Endothelial nitric oxide synthase is involved in calcium-induced Akt signaling in mouse skeletal muscle Jason A. Drenning, Vitor A. Lira, Quinlyn A. Soltow, Claire N. Canon, Lauren M. Valera, Dana L. Brown, David S. Criswell * Center for Exercise Science, Department of Applied Physiology & Kinesiology, University of Florida, Gainesville, FL, USA article info Article history: Received 12 May 2009 Revised 20 July 2009 Available online 12 August 2009 Keywords: Nitric oxide Skeletal muscle Myotubes Protein kinase B Fiber type abstract We hypothesized that targeted mutation of the endothelial nitric oxide synthase (eNOS) gene would reduce Akt-related signaling events in skeletal muscle cells, compared to wild type (WT) controls. Results show that slow myosin heavy chain (type I/b) expression and the abundance of slow-twitch fibers are reduced in plantaris muscle of eNOS / mice, compared to WT. Further, basal phosphorylation of Akt (p-Akt (Ser-473)/total Akt) and GSK-3b (GSK-3b (Ser-9)/total GSK-3b) are reduced 60–70% in primary myotubes from eNOS / mice. Treatment with the calcium ionophore, A23187 (0.4 lM, 1 h), increased phosphorylation of Akt and GSK-3b by 2-fold (P < 0.05) in myotubes from WT mice, but had no effect on phosphorylation of these proteins in eNOS / myotubes. Additionally, A23187 treatment failed to induce nuclear translocation of the transcription factor, NFATc1, in eNOS / myotubes. Treatment with the nitric oxide donor, propylamine propylamine NONOate (PAPA-NO; 1 lM for 1 h) increased Akt and GSK-3b phosphorylation, and induced NFATc1 nuclear translocation in WT and eNOS / myotubes, and eliminated differences from WT in the NOS knockout cultures. Parallel experiments in C2C12 myotubes found that Akt phosphorylation induced by NO or the guanylate cyclase activator, YC-1, is prevented by co-treatment with either a guanylate cyclase or PI3K inhibitor (10 lM ODQ or 25 lM LY2904002, respec- tively). These data suggest that eNOS activity is necessary for calcium-induced activation of the Akt path- way, and that nitric oxide is sufficient to elevate Akt activity in primary myotubes. NO appears to influence Akt signaling through a cGMP, PI3K-dependent pathway. Ó 2009 Elsevier Inc. All rights reserved. Introduction Skeletal muscle fiber type greatly influences its functional char- acteristics as well as impacting overall metabolic health [1–3]. Muscle phenotype ranges from fast glycolytic fibers, expressing type IIb or IIx myosin heavy chain (MHC) to slow oxidative fibers expressing type I/b MHC [3,4]. Fiber type proportions in adult mus- cle are generally recognized to be plastic, with chronic activity increasing expression of slow, oxidative genes, and giving muscle fibers an insulin sensitive and metabolically active phenotype [5]. We have recently reported a potential role of nitric oxide (NO) in the regulation of load-induced fast-to-slow fiber type conversion in rat skeletal muscle [6,7], and in the activation of the transcrip- tion factor, nuclear factor of activated T-cells (NFAT), in cultured myotubes exposed to a calcium ionophore [8]. NFAT is a key initi- ator of the slow-twitch phenotype. Intracellular calcium transients during muscle recruitment induce calcineurin-dependent dephos- phorylation of NFAT, allowing its nuclear translocation and DNA binding [9]. This signal is suppressed by glycogen synthase ki- nase-3b (GSK-3b) and casein kinase, which re-phosphorylate NFAT [10]. Since GSK-3b can be inhibited by Akt-dependent phosphory- lation, we postulated that NO affects Akt activation in skeletal muscle cells. Of the three nitric oxide synthase (NOS) isoforms, the endothelial and neuronal isoforms (eNOS and nNOS, respectively) are calcium- sensitive and constitutively expressed in skeletal muscle [11]. These enzymes synthesize NO at low levels compared to the inducible NOS isoform [12], and are activated by skeletal muscle contractions [13]. NO is associated with inhibitory phosphorylation of GSK-3b, facilita- tion of NFAT-dependent transcription, and expression of slow myo- sin heavy chain (MHC I/b) in C2C12 myotubes [8]. Hence, these experiments sought to investigate the significance of NO involve- ment in the GSK-3b/NFAT pathway in adult skeletal muscle and primary mouse myotube cultures, and furthermore, to determine NOS isoform specificity by studying the GSK-3b/NFAT pathway in mice harboring a targeted mutation of the eNOS gene. The phosphoinositide 3-kinase (PI3K)/Akt pathway is a commonly described pathway for inhibitory phosphorylation of 1089-8603/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2009.08.001 * Corresponding author. Address: P.O. Box 118206, Center for Exercise Science, University of Florida, Gainesville, FL 32611, USA. Fax: +1 352 352 0316. E-mail address: dcriswell@hhp.ufl.edu (D.S. Criswell). Nitric Oxide 21 (2009) 192–200 Contents lists available at ScienceDirect Nitric Oxide journal homepage: www.elsevier.com/locate/yniox