Impact of Rad-mediated inhibition of Cav1.1 on muscle composition and contractile function

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Expression of the monomeric G protein Rad (Ras-like Associated with Diabetes) is elevated in skeletal muscle of Amyotrophic Lateral Sclerosis (ALS) patients and mouse models of neuromuscular disease. The elevated expression of Rad is likely to impact muscle function because Rad is a constitutively-active inhibitor of the skeletal muscle L-type Ca2+ channel (CaV1.1), which serves as the voltage-sensor for excitation-contraction (EC) coupling. It has been established that impaired EC coupling contributes to muscle weakness in multiple neuromuscular disorders such as ALS. This depression of excitability (termed “EC un-coupling”) is a direct consequence of a fewer number of functional CaV1.1 channels present in the plasma membrane. In addition, targeted knock-down of CaV1.1 in vivo causes significant muscle atrophy. Taken together, these observations suggest that Rad blunts force generation and can initiate morphological changes in skeletal muscle. Indeed, preliminary data indicate that overexpression of Rad in normal mouse skeletal muscle impairs EC coupling, causes profound atrophy and changes muscle metabolic profile. Thus, the purpose of this R03 small grant proposal is to investigate the broader consequences of chronic inhibition of CaV1.1 in skeletal muscle. Specific Aim 1 will determine whether long-term Rad-induced impairment of EC coupling manifests in reduced muscle force generation. To do so, otherwise normal gastrocnemius muscle will be infected with an Adeno-Associated Virus (AAV1) encoding a skeletal muscle-targeted Venus fluorescent protein-Rad fusion construct (tMCK-V-Rad). Two, four, and six months post-infection, the ability of tMCK-V-Rad to reduce absolute and/or specific muscle force generation in response to nerve and direct muscle stimulation will be evaluated using an in vivo force assay. Specific Aim 2 will investigate the idea that elevated Rad expression can drive changes in muscle mass and composition similar to those observed in progressive neurodegenerative disease. In these experiments, normal mouse hindlimb muscles infected with tMCK-V-Rad will be harvested, frozen, sectioned and probed with antibodies directed to Type I, Type IIA, Type IIX and Type IIB myosin heavy chains to test the hypothesis that elevated Rad expression causes a shift in muscle fiber-type profile from more glycolytic fibers towards more oxidative fibers.