Endothelial nitric oxide synthase (eNOS) activation in the heart plays a key role in exercise-induced cardioprotection during ischemia–reperfusion, but the underlying mechanisms remain unknown. We hypothesized that the cardioprotective effect of exercise training could be explained by the re-localization of eNOS-dependent nitric oxide (NO)/S-nitrosylation signaling to mitochondria. By comparing exercised (5 days/week for 5 weeks) and sedentary Wistar rats, we found that exercise training increased eNOS level and activation by phosphorylation (at serine 1177) in mitochondria, but not in the cytosolic subfraction of cardiomyocytes. Using confocal microscopy, we confirmed that NO production in mitochondria was increased in response to H₂O₂ exposure in cardiomyocytes from exercised but not sedentary rats. Moreover, by S-nitrosoproteomic analysis, we identified several key S-nitrosylated proteins involved in mitochondrial function and cardioprotection. In agreement, we also observed that the increase in Ca²⁺ retention capacity by mitochondria isolated from the heart of exercised rats was abolished by exposure to the NOS inhibitor L-NAME or to the reducing agent ascorbate, known to denitrosylate proteins. Pre-incubation with ascorbate or L-NAME also increased mitochondrial reactive oxygen species production in cardiomyocytes from exercised but not from sedentary animals. We confirmed these results using isolated hearts perfused with L-NAME before ischemia–reperfusion. Altogether, these results strongly support the hypothesis that exercise training increases eNOS/NO/S-nitrosylation signaling in mitochondria, which might represent a key mechanism of exercise-induced cardioprotection.

心脏中的内皮一氧化氮合酶 (eNOS) 激活在缺血再灌注期间运动诱导的心脏保护中起关键作用，但其潜在机制仍不清楚。我们假设运动训练的心脏保护作用可以通过 eNOS 依赖性一氧化氮 (NO)/ S-亚硝基化信号重新定位到线粒体来解释。通过比较运动（每周 5 天，共 5 周）和久坐的 Wistar 大鼠，我们发现运动训练增加了线粒体中的 eNOS 水平和磷酸化（在丝氨酸 1177 处），但在心肌细胞的胞质亚组分中没有。使用共聚焦显微镜，我们证实线粒体中 NO 的产生随着 H ₂ O _{2 的}增加而增加来自运动但不是久坐的大鼠的心肌细胞暴露。此外，通过S-亚硝基蛋白质组学分析，我们确定了几种参与线粒体功能和心脏保护的关键S-亚硝基化蛋白。一致地，我们还观察到 Ca ^2+的增加通过暴露于 NOS 抑制剂 L-NAME 或还原剂抗坏血酸盐（已知可将蛋白质脱硝基化）消除了从运动大鼠心脏分离的线粒体的保留能力。与抗坏血酸盐或 L-NAME 预孵育也增加了来自运动但不来自久坐动物的心肌细胞中线粒体活性氧的产生。我们使用在缺血再灌注前灌注 L-NAME 的离体心脏证实了这些结果。总之，这些结果强烈支持运动训练增加线粒体中 eNOS/NO/ S-亚硝基化信号传导的假设，这可能代表运动诱导的心脏保护的关键机制。