Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe
Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe
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DOI：
10.1073/pnas.1009168108
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发表时间：
2010-12-21
影响因子：
11.1
通讯作者：
Stienen, G. J. M.
中科院分区：
文献类型：
--
作者：
Canato, M.;Scorzeto, M.;Stienen, G. J. M.
由肌纤维激发引起的胞质游离Ca2+瞬态是很好的表征，但对肌浆网（SR）内的游离Ca2+动力学知之甚少。一个可靶向的基于比例fret的钙指示剂（D1ER Cameleon）使我们能够研究SR Ca2+动力学，并分析钙sequestrin （CSQ）对酶解的指屈肌短肌纤维SR [Ca2+]的影响，这些肌纤维来自缺乏1型异构体（CSQ-KO）或两种异构体[CSQ-双KO (DKO)]的WT和CSQ-KO小鼠。静止时，游离SR [Ca2+]在WT、CSQ-KO和CSQ-DKO纤维之间没有差异。在持续收缩期间，WT的变化相当小，反映了CSQ的强大缓冲，而在CSQ- ko纤维中，SR [Ca2+]发生了显著下降。在1 ~ 60 Hz的刺激频率范围内，其振幅随刺激频率的增加而增加。在60 Hz时，CSQ-KO和CSQ-DKO纤维中的SR几乎耗尽了Ca2+。在CSQ-KO纤维中，Fura-2检测到的胞质游离钙在重复刺激期间下降，表明SR钙含量不足以维持持续的收缩活动。在刺激训练期间和之后，SR Ca2+再摄取似乎受到三个时间上不同的过程的控制，其速率常数为50、1-5和0.3 s(-1)（26℃），反映了SR Ca2+泵的活性以及腔内和细胞质Ca2+缓冲液的相互作用，并指向储存操作的钙进入（SOCE）。在缺乏CSQ的小鼠中，SOCE可能在导致恶性高热样综合征的肌肉挛缩中发挥重要作用。
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 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 [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 degrees 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.