Cardiac-specific Caveolin-3 Overexpression Prevents Post-myocardial Infarction Ventricular Arrhythmias By Inhibiting Ryanodine Receptor-2 Hyperphosphorylation

Background:Ventricular arrhythmia is the most important risk factor for sudden cardiac death(SCD)after acute myocardial infarction(MI)worldwide.Although a large number of studies have been conducted to explore the molecular mechanisms of it,those mechanisms are not completely understood and there is still a lack of effective treatments for these arrhythmias.Therefore,it is of great significance to further understand the mechanism of ventricular arrhythmia and to find more effective targets and intervention methods.Caveolin-3(Cav3)is the main component protein of caveolae in the cardiomyocyte.A large number of studies have shown that Cav3 plays an important role in the regulation of cardiovascular system and overexpression of Cav3 can have a protective effect in various cardiovascular diseases,such as hypertrophic cardiomyopathy,myocardial ischemia reperfusion injury and diabetic cardiomyopathy.However,the effect of Cav3 on ventricular arrhythmia after myocardial infarction remains unclear.Here,we evaluated whether Cav3 played an important role in the strategy for reducing ventricular arrhythmia and sudden cardiac death after myocardial infarction.Methods and Results:1.To explore the effect of Cav3 on ventricular arrhythmia after myocardial infarction in vivo experiment,the mice were randomly divided into four groups: r AAV-c TNT-RFP+sham,r AAV-c TNT-RFP+MI,r AAV-c TNT-Cav3+sham,r AAV-c TNT-Cav3+MI.The recombinant virus(r AAV-c TNT-RFP and r AAV-c TNT-Cav3)were injected into these mice by caudal vein injection to obtain blank control mice and cardiac-specific overexpression of Cav3 mice.Four weeks after injection,the mice in MI groups were subjected to MI by ligation of the left anterior descending artery(LAD).After 24 hours after LAD ligation,the incidence of ventricular arrhythmias was detected in each group.The results showed that cardiac-specific overexpression of Cav3 could reduce ventricular arrhythmia and sudden cardiac death after MI in mice.2.To further explore the mechanism how Cav3 affects ventricular arrhythmia,we transfected the Cav3 expression plasmids into cultured myocardial cells to overexpress Cav3 and then detected m RNA levels of some arrhythmogenic molecules after Cav3 transfection by real-time PCR.We found that Cav3 could regulate the expression of Plakophilin-2(Pkp2)and proved this result in heart tissues by western blot.Furthermore,through immunofluorescence and immunoprecipitation,we also found Cav3 and Pkp2 may interact directly.3.Then the langendorff perfusion system was used to extract adult mice cardiomyocytes and the susceptibility of ventricular myocytes to arrhythmia was evaluated by calcium imaging.The results showed that MI increased the proportion of spontaneous calcium release in cardiomyocytes,while cardiac-specific overexpression of Cav3 decreased it.To further explore the mechanism of this effect,we detected the phosphorylation of Ry R2 in cardiomyocytes by western blot and found that MI increased the phosphorylation at Ser2814 and Ser2808 of Ry R2.However,cardiac-specific overexpression of Cav3 could inhibit the hyperphosphorylation at Ser2814 of Ry R2 after MI.4.Finally,in order to explore the role of Pkp2 in the regulation of Cav3 to calcium activity in cardiomyocytes,the expression plasmid and si RNA were transfected to the cultured cardiac myocytes to overexpress Cav3 and knock down Pkp2.After 24 hours of transfection,hypoxia treatment was performed,and changes in the phosphorylation level of Ry R2Ser2814 were detected by western blot.We found that the inhibition of overphosphorylation at Ry R2Ser2814 by Cav3 was reduced after the knockdown of Pkp2.Conclusions:In conclusion,we found that cardiac-specific overexpression of Cav3 could reduce the incidence of ventricular arrhythmia and sudden cardiac death by reducing the proportion of spontaneous calcium release of cardiomyocytes after myocardial infarction,this effect was conducted by the inhibition of hyperphosphorylation of Ry R2Ser2814 which relied on Pkp2.Our results suggested a novel role for Cav3 in the prevention of ventricular arrhythmias,thereby identifying a new target for preventing SCD after MI.