Functional loss of DHRS7C induces intracellular Ca2+ overload and myotube enlargement in C2C12 cells via calpain activation
Functional loss of DHRS7C induces intracellular Ca2+ overload and myotube enlargement in C2C12 cells via calpain activation
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DOI：
10.1152/ajpcell.00090.2016
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发表时间：
2017-01-01
影响因子：
5.5
通讯作者：
Tsutsui, Hiroyuki
中科院分区：
文献类型：
--
作者：
Arai, Shinobu;Ikeda, Masataka;Tsutsui, Hiroyuki
脱氢酶/还原酶成员7 C（DHRS 7 C）是新近发现的一种NAD/NADH依赖性脱氢酶，在心肌和骨骼肌中表达，定位于内质网/肌浆网（ER/SR）。然而，其在肌肉细胞中的功能作用仍有待充分阐明。在此，我们通过分析DHRS 7 C缺陷的小鼠C2 C12成肌细胞（DHRS 7 C-KO细胞）、过表达野生型DHRS 7 C的小鼠C2 C12成肌细胞（DHRS 7 C-WT细胞）或表达突变型DHRS 7 C的小鼠C2 C12成肌细胞[DHRS 7 C-Y191 F或DHRS 7 C-K195 Q细胞，在NAD/NADH依赖性脱氢酶催化核心结构域（YXXXK）中携带点突变]，研究了DHRS 7 C的作用。当C2 C12成肌细胞分化成成熟肌管时，诱导DHRS 7 C表达，而DHRS 7 C-KO肌管在分化后表现出扩大的细胞形态。值得注意的是，DHRS 7 C-Y191 F和DHRS 7 C-K195 Q细胞也显示出相似的增大的细胞形态，表明NAD/NADH依赖性脱氢酶催化核心结构域对于DHRS 7 C功能是关键的。在DHRS 7 CKO、DHRS 7 C-Y191 F和DHRS 7 C-K195 Q细胞中，分化后ER/SR中的胞质Ca 2+静息水平和Ca 2+储存总量显著高于对照C2 C12和DHRS 7 C-WT细胞。此外，在这些细胞中，由毒胡萝卜素和4-氯-间甲酚诱导的来自ER/SR的Ca 2+释放增加，并且钙蛋白酶（一种钙依赖性蛋白酶）在DHRS 7 C-KO、DHRS 7 C-Y191 F和DHRS 7 C-K195 Q肌管中显著活化，这与细胞溶质Ca 2+浓度的较高静息水平和分化后的扩大形态一致。此外，用钙蛋白酶抑制剂处理废除了扩大的细胞形态。综上所述，我们的研究结果表明，DHRS 7 C维持细胞内Ca 2+稳态，涉及ER/SR和DHRS 7 C的功能丧失导致细胞质和ER/SR中的Ca 2+过载，通过钙蛋白酶激活导致扩大的细胞形态。
Dehydrogenase/reductase member 7C (DHRS7C) is a newly identified NAD/NADHdependent dehydrogenase that is expressed in cardiac and skeletal muscle and localized in the endoplasmic/sarcoplasmic reticulum (ER/SR). However, its functional role in muscle cells remains to be fully elucidated. Here, we investigated the role of DHRS7C by analyzing mouse C2C12 myoblasts deficient in DHRS7C (DHRS7C-KO cells), overexpressing wild-type DHRS7C (DHRS7C-WT cells), or expressing mutant DHRS7C [ DHRS7C-Y191F or DHRS7C-K195Q cells, harboring point mutations in the NAD/NADH-dependent dehydrogenase catalytic core domain (YXXXK)]. DHRS7C expression was induced as C2C12 myoblasts differentiated into mature myotubes, whereas DHRS7C-KO myotubes exhibited enlarged cellular morphology after differentiation. Notably, both DHRS7C-Y191F and DHRS7C-K195Q cells also showed similar enlarged cellular morphology, suggesting that the NAD/NADH-dependent dehydrogenase catalytic core domain is pivotal for DHRS7C function. In DHRS7CKO, DHRS7C-Y191F, and DHRS7C-K195Q cells, the resting level of cytosolic Ca2+ and total amount of Ca2+ storage in the ER/SR were significantly higher than those in control C2C12 and DHRS7C-WT cells after differentiation. Additionally, Ca2+ release from the ER/SR induced by thapsigargin and 4-chloro-m-cresol was augmented in these cells and calpain, a calcium-dependent protease, was significantly activated in DHRS7C-KO, DHRS7C-Y191F, and DHRS7C-K195Q myotubes, consistent with the higher resting level of cytosolic Ca2+ concentration and enlarged morphology after differentiation. Furthermore, treatment with a calpain inhibitor abolished the enlarged cellular morphology. Taken together, our findings suggested that DHRS7C maintains intracellular Ca2+ homeostasis involving the ER/SR and that functional loss of DHRS7C leads to Ca2+ overload in the cytosol and ER/SR, resulting in enlarged cellular morphology via calpain activation.