Nitric oxide synthase inhibition prevents activity-induced calcineurin-NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions.
The 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.
Exercise Biochemistry Laboratory, Faculty of Physical Education and Recreation, University of Alberta, Edmonton, AB, Canada T6G 2H9.
Karen J B
KJ
Martins
Mathieu
M
St-Louis
Gordon K
GK
Murdoch
Ian M
IM
MacLean
Pamela
P
McDonald
Walter T
WT
Dixon
Charles T
CT
Putman
Robin N
RN
Michel
D000818:Animals; D019703:Calcineurin / Q000502:physiology*; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D018656:Muscle Fibers, Fast-Twitch / Q000502:physiology*; D018657:Muscle Fibers, Slow-Twitch / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000502:physiology; D050778:NFATC Transcription Factors / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000502:physiology; D020033:Protein Isoforms / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction
D004791:Enzyme Inhibitors; D050778:NFATC Transcription Factors; D020033:Protein Isoforms; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019703:Calcineurin; D018995:Myosin Heavy Chains; D019331:NG-Nitroarginine Methyl Ester