Abstract-Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKIIdependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF. Key Words: ryanodine receptor Ⅲ CaMKII Ⅲ phosphorylation Ⅲ heart failure Ⅲ arrhythmia C ontractile dysfunction in HF is caused by diminished sarcoplasmic reticulum (SR) Ca load that could arise from enhanced activity of Na/Ca exchange (NCX), reduced SR Ca ATPase (SERCA) function, and increased diastolic SR Ca leak via ryanodine receptors (RyR), 1-5 all of which we have demonstrated to occur in our arrhythmogenic rabbit model of nonischemic HF. [1][2][3] HF is also associated with a nearly 50% incidence of sudden cardiac death from ventricular tachycardia (VT) that degenerates to ventricular fibrillation (VF). 6 In 3D cardiac mapping studies in our HF rabbit model, we showed that spontaneously occurring VT initiates by nonreentrant mechanisms 7 associated with delayed afterdepolarizations. 2 These arise from spontaneous SR Ca release that activates a transient inward current (I ti ) carried primarily by NCX. 2 Thus abnormal SR Ca release via RyR may contribute to both contractile dysfunction and arrhythmogenesis.The cardiac RyR (RyR2) is the center of a large macromolecular protein complex that directly or indirectly interacts with RyR2 and modulates its function. The complex includes FK506 binding protein 12.6 (FKBP12.6), calmodulin (CaM), protein kinase A (PKA), Ca/CaM-dependent protein kinase (CaMKII), protein phosphatases PP1 and PP2A, mAKAP, and other associated proteins such as spinophilin, calsequestrin, and sorcin. 8 It was shown 9 in HF that PKA mediates RyR2 hyperphosphorylation at the RyR2-Ser2809 site (th...

Abstract-In nonischemic heart failure (HF), ventricular tachycardia initiates by a nonreentrant mechanism, but there is altered conduction (that could lead to re-entry) that could arise from changes in gap junctional proteins, especially connexin43 (Cx43). We studied Cx43 expression and phosphorylation state in the left ventricle (LV) from an arrhythmogenic rabbit model of nonischemic HF and from patients with HF attributable to idiopathic dilated cardiomyopathy. We also investigated the role of protein phosphatases that dephosphorylate Cx43-PP1 and PP2A. In HF rabbit LV, Cx43 mRNA and total protein were decreased by 29% and 34%, respectively (PϽ0.05 and PϽ0.001).In controls, Cx43 was primarily in the phosphorylated state, but with HF there was a 64% increase in nonphosphorylated Cx43 (Cx43-NP, normalized to total Cx43; PϽ0.05). Similar results were noted in HF rabbit myocytes (PϽ0.05) and in human idiopathic dilated cardiomyopathy LV (PϽ0.05). We found that PP1 and PP2A colocalized with Cx43 in rabbit LV. With HF, the level of colocalized PP2A increased Ͼ2.5-fold (PϽ0.002), whereas colocalized PP1 was unchanged. We also found intercellular coupling (assessed by Lucifer Yellow dye transfer) was markedly reduced in HF. However, okadaic acid (10 nmol/L) reduced the amount of Cx43-NP and significantly improved cell coupling in HF. Thus, in nonischemic HF in rabbits and humans, there is a decrease in both Cx43 expression and phosphorylation that contributes to uncoupling. Increased levels of PP2A that colocalize with Cx43 can underlie enhanced levels of Cx43-NP in HF. Modulation of Cx43 phosphorylation may be a potential therapeutic target to improve conduction in HF. Key Words: gap junctions Ⅲ phosphorylation Ⅲ phosphatases Ⅲ heart failure Ⅲ arrhythmia H eart failure (HF), whether nonischemic or ischemic, is associated with a nearly 50% incidence of sudden death, primarily from ventricular tachycardia (VT) degenerating to ventricular fibrillation (VF). 1 Whereas VT in nonischemic HF initiates primarily by a nonreentrant mechanism, 2 myocardium from patients with idiopathic dilated cardiomyopathy (IDCM) exhibits nonuniform anisotropy, slow conduction, and conduction block 3 that could underlie reentry during the transition from VT to VF. Conduction slowing could arise from decreased depolarizing currents and/or decreased gap junctional coupling. 4 However, the degree of slow conduction and block in failing myocardium appears to be out of proportion to the changes in active membrane properties. 5 Moreover, LV myocytes from an animal model of nonischemic HF exhibit markedly decreased gap junctional conductance. 6 Thus, alterations in intercellular coupling involving cardiac gap junctions may underlie slow conduction in nonischemic HF.Gap junctions are specialized membrane structures consisting of arrays of intercellular channels that directly connect adjacent cells by providing chemical and electrical communication. They are composed of connexins, a multigene family of conserved proteins. The relative amounts, com...

Rationale Increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in heart failure (HF) development and associated diastolic sarcoplasmic reticulum (SR) Ca2+ leak is unclear. Objective Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with non-ischemic and ischemic forms of HF. Methods and Results Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with non-ischemic but not with ischemic HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development following transverse aortic constriction (TAC) compared to wildtype (WT) littermates. After TAC, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor (ANF). Cardiomyocytes from S2814A mice exhibited significantly lower SR Ca2+ leak and improved SR Ca2+ loading compared to WT mice after TAC. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice following experimental myocardial infarction. Conclusions Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological SR Ca2+ leak and heart failure development in non-ischemic forms of HF such as transverse aortic constriction in mice.