Dept. of Zoology, La Trobe Univ., VIC 3086, Australia.
J Appl Physiol (1985). 2011 Nov;111(5):1448-58. doi: 10.1152/japplphysiol.00441.2011. Epub 2011 Aug 11.
The skeletal muscle-specific calpain-3 protease is likely involved in muscle repair, although the mechanism is not known. Physiological activation of calpain-3 occurs 24 h following eccentric exercise in humans. Functional consequences of calpain-3 activation are not known; however, calpain-3 has been suggested to be involved in nuclear signaling via NF-κB. To test this and help identify how/where calpain-3 acts, we investigated whether calpain-3 autolysis (hence, activation) following eccentric exercise results in translocation from its normal myofibrillar location to the nucleus or the cytosol. In resting human skeletal muscle, the majority (87%) of calpain-3 was present in myofibrillar fractions, with only a small proportion (<10%) in an autolyzed state. Enriched nuclear fractions contained ∼8% of the total calpain-3, which was present in a predominantly (>80%) autolyzed state. Using freshly dissected human muscle fibers to identify freely diffusible proteins, we showed that only ∼5% of the total calpain-3 pool was cytosolic. At 3 and 24 h following eccentric step exercise, there was an ∼70% increase in autolysis in whole muscle samples (n = 11, P < 0.05, by 1-way ANOVA with repeated measures and Newman-Keuls post hoc analysis). This exercise-induced autolysis was attributed to myofibrillar-bound calpain-3, since neither the amount of calpain-3 nor the proportion autolyzed was significantly changed in enriched nuclear or cytosolic fractions following the exercise intervention. We present a model for calpain-3 localization at rest and following activation in human skeletal muscle and suggest that the functional importance of calpain-3 remains predominantly tightly associated with its localization within the myofibrillar compartment.
骨骼肌特异性钙蛋白酶-3 蛋白酶可能参与肌肉修复，尽管其机制尚不清楚。在人类中，离心运动后 24 小时会发生钙蛋白酶-3 的生理激活。钙蛋白酶-3 激活的功能后果尚不清楚；然而，钙蛋白酶-3 已被提议通过 NF-κB 参与核信号转导。为了验证这一点并帮助确定钙蛋白酶-3 的作用方式和位置，我们研究了离心运动后钙蛋白酶-3 的自溶（因此是激活）是否导致其从正常的肌原纤维位置转移到核或细胞质。在静息的人类骨骼肌中，大部分（87%）钙蛋白酶-3 存在于肌原纤维部分，只有一小部分（<10%）处于自溶状态。富含核的部分包含约 8%的总钙蛋白酶-3，其主要处于自溶状态（>80%）。使用新分离的人类肌肉纤维来鉴定可扩散的蛋白质，我们发现只有约 5%的总钙蛋白酶-3 池位于细胞质中。在离心台阶运动后 3 小时和 24 小时，整个肌肉样本中的自溶增加了约 70%（n=11，P<0.05，通过单向方差分析和重复测量以及 Newman-Keuls 事后分析）。这种运动引起的自溶归因于肌原纤维结合的钙蛋白酶-3，因为在运动干预后，富含核或细胞质的部分中钙蛋白酶-3 的量或自溶比例均没有显著变化。我们提出了一种钙蛋白酶-3 在静息和激活状态下在人类骨骼肌中的定位模型，并表明钙蛋白酶-3 的功能重要性仍然主要与其在肌原纤维隔室中的定位紧密相关。
