刘艳丽, 刘奔, 屈扬扬, 柴慧娟, 李锐, 张玲*
持久激动β肾上腺素受体(β adrenergic receptor, βAR)可导致病理性心肌肥厚，但其机制尚不明确。本研究观察抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine, NAC)对异丙肾上腺素(isoproterenol, ISO)诱导的心肌肥厚大鼠的心肌氧化应激水平及钙/钙调蛋白依赖性蛋白激酶II (calcium/calmodulin-dependent protein kinase II, CaMKII)表达的影响，探讨βAR持久激动诱发心肌肥厚机制中CaMKII和氧化应激的作用。健康雄性Wistar大鼠，随机分成4组：正常对照组(CTRL)，ISO处理组(ISO)，正常NAC组(CTRL+NAC)和ISO处理+NAC组(ISO+NAC)，每组6只。ISO及ISO+NAC组动物每天腹腔注射3 mg/kg ISO，CTRL及CTRL+NAC组腹腔注射相同体积的生理盐水；CTRL+NAC及ISO+NAC组动物每日自由饮用含15 g/L NAC的饮用水。每周用无创动脉血压仪检测鼠尾动脉血压，连续2周；以心重指数(HW/BW)和左心室组织HE染色检测心肌肥厚情况；荧光测定法检测心肌线粒体活性氧(reactive oxygen species, ROS)水平；Western blot检测左室心肌NADPH氧化酶4 (NADPH oxidase 4, NOX4)以及有活性的CaMKII (p-CaMKII/CaMKII)的表达变化。结果显示，与CTRL组相比，ISO组大鼠出现明显的心肌肥厚，心肌线粒体ROS水平升高(P < 0.05)，心肌组织NOX4和p-CaMKII的表达均显著增加(分别为CTRL组的1.4倍和1.6倍，P < 0.05)；NAC显著改善了ISO诱导的心肌肥厚，降低了ISO诱导的心肌线粒体ROS过度生成(P < 0.05 vs ISO)，有效抑制了ISO诱发的NOX4及p-CaMKII表达上调(P < 0.05 vs ISO)；CTRL+NAC组各项指标与CTRL组大鼠无差异；各组动物尾动脉血压亦无显著性差异。上述结果提示，氧化应激和CaMKII在βAR持久兴奋诱发心肌肥厚机制中起重要作用，NAC可以通过抑制心肌细胞线粒体及NADPH氧化酶应激途径降低氧化应激水平，抑制CaMKII激活，从而改善βAR持久激动引起的心肌肥厚。
关键词： 氧化应激; 钙/钙调蛋白依赖性蛋白激酶; β肾上腺素受体; 心肌肥厚
[Oxidative stress and CaMK II contribute to the development of sustained-βAR-stimulated cardiac hypertrophy in rats] [Article in Chinese]
LIU Yan-Li, LIU Ben, QU Yang-Yang, CHAI Hui-Juan, LI Rui, ZHANG Ling*
Department of Physiology and Pathophysiology, Basic Medical School, Tianjin Medical University, Tianjin 300070, China
Sustained activation of β adrenergic receptor (βAR) leads to pathologic cardiac hypertrophy. However, the related mechanisms still remain unclear. In this study, we observe how N-acetylcysteine (NAC) affects the oxidative stress and calcium/calmodulin-dependent protein kinase II (CaMKII) expression in heart of isoproterenol (ISO)-stimulated rats, and investigate whether oxidative stress and CaMKII contribute to the development of sustained βAR-stimulated cardiac hypertrophy. Healthy male Wistar rats were randomly separated into 4 groups: control (CTRL), ISO-treated (ISO), control with NAC supplement (CTRL+NAC) and ISO-treated with NAC supplement (ISO+NAC) groups (6 rats in each group). Systolic blood pressure (SBP) was measured in awake rats with the tail-cuff method every week for two weeks. Heart weight/body weight ratio (HW/BW) and HE staining were used for the detection of myocardial hypertrophy. Myocardial mitochondrial reactive oxygen species (ROS) levels were measured by DCF fluorometry. The expressions of activated-CaMKII (p-CaMKII/CaMKII) and NADPH oxidase 4 (NOX(4)) were determined by Western blot analysis. The results showed that ISO-treated (i.p., daily 3 mg/kg, 2 weeks) rats developed an obvious cardiac hypertrophy as expressed by increases of HW/BW and myocyte cross-section area. Cardiac mitochondrial ROS level was significantly enhanced in ISO group as compared to CTRL group (P < 0.05). The expressions of NOX(4) and p-CaMKII in ISO group were also up-regulated as compared to CTRL group (1.4 and 1.6 times of CTRL, respectively, P < 0.05). NAC supplement significantly suppressed the hypertrophic development of heart in ISO-stimulated rats. The cardiac mitochondrial ROS level showed a significant decrease in rats of ISO+NAC group (P < 0.05 vs ISO). In accordance with this, ISO+NAC group rats also showed marked reductions in the expressions of NOX(4) and p-CaMKII/CaMKII compared to ISO group rats (P < 0.05). There were no significant differences of the detected indices between the rats from CTRL+NAC and CTRL groups. SBP showed no differences among four groups. These results suggest that both oxidative stress and CaMKII play important roles in sustained βAR-stimulated cardiac hypertrophy. NAC may suppress ISO-induced cardiac hypertrophy by down-regulating the expression of activated-CaMKII, and by reducing the level of oxidative stress originated from mitochondria and NADPH oxidase pathways.
Key words: oxidative stress; calcium/calmodulin-dependent protein kinase II; β adrenergic receptor; cardiac hypertrophy
通讯作者：张玲 E-mail: firstname.lastname@example.org
刘艳丽, 刘奔, 屈扬扬, 柴慧娟, 李锐, 张玲. 氧化应激和CaMKⅡ参与β-肾上腺素受体持久激动引起的大鼠心肌肥厚 [J]. 生理学报 2013; 65 (1): 1-7.
LIU Yan-Li, LIU Ben, QU Yang-Yang, CHAI Hui-Juan, LI Rui, ZHANG Ling. [Oxidative stress and CaMK II contribute to the development of sustained-βAR-stimulated cardiac hypertrophy in rats] [Article in Chinese]. Acta Physiol Sin 2013; 65 (1): 1-7 (in Chinese with English abstract).