Role Of PKA And CaMKⅡ Mediated RyR2 Phosphorylation In Ventricular And Atrial Arrhythmias

BackgroundTriggered activity and reentry are the two mechanisms of arrhythmias.The impaired of calcium cycling in cardiomyocytes would facilitate triggered activity.The depression function of sarcoplasmic reticulum Ca2+ ATPases(SERCA2a)and the dysfunction of cardiac ryanodine receptor type 2(RyR2)are the two factors which contributed to the impaired of calcium cycling.Therefore,it was widely accepted that the dysfunction of RyR2 was closely related to the genesis of arrhythmias.Cardiac RyR2 plays a key role in excitation-contraction coupling and it coupled with L type calcium channel in structure.During cardiac repolarization,extracellular Ca2+ enter into intracellular through L type calcium channel which would lead RyR2 open to release calcium.This process is called calcium-induced calcium release.RyR2 remains closed and there are very few of calcium would release to cytoplasm from sarcoplasmic reticulum during diastole in the physiological state.However,in cardiomyocytes form hypertrophy or heart failure,there would more calcium leak from SR as the function of RyR2 was impaired.On one hand,it would reduced sarcoplasmic reticulum calcium content and thus recued the amplitude of calcium transient and myocardial contractility.On the other hand,the increased cytoplasm calcium concentration would enhanced the activity of Na+-Ca2+ exchanger which extrude one Ca2+ to extracellular and transfer three Na+ into intracellular.This process would generate inward currents thus lead to afterdepolarization.Cardiac RyR2 channel protein in modulated by various post-translational modifications,including phosphorylation by protein kinase A(PKA)and Ca2+/calmodulin protein Ⅱ.Serine-2808(S2808)was the first RyR2 residue identified as a phosphorylation site and is thought to be the primary target of PKA phosphorylation.Serine-2814(S2814)was the second RyR2 residue identified as a primary CaMKII target.The degree of steady-state phosphorylation of each site depends on a dynamic balance between multiple protein kinases and phosphatases,allowing precise control of RyR2 phosphorylation and,consequently,channel activity.Alerations in RyR2 phosphorylation play a critical role in various cardiac diseases,including heart failure as well as atrial and ventricular arrhythmias.Despite extensive research in this area,the functional effects of RyR2 phosphorylation remain disputed.In particular,the potential involvement of increased RyR2-S2808 phosphorylation in the pathogenesis of arrhythmias remains a controversial area.In the present study,we would explore the alterations of RyR2-S2808 and RyR2-S2814 phosphorylation level in rat hearts subjected to constricting of abdominal aorta,and whether these changes would contribute to the pathogenesis of ventricular and atrial arrhythmias.In addition,we would evaluate the effect of chronic administraton of β receptor blocker on the phosphorylation of RyR2-S2808 and RyR2-S2814 in rat hearts subjected to constricting of abdominal aorta,and this would help us to understand the antiarrhythmia mechanisms of P receptor blocker.It would provide more evidence in this conterversial area.Part I:The role of PKA and CaMKII mediated phosphorylation of RyR2 on pathogenesis of ventricular arrhythmias in hypertrophy and early stage of heart failure induced by constricting of abdomonial aortaObjective:to explore role of PKA and CaMKII mediated phosphorylation of RyR2 on pathogenesis of ventricular arrhythmias in hypertrophy and early stage of heart failure induced by constricting of abdomonial aorta.Methods:SD rats were randomly divided into sham group(Sham),hypertrophy group(Hy),and early stage of heart failure group(HF).A rat model of cardiac Hy and HF in vivo was established by constriction of abdominal aorta.Cardiac function was evaluated via echocardiography.The concentration of cAMP in ventricular tissue was determined by Elisa.And the protein expression was tested by western blot.The Ca2+sparks and Ca2+ waves of cardiomyocytes during diastolic were detected by immunofluorescence laser scanning confocal.Afterdepolarization and induced ventricular arrhythmias were tested in perfused hearts.Results:Compared with sham group,the left ventricular diastolic wall thickness(LVPWd)and ventricular septal diastolic thickness(IVSd)were significantly increased after 8w of abdominal aortic constriction(P<0.01)and left ventricular ejection fraction(LVEF)and fractional shortening(EF)were slightly reduced after 20w(P<0.01).Therefore,8w and 20w were chosen as the research points.The concentration of cAMP in ventricular tissue of Hy and HF were significantly increased compared with Sham group(P<0.01),and the concentraton of cAMP in HF was reduced compared with Hy(P<0.05).In addition,the expression of CaMKII,p-CaMKII,RyR2-S2808 and RyR2-S2814 were also elevated in both Hy and HF compared with Sham group(P<0.05),and the level of p-CaMKII in HF was increased compared with Hy(P<0.05).KN-93,the inhibitor of CaMKII could suppressed the frequency and amplitude of Ca2+ sparks as well as spontentous of Ca2+ waves in Hy and HF(P<0.01).Similar to the role of KN-93,the PKA inhibitor H89 could also inhibit the Ca2+ sparks and waves(P<0.01).In addition,the frequent afterdepolarizations can be observed in HF.And these abnormalities can be alleviated by KN-93 and H89.Moreover both KN-93 and H89 could reduced the the induction rate of ventricuarl tachyarrhythmia(VT)or ventricular fibrillation(VF)in Hy and HF(P<0.05).Conclusion:The hyperphosphorylation of RyR2 by PKA and CaMKII in cardiac hypertrophy and early stage of heart failure contributed to pathogenesis of ventricular arrhythmiasPart Ⅱ:The alterations of PKA and CaMKⅡ mediated phosphorylation of RyR2 and its effects on atrial arrhythmias after chronic pressure overloadObjective:to explore the alterations of PKA and CaMKⅡ mediated phosphorylation of RyR2 and its effects on atrial arrhythmias after chronic pressure overload.Methods:SD rats were randomly divided into sham group(Sham)and chronic pressure overload group(HF).A rat model of chronic pressure overload in vivo was established by constriction of abdominal aorta 20 weeks.Cardiac function was evaluated via echocardiography.The concentration of cAMP in ventricular tissue was determined by Elisa.And the protein expression was tested by western blot.The Ca2+sparks and Ca2+ waves of cardiomyocytes during diastolic were detected by immunofluorescence laser scanning confocal.Afterdepolarization and induced ventricular arrhythmias were tested in perfused hearts.Results:The concentration of cAMP in atrial tissue of HF were significantly increased compared with Sham group(P<0.01).In addition,the expression of CaMKⅡ,p-CaMKⅡ,RyR2-S2808 and RyR2-S2814 were also elevated in HF compared with Sham group(P<0.05).Both of H89,the inhibitor of PKA,and KN-93,the inhibitor of CaMKⅡ,could suppressed the increased frequency and amplitude of Ca2+ sparks as well as spontaneous of Ca2+ waves in HF(P<0.05).Furthermore,the decreased effective refractory period(ERP)in HF were significantly prolong by H89 and KN-93.Both of H89 and KN-93 can also decrease atrial afterdepolarizations in HF.The rate of induction atrial arrhythmias in HF were reduced by H89 and KN-93(P<0.05).Conclusion:The hyperphosphorylation of RyR2 by PKA and CaMKⅡ in atrium subjected to chornic pressure overload contributed to the dysfunction of RyR2,the Ca2+ leakage of SR and pathogenesis of atrial arrhythmias.Part Ⅲ:the effect of β-adrenergic blocker on RyR2 hyperphosphorylation by PKA and CaMKⅡ in cardiac hypertrophy and heart failureObject:to explore the effect of metoprolol on RyR2 hyperphosphorylation by PKA and CaMKⅡ in cardiac hyprtrophy and heart failureMethods:Rats were randomly divided into Hy,HF,Hy+MT,HF+MT.The Hy+MT and HF+MT group were gavaged with metoprolol(20mg/kg/d)in following day of surgery,while the Hy and HF gourp were gavaged with saline.The concentration of cAMP in ventricular and atrial tissue was determined by Elisa.And the protein of t-CaMKII,p-CaMKⅡ,RyR2,RyR2-S2808 and RyR2-S2814 expression was tested by western blot.Results:The metoprolol significantly decreased the concentration of cAMP in ventricular tissue of Hy and HF(P<0.01)and in atrial tissue of HF(P<0.01).The elevated expression of t-CaMKⅡ,p-CaKⅡ in ventricular tissue of Hy and HF and in atrial tissue of HF were also decreased by metoprolol(P<0.01).In addition,both the expression of atrial and ventricular tissue of RyR2-S2808 and RyR2-S2814 were significantly reduced by metoprolol.Conclusion:The hyperphosphorylation of RyR2 by PKA and CaMKⅡ were depressed by metoprolol,which may be an vital mechanism for metoprolol to exert beneficial of hearts.