The Study Of The Effect Of LKB1 On The Proliferation Of Cardiomyocytes Through Hippo-Yap Signal And The Protective Effect Of Irisin On Myocardial Infarction

Effects of LKB1 on Cardiomyocyte Proliferation through Hippo-Yap SignalingBackground Myocardial infarction(MI)has been a worldwide problem which lead to an increase of inmorbidity and mortality.It occurs as the results of insufficient myocardial perfusion leading to loss of cardiomyocytes death.The inability to restore the damaged cardiomyocytes causes the major pathologies of MI.A traditional view holds that heart used to be regarded as a terminally differentiated organ,adult mammalian cardiomyocytes exit cell cycle and lose its ability to proliferate.However,recent studies found that cardiomyocytes can proliferate after birth.But the rate is too low to replenish the lost cardiomyocytes after MI.How to increase to proliferation ability become the main target to treat MI.Researchers show that the cell cycle ability of cardiomyocytes declined rapidly during the first week of postnatal life and almost lost its abiltity after 3 weeks of postnatal life in mice.Liver kinase B1(LKB1)is a kind of serine/threonine kinase encoded by STK11 gene which plays an important role in cell polarity,cell proliferation and function as a tumour suppressor.The expression of LKB1 is related to the proliferation ability of cardiomyocytes.The lowest level of LKB1 expression was found in the first day after birth and the expression level of LKB1 increased as the proliferation ability of cardiomyocyte is reduced.Therefore,we speculate that when we inhibit the expression of LKB1,the proliferation of cardiomyocytes is increased.Hippo/ Yes-associated protein(YAP)is an important pathway in heart regeneration.YAP is involved in cell cycle regulation as a transcription factor.Cell cycle regulation is a complex process involving a variety of signaling molecules.However,the role which YAP played in the regulation of LKB1 in the proliferation of cardiomyocytes has not been reported yet.Our study proposed the hypothesis that the inhibtion of LKB1 can promote cardiomyocytes proliferation through the regulation of Hippo/YAP pathway.To prove this hypothesis siRNA treatment model in vitro was established to verify the proliferation of cardiomyocytes when the expression of LKB1 is reduced.And the detection and regulation of YAP were performed to illustrate the mechanism of proliferation induce by LKB1 decline in cardiomyocytes.Methods 1.The RNA of mice hearts was extracted at different time point after birth.The qPCR was performed to detect the expression level of LKB1.2.Neonatal rat ventricular myocytes(NRVMs)were used as the research object,an LKB1 siRNA treatment model of NRVMs in vitro by formulating 10% fetal bovine serum(FBS)dulbecco’s modified eagle medium(DMEM)with high glucose.Immunofluorescence(IF)was used to evaluated the proliferation rate of cardiomyocytes after LKB1 siRNA treatment.3.Adult C57/BL6 male mice were subjected to MI by ligation of the left anterior descending(LAD)coronary artery and AAV9-LKB1-GFP-shRNA was injected to mice heart post-MI.4.Westernblot assay was used to detect the change of YAP,phosphorylated YAP and Cyclin D after LKB1 siRNA treatment on cardiomyocytes.The changes in the YAP and phospho-YAP level and the expression of Cyclin D were observed to clarify the regulatory effect on the proliferation of cardiomyocytes.Results 1.The expression of LKB1 was increased after birth.As the proliferation ability is decreased,the expression of LKB1 is increased,which indicated that the expression of LKB1 may has relationship with cardiomyocytes abilities.2.The low expression of LKB1 may lead to high proliferation ability of NRVMs.By using LKB1 siRNA to decrease the expression level of LKB1,the IF shows that the expression of Ki67 and pH3 in cardiomyocytes were increased after siRNA treatment.3.After AAV9-LKB1-GFP-shRNA was injected to mice heart post-MI,the proliferation of cardomyocytes is increased.4.After treatment of LKB1 siRNA,the expression of YAP and cyclin D were increased,the level of phospho-YAP was decreased.Conclusions When the expression of LKB1 is declined,the proliferation rate of cardiomyocytes is increased,which is the result of decreasing expression of phospho-YAP and increasing expression of YAP and Cyclin D.The results above suggest that LKB1 is involved in the regulation of cardiomyocyte proliferation ability induced by YAP upregulation,and the regulation of Cyclin D mediated by YAP/p-YAP is a crucial regulatory mechanism.This conclusion further indicates that the expression of LKB1 is involved in the regulation of cardiomycyte cell cycle,and is helpful for us to explore new treatment of cardiomyocyte loss after diseases such as MI.Irisin exerts a therapeutic effect against myocardial infarction via promoting angiogenesisBackground: The incidence and mortality of myocardial infarction(MI)are increasing worldwide.MI usually refers to myocardial ischemic necrosis caused by the sharp decrease or interruption of coronary blood flow.MI will result in the death of a large number of cardiomyocytes,the formation of fibrous scar,and the obvious impairment of cardiac function.Irisin,as a myokine,is cleaved from the extracellular portion of fibronectin domain-containing 5 protein in skeletal muscle and myocardium and secreted into circulation as a hormone during exercises.Irisin has been found to exert protective effects against lung and heart injuries.However,whether irisin influences myocardial infarction(MI)remains unclear.In this study we investigated the therapeutic effects of irisin in an acute MI model and its underlying mechanisms.Methods: 1.Adult C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery and treated with irisin for 2 weeks after MI.Cardiac function was assessed using echocardiography at 7 days,14 days,28 days post-MI.And Masson staining shows the degree of cardiac fibrosis.2.To observe the effect of irisin MI,we compared the number of Tunel positive cardiomyocytes by Tunel staining at 2 days post-MI,and observed the effect of angiogenesis in the infarct border zone at 14 days post-MI by immunofluorescence staining.3.To observe the effect of irisin on angiogenesis,human umbilical vein endothelial cells(HUVEC)were treated with irisin and U0126 respectively,and their effects on ERK were observed through Westernblot and the ability of migration were observed through transewell migration assay.Results: 1.We found that irisin administration significantly alleviated MI-induced cardiac dysfunction and ventricular dilation at 4 weeks post-MI.Irisin significantly reduced fibrosis in post-MI hearts.2.The decreasing number of Tunel positive cardiomyocytes and increasing expression of CD31 indicates that irisin administration decreased cardiomyocyte apoptosis and significantly increased angiogenesis in the infarct border zone.3.In human umbilical vein endothelial cells(HUVEC),irisin significantly increased the phosphorylation of ERK,and promoted the migration of HUVEC detected in transwell chamber migration assay.The effects of irisin were blocked by the ERK inhibitor U0126.Conclusion: Irisin improves cardiac function and reduces infarct size in post-MI mouse heart.The therapeutic effect is associated with its pro-angiogenic function through activating ERK signaling pathway.