Skeletal Muscle Chloride Channel, a Biophysical Sensor of Dystrophic Progression in Mdx Mouse, is a Potential Target of Pro-inflammatory Mediators

Abstract

A decrease in resting chloride channel conductance (gCl) characterizes myofibers of dystrophic mdx mouse in relation to both spontaneous degeneration, as in diaphragm, or exercise-induced damage as in fast-twitch EDL muscle (De Luca et al., J. Pharmacol. Exp. Ther. 2003). The molecular mechanism underlying gCl impairment might involve change in ClC-1 channel expression/turnover and/or function. Considering the role of inflammation in dystrophic damage, we tested if pro-inflammatory mediators may have ClC-1 channel as a target, through phosphorylating/dephosphorylating pathways. Two microelectrodes current clamp recordings were used to measure resting gCl in EDL and diaphragm muscle fibers from adult wild-type (wt) and mdx mice. In line with previous evidences, the in vitro application of phorbol dibutyrate (50 microM) reduced gCl of wt EDL myofibers from 2610±240 microS/cm2 (n=30) to 1265±180 microS/cm2 (n=15). The application of TNF-alpha (1-30 ng/ml), a cytokine highly expressed in dystrophic muscle, to wt EDL fibers reduced gCl in a concentration-dependent manner with a maximal significant 20% decrease at 10-30 ng/ml. Angiotensin-II (10-100nM), possibly involved in muscle degeneration and oxidative stress, produced a concentration-dependent decrease of gCl in wt EDL myofibers, with a 40 % decrease at 100 nM. The PKC-inhibitor chelerythrine (1 microM) contrasted the effect of either phorbol ester, TNF-alpha or angiotensin-II. The application of 3.3 nM IGF-1 to diaphragm and EDL muscle fibers from exercised mdx mice, significantly counteracted the 40% impairment of gCl. Okadaic acid (0.25 microM) fully prevented IGF-1 effect, supporting the involvement of a serine-threonine phosphatase in IGF-1 activity (De Luca et al., Br. J. Pharmacol. 1998). Angiotensin receptor antagonists and other tools are in use to gain further insight in the mechanisms involved in inflammation-sensitive ClC-1 impairment in muscular dystrophy (Telethon-Italy GGP05130).