Ca2+/Calmodulin Kinase II–Dependent Phosphorylation of Ryanodine Receptors Suppresses Ca2+ Sparks and Ca2+ Waves in Cardiac Myocytes

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Abstract

Abstract—

The multifunctional Ca2+/calmodulin-dependent protein kinase II δC (CaMKIIδC) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca2+ 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 CaMKIIδC 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 CaMKIIδC 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 Ca2+ transients was slowed, accelerated, or unchanged, whereas spontaneous Ca2+ spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIδC groups, respectively. When challenged by high extracellular Ca2+, both wild-type and constitutively active CaMKIIδC protected the cells from store overload–induced Ca2+ release, manifested by a ≈60% suppression of Ca2+ waves (at 2 to 20 mmol/L extracellular Ca2+) in spite of an elevated sarcoplasmic reticulum Ca2+ content, whereas dominant-negative CaMKIIδC promoted Ca2+ wave production (at 20 mmol/L Ca2+) with significantly depleted sarcoplasmic reticulum Ca2+. Taken together, our data support the notion that CaMKIIδC negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca2+ release, thereby affording a negative feedback that stabilizes local and global Ca2+-induced Ca2+ release in the heart.

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