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

• Published in: American Journal of Physiology: Cell Physiology 2017-07, Vol.313 (1), p.C11-C26
• Main Authors: Yamashita, Aline M S, Ancillotti, Maryana T C, Rangel, Luciana P, Fontenele, Marcio, Figueiredo-Freitas, Cicero, Possidonio, Ana C, Soares, Carolina P, Sorenson, Martha M, Mermelstein, Claudia, Nogueira, Leonardo
• Format: Article
• Language: English
• Subjects: Aldehyde Oxidoreductases - antagonists & inhibitors; Aldehyde Oxidoreductases - genetics; Aldehyde Oxidoreductases - metabolism; Animals; Cell Differentiation; Cell Fusion; Cell proliferation; Cells; Chick Embryo; Cyclic AMP - metabolism; Cyclic AMP - pharmacology; Cyclic GMP; Cyclic GMP - analogs & derivatives; Cyclic GMP - pharmacology; Cysteine - analogs & derivatives; Cysteine - metabolism; Cysteine - pharmacology; Cytology; Enzyme Inhibitors - pharmacology; Fibroblasts; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - metabolism; Fusion; Gene Expression Regulation, Developmental; Guanylate cyclase; Morphology; Muscle Development - drug effects; Muscle Development - genetics; Muscle Fibers, Skeletal - cytology; Muscle Fibers, Skeletal - drug effects; Muscle Fibers, Skeletal - metabolism; Myoblasts; Myoblasts - cytology; Myoblasts - drug effects; Myoblasts - metabolism; Myogenesis; Myotubes; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Nitric-oxide synthase; Primary Cell Culture; Reductase; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; S-Nitrosoglutathione - metabolism; S-Nitrosothiols - metabolism; S-Nitrosothiols - pharmacology; Signal Transduction; siRNA; Skeletal muscle; Soluble Guanylyl Cyclase - genetics; Soluble Guanylyl Cyclase - metabolism; Soluble Guanylyl Cyclase - pharmacology; Studies; Supplements; Thionucleotides - pharmacology; Triazenes - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00140.2016

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.