Correlation Between SK2 Expression And Myocardial Fibrosis In Cardiac Hypertrophy Mice

Bachground and PurposeMyocardial hypertrophy is an adaptive response to hemodynamic stress caused by physiological or pathological stimuli,including physiological and pathological hypertrophy.By physiological hypertrophy,the pumping force of the heart is enhanced in the course of systematic exercise training to maintain the cardiac normal function.The prominent feature of pathological hypertrophy which is induced by hypertension,coronary heart disease,myocardial infarction and other diseases is the augmented cardiomyocytes and increased protein synthesis,accompanied by decreased myocardial cells and increased fibrosis,leading to cardiac systolic dysfunction,ventricular dilation,heart failure,and even sudden death.Myocardial fibrosis（MF）characterized by the accumulation and differentiation of fibroblasts and the excessive accumulation of extracellular matrix（ECM）,which leads to the destruction of normal tissue structure and progressive organ dysfunction is the main pathological process of heart remodeling and heart failure.Thus,to reduce or prevent the happening of the MF has important clinical significance.Studies have shown that some ion channels expressed by the heart are related to the proliferation,differentiation and secretion of fibroblasts,wich have potential effects in myocardial fibrosis or remodeling of cardiac hypertrophy.Small conductance calcium activated K⁺channel（SK）belongs to a voltage independent,Ca²⁺activated K⁺channel family,which can be divided into four subtypes SK1 to SK4 according to its genetic coding.Studies have shown that SK2 and SK3 channel proteins are mainly express in the mammalian hearts,in which SK2 channel is expressed mainly in the atrium and is involved in the formation of action potential of cardiomyocytes in physiological conditions.The expression of SK2 channel in the ventricle is significantly increased during cardiac hypertrophy,which is also closely related to the molecular mechanism of heart failure and arrhythmia.Whether the up-regulated expression of SK2 channel in the ventricle of cardiac hypertrophy is related to fibrosis caused by proliferation and differentiation of fibroblasts has not been reported.Therefore,in the experiment,we plan to construct a pressure-overloaded myocardial hypertrophy model mouse through the transverse aortic constriction（TAC）,to explore the relationship between SK2 channel reconstruction and fibrosis in the process of myocardial hypertrophy development.The experiment’results will theoretically elucidate the role of SK2 channel in myocardial hypertrophy fibrosis,which provides important experimental basis for further study on the pathogenesis of pathological myocardial hypertrophy.Methods1.Pressure-overloaded myocardial hypertrophy induced by TAC in miceC57BL/6JNifdc mice（male and female,weight about 20-25g）were selected for TAC operation to construct pressure overload myocardial hypertrophy.The isolated aorta was not narrowed as sham operation（sham group）.Echocardiography was used to detect the changes of diastolic left ventricular posterior wall thickness（LVPWd）,diastolic left ventricular internal diameter（LVIDd）,left ventricular ejection fraction（LVEF）,systolic left ventricular internal diameter（LVIDs）and systolic left ventricular posterior wall thickness（LVPWs）during 1-4w after TAC.Using heart weight/body weight（HW/BW）,heart weight/the length of the tibia（HW/TL）to evaluate the changes in heart quality after TAC.The HE and Masson staining technique was used to detect the changes of volume in the myocardial cells and fibrosis in myocardial interstitial after TAC.2.To explore the relationship between SK2 remodeling and fibrosis in the development of myocardial hypertrophy after TACWestern blot was used to detect the expression of fibrosis-related proteins,including CollagenⅢand CollagenⅠ,to detect the expression ofα-SMA（myofibroblast marker）and SK2 channel protein in the mice heart 1w,2w,3w,4w after TAC.Pearson product-Moment correlation coefficient analysises of the correlation between SK2 channel protein and fibrosis-related protein 1～4w after TAC.3.To explore the effect of AAV9-SK2-RNAi on cardiac structure and function after TACOne week after TAC,the small interfering RNA adeno-associated virus（AAV9-SK2-RNAi）carrying EGFP green fluorescent targeting SK2 channel protein was injected into the mice through the tail vein,and the changes in cardiac structure and function were detected by echocardiography 4w after the operation.The changes of cardiac structure and tissure in TAC+AAV9-SK2-RNAi mice were detected by HW/BW,HW/TL,HE and Masson staining techniques.4.To explore the effect of SK2 knockdownon myocardial fibrosis after TACWestern blot was used to detect the expression of SK2 channel protein in TAC+AAV9-SK2-RNAi mice,and to observe SK2 knockdown on the expression of CollagenⅢ,Collagen I,andα-SMA protein.Results1.TAC induced some changes of heart structure and function in miceThe echocardiographic results showed that compared with the Sham group,LVIDd and LVIDs increased significantly from 2 week after TAC,and the P value was less than 0.0001.At 1 week,LVPWd and LVPWs increased significantly,and LVEF decreased significantly.It indicates that the ventricular wall of the mice after TAC is hypertrophy,the inner diameter gradually increases,and the cardiac ejection function is weakened.The mice heart and body weight were weighed after the animals were killed.The calculated HW/BW and HW/TL were larger than that in the Sham group.HE and Masson staining further showed that the volume of myocardial cells was increased and stromal fibrosis was caused by TAC,and the degree of fibrosis tended to aggravate with the following weeks after operation.The above results all indicate that myocardial hypertrophy occurs in mice after TAC,and eccentric hypertrophy may appear at the end of 4 weeks,and the remodeling of myocardial fibrosis is aggravated.2.SK2 channel protein is positively correlated with fibrosis-associated protein in mice heart after TACAfter TAC,SK2 channel protein,fibrosis-related proteins CollagenⅠand CollagenⅢ,and myofibroblast markerα-SMA in heart all increased significantly compared with the Sham group,the P values were all less than 0.05,and there is a trend of increases with the following weeks after operation.Therefore,Pearson product-moment correlation coefficient was used to detect the correlation between fibrosis-associated protein and SK2 channel protein,and the results showed that SK2channel protein was positively correlated with Collagen I and CollagenⅢafter1-4w.This result suggests that there is a positive correlation between myocardial fibrosis and SK2 channel protein expression during the development of myocardial hypertrophy after TAC.3.Injection of AAV9-SK2-RNAi after TAC can improve cardiac structure and functionAAV9-SK2-RNAi was injected into the tail vein at 1 week after TAC,and myocardial cells and fibroblasts were separated 4 weeks after the operation.EGFP green fluorescence seen indicated that the viral vector was successfully transfected into the cells.Ultrasonic testing showed that LVIDs,LVIDd,LVPWs,and LVPWd were significantly lower than those in the TAC+AAV9-Con group,and LVEF was significantly increased,but there was no significant difference in these parameters between the TAC+AAV9-Con group and the TAC group.At the same time,compared with the TAC+AAV9-Con group,the TAC+AAV9-SK2-RNAi group showed reduced myocardial cell volume and reduced fibrosis,but there was no difference between the TAC+AAV9-Con group and the TAC group.These results suggest that AAV9-SK2-RNAi injection through caudal vein in mice can improve cardiac function,reduce the remodeling of myocardial fibrosis during cardiac hypertrophy,and slow down the development of cardiac hypertrophy.4.Knockdown of the SK2 protein in TAC mice could reduce the expression of fibrosis related proteins in heartAfter AAV9-SK2-RNAi was injected into the tail vein 1 W after TAC,the expression of SK2 in mice heart at 4 weeks after TAC was reduced by about 70%compared with that of the negative control group（TAC+AAV9-Con）,while there was no significant difference in the protein expression of SK2 in the TAC+AAV9-Con group and the TAC group,suggesting that the protein expression of SK2 in the myocardium was successfully knocked down 4 weeks after TAC.Compared with the TAC+AAV9-Con group,the expression of CollagenⅢ,Collagen I andα-SMA protein in heart were significantly reduced,but there was no statistical difference between the TAC+AAV9-Con group and the TAC group.It is suggested that the decreased expression of SK2 channel protein in heart can reduce the degree of fibrosis in cardiac hypertrophy.ConclusionThere are positively correlated between SK2 expression and fibrosis remodeling in mice heart during the occurrence and development of myocardial hypertrophy after TAC.Knockdown of SK2 expression can improve myocardial function and the degree of fibrosis remodeling in mice with cardiac hypertrophy.