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

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.

The role of activation of two different sGC binding sites by NO‐dependent and NO‐independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes

“…Recent works in this area are not related to cell elongation. For example, in studies on isolated murine cardiomyocytes loaded with a highly specific copper dye for NO, the authors observed a single transient signal of NO production after any particular event of electrical stimulation (Mosqueira et al, 2021 ). Application of specific NOS isoform blockers or NO scavengers causes significant NO transient inhibition.…”

nNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways

“…These aspects mentioned above were relevant owing to the possibility of independent evaluation without interference of two known pathways, through which NO exerts its effect: either via NO-sGC-PKG or post-translational modification from peroxynitrite nitrosylating thiol groups of cysteines, well-known as S-nitrosylation (SNO) ( Eu et al, 2000 ; Mosqueira et al, 2013 ) or via nitration of tyrosine rings ( Viner et al, 1999 ; Squier and Bigelow, 2000 ; Uda et al, 2020 ). Taking advantage of the standard pharmacological tools, we first used the sGC’s activator Bay41-2272 and the blocker ODQ ( Drenning et al, 2008 ; Makris et al, 2010 ; Mosqueira et al, 2021 ). Leaving aside the results of excitability parameters, the results from force-frequency and single twitch biophysical parameters showed that Bay41-2272 and ODQ induced similar effects as observed with SNAP and L-NAME, respectively, suggesting that NO would modulate force production via sGC.…”

“…Leaving aside the results of excitability parameters, the results from force-frequency and single twitch biophysical parameters showed that Bay41-2272 and ODQ induced similar effects as observed with SNAP and L-NAME, respectively, suggesting that NO would modulate force production via sGC. The next point of investigation addressed PKG, which is activated by cGMP-produced sGC, using a specific agonist [8-pCPT-cGMP Ishikawa et al (2019) , Mosqueira et al (2021) ] and a blocker (DT-3 ( Takahashi et al, 2008 )). Supporting previous results, the analysis of rheobase differed from SNAP and L-NAME, but no effect was seen in the analyses of rheobase for Bay41-2272 and ODQ.…”

“…Next, we evaluated whether the post-translational modification from peroxynitrite production to form either SNO or nitration would take part in the modulatory effect observed above using two compounds in skeletal muscle ( Porter Moore et al, 1999 ; Salanova et al, 2013 ; Dutka et al, 2017 ): N-ethylmaleimide [NEM ( Xiyuan et al, 2017 ; Mosqueira et al, 2021 )] and ascorbic acid [AA ( Holmes and Williams, 2000 ; Ferrer-Sueta et al, 2018 ; Mosqueira et al, 2021 )]. Surprisingly, both antioxidants produced the same effect on rheobase, increasing it to the same level as it was observed with L-NAME.…”

Substrate Stiffness Influences Structural and Functional Remodeling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes

“…In addition to reactive oxygen and nitrogen species, Ca 2+ -dependent RyR2 activation is physiologically regulated by phosphorylation via protein kinase A (PKA) and Ca 2+ /calmodulin-dependent kinase II (CaMKII; Niggli et al, 2013 ). Interestingly, increased RyR2 sensitivity mediated by the neuronal isoform of the nitric oxide synthase (nNOS) and CaMKII was shown to be induced by multiaxial mechanical stress during cardiomyocyte contraction ( Jian et al, 2014 ; Mosqueira et al, 2021 ). As cellular afterload response results in modulation of RyR2 activity in adult cardiomyocytes, substrates with different stiffnesses may influence signaling pathways leading to altered Ca 2+ sensitivity of RyR2s in iPSC-CMs as well.…”