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Abstract：
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.
摘要：
心脏兴奋-收缩偶联以及代谢和信号活动由一氧化氮（NO）集中调节，它是由三种NO合成酶（NOSs）之一产生的。尽管在不同病理生理条件下，NO在心脏Ca2+稳态调节中具有重要作用，例如杜氏肌营养不良症（DMD），没有精确的方法描述生产，NO的来源或作用通过两个NO信号通路:可溶性鸟苷酸环化酶-蛋白激酶G(NO-sGC-PKG)和S-亚硝基化(SNO)。使用一种新策略，涉及分离的鼠心肌细胞，其中装载了对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.NO瞬态信号在Rhod-2Ca2+瞬态信号开始之后67.5ms开始,并持续大约430ms。特定的NOS同工型阻断剂或NO清除剂显著抑制NO瞬时，表明野生型（WT）心肌细胞产生依赖nNOS的NO瞬变。相反，mdx心肌细胞中的NO瞬时，DMD的小鼠模型,依赖于诱导型NOS（iNOS）和内皮（eNOS）。在连续的刺激方案中，WT心肌细胞中nNOS依赖的NO瞬变通过NO-sGC-PKG显着降低了下一个Ca2瞬变。在mdx心肌细胞中，这种抑制作用是iNOS和eNOS依赖性的，并通过SNO途径发生。WT心肌细胞中的基础NO产生是nNOS和iNOS依赖性的，mdx心肌细胞中的eNOS和iNOS依赖性的。这些结果表明，在毫秒级的膜去极化后，心肌细胞会产生NO同种型依赖性瞬变，从而激活特定的信号传导途径，从而负向调节随后的Ca2瞬态。
