Intra-sarcoplasmic Reticulum Ca2+ Depletion In Adult Skeletal Muscle Fibers Measured With The Biosensor D1ER

Abstract

The endoplasmic/sarcoplasmic reticulum (ER/SR) plays a crucial role in cytoplasmic signaling in a variety of cells. It is particularly relevant for skeletal muscle fibers, where this organelle constitutes the main Ca2+ store for essential functions, such as contraction. In this work, we expressed the cameleon biosensor D1ER by in vivo electroporation in the mouse flexor digitorum brevis (FDB) muscle to directly assess intra-SR Ca2+ depletion in response to electrical and pharmacological stimulation. The main conclusions are: (1) D1ER is expressed in the SR of FDB fibers according to both di-8-ANEPPs staining and reductions in FRET; (2) the amplitude of intra-SR Ca2+ release evoked by either 4-CmC or electrical stimulation is directly proportional to resting SR Ca2+, which indicates that intra-Ca2+ modulates RyR1-meditated SR Ca2+ release in the intact muscle fiber; (3) intra-SR Ca2+ release, as measured with D1ER, is voltage-dependent and follows a Boltzmann function; (4) average SR Ca2+ depletion is 13% in response to 4-CmC and 4.5% in response to prolonged and maximal sarcolemmal depolarization; and (5) the time-to-peak of intra-SR Ca2+ release and cytoplasmic Ca2+ transient at maximal sarcolemmal depolarization do not differ significantly.View Large Image | View Hi-Res Image | Download PowerPoint Slide