Pretreatment with cilnidipine attenuates hypoxia/reoxygenation injury in HL-1 cardiomyocytes through enhanced NO production and action potential shortening
Pretreatment with cilnidipine attenuates hypoxia/reoxygenation injury in HL-1 cardiomyocytes through enhanced NO production and action potential shortening
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DOI：
10.1038/s41440-019-0391-7
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发表时间：
2020-01
影响因子：
5.4
通讯作者：
H. Minato;I. Hisatome;Y. Kurata;T. Notsu;Naoe Nakasone;H. Ninomiya;T. Hamada;Takuya Tomomori;Akihiro Okamura;J. Miake;M. Tsuneto;Y. Shirayoshi;R. Endo;A. Otsuki;F. Okada;Y. Inagaki
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作者：
H. Minato;I. Hisatome;Y. Kurata;T. Notsu;Naoe Nakasone;H. Ninomiya;T. Hamada;Takuya Tomomori;Akihiro Okamura;J. Miake;M. Tsuneto;Y. Shirayoshi;R. Endo;A. Otsuki;F. Okada;Y. Inagaki
心肌缺血/再灌注损伤在一氧化氮合酶（NOS）缺乏的情况下发生。西尼地平是一种钙离子通道阻滞剂，有报道可激活血管内皮细胞的内皮型一氧化氮合酶（eNOS），增加一氧化氮（NO）。我们研究了西尼地平预处理是否可以减轻缺氧/复氧（H/R）损伤引起的心肌细胞死亡，包括凋亡。HL-1小鼠心房肌细胞以及H9 c2大鼠心室肌细胞暴露于H/R，细胞活力进行了评价，通过自动分析仪和流式细胞仪; eNOS表达，NO的生产，和电生理特性也进行了评估，分别通过Western印迹法，比色法，膜片钳，西尼地平的存在和不存在。西尼地平以浓度依赖性的方式增强eNOS的磷酸化和NO的产生，这被针对eNOS的siRNA或Hsp 90抑制剂格尔德霉素废除。预处理西尼地平衰减细胞死亡，包括细胞凋亡在H/R，这种效果是由NO供体和黄嘌呤氧化酶抑制剂再现。NOS抑制剂L-NAME可阻断西尼地平的保护作用。西尼地平预处理也能减轻H/R期间H9 c2细胞的死亡。在H/R期间额外的西尼地平治疗并没有显著增强其保护作用。西尼地平在正常和高钙条件下的保护作用无显著差异。西尼地平可缩短HL-1细胞动作电位时程（APD），H/R后APD缩短幅度增大。L-NAME可明显抑制西尼地平引起的APD缩短。这些结果表明西尼地平通过阻断L-型Ca ~（2+）通道，增加NO的产生，缩短APD，从而阻止HL-1细胞在H/R过程中的死亡。
Myocardial ischemia/reperfusion injury worsens in the absence of nitric oxide synthase (NOS). Cilnidipine, a Ca2+channel blocker, has been reported to activate endothelial NOS (eNOS) and increases nitric oxide (NO) in vascular endothelial cells. We examined whether pretreatment with cilnidipine could attenuate cardiac cell deaths including apoptosis caused by hypoxia/reoxygenation (H/R) injury. HL-1 mouse atrial myocytes as well as H9c2 rat ventricular cells were exposed to H/R, and cell viability was evaluated by an autoanalyzer and flow cytometry; eNOS expression, NO production, and electrophysiological properties were also evaluated by western blotting, colorimetry, and patch clamping, respectively, in the absence and presence of cilnidipine. Cilnidipine enhanced phosphorylation of eNOS and NO production in a concentration-dependent manner, which was abolished by siRNAs against eNOS or an Hsp90 inhibitor, geldanamycin. Pretreatment with cilnidipine attenuated cell deaths including apoptosis during H/R; this effect was reproduced by an NO donor and a xanthine oxidase inhibitor. The NOS inhibitor L-NAME abolished the protective action of cilnidipine. Pretreatment with cilnidipine also attenuated H9c2 cell death during H/R. Additional cilnidipine treatment during H/R did not significantly enhance its protective action. There was no significant difference in the protective effect of cilnidipine under normal and high Ca2+conditions. Action potential duration (APD) of HL-1 cells was shortened by cilnidipine, with this shortening augmented after H/R. L-NAME attenuated the APD shortening caused by cilnidipine. These findings indicate that cilnidipine enhances NO production, shortens APD in part by L-type Ca2+channel block, and thereby prevents HL-1 cell deaths during H/R.