S100A1 modulation of skeletal muscle excitation-contraction coupling

Abstract

S100A1, a 21 kDa dimeric Ca2+ binding protein, enhances cardiac Ca2+ release and contractility, and is a potential therapeutic agent for the treatment of cardiomyopathy. A role of S100A1 in skeletal muscle is less well defined. Additionally, the molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release has not been fully elucidated. Here, utilizing a genetic approach to knock out (KO) S100A1, I demonstrate a physiologic role of S100A1 in skeletal muscle excitation-contraction (EC) coupling. Using high-speed confocal microscopy, I show that ablation of S100A1 leads to delayed myoplasmic Ca2+ transients with decreased amplitude following an action potential in isolated flexor digitorum brevis (FDB) muscle fibers. Through binding assays and competition experiments, I identify a novel S100A1 binding site on the cytoplasmic face of the ryanodine receptor (RyR1) that corresponds to a previously identified calmodulin (CaM) binding domain (CaMBD). I find that S100A1 competes with CaM for this site, which also interacts with the voltage sensor of EC coupling, the dihydropyridine receptor. To investigate effects of S100A1 on the voltage sensor, I utilized whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in WT and KO fibers. In contrast to recent reports, I find that FDB fibers exhibit two distinct components of charge movement, an initial rapid component (Qgamma) and a delayed, steeply voltage dependent "hump" component (Qbeta;) previously recorded primarily in amphibian but not mammalian fibers. Surprisingly, I find that Qgamma is selectively suppressed in S100A1 KO fibers. Finally, I explore the effects of S100A1 on whole muscle contractile force, to test if S100A1's modulation of single fiber Ca2+ release translates to altered contractile performance in vivo. I find that tibialis anterior muscles of S100A1-/- mice generate less contractile force and exhibit a greater rate of fatigue than WT counterparts. Taken together, these data suggest S100A1 binds to the CaMBD of RyR1 and enhances voltage-gated Ca2+ release, leading to elevated myoplasmic Ca2+ and increased contractile force following muscle fiber excitation. This thesis sheds light on voltage sensor activation of Ca2+ release in skeletal muscle, and supports S100A1 as a positive regulator of EC coupling.