Ca2+/calmodulin kinase II-dependent phosphorylation of ryanodine receptors suppresses Ca2+ sparks and Ca2+ waves in cardiac myocytes

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKIIdelta(C)) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca(2+) release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIdelta(C) via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIdelta(C) expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing beta-galactosidase (beta-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca(2+) transients was slowed, accelerated, or unchanged, whereas spontaneous Ca(2+) spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIdelta(C) groups, respectively. When challenged by high extracellular Ca(2+), both wild-type and constitutively active CaMKIIdelta(C) protected the cells from store overload-induced Ca(2+) release, manifested by a approximately 60% suppression of Ca(2+) waves (at 2 to 20 mmol/L extracellular Ca(2+)) in spite of an elevated sarcoplasmic reticulum Ca(2+) content, whereas dominant-negative CaMKIIdelta(C) promoted Ca(2+) wave production (at 20 mmol/L Ca(2+)) with significantly depleted sarcoplasmic reticulum Ca(2+). Taken together, our data support the notion that CaMKIIdelta(C) negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca(2+) release, thereby affording a negative feedback that stabilizes local and global Ca(2+)-induced Ca(2+) release in the heart.