Mechanism Of Myosin Light Chain 3 Promoting Myocardial Ischemia-Reperfusion Injury

Objective:The aim of this study was to investigate the role of myosin light chain 3(MYL3)in MIRI by using in vivo and in vitro models of myocardial ischemia-reperfusion,and to reveal the molecular mechanism and key signal transduction pathways of MYL3 in regulating the pathogenesis of myocardial ischemia-reperfusion injury(MIRI).Methods:(1)In vivo animal experiment:The MIRI in vivo model was established by ligating the left anterior descending coronary artery of C57BL/6J mice for 45 min and cutting off the ligature line to restore blood flow and reperfusion for 2 h.The ST segment of lead II electrocardiogram(ECG)was elevated and the myocardial tissue below the ligature turned white to determine the successful modeling.Myocardial infarct size was detected by TTC staining,and myocardial cell morphological changes and muscle fiber morphological changes were detected by HE staining.Cardiac function was measured by ultrasound system(main parameters:left ventricular shortening fraction and left ventricular ejection fraction).The contents of cTnT,IL-1β,IL-6,TNF-α and LDH in mouse serum were detected by kit;the expression changes of MYL3,YAP,p-YAP,MST1,p-MST1,LATS1 and p-LATS1 proteins were detected by Western blotting;the mRNA expression levels of genes related to glycolysis and tricarboxylic acid(TCA)cycle were detected by quantitative real-time PCR(RT-PCR);and the expression changes of MYL3,YAP and p-CaM proteins in heart tissues were detected by immunofluorescence.In vitro cell experiments:Mouse myocardial cell line(HL-1 cells),rat myocardial cell line(H9c2 cells)and human myocardial cell line(AC 16 cells)were deprived with oxygen-glucose for 2 h,and then cultured with normal medium for different time periods to construct oxygen-glucose deprivation/reoxygenation(OGD/R)cell models.Western blotting and immunofluorescence were used to detect the protein expression levels of MYL3 and YAP;real-time PCR was used to detect the mRNA expression levels of Myl3,genes involved in glycolysis and TCA cycle processes.The kit detects the survival rate and LDH release of cells.Results:(1)The protein and mRNA expression levels of MYL3 were increased in vivo and in vitro models of myocardial ischemia-reperfusion injury.(2)Increased MYL3 expression exacerbates MIRI,and knockdown of Myl3 or inhibition of Myl3 expression promotes cardiomyocyte survival and prevents MIRI.(3)Increased MYL3 expression induces inflammatory responsesand knockdown of Myl3 or inhibition of Myl3 expression inhibits the production of inflammatory factors.(4)Myocardial ischemia-reperfusion injury leads to abnormal expression of genes and proteins involved in glycolysis and TCA cycle processes and reduces the production of APT.Increased MYL3 expression can lead to glycolysis and TCA cycle dysfunction and promote MIRI.(5)Myocardial ischemia-reperfusion injury leads to activation of the Hippo signaling pathway,and overexpression of the Hippo signaling effector molecule YAP inhibits the expression of MYL3 and prevents MIRI.(6)Myocardial ischemia-reperfusion injury can lead to activation of Ca2+-CaM signal,and inhibition of Cam expression can block Hippo signaling and down-regulate MYL3,thereby alleviating MIRI.Conclusion:Increased MYL3 expression aggravates MIRI.Activation of Ca2+-CaM signaling can lead to activation of Hippo signaling and prevent YAP nuclear translocation,thereby up-regulating MYL3 expression,disturbing glycolysis and TCA cycle,reducing ATP production,and exacerbating MIRI.