Ca 2+ /Calmodulin Kinase II-Dependent Phosphorylation of Ryanodine Receptors Suppresses Ca 2+ Sparks and Ca 2+ Waves in Cardiac Myocytes

The multifunctional Ca 2+ /calmodulin-dependent protein kinase II δ C (CaMKIIδ 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 CaMKIIδ 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 CaMKIIδ 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 β-galactosidase (β-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 CaMKIIδ C groups, respectively. When challenged by high extracellular Ca 2+ , both wild-type and constitutively active CaMKIIδ C protected the cells from store overload-induced Ca 2+ release, manifested by a ≈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 CaMKIIδ 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 CaMKIIδ 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.