Reduced resting potentials in dystrophic (mdx) muscle fibers are secondary to NF-κB-dependent negative modulation of ouabain sensitive Na+–K+ pump activity

Abstract

To examine potential mechanisms for the reduced resting membrane potentials (RP) of mature dystrophic (mdx) muscle fibers, the Na+ - K+ pump inhibitor ouabain was added to freshly isolated nondystrophic and mdx fibers. Ouabain produced a 71% smaller depolarization in mdx fibers than in nondystrophic fibers, increased the [Na+]i in nondystrophic fibers by 40%, but had no significant effect on the [Na+]i of mdx fibers, which was approximately double that observed in untreated nondystrophic fibers. Western blots indicated no difference in total and phosphorylated Na+ - K+ ATPase catalytic α1 subunit between nondystrophic and mdx muscle Examination of the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) indicated that direct application of the drug slowly hyperpolarized mdx fibers (7 mV in 90 minutes) but had no effect on nondystrophic fibers. Pretreatment with ouabain abolished this hyperpolarization, and pretreatment with PDTC restored ouabain-induced depolarization and reduced [Na+]i Administration of an NF-κB inhibitor that utilizes a different mechanism for reducing nuclear NF-κB activation, ursodeoxycholic acid (UDCA), also hyperpolarized mdx fibers. These results suggest that in situ Na+ - K+ pump activity is depressed in mature dystrophic fibers by NF-κB dependent modulators, and that this reduced pump activity contributes to the weakness characteristic of dystrophic muscle.

Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes

“…Together, this suggests that the filling state of the SR contributes significantly to the dysregulation [Ca 2ϩ ] rest observed in mdx muscles. Several reports indicate that resting membrane potentials are more positive in mdx muscles fibers than WT (27,(63)(64)(65). We have found that mdx myotubes showed a partial membrane depolarization compared with WT.…”

Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

“…Moreover, blocking this known increase in the activity of TRP channels with either a dnTRPC or a dnTRPV construct abrogated MD in mouse models of disease, suggesting that unregulated or greater activation of the TRP class of cation channels is a primary disease determinant. These channels also permeate Na ϩ , which would enhance Ca 2ϩ entry through NCX1 by favoring reverse-mode activity (7,(17)(18)(19)(20). Finally, we also previously showed that overexpression of SERCA1 in skeletal muscle, which produces higher rates of Ca 2ϩ clearance back into the SR, was protective against MD in the mouse (16).…”

“…Importantly, cytosolic Na ϩ levels appear to be elevated in myofibers from the mdx mouse, a model of Duchenne MD (7,(17)(18)(19)(20). Indeed, recent evidence suggests that intracellular Na ϩ levels are also elevated in Duchenne MD patients (21,22).…”

Pharmacologically Targeting the Primary Defect and Downstream Pathology in Duchenne Muscular Dystrophy

“…Whilst neither agent is suitable for clinical development, treatment of the mdx mouse has demonstrated that inhibition of NF-B signalling can improve strength, reduce necrosis and enhance muscle regeneration [216,221,225]. PDTC and UDCA hyperpolarise mdx fibres by counteracting negative modulation of the resting Na + -K + pump activity and, encouragingly, PDTC had no effect in non-dystrophic mice [226]. These positive results have led to further tests in the mdx with more clinically suitable substances including curcumin, the principal curcuminoid found in turmeric and a potent NF-B inhibitor [227], and flavocoxid, a mixed flavonoid extract with anti-inflammatory, antioxidant and NF-B inhibiting properties that is marketed as Limbrel® (Primus Pharamceuticals, AZ, USA) for management of osteoarthritis [228].…”