Imaging Studies of Calsequestrin Structure in Skeletal Muscle. Effects of Calcium Release

Abstract

Studying Ca2+ release flux and FRAP of fluorescently tagged proteins, Manno et al (this meeting) showed that SR Ca2+ depletion results in impairment of Ca2+ signaling and disassembly of the calsequestrin polymer that fills SR terminal cisternae (TC) in resting myofibers. We sought evidence of depolymerization in images of fluorescence of D4cpV-calsequestrin, a FRET biosensor that targets TC and measures [Ca2+]SR. Vertical (z-) stacks of fluorescence images allowed for identification of subcellular structures (resolution was ca. 0.3 μm in xy and 0.6 μm in z, after deblurring). Depletion was induced by solutions with high K+, 0.5-5 mM chloro-M-cresol (CMC), plus pump inhibitors, and was verified by FRET of D4cpV. Upon depletion, the fluorescence transitioned from a pattern limited to TC to a more diffuse one, consistent with mobilization of calsequestrin away from TC. Fourier analysis of these images showed migration of power away from the fundamental and into the lower harmonics of the power spectrum. Muscles depleted by solutions with 5 mM CMC, imaged by electron microscopy, showed electron-dense content —calsequestrin-- in longitudinal sacs that normally appear empty. These images also showed major changes in membrane structure, which raise the possibility that the high CMC resulted in irreversible changes. After SR depletion by fatiguing tetanic stimulation of intact muscles the fluorescence of D4cpV-calsequestrin redistributed to a lesser extent. This first demonstration of calsequestrin depolymerization in vivo introduces the loss of Ca2+ buffering associated with this change as a previously unrecognized mechanism for functional impairment in muscle fatigue, suggests ways in which calsequestrin could mediate gating control of RyR channels by [Ca2+]SR and justifies disease phenotypes linked to mutations of calsequestrin that prevent its correct polymerization (see poster by Lewis et al, this meeting). Supported by NIGMS/NIH grant GM111254.