Cardiac excitation–contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca²⁺ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca²⁺ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca²⁺ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca²⁺ transient.

心脏兴奋-收缩耦合以及代谢和信号传导活动由一氧化氮 (NO) 集中调节，一氧化氮由三种 NO 合酶 (NOS) 中的一种产生。尽管 NO 在不同病理生理条件下（如杜氏肌营养不良症 (DMD)）在心脏 Ca ²⁺稳态调节中发挥重要作用，但没有精确的方法描述 NO 通过两种 NO 信号通路产生、来源或作用：可溶性鸟苷酸环化酶蛋白激酶 G (NO-sGC-PKG) 和 S-亚硝基化 (SNO)。使用一种涉及分离的鼠心肌细胞的新策略，该细胞装载了对 NO 具有高度特异性的铜基染料，我们在每次电刺激事件后观察到单个瞬时 NO 产生信号。在 Rhod-2 Ca 开始后 67.5 ms 开始出现 NO 瞬态信号²⁺瞬态信号，持续约 430 毫秒。特定的 NOS 亚型阻滞剂或 NO 清除剂显着抑制 NO 瞬变，表明野生型 (WT) 心肌细胞产生 nNOS 依赖性 NO 瞬变。相反，mdx 心肌细胞（DMD 的小鼠模型）中的 NO 瞬态依赖于诱导型 NOS (iNOS) 和内皮细胞 (eNOS)。在连续刺激方案中，WT 心肌细胞中 nNOS 依赖性 NO 瞬变显着降低了下一个 Ca ²⁺通过 NO-sGC-PKG 瞬变。在 mdx 心肌细胞中，这种抑制作用是 iNOS 和 eNOS 依赖性的，并且通过 SNO 途径发生。基础 NO 产生在 WT 心肌细胞中是 nNOS 和 iNOS 依赖性的，而在 mdx 心肌细胞中是 eNOS 和 iNOS 依赖性的。这些结果表明，心肌细胞在膜去极化后在毫秒时间尺度上产生 NO 异构体依赖性瞬变，激活特定的信号通路以负调节随后的 Ca ²⁺瞬变。