Abstract 409: Ca2+/Calmodulin Kinase II -Dependent Phosphorylation of Ryanodine Receptors Stabilizes Ca2+ Signaling in Intact Cardiac Myocytes
The multifunctional Ca2+/calmodulin-dependent protein kinase II ΔC (CaMKIIΔC) is found in the macromolecular complex of ryanodine receptor (RyR) Ca2+ release channels in the heart. However, the functional role of CaMKII-dependent RyR phosphorylation remains controversial. To address this issue, we expressed wild type (WT), constitutively active (CA), or dominant negative (DN) CaMKIIΔC via acute adenovirus gene transfer in cultured adult rat ventricular myocytes. CaMKII activity was examined by determining the kinase activity and the level of CaMKII-specific phosphorylation of phospholamban (PLB) at Thr-17. Compared with β-gal adenovirus control, CaMKII-mediated phosphorylation of RyR, assessed by back phosphorylation, was reduced by DN-CaMKIIΔC, enhanced by CA-CaMKIIΔC, or unaltered by WT-CaMKIIΔC expression, which is consistent with the CaMKII activities and the corresponding PLB phosphorylation under these conditions. Concomitantly, spontaneous Ca2+ sparks at 1mM Ca2+ was hypoactive, hyperactive, or unchanged in CA-, DN- or WT-CaMKIIΔC groups, respectively; Ca2+ transients elicited by action potentials displayed accelerated, slowed or unchanged rate of relaxation in CA-, DN- or WT-CaMKIIΔC groups, respectively, without affecting the amplitudes. Both WT- and CA-CaMKIIΔinstability as manifested protected the cells from Ca2+ by ~60% attenuation of the frequency of Ca2+ waves induced by elevating extracelular Ca2+ over a wide range (2–20 mmol/L), whereas DN-CaMKIIΔcwave frequency at 20 mmol/L Ca2+. Furthermore, activation of endogenous CaMKII during sustained α1-adrenergic receptor stimulation (norepinephrine 100 nmol/L, 24h in the presence of α-adrenergic blocker) did not alter Ca2+ spark frequency in spite of elevated caffeine-liable Ca2+ store at 1 mmol/L Ca2+, and reduced Ca2+ wave frequency at high Ca2+concentrations. Taken together, our data support the notion that CaMKIIΔC negatively regulates the RyR channel activity in intact cells (particularly under Ca2+ overload conditions), which counteracts the inherent positive feedback of Ca2+ induced Ca2+ release, enhancing the stability of Ca2+ signaling in the heart.