Grant-funded Project Nr. 111/2005/C/PřF
Final Report

Project title:Effect of NO synthases activity on voltage-gated sodium channels expression in skeletal muscle.
Research leader:Mgr. Ivana Švandová
Co-researcher: Pavel Ostašov; Zdeňka Drastichová
Period of project:2005-2005
Overall grant:170 000 CZK

Project Results

  Denervation of skeletal muscle is associated with some substantial functional and pharmacological changes in the sarkolemma. It is now well established that in adult innervated skeletal muscle, voltage-gated sodium channels 1.4 (NaV 1.4 ICs) isoforms are dominant whereas 1.5 isoforms (NaV 1.5 ICs) become highly abundant after denervation. Expression of NaV 1.4 ICs mRNA is unchanged at the neuromuscular junction after denervation although the endplate NaV 1.4 ICs density is reduced by 40-50%. On the contrary, NaV 1.5 ICs mRNA level increases up to 50-fold after denervation, with channel endplate density increase of about 43%.  NaV 1.5 ICs expression can be down-regulated by direct muscle stimulation, thus simulating normal motor nerve activity. It is also established that first typical attribute of denervation, early postdenervation depolarization, is controlled via nitric oxide synthase activity and production of nitric oxide, which can diminish this phenomenon.
  The experiments in this project were carried out with the aim to find whether (1) administration of NO donors to the denervated animals could diminish/revert characteristic postdenervation expresion of 1.5 isoform of NaV ICs and (2) whether inhibition of NO synthases (thus simulating failure of NO signal system) could elicit changes in expression of 1.5 and 1.4  isoforms of NaV ICs similar to those observed in denervated muscles, respectively.
  In the first series of experiments, we tested the hypothesis that increased NO synthases activity mimicked by nitric oxide donor SNP (sodium nitroprusside) addition can revert NaV 1.4/NaV 1.5 ICs ratio after denervation. Sciatic nerves of male Wistar rats (body weight 120-150 g) were dissected and animals were denervated for 7 days. Denervated animals were daily treated with i.p. injections of sodium nitroprusside (SNP; 1.5 mg/kg). Then, extensor digitorum longus (EDL) muscles of control, denervated and denervated/SNP-treated animals were removed and crude membranes for NaV 1.4/1.5 ICs immunodetection were prepared. We did not separate junctional and extra-junctional membranes. In a slight contrast with literary evidence, we were able to detect very low but not completely insignificant levels of NaV 1.5 ICs in control innervated muscles, too, with NaV 1.5 ICs level 4.9-fold higher in denervated than in control muscles. After SNP treatment, NaV 1.5 ICs level reached 54% of NaV 1.5 ICs level in denervated muscles. Ratio of NaV 1.4 ICs level remained constant in control/denervated and SNP-treated/denervated animals, respectively. We conclude that NO-donor supply diminished NaV 1.5 IKs expression in denervated muscles.
  In the second series of experiments, the NaV 1.4/NaV 1.5 ICs ratio was examined in the EDL muscle of intact rats treated  for 7 days with intraperitoneal injections of NO syntahses inhibitors, L-NAME, in the daily dose of 50 mg/kg, and with S-ethylthiourea, in the daily dose of 12.5 mg/kg, and compared with  NaV 1.4/NaV 1.5 ICs ratio in control EDL muscles. After NO synthases inhibition, NaV 1.4 ICs level reached 41.4% of NaV 1.4 ICs level in control muscles. NaV 1.5 ICs level in animals treated with NO synthases inhibitors was 4.3-fold higher than in control animals. We conclude that inhibition of NO synthases stimulates NaV 1.5 ICs and decreases 1.4 NaV ICs expression in skeletal muscles, respectively.
  The data are in accordance with our previous observations and further support the view that the activity of NO cascade significantly participates in regulation of the function state of the neuromuscular junction. Results were presented at 82. Fyziologické dny, Prague, and abstract was published in Physiological Research.