Ca sub()V.2 b-subunit coordinates CaMKII-triggered cardiomyocyte death and afterdepolarizations

Excessive activation of calmodulin kinase II (CaMKII) causes arrhythmias and heart failure, but the cellular mechanisms for CaMKII-targeted proteins causing disordered cell membrane excitability and myocardial dysfunction remain uncertain. Failing human cardiomyocytes exhibit increased CaMKII and voltage-gated Ca super(2+) channel (Ca sub()V.2) activity, and enhanced expression of a specific Ca sub()V.2 b-subunit protein isoform (b sub(2a)). We recently identified Ca sub()V.2 b sub(2a) residues critical for CaMKII phosphorylation (Thr 498) and binding (Leu 493), suggesting the hypothesis that these amino acids are crucial for cardiomyopathic consequences of CaMKII signaling. Here we show WT b sub(2a) expression causes cellular Ca super(2+) overload, arrhythmia-triggering cell membrane potential oscillations called early afterdepolarizations (EADs), and premature death in paced adult rabbit ventricular myocytes. Prevention of intracellular Ca super(2+) release by ryanodine or global cellular CaMKII inhibition reduced EADs and improved cell survival to control levels in WT b sub(2a)-expressing ventricular myocytes. In contrast, expression of b sub(2a) T498A or L493A mutants mimicked the protective effects of ryanodine or global cellular CaMKII inhibition by reducing Ca super(2+) entry through Ca sub()V.2 and inhibiting EADs. Furthermore, Ca sub()V.2 currents recorded from cells overexpressing CaMKII phosphorylation- or binding-incompetent b sub(2a) subunits were incapable of entering a CaMKII-dependent high-activity gating mode (mode 2), indicating that b sub(2a) Thr 498 and Leu 493 are required for Ca sub()V.2 activation by CaMKII in native cells. These data show that CaMKII binding and phosphorylation sites on b sub(2a) are concise but pivotal components of a molecular and biophysical and mechanism for EADs and impaired survival in adult cardiomyocytes.