ischemia injury is not clear.Transcription factor Yin-Yang 1(YY1)is an essential regulator of apoptosis and angiogenesis,if YY1 also play a role in myocardial infarction(MI)remains unknow.The aim of this study is to investigate the effects of GRK4 and YY1 on MI and the underlying mechanisms.Methods: In C57/BL6 mice,we assessed the expression and distribution of GRK4 in the basal state and at different time after MI.GRK4 A486V(GRK4 486 glycine mutation to valine)is a variant of GRK4 in human.Its distribution frequency in Asian people is about66.4%,and its enzyme activity is higher than that of GRK4 wild type(GRK4 WT).We generated GRK4 WT and GRK4 A486 V transgenic mice,as well as cardiac GRK4 knockout mice.We measured the cardiac function,infarct size,cardiomyocyte apoptosis and autophagy after MI,and examined the interaction between GRK4 and HDAC4 and its effect on Beclin-1expression.We also analyzed the correlation between cardiac function and GRK4 A486 V variant in 550 patients with AMI.To investigate the role of YY1 in MI,we delivered AAV9-sh YY1 by intramyocardial injection immediately after MI,and assessed the cardiac function and scar size.Results: The m RNA and protein levels of GRK4 in the heart of C57BL/6 mice increased significantly 48 hours after MI,and were mainly distributed in the nucleus.Hypoxia induced the increase of GRK4 m RNA and protein levels and nuclear entry of primary cardiomyocytes.Compared with control group,the GRK4 WT transgenic mice exhibited impaired cardiac function of,enlarged infarct size and increased mortality,these were more significant in GRK4 A486 V transgenic mice.On the contrary,cardiac GRK4 knockout mice had better cardiac function and smaller infarct size than the control group.TUNEL assays showed that overexpression of GRK4 WT and GRK4 A486 V increased cardiomyocyte apoptosis.Overexpression of GRK4 decreased the LC3 II levels and the GFP puncta in primary cardiomyocyte transfected with GFP-LC3 adenovirus.Rapamycin,an autophagy agonist,partially rescued the autophagy inhibition and apoptosis induced by GRK4 overexpression in primary cardiomyocytes.Overexpression of GRK4 reduces the expression of Beclin-1,which is positively regulated by histone deacetylase 4(HDAC4)in the nucleus,while GRK4 reduces the nuclear entry of HDAC4 after hypoxia.HDAC4 S3A(HDAC4 246/467/632 serine to alanine mutation,which leads to phosphorylation resistance)ameliorated inhibition of autophagy and promotion of apoptosis induced by GRK4.The phosphorylation of HDAC4632 serine was further confirmed by immunoprecipitation and Western blot after myocardial infarction.HDAC4 S632A(serine to alanine mutation at HDAC4 632,resistant to phosphorylation)decreased the inhibition of GRK4 on autophagy.Immunoprecipitation and proximal ligation assay indicated that GRK4 interacted with HDAC4 in mouse heart.Chip assay showed that HDAC4 bound to the promoter region of Beclin-1,while GRK4 weakened this binding.The multiple regression analysis of 550 patients diagnosed with acute myocardial infarction showed that,the cardiac function of GRK4 A486 V carriers were worse than that of GRK4 WT genotype carriers,GRK4 A486 V genotype is an independent factor for predicting lower cardiac function after myocardial infarction.In MI mice delivererd with AAV9-sh YY1,the worsened cardiac function,survival rate,and scar size were obversed compared with that in mice delivererd with AAV9-scramble control.Conclusion: The expression of GRK4 increases in infarcted mice heart and it translocates into the cardiomyocyte nuclei.GRK4 inhibits autophagy and promotes apoptosis in cardiomyocyte after MI.This effect is depends on the phosphorylation of HDAC4 on 632 serine by GRK4 and the subsequent nuclear-export of HDAC4 which leads to down regulation of Beclin-1.In addition,the deficiency of YY1 aggrevates the cardiac ischemic injury and remodeling,suggesting a protective role for YY1 in MI.