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

Abstract

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 caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific 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.<b>NEW & NOTEWORTHY</b> While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing due to lack of tools to assess caveolae-specific signaling. This is the first study to demonstrate caveolae-specific activation of CaMKII signaling in cardiac hypertrophy induced by α₁-adrenergic stimulation using an originally developed tool.