Ca ²⁺ /Calmodulin–Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca ²⁺ Leak in Heart Failure

Abstract

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...

Genetic inhibition of PKA phosphorylation of RyR2 prevents dystrophic cardiomyopathy

“…Whereas PKA phosphorylation of RyR2 is often elevated, reduced phosphorylation levels of PLN (27), cardiac myosin-binding protein C, and troponin I have been reported in failing hearts (28). In the case of dystrophic hearts, Williams et al (5) reported a reduction in PKA phosphorylation of S16 on PLN in mdx mice.…”

“…Finally, there is emerging evidence that phosphorylation of RyR2 by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) also plays an role in the regulation of SR Ca 2+ homeostasis and cardiac function in heart failure (27). Because cytosolic Ca 2+ levels are elevated in dystrophic hearts, it is possible that CaMKII is activated and contributes to SR Ca 2+ leak.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…However, other groups have not found PKA-dependent hyperphosphorylation in failing hearts (Xiao et al 2005). In addition, other laboratories suggest that CamKII-dependent phosphorylation of RyR2 is involved in enhanced SR Ca 2þ leak and reduced SR Ca 2þ load in heart failure and may contribute to arrhythmias and contractile dysfunction (Ai et al 2005;Chelu et al 2009;Curran et al 2010;Neef et al 2010). Phosphorylation of other targets of these kinases (troponin I, sarcolemmal Ca 2þ channels, and phospholamban) could also alter the Ca 2þ handling in cardiac and skeletal muscle.…”

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

“…In animal models of heart failure, changes in phosphoinositide metabolism occur, including alterations in the abundance, expression and/or activity of PIP 2 , PI-4 kinase, PIP-5 kinase, PLC isoforms and inositol phosphates [200,201]. Expression of IP 3 Rs is increased in both human and animal heart failure, whereas RyR levels are reduced, indicating a shift toward more IP 3 R-mediated Ca 2+ release in the failing heart [19,202]. These data indicate that, in heart failure, there are complex changes in the transmitter and receptor systems coupling to IP 3 formation as well as in the regulation of IP 3 Rs suggesting that IP 3 -induced Ca 2+ release is involved in the development and/or progression of the disease.…”