Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, Japan.
PLoS Genet. 2013;9(8):e1003668. doi: 10.1371/journal.pgen.1003668. Epub 2013 Aug 1.
Calpains are Ca(2+)-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.
钙蛋白酶是 Ca(2+)-依赖性调节半胱氨酸蛋白酶，在几乎所有真核生物中都具有多种功能。哺乳动物中有超过 10 种高度保守的钙蛋白酶，其中真哺乳亚纲钙蛋白酶-6（CAPN6）的独特之处在于其活性位点半胱氨酸残基（在人类中突变为赖氨酸）发生了氨基酸取代，强烈提示其失去了蛋白水解活性。CAPN6主要在胚胎肌肉、胎盘和几种培养细胞系中表达。我们之前报道过，CAPN6参与调节培养细胞中的微管动力学和肌动蛋白重组。然而，CAPN6 的生理功能仍不清楚。在这里，为了阐明 CAPN6 的体内作用，我们生成了 Capn6 缺陷型小鼠，其中一个 lacZ 表达盒被整合到 Capn6 基因中。这些 Capn6 缺陷型小鼠胚胎在骨骼肌以及软骨和心脏中主要表达 lacZ。组织学和生化分析表明，CAPN6 缺失促进了胚胎骨骼肌的发育。在诱导分化为肌管的原代培养骨骼肌细胞中，CAPN6 在骨骼肌细胞中表达，Capn6 缺陷型培养物显示出分化增加。此外，我们发现 CAPN6 在成年小鼠心脏毒素诱导退变后的再生骨骼肌中表达。在这个实验系统中，与杂合子或野生型小鼠相比，Capn6 缺陷型小鼠在同一时间点显示出更先进的骨骼肌再生。这些结果共同表明，CAPN6 的缺失促进了发育和再生过程中的骨骼肌分化，提示 CAPN6 作为骨骼肌分化抑制因子具有新的生理功能。
