Research On The Regulation Mechanism Of Ca_V1.2 C Hannel By CaM And CaMKⅡ In Pathological Myocardial Hypertrophy

Background:In the face of various cardiovascular stimuli,the heart will undergo an adaptive compensatory response-myocardial hypertrophy to maintain early cardiac output.However,if the heart is under stress for a long time,it may lead to pathological myocardial hypertrophy and may develop into diseases such as heart failure and sudden death.Pathological myocardial hypertrophy has become an independent risk factor for cardiovascular disease.Pathological myocardial hypertrophy has the characteristics of continuous progress and difficult to reverse,but the mechanism of difficult to reverse is still unclear.The change of calcium homeostasis in myocardial cells has a very important effect on the occurrence of myocardial hypertrophy,and Ca²⁺mainly enters myocardial cells through Ca_V1.2channels.Therefore,it is important to study the changes of Ca_V1.2 function during pathologicalmyocardialhypertrophy.Calmodulin（Ca M）and Ca²⁺/calmodulin-dependent protein kinase Ⅱ（CaMKⅡ）can regulate the intracellular calcium content by regulating the function of Ca_V1.2 channels,thereby changing the state of myocardium.However,there are still differences in the ability of CaM and CaMKⅡ to regulate Ca_V1.2 in pathological myocardial hypertrophy,and the specific regulatory mechanism is not clear.Therefore,CaM and CaMKⅡ are worth studying as drug targets.Objective:To observe the changes of Ca²⁺concentration in cardiac myocytes during pathological cardiac hypertrophy,and to detect the expression of Ca_V1.2 channels in cardiomyocytes during this process,to clarify the role of CaM and CaMKⅡ in the regulation of Ca_V1.2 channels in pathological cardiac hypertrophy,and to explore the regulatory mechanism of CaM and CaMKⅡ on Ca_V1.2 channels.Methods:1.The pathological cardiac hypertrophy model of SD rats was established by subcutaneous injection of isoproterenol（ISO）every 24 hours.2.The cardiac changes of SD rats were measured by heart weight（HW）and left ventricular weight（LVW）/body weight（BW）ratio.3.HE staining was used to detect the changes of cross-sectional area of cardiomyocytes and Masson staining was used to detect the changes of myocardial fibrosis.4.The expression of cardiac hypertrophy related protein was detected by Western blot.5.Co-IP test is used to detect the binding ability of CaM and CaMKⅡ to Ca_V1.2 channel.6.Primary cardiomyocytes were induced by ISO to establish an in vitro model of pathological cardiac hypertrophy,and the effects of CaM and CaMKⅡ on cardiomyoc ytes were detected by inhibitors.We used AIP to inhibit the expression of CaMKⅡ and CMZ to inhibit the expression of CaM.7.The intracellular calcium concentration of cardiomyocytes was measured by flow cytometry（（FCM））.Results:1.In vivo experiment,ISO was used to induce cardiac hypertrophy in SD rats.Compared with normal group,LVW/BW,HW/BW,cardiac cross-sectional area and myocardial fibrillation rate increased in ISO group.Western blotting showed that the protein expression levels of ANP,CaMKⅡ and their phosphorylated form p-CaMKⅡ increased in ISO group.However,the expression of CaM protein decreased,and there was no significant difference in the expression of Ca_V1.2 channel.We found that the binding ability of CaMKⅡ to Ca_V1.2 channel in ISO group was higher than that in normal group,while the binding ability of CaM was decreased in ISO group.2.In the experiment in vitro,we found that the level of protein expression was consistent with that in vivo by Western blotting.Flow cytometry showed that ISO stimulation increased the activity of Ca_V1.2 channel and intracellular calcium concentration.3.In vitro experiments,we found that the expression of ANP,CaMKⅡ and their phosphorylated form p-CaMKⅡ in ISO group decreased after adding AIP.The expression of HDAC4,p-HDAC and MEF2C in AIP group also decreased,but no significant effect was found after adding CMZ.Conclusions:1.In the rat cardiac hypertrophy model,the expression of CaMKⅡ in cardiomyocytes increased and the expressio n of CaM decreased;the binding capacity of CaMKⅡ and Ca_V1.2 channels increased,while the binding capacity of CaM decreased.2.In the model of cardiomyocyte hypertrophy in neonatal rats,the changes of CaMKⅡ and CaM were consistent with in vivo experiments.3.In the model of cardiomyocyte hypertrophy in neonatal rats,the increase of calcium ion concentration in cardiomyocytes may be due to the change in the binding capacity of CaM and CaMKⅡ to Ca_V1.2.4.In the model of neonatal rat cardiomyocyte hypertrophy,AIP decreased the expression of ANP,CaMKⅡ and p-CaMKⅡ;meanwhile,the expression of HDAC4,p-HDAC and MEF2C also decreased;however,CMZ did not find any significant effect.5.CaM and CaMKⅡ have different degrees of regulation of Ca_V1.2channel.Compared with CaM,CaMKⅡ may be a more important drug target to reverse myocardial hypertrophy.