Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle

Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3-/-Sgcd-/- double targeted mice. Mechanistically, Nol3-/-Sgcd-/- mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.

肌营养不良症中的肌纤维萎缩在很大程度上被归因于坏死性细胞死亡，尽管已有研究在营养不良性肌肉中检测到凋亡标志物。本研究旨在通过在营养不良性小鼠模型中基因敲除已知的凋亡抑制剂——含CARD结构域的凋亡抑制因子（Apoptosis Repressor with a CARD Domain, Arc），明确经典凋亡通路在骨骼肌变性中的作用。在营养不良性Sgcd或Lama2敲除背景中敲除Nol3（Arc蛋白编码基因），与单基因敲除的营养不良性同窝对照小鼠相比，其骨骼肌病理损伤加剧，肌肉功能也有所下降。与Nol3缺失相关的营养不良表型加重不依赖于半胱天冬氨酸蛋白酶（caspase），但依赖于线粒体通透性转换孔（mitochondrial permeability transition pore, MPTP）：抑制剂Debio-025可部分挽救Nol3-/-Sgcd-/-双敲除小鼠的骨骼肌病理损伤。从机制层面来看，Nol3-/-Sgcd-/-小鼠的总Bax蛋白及线粒体Bax蛋白水平均升高，同时线粒体肿胀程度更为显著，这提示Arc在正常生理状态下可抑制骨骼肌中Bax介导的细胞死亡效应。确实，在小鼠胚胎成纤维细胞中敲低Arc可提升细胞死亡敏感性，而这种敏感性在Bax与Bak1双敲除的成纤维细胞中被完全阻断。因此，营养不良性肌肉中Arc的缺失通过增强Bax介导的线粒体依赖性死亡机制，加剧了疾病的发病进程。