In situ measurements of calpain activity in isolated muscle fibres from normal and dystrophin-lacking mdx mice
In situ measurements of calpain activity in isolated muscle fibres from normal and dystrophin-lacking mdx mice
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DOI：
10.1113/jphysiol.2007.132191
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发表时间：
2007-08-01
影响因子：
5.5
通讯作者：
Gillis, J. M.
中科院分区：
文献类型：
--
作者：
Gailly, P.;De Backer, F.;Gillis, J. M.
钙蛋白酶是一种钙激活的蛋白酶，被认为与肌肉退行性疾病有关，如Duchenne肌营养不良症。钙调蛋白在成人肌肉纤维中的状态和活性很少有文献记载。我们在此报道了来自C57小鼠和形成两种营养不良模型的胶原酶分离的纤维中的calain活性的原位测量：dystrophin缺陷的mdx和calain-3基因敲除的小鼠。用一种永久的荧光底物测定了钙蛋白酶的活性，并研究了它的钙依赖性。在最低和最高稳态钙利用率之间，观察到活性变化了30倍。电刺激或KCl2依赖的去极化引起的钙离子快速瞬变对钙激活酶无作用。钙库耗尽、钙库再耗尽和低渗肿胀引起的慢[钙]瞬变能够激活钙蛋白酶。恢复静息状态后，Calain活性在10min内恢复到基础水平。在静止的完整肌肉中，Mu-calain主要以80 kDa的天然形式存在，少量以78 kDa的自溶形式存在。后者被认为是在我们的条件下衡量的活动的责任。与C57纤维相比，MDX纤维中的钙蛋白酶活性平均增加了1.5倍。这种活性因[Ca~(2+)](O)降低10倍而降低。缺乏calain-3的纤维表现出大致相同的增加，因此calain-3对此处测量的活性没有贡献，而且calain的激活并不是dystrophin缺乏所特有的。在过度表达calastatin的转基因小鼠的纤维中，观察到与合成药物(Z-Leu-Leu-Cho和SNT198438)一样，Calain活性降低了40%-50%。我们提供了关于在原位控制钙蛋白酶活性的生理因素的新信息，特别是发生在兴奋-收缩偶联、钙库耗竭和再充盈以及机械敏感钙通道激活的细胞内钙瞬变的影响。
Calpains are Ca2+-activated proteases that are thought to be involved in muscle degenerative diseases such as Duchenne muscular dystrophy. Status and activity of calpains in adult muscle fibres are poorly documented. We report here in situ measurements of calpain activity in collagenase-isolated fibres from C57 mice and form two models of dystrophy: dystrophin-deficient mdx and calpain-3 knocked-out mice. Calpain activity was measured using a permeant, fluorogenic substrate and its Ca2+ dependence was studied. A 30-fold change of activity was observed between the lowest and the highest steady-state Ca2+ availability. Fast transient changes of [Ca2+], induced by electrical stimulation or KCl-dependent depolarization were ineffective in activating calpain. Slow [Ca2+] transients, as elicited during depletion of Ca2+ stores, Ca2+ store repletion and hypo-osmotic swelling were able to activate calpain. On return to resting conditions, calpain activity recovered its basal rate within 10 min. In resting intact muscle, mu-calpain was predominantly in the 80 kDa native form, with a small fraction in the 78 kDa autolysed form. The latter is thought to be responsible for the activity measured in our conditions. Calpain activity in mdx fibres showed an average 1.5-fold increase compared to activity in C57 fibres. This activity was reduced by a 10-fold lowering of [Ca2+](o). Calpain-3-deficient fibres showed about the same increase, thus calpain-3 did not contribute to the activity measured here and calpain activation is not specific to dystrophin deficiency. In fibres from transgenic mice over-expressing calpastatin, a 40-50% reduction of calpain activity was observed, as with synthetic drugs (Z-Leu-Leu-CHO and SNT198438). We provide novel information on the physiological factors that control calpain activity in situ, particularly the effect of intracellular Ca2+ transients that occur in excitation-contraction coupling, Ca2+ store depletion and refilling, and activation of mechanosensitive Ca2+ channels.