Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation
Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation
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DOI：
10.1080/15548627.2019.1591672
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发表时间：
2019-04-08
期刊：
影响因子：
13.3
通讯作者：
Quadrilatero, Joe
中科院分区：
文献类型：
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作者：
Baechler, Brittany L.;Bloemberg, Darin;Quadrilatero, Joe
巨自噬/自噬是各种细胞过程所必需的降解过程。我们以前证明，自噬缺陷导致成肌细胞凋亡和损害肌管形成。在这项研究中，我们继续这项工作，特别强调线粒体重塑和应激/凋亡信号。我们发现自噬增加（p < 0.05）（例如，改变的LC 3B水平，增加的ATG 7，减少的SQSTM 1）和线粒体吞噬（例如，BNIP 3上调，线粒体定位的GFP-LC 3斑点，和升高的线粒体LC 3B-II）信号传导。shRNA介导的ATG 7敲低（shAtg 7）降低了这些自噬和线粒体吞噬反应，同时增加了分化成肌细胞中的CASP 3活性和ANXA 5/膜联蛋白V染色;最终导致肌生成显著受损。进一步证实了线粒体自噬在这些反应中的重要性，CRISPR-Cas9介导的Bnip 3敲除（bnip 3（-/-））导致CASP 3活性和DNA片段化增加以及成肌细胞分化受损。此外，shAtg 7成肌细胞显示出更大的内质网（例如，增加的CAPN活性和HSPA）和线粒体（例如，mPTP形成、降低的线粒体膜电位、升高的线粒体4-HNE）应激。shAtg 7和bnip 3（-/-）成肌细胞也显示出改变的与ESTA相关的信号传导（例如，PPARGC 1A、DNM 1 L、OPA 1）和蛋白质含量（例如，SLC25A4、VDAC1、CYCS）。此外，shAtg 7成肌细胞显示CYCS和AIFM 1从线粒体释放，以及CASP 9活化。类似地，bnip 3（-/-）成肌细胞在分化期间具有显著更高的CASP 9活化。重要的是，给予CASP 9（Ac-LEHD-CHO）或显性阴性CASP 9（ad-DNCASP 9）的化学抑制剂部分恢复了shAtg 7成肌细胞的分化和肌发生。总之，这些数据证明了自噬在保护成肌细胞免受分化过程中线粒体氧化应激和凋亡信号的影响以及在线粒体网络重塑和肌发生的调节中的重要作用。
Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/apoptotic signaling. We found increased (p < 0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (shAtg7) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9-mediated knockout of Bnip3 (bnip3(-/-)) resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, shAtg7 myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. shAtg7 and bnip3(-/-) myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, shAtg7 myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, bnip3(-/-) myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in shAtg7 myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis.