title: 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 formS-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 ofS-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 withoutS-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.
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Aline M S
authors.initials: AMS
authors.lastname: Yamashita
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Maryana T C
authors.initials: MTC
authors.lastname: Ancillotti
authors.affiliation: Departamento de Análise Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and.
authors.forename: Luciana P
authors.initials: LP
authors.lastname: Rangel
authors.affiliation: Laboratório de Biologia Molecular do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marcio
authors.initials: M
authors.lastname: Fontenele
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Cicero
authors.initials: C
authors.lastname: Figueiredo-Freitas
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Possidonio
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Carolina P
authors.initials: CP
authors.lastname: Soares
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Claudia
authors.initials: C
authors.lastname: Mermelstein
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; lnogueira@bioqmed.ufrj.br.
authors.forename: Leonardo
authors.identifier: http://orcid.org/0000-0001-5194-9126
authors.initials: L
authors.lastname: Nogueira
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D002459:Cell Fusion; D002642:Chick Embryo; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D024510:Muscle Development / Q000187:drug effects / Q000235:genetics*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000378:metabolism; D026403:S-Nitrosothiols / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014226:Triazenes / Q000494:pharmacology
chemical_list: C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D026403:S-Nitrosothiols; D013873:Thionucleotides; D014226:Triazenes; C083763:8-bromoguanosino-3',5'-cyclic monophosphorothioate; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; C021315:S-nitrosocysteine; D000242:Cyclic AMP; D052247:Nitric Oxide Synthase Type II; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D003545:Cysteine
keywords: S-nitrosothiols; denitrosylation; myogenesis; soluble guanylyl cyclase