The cytosolic free Ca2+ transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca2+] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca2+ dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca2+] in enzymatically dissociated FDB muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca2+] 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 [Ca2+] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca2+, 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 Ca2+ 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 Ca2+ pump and interplay of luminal and cytosolic Ca2+ 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.
Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe
CANATO, MARTA;SCORZETO, MICHELE;GIACOMELLO, MARTA;REGGIANI, CARLO;
2010
Abstract
The cytosolic free Ca2+ transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca2+] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca2+ dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca2+] in enzymatically dissociated FDB muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca2+] 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 [Ca2+] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca2+, 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 Ca2+ 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 Ca2+ pump and interplay of luminal and cytosolic Ca2+ 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.
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