Key points• Exercise is known to trigger skeletal muscle structural and functional adaptations.• Control of these adaptive alterations is a complex process involving multiple signalling pathways and levels of regulation.• The well-characterized calcineurin-nuclear factor of activated T-cells (NFATc1) signalling pathway is involved in the regulation of activity-dependent alterations in skeletal muscle myosin heavy chain expression. Myosin heavy chain is a contractile protein that largely dictates a muscle's speed of contraction.• We show that a signalling molecule called nitric oxide may be regulating alterations in myosin heavy chain expression via activity-modulated calcineurin-NFATc1 signalling.• These findings increase our understanding of how skeletal muscle adaptive alterations are regulated. AbstractThe calcineurin-NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin-NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml −1 ) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day −1 ) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3β phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3β-induced facilitation of calcineurin-NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling.

Phex (a phosphate-regulating gene with homologies to endopeptidases on the X chromosome) is expressed predominantly in bone in which it has been implicated in the mineralization process. Multiple factors and hormones, including PTHrP, regulate formation, development, and/or homeostasis of bone. The purpose of the present study was to determine whether PTHrP(1-34) regulates Phex expression and identify the signaling pathway used. Phex mRNA and protein levels were analyzed by RT-PCR and immunoblotting, respectively. In UMR-106 cells, PTHrP(1-34) caused a time- and concentration-dependent decrease in Phex expression. Forskolin, an adenylate cyclase activator, had the same effect. Dibutiryl cAMP also decreased Phex expression, and its effect was blocked by H89, a protein kinase A (PKA) inhibitor. In contrast, 12-O-tetradecanoyl phorbol-13-acetate, a protein kinase C (PKC) activator, increased Phex expression in a time- and dose-dependent manner. This effect was reversed by bisindolylmaleimide Iota, a PKC inhibitor. Bovine PTH(3-34), which activates PKC but not PKA, had no effect. On the contrary, human PTH(1-31), which activates PKA but not PKC, decreased Phex expression. H89 but not bisindolylmaleimide Iota blocked the effect of PTHrP(1-34). PTHrP(1-34) also decreased Phex expression in cultures of fetal rat calvaria cells at d 7 of culture but not at later stages. These data demonstrate that PTHrP(1-34), through PKA, down-regulates Phex expression in osteoblasts.

PHosphate-regulating gene with homology to Endopeptidase on the X chromosome (PHEX) has been identified as the gene mutated in X-linked hypophosphatemia (XLH) syndrome, the most prevalent form of rickets in humans. The predominant expression of PHEX in bones and teeth, and the defective mineralization of these tissues in XLH patients indicate that PHEX is an important regulator of mineralization. Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are known to regulate the expression of numerous genes in osteoblastic cells through activation of the protein kinase A pathway, including repression of PHEX. PTH also activates the transcriptional repressor E4BP4 through the same pathway, suggesting that PTH or PTHrP-mediated repression of PHEX expression could involve E4BP4. To evaluate this possibility, we treated UMR-106 osteoblastic cells with PTHrP(1-34), and used RT-PCR and immunoblotting to analyze PHEX and E4BP4 expression. E4BP4 mRNA and protein levels were rapidly increased in cells treated with PTHrP(1-34), with a concomitant decrease in PHEX expression. This downregulation of PHEX could be reproduced by overexpression of E4BP4. Moreover, PTHrP(1-34)-mediated PHEX repression was blocked when cells were transfected with a siRNA targeting E4BP4 mRNA. Finally, DNA pull-down and luciferase assays showed that two E4BP4 response elements located in PHEX promoter were functional. These results underline the important role of E4BP4 in osteoblastic cells and further define the repression mechanism of PHEX gene by PTHrP(1-34).

Involvement of calcineurin/NFAT signaling in the regulation of skeletal muscle myosin heavy chain (MHC)‐based fast‐to‐slower isoform adaptations (F‐S) is recognized. Less is known about the role nitric oxide (NO) plays in this process.OBJECTIVETo investigate the necessity of NO in activity‐induced skeletal muscle F‐S in vivo.METHODSEndogenous NO production was blocked by administering L‐NAME (0.75 mg ml−1; ~100 mg kg−1 day−1) during 0, 1, 2, 5 or 10 days of chronic low‐frequency stimulation (CLFS; 10 Hz, 12 h d−1) applied to the tibialis anterior and extensor digitorum longus muscles of rats (L‐Stim; n=6 each group). Control rats received only CLFS (Stim; n=6 each group).RESULTSCLFS induced increases in NFATc1 nuclear localization in Stim, which did not occur in L‐Stim at any time point. These results were confirmed by western blot analyses. Moreover, MHC mRNA, protein and fibre type analyses revealed CLFS‐induced F‐S occurred in Stim, but were completely abolished in L‐Stim.CONCLUSIONSNO may be regulating activity‐induced MHC‐based F‐S at the transcriptional level via NFAT nuclear accumulation in vivo. *Authors contributed equally. Funded by NSERC, AHFMR, CIHR and CRC.

Involvement of Cn/NFAT signaling in the regulation of the skeletal muscle fiber phenotype is recognized, however, the role of the various Cn‐regulated NFAT isoforms in this process remains rudimentary. We thus assessed the expression, localization and function of NFAT isoforms in muscle fibers of wild type or transgenic NFATc2 or NFATc3 deficient mice under normal weightbearing or functional compensatory work overload conditions. Immunohistochemical analyses indicated that NFAT isoforms have distinct roles in the regulation of muscle remodeling associated with compensatory growth. Semi‐quantitative RT‐PCR did not reveal compensatory up‐regulation of surviving NFAT isoform mRNA levels in transgenic mice. On the other hand, western blotting and immunofluorescence data showed NFAT protein localization to be differentially regulated during compensatory growth. Moreover, gene array analyses showed distinct gene expression patterns across conditions. Taken together, these results show that members of the NFAT family of transcription factors have distinct roles in the expression of the muscle fiber phenotype and a limited capacity to substitute for one another during conditions of rapid fiber growth. Supported by NSERC, CIHR and CRC to RNM. EMK is a CIHR scholar.