Abstract 11273: Caveolae-Specific Phosphorylation of L-type Calcium Channel β2a Subunit Exacerbates Cardiac Hypertrophy

Abstract

Background: L-type calcium channel (LTCC) localizes at T-tubules and caveolae in cardiomyocytes, and plays major roles in excitation-contraction coupling and cardiac hypertrophy. The expression of β2a subunit of LTCC (β2a) is increased in human failing heart. Recently, it was reported that phosphorylation of β2a by CaMKII enhanced LTCC activity. However, the functional role of β2a phosphorylation in heart failure remains to be elucidated. Objective: To make clear the functional role of β2a phosphorylation in cardiomyocytes. Methods and Results: Adenoviral overexpression of β2a demonstrated its constitutive phosphorylation by CaMKII and induced neonatal cardiomyocyte hypertrophy. Interestingly, phosphorylated β2a was co-localized with caveolin3. To assess caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin3 (PLN-Cav3). This protein localized at caveolae, and caveolae-specific activation of CaMKII was detected using phospho-specific antibody for PLN (Thr17). In addition, to inhibit caveolae-specific CaMKII activity, we developed a GFP fusion protein with caveolin binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP). We identified that this protein co-localized with caveolin3, and inhibited activation of CaMKII specifically at caveolae using PLN-Cav3 method. Moreover, CBD-GFP-AIP abolished β2a phosphorylation and attenuated β2a-induced cardiac hypertrophy. We found that phenylephrine (PE) stimulation activated caveolae-CaMKII and β2a in vitro and in vivo. Finally, we generated transgenic mice overexpressing wild-type (w-TG) or non-phospho mutant β2a (m-TG) in cardiomyocyte and evaluated cardiac hypertrophy after two weeks of chronic PE stimulation. The expression of β2a in both TG increased approximately 2.5 fold compared to control mice. PE-induced cardiac hypertrophy was attenuated in m-DTG compared to w-DTG mice (heart weight-body weight ratio: control; 4.8±0.2, *w-TG; 5.5±0.3, m-TG; 4.8±0.4, n=5-6, *p<0.05 vs others). Conclusion: We developed novel methods to evaluate and inhibit caveolae-specific activation of CaMKII. Using these methods, we revealed that phosphorylated β2a localized at caveolae, and exaggerates cardiac hypertrophy.