The Effects Of Rho-Kinase On Cardiac Apoptosis Inducedy By Ischemia/Reperfusion Injury In Neonatal Rat Cardiomyocytes

ObjectiveTo investigate the effects of Rho-kinase on ischemia/reperfusion-induced cardiac apoptosis and the effects of Rho-kinase inhibitor,fasudil on cardiac apoptosis in cultured neonatal rat cardiomyocytes.MethodsNeonatal rat ventricular myocytes were prepared from hearts of 1～2 days old Sprague Dawley rat pups.To observe the morphological characteristics of the cultured cardiomyocytes by means of phase contrast microscopy.Cultured neonatal cardiomyocytes were immunostained with antibody against troponin T and FITC,and visualized by Laser Scanning Confocal Microscope(LSCM)to evaluate the purity of the myocardial cell preparation.The cardiomyoctyes were cultured in hypoxic condition made with nitrogen to establish a model of hypoxia-reoxygenated cardiomyoctyes. The cultured cardiomyocytes were divided randomly into three groups: control group,I/R group,I/R+fasudil group(treated with three concentration 10μmol/L,30μmol/L,50μmol/L).The extent of phosphorylation of myosin phosphatase target subunit 1(MYPT1)was quantified by Western blot analysis,and it was used to evaluate the activity of Rho-kinase.Cardiac myocyte apoptosis ratio was determined by flow cytometry with Annexin-V and propidium iodide(PI)stainings respectively.Results1.Morphological characteristics of cadiomyocytes:12～24 hours after culture,the cardiomyocytes became adherent to bottal wall.The shape of cardiomyocyte initially was round,then became fusiform or irregular triangle.Cardiomyocytes stretch out pseudopodium and interlace networks, and formed a single cell layer after 3～5d.Single or clumps of myocardial cells pulsated spontaneously at 80～100.2.The identity of cultured cardiomyocytes:The slide of myocytes was visualized by Laser Scanning Confocal Microscopy.The endocchylema was stained with green fluorescence and the nucleus was not stained.The purity of myocardial cells in culture cell population was more than 90%.3.Analysis of Rho-kinase:Cardiomyocyte ischemia reperfusion resulted in a 5.7-fold increase in the amount of phosphorylated MYPT1 compared with control group(P＜0.01),indicating the activation of Rho-kinase following ischemia/reperfusion.In contrast,treatment with fasudil at 10μmol/L,30μmol/L and 50μmol/L attenuated the amount of phosphorylated MYPT1 by24.6%,40.1%,60.1%respectively(P＜0.05)4.Results of cardiac myocyte apoptosis ratio:Cardiac myocyte apoptosis ratio increased apparently in I/R group compared with control group;The apoptosis ratio decreased with the raising of drug concentration.With the lasting of reperfusion,the apoptosis ratio in I/R and drug intervention group with 6 hours was significantly higher than the two groups with 3 hours.ConclusionsRho-kinase induces cardiomyocyte apoptosis,however Rho-kinase inhibitor, fasudil can decrease cardiomyocyte apoptosis and protect cardiac cells.