Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) transient. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00395-021-00860-0.