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 Ca 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 (Thr). Furthermore, adenoviral overexpression of LTCC β-subunit (β) 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 phosphorylation occurs specifically in caveolae. Finally, β phosphorylation was observed after phenylephrine stimulation in β-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of β-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated β dominantly localizes to caveolae and induces cardiac hypertrophy after α-adrenergic stimulation in mice. 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 α-adrenergic stimulation using an originally developed tool.

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.

Background: Cardiac fibroblasts (CFs) are the most prevalent cell types in heart and play important roles in cardiac remodeling. While the roles of β-adrenergic receptor (βAR) signaling in cardiomyocytes (CMs) are well characterized, those in CFs remain to be elusive due to lack of convenient method to assess those signaling. There are three subtypes of, βAR β1, β2, β3 and β2AR is reported to be expressed in CFs by which enhances cell proliferation and production of inflammatory cytokines. Clinical efficacy of non-selective β blocker carvedilol for heart failure (HF) surpasses that of β1 selective blocker metoprolol, suggesting critical roles of β2 and β3AR in the pathogenesis of HF. Objective: To elucidate the signaling downstream βARs in CFs in heart. Methods and Results: Caveolae is an important microdomain for signal transduction, such as βAR, present in CMs or CFs. To elucidate βAR signaling of caveolae in CFs, we generated a fusion protein composed of phospholamban (PLN) and caveolin3 (Cav3) representing PKA activation as phosphorylation at S16 of PLN and CaMKII as that at T17 in caveolae. Thus, activation of PKA or CaMKII is detectable by anti-phospho-S16 or T17 antibody, respectively. In neonatal rat CFs (NRCFs) infected PLN-Cav3 adenovirus, stimulation by isoproterenol (ISO) led to enhanced phosphorylation of both S16 and T17, suggesting PKA and CaMKII activation in caveolae of CFs. RT-PCR analyses showed β2AR and β3AR were present in NRCFs. Stimulation with β2AR selective agonists activated both PKA and CaMKII, while β3AR elicited solely PKA activation, analyzed by using β3AR selective agonist/antagonist. In addition, in order to examine the significance of βAR stimulation for heart failure, we administered ISO continuously for two weeks in β2ARKO mice. As a result, fibrosis was suppressed in β2ARKO mice compared with wild-type mice (0.35% vs 2.37%, p<0.05) suggesting critical roles of β2AR in development of cardiac fibrosis caused by βAR stimulation in mice. Conclusions: Both β2 and β3AR are expressed in NRCFs and transduce distinct signaling and β2AR selective stimulation elicit development of cardiac fibrosis via activation of CaMKII signaling. Thus, selective βAR regulation could be potential novel anti-fibrotic therapeutics in HF.

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.