Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe

摘要

The cytosolic free Ca~(2+) transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca~(2+)] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca~(2+) dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca~(2+)] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca~(2+)] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca~(2+)] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca~(2+), both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca~(2+) reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50,1-5, and 0.3 s~(-1) (at 26 ℃), reflecting activity of the SR Ca~(2+) pump and interplay of luminal and cytosolic Ca~(2+) buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contrac-tures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.