Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe.
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 °C), 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 contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.
Department of Human Anatomy and Physiology, University of Padua, 35121 Padua, Italy.
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Canato
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Scorzeto
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Giacomello
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Protasi
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Reggiani
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Stienen
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009119:Muscle Contraction / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*
D002155:Calsequestrin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium