Huang Yuan, Wright Casey D, Merkwan Chastity L, Baye Nichole L, Liang Qiangrong, Simpson Paul C, O'Connell Timothy D
Cardiovascular Research Institute, Sanford Research/USD, and Department of Medicine, The University of South Dakota School of Medicine, Sioux Falls, SD 57105, USA.
Circulation. 2007 Feb 13;115(6):763-72. doi: 10.1161/CIRCULATIONAHA.106.664862. Epub 2007 Feb 5.
In alpha1-AR knockout (alpha1ABKO) mice that lacked cardiac myocyte alpha1-adrenergic receptor (alpha1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte alpha1-ARs and whether the alpha1A, alpha1B, or both subtypes mediated protection. Therefore, we investigated alpha1A and alpha1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of alpha1-ARs in cardiac myocytes.
We cultured alpha1ABKO myocytes and reconstituted alpha1-AR signaling with adenoviruses expressing alpha1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H2O2 and was measured by annexin V/propidium iodide staining. In alpha1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the alpha1A subtype, but not the alpha1B, rescued alpha1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined alpha1-AR activation of extracellular signal-regulated kinase (ERK). In alpha1ABKO hearts, aortic constriction failed to activate ERK, and in alpha1ABKO myocytes, expression of a constitutively active MEK1 rescued alpha1ABKO myocytes from norepinephrine-induced death. In addition, only the alpha1A-AR activated ERK in alpha1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked alpha1A survival signaling in alpha1ABKO myocytes.
Our results demonstrate a direct protective effect of the alpha1A subtype in cardiac myocytes and define an alpha1A-ERK signaling pathway that is required for myocyte survival. Absence of the alpha1A-ERK pathway can explain the failure to activate ERK after aortic constriction in alpha1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.
在缺乏心肌细胞α1 - 肾上腺素能受体（α1 - AR）结合的α1 - AR基因敲除（α1ABKO）小鼠中，主动脉缩窄可诱导细胞凋亡、扩张型心肌病和死亡。然而，尚不清楚这些效应是否归因于心肌细胞α1 - AR的缺乏，以及α1A、α1B亚型或两者是否介导保护作用。因此，我们研究了培养心肌细胞中α1A和α1B亚型特异性的存活信号，以测试α1 - AR对心肌细胞的直接保护作用。
我们培养了α1ABKO心肌细胞，并用表达α1 - GFP融合蛋白的腺病毒重建α1 - AR信号。通过膜联蛋白V/碘化丙啶染色检测去甲肾上腺素、阿霉素或过氧化氢诱导的心肌细胞死亡。在α1ABKO心肌细胞中，所有这三种刺激均显著增加细胞凋亡和坏死。重建α1A亚型而非α1B亚型可使α1ABKO心肌细胞免受每种刺激诱导的细胞死亡。为了探究其机制，我们检测了细胞外信号调节激酶（ERK）的α1 - AR激活情况。在α1ABKO心脏中，主动脉缩窄未能激活ERK，而在α1ABKO心肌细胞中，组成型活性MEK1的表达使α1ABKO心肌细胞免受去甲肾上腺素诱导的死亡。此外，只有α1A - AR在α1ABKO心肌细胞中激活ERK，而显性负性MEK1的表达完全阻断了α1ABKO心肌细胞中的α1A存活信号。
我们的结果证明了α1A亚型对心肌细胞具有直接保护作用，并确定了心肌细胞存活所需的α1A - ERK信号通路。α1A - ERK通路的缺失可以解释α1ABKO小鼠主动脉缩窄后未能激活ERK的原因，并且可能导致细胞凋亡、扩张型心肌病和死亡的发生。
