Calcium/calmodulin-dependent protein kinase IIdelta associates with the ryanodine receptor complex and regulates channel function in rabbit heart

Cardiac ryanodine receptors (RyR2s) play a critical role in excitation–contraction coupling by providing a pathway for the release of Ca2+ from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca2+ and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIδ. Quantitative assessment of RyR2 protein was performed by [3H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca2+ release (Ca2+ sparks) using confocal microscopy and (ii) Ca2+-sensitive [3H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 µM AIP Ca2+ spark duration, frequency and width were decreased significantly. Similarly, 1 µM AIP decreased [3H]ryanodine binding. At 5 µM AIP, a more profound inhibition of Ca2+ sparks and a decrease in [3H]ryanodine binding was observed. Separate measurements showed that AIP (1–5 µM) did not affect sarcoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake. These results suggest the existence of an endogenous CaMKIIδ that associates directly with RyR2 and specifically modulates RyR2 activity.