Tonegawa Kota, Otsuka Wataru, Kumagai Shohei, Matsunami Sachi, Hayamizu Nao, Tanaka Shota, Moriwaki Kazumasa, Obana Masanori, Maeda Makiko, Asahi Michio, Kiyonari Hiroshi, Fujio Yasushi, Nakayama Hiroyuki
Laboratory of Clinical Science and Biomedicine, Graduate School of Pharmacological Sciences, Osaka University, Suita, Osaka, Japan.
Faculty of Medicine, Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka, Japan; and.
Am J Physiol Heart Circ Physiol. 2017 Mar 1;312(3):H501-H514. doi: 10.1152/ajpheart.00601.2016. Epub 2016 Dec 30.
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.
钙调蛋白依赖性蛋白激酶II（CaMKII）的激活可诱导众多生物学过程，并在心肌肥大中起主导作用。小窝微区包含许多钙/钙调蛋白依赖性激酶II（CaMKII）靶点，包括L型钙通道（LTCC）复合体，并作为一个信号平台。CaMKII激活的位置被认为至关重要；然而，由于缺乏评估小窝特异性激活的方法，CaMKII在小窝中的作用仍不清楚。我们的目标是开发一种用于特异性分析小窝中CaMKII激活的新型工具，并确定小窝CaMKII在心肌肥大中的功能作用。为了评估小窝特异性的CaMKII激活，我们构建了一种由受磷蛋白和小窝蛋白-3组成的融合蛋白（cPLN-Cav3）以及与小窝蛋白结合域融合的绿色荧光蛋白（GFP）融合蛋白，该结合域与CaMKII抑制肽（CBD-GFP-AIP）相连，其可特异性抑制小窝中的CaMKII激活。使用针对受磷蛋白（苏氨酸）的磷酸特异性抗体检测小窝特异性的CaMKII激活。此外，在新生大鼠心肌细胞（NRCMs）中腺病毒过表达LTCCβ亚基（β）显示其被CaMKII组成性磷酸化，这会诱导肥大，并且CBD-GFP-AIP的表达消除了磷酸化和肥大，表明β磷酸化特异性发生在小窝中。最后，在β过表达小鼠中苯肾上腺素刺激后观察到β磷酸化，并且在β过表达小鼠的非磷酸化突变体中观察到慢性苯肾上腺素刺激后心肌肥大减轻。我们开发了用于评估和抑制小窝特异性CaMKII激活的新型工具。我们证明磷酸化的β主要定位于小窝，并在小鼠α-肾上腺素能刺激后诱导心肌肥大。虽然小窝中的信号传导在心肌肥大中被认为很重要，但由于缺乏评估小窝特异性信号传导的工具，直接证据仍然缺失。这是第一项使用最初开发的工具证明在α-肾上腺素能刺激诱导的心肌肥大中小窝特异性激活CaMKII信号传导的研究。
