Calcium Leak-Dependent Muscle Function Loss in Aged C elegans

? DESCRIPTION (provided by applicant): Sarcopenia, defined as an age-related loss of muscle mass and function, is a burgeoning health problem with a prevalence as high as 30% in the geriatric population. Current treatments have low long- term efficacy and do not target any underlying causes of muscle function loss. Recent research in aged mice in our lab has implicated the Ryanodine Receptor (RyR1), an intracellular calcium release channel important for excitation-contraction coupling, as a potential therapeutic target. Over time, RyR1 undergoes posttranslational modification ('remodeling') that causes calstabin1, an important RyR1 modulatory subunit, to dissociate from the channel; this results in increased RyR1 open probability and Ca2+ 'leak'. This Ca2+ 'leak' cause nearby mitochondria to overload with calcium and produce excess reactive oxygen species (ROS), which exacerbate the leak by further remodeling RyR1. Due to the relatively long lifespan of mice we were testing if C. elegans could serve as a better model of age- dependent muscle function loss. C. elegans contain UNC-68, the RyR1 homologue, and FKB-2, a calstabin homologue; therefore, we hypothesize that 1) FKB-2 dissociation destabilizes UNC-68 channels, resulting in pathogenic sarcoplasmic reticulum (SR) calcium leak and subsequent muscle weakness in aged nematodes and 2) mitochondrial ROS oxidizes nearby UNC-68 channels and exacerbates SR calcium leak in body wall muscle. Aim One will test whether leaky UNC-68 channels play a role in C. elegans muscle weakness. We first established that UNC-68 remodeling increases with age in WT worms, but is accelerated in FKB-2 KO worms using immunoprecipitation and immunoblot. We have determined that FKB-2 depletion results in reduced peak Ca2+ transients and blunted UNC-68 caffeine activation by crossing WT and FKB-2 KO worms with Pmyo-3: GCaMP2 worms. In Aim Two, we will assess the contribution of mitochondrial ROS in UNC-68 dysfunction by measuring mitochondrial free radicals in two electron transport chain (ETC) mutant strains, clk-1 and mev-1. We have found that the timing of UNC-68 remodeling correlates with their lifespan differences; clk-1 is long-lived and lacks UNC- 68 remodeling until old age, while mev-1 is short-lived and has remodeled UNC-68 channels earlier in life. Successful completion of the proposed studies will define the contributions of pathological SR calcium leak in age-dependent muscle function loss.