Balance betweenS-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation

Abstract

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form -nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without -nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

nNOS/GSNOR interaction contributes to skeletal muscle differentiation and homeostasis

“…These results strongly argue for GSNOR playing a role in myogenesis, this being in perfect agreement with recent data 30 and previous evidence showing that Gsnor −/− mice show a delayed muscle regeneration following injury 12 .…”

“…In line with data arguing for a pivotal role of GSNOR and S -nitrosylation in myoblast differentiation 30 and muscle regeneration 12,31 , we also observed that GSNOR is involved in myogenesis. This is probably due to its recruitment in the same complex with nNOS, which becomes detectable at day 4 when other markers of differentiation are still expressed (Fig.…”

The Role of Embryonic Chick Muscle Cell Culture in the Study of Skeletal Myogenesis

“…Importantly, chick skeletal muscle cell cultures have some disadvantages, such as the lack of widespread genetic manipulations, which are cutting edge approaches in other animal models of myogenesis, such as the fruit fly Drosophila ( Bryantsev et al, 2019 ). Nevertheless, the genetic approach weakness of the chick muscle culture model has been, in part, surpassed by some recent advances in the transfection of chick cultured cells with siRNA targeted to specific proteins and fluorescent/luminescent protein reporter systems ( Mermelstein et al, 2007 ; Possidonio et al, 2016 ; Yamashita et al, 2017 ; Bagri et al, 2020 ; de Andrade Rosa et al, 2020 ).…”

Anti-asthmatic effect of nitric oxide metallo-donor FOR811A [cis-[Ru(bpy)2(2-MIM)(NO)](PF6)3] in the respiratory mechanics of Swiss mice

“…It is known that NO can control the proliferation and differentiation of muscle cells through the activation of sGC and, consequently, increased levels of cGMP. On the other hand, it is also believed that NO can control some of these events through an independent mechanism, PLOS ONE such as the formation of S-nitrosothiols, which can modulate the formation of muscle fibers [41].…”