Caveolae-specific activation loop between CaMKII and L-type Ca2+ channel aggravates cardiac hypertrophy in α1-adrenergic stimulation.

Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin- dependent kinase II (CaMKII) targets, including L-type Ca2⁺ channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolinbinding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolaespecific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr¹⁷). Furthermore, adenoviral overexpression of LTCC β_2a-subunit (β_2a) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that β_2a phosphorylation occurs specifically in caveolae. Finally, β_2a phosphorylation was observed after phenylephrine stimulation in β_2a-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of β_2a-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated β_2a dominantly localizes to caveolae and induces cardiac hypertrophy after α1-adrenergic stimulation in mice. [ABSTRACT FROM AUTHOR]