Rad is an Agent of Skeletal Muscle Atrophy

Rad, a member of the RGK (Rad, Rem, Rem2, Gem/Kir) family of small GTP binding proteins, is expressed at very low levels in “normal” skeletal muscle. However, Rad expression is substantially upregulated in muscle of sporadic ALS patients and two established familial ALS mouse models just prior to presentation of symptoms. Rad is a potent, endogenous inhibitor of skeletal muscle L-type Ca2+ channels (CaV1.1). Recently, downregulation of CaV1.1 expression via exon skipping was reported to cause profound atrophy and fibrosis. In light of the increased Rad expression in ALS muscle, we investigated the effect(s) of long-term elevated Rad expression in otherwise normal skeletal muscle. To do so, we injected a serotype 1 Adeno-Associated Virus encoding a muscle-specific, tMCK promoter-driven Venus-Rad fusion construct into various mouse hindlimb muscles. Five months post-injection, we observed profound atrophy of V-Rad-expressing tibialis anterior (TA) muscles. These TA muscles displayed extensive fibrosis and considerable reductions in fiber diameter and area relative to naïve and GFP-expressing control TA muscles. Examination of V-Rad-expressing extensor digitorum longus fibers in thin sections revealed a general disruption of muscle ultrastructure. L-type currents and intramembrane charge movement were both reduced (∼55% and ∼34%, respectively) in V-Rad-expressing flexor digitorum brevis (FDB) fibers in comparison to naïve and GFP-expressing fibers. Likewise, depolarization-induced myoplasmic Ca2+ transients were reduced (∼55%) in V-Rad-expressing FDB fibers. The reduction in transient amplitude appeared not be a consequence of a reduced SR Ca2+ store because control and V-Rad-expressing FDB fibers had similar responses to 4-cholo-m-cresol (p > 0.05). Taken together, our results indicate that Rad can engage atrophic signaling in skeletal muscle, perhaps as an effect of inactivity due to impaired EC coupling. Supported by the Boettcher Foundation (R.A.B.), 2T32AG000279-11 (R.S. Schwartz) and 2PO1 AR052354 (P.D. Allen PI; CFA Core D).