Aims: Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many CaMKII targets, including L-type Ca2+ channel (LTCC) complex, and serves as a signalling 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. Methods and results: 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 phospho-specific antibody for PLN threonine 17. Furthermore, adenoviral overexpression of LTCC β2a subunit (β2a) 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 β2a phosphorylation occurs specifically in caveolae. Finally, β2a phosphorylation was observed after phenylephrine (PE) stimulation in β2a-overexpressing mice, and attenuation of cardiac hypertrophy after chronic PE stimulation was observed in non-phosphorylated mutant of β2a-overexpressing mice. Conclusions: We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated β2a dominantly localizes to caveolae and induces cardiac hypertrophy after α1 adrenergic stimulation in mice.
- caveolae microdomain
- Ca2+/calmodulin dependent kinase II
- L-type calcium channel
- cardiac hypertrophy
- Copyright © 2016, American Journal of Physiology-Heart and Circulatory Physiology