Abstract 68: Caveolae-specific Phosphorylation of L-type Calcium Channel β2a Subunit exaggerates Cardiac Hypertrophic Responses after α1 Adrenergic Stimulation in Mice

Abstract

Rationale: Ca2+ influx via L-type Ca2+ channels (LTCCs) plays a pivotal role in excitation-contraction coupling and cardiac hypertrophy. Phosphorylation of LTCC β2a subunit (β2a) by CaMKII enhances channel activity. LTCCs are localized in both T-tubules and caveolae, but the functional role of phosphorylation of β2a in caveolae remains unelucidated. Objective: To develop a novel tool to analyze caveolae specific activation of CaMKII and to determine the functional roles of caveolae-specific CaMKII signaling in LTCC-related cardiac hypertrophy. Methods and Results: To evaluate caveolae-specific activation of CaMKII, we generated a fusion protein composed of the cytosolic domain of phospholamban (PLN) as a phosphopeptide tag and caveolin3 (cPLN-Cav3). Activation of CaMKII was assessed by phospho-specific antibody for PLN (Thr17). To inhibit caveolae-specific activation, we generated a GFP fusion protein with caveolae-targeting sequences fused to CaMKII inhibitory peptide (CTS-GFP-AIP). In neonatal rat cardiomyocytes (NRCM), adenoviral expression revealed that CTS-GFP-AIP co-localizes with caveolin3 and mediates caveolae specific inhibition of CaMKII, thus validating this novel method. CTS-GFP-AIP inhibited CaMKII phosphorylation of β2a in NRCM, thus suggesting that phosphorylation of β2a occurs exclusively in caveolae. Phenylephrine stimulation mediates CaMKII activation in caveolae, which leads to CaMKII-specific phosphorylation of β2a in vitro and in vivo. Finally, we generated non-phospho mutant β2a -overexpressing mice and assessed hypertrophic responses in both wild-type and mutant β2a transgenic animals (TG). Protein expression by transgenes in mutant TG was similar to those previously reported in wild-type β2a overexpressing mice. Wild-type β2a TG showed exaggerated cardiac hypertrophic responses at two weeks after phenylephrine stimulation when compared to controls (4.8 ± 0.2 vs. 5.5 ± 0.3 for heart weight to body weight ratio; p<0.05), which was completely abolished in mutant TG. Conclusions: We developed a novel method to analyze caveolae-specific activation of CaMKII and confirmed that caveolae-specific phosphorylation of β2a exaggerates cardiac hypertrophy caused by phenylephrine stimulation.