由线粒体(mtDNA)缺失引起的线粒体功能障碍与骨骼肌萎缩和肌纤维丢失有关。然而,肌纤维中的这种缺陷是否会导致肌少症尚不清楚。此外,肌肉干细胞(MuSCs)中mtDNA改变对肌肉减少症的贡献还有待研究.
我们表达了线粒体解旋酶的显性阴性变体,诱导mtDNA改变,特别是在分化的肌纤维(K320Eskm小鼠)和MuSC(K320Emsc小鼠)中,分别,并通过免疫组织化学研究了它们对肌肉结构和功能的影响,分析mtDNA和呼吸链含量,肌肉转录组和功能测试。
与比目鱼对照相比,24月龄的K320Eskm小鼠的mtDNA缺失水平更高(SOL,0.07673%与0.00015%,P=0.0167),趾长伸肌(EDL,0.0649vs.0.000925,P=0.0015)和腓肠肌(气体,0.09353vs.0.000425,P=0.0004)。K320Eskm小鼠显示骨骼肌横截面中细胞色素c氧化酶缺陷(COX-)纤维的比例逐渐增加,达到最高3.03%,4.36%,13.58%,EDL占17.08%,SOL,胫骨前肌(TA)和气体,分别。然而,小鼠没有表现出加速的肌肉质量损失,肌肉力量或身体表现。组织学分析显示红色纤维参差不齐,但也刺激了再生,指示MuSC的激活。RNAseq证明与蛋白质合成相关的基因表达增强,但也退化,以及肌纤维分化和细胞增殖。相比之下,心脏毒素破坏后7天,K320Emsc小鼠再生TA显示30%的COX-纤维。值得注意的是,再生肌肉表现出营养不良的变化,纤维化增加(2.5%vs.1.6%,P=0.0003),脂肪细胞丰度增加(2.76%vs.0.23%,P=0.0144)和减少的肌肉质量(再生TA:40.0mgvs.60.2mg,P=0.0171)。与K320Eskm小鼠的肌肉相反,从老年K320Emsc小鼠中新鲜分离的MuSCs完全没有mtDNA改变。然而,传代后,mtDNA拷贝数以及呼吸链亚基和p62水平逐渐降低。
合照,仅在肌纤维中大规模mtDNA改变的积累不足以引起肌肉减少症。K320E-Twinkle的表达在静止的MuSCs中是耐受的,但激活后会逐渐导致mtDNA和呼吸链耗尽,体内和体外,可能是由线粒体去除增加引起的。总之,我们的结果表明,肌纤维中mtDNA改变的积累激活了衰老过程中的再生,如果这种改变也在MuSC中扩展,则会导致肌肉减少症。
Mitochondrial dysfunction caused by mitochondrial (mtDNA) deletions have been associated with skeletal muscle atrophy and myofibre loss. However, whether such defects occurring in
myofibres cause sarcopenia is unclear. Also, the contribution of mtDNA alterations in muscle stem cells (MuSCs) to sarcopenia remains to be investigated.
We expressed a dominant-negative variant of the mitochondrial helicase, which induces mtDNA alterations, specifically in differentiated
myofibres (K320Eskm mice) and MuSCs (K320Emsc mice), respectively, and investigated their impact on muscle structure and function by immunohistochemistry, analysis of mtDNA and respiratory chain content, muscle transcriptome and functional tests.
K320Eskm mice at 24 months of age had higher levels of mtDNA deletions compared with controls in soleus (SOL, 0.07673% vs. 0.00015%, P = 0.0167), extensor digitorum longus (EDL, 0.0649 vs. 0.000925, P = 0.0015) and gastrocnemius (GAS, 0.09353 vs. 0.000425, P = 0.0004). K320Eskm mice revealed a progressive increase in the proportion of cytochrome c oxidase deficient (COX- ) fibres in skeletal muscle cross sections, reaching a maximum of 3.03%, 4.36%, 13.58%, and 17.08% in EDL, SOL, tibialis anterior (TA) and GAS, respectively. However, mice did not show accelerated loss of muscle mass, muscle strength or physical performance. Histological analyses revealed ragged red fibres but also stimulated regeneration, indicating activation of MuSCs. RNAseq demonstrated enhanced expression of genes associated with protein synthesis, but also degradation, as well as muscle fibre differentiation and cell proliferation. In contrast, 7 days after destruction by cardiotoxin, regenerating TA of K320Emsc mice showed 30% of COX- fibres. Notably, regenerated muscle showed dystrophic changes, increased fibrosis (2.5% vs. 1.6%, P = 0.0003), increased abundance of fat cells (2.76% vs. 0.23%, P = 0.0144) and reduced muscle mass (regenerated TA: 40.0 mg vs. 60.2 mg, P = 0.0171). In contrast to muscles from K320Eskm mice, freshly isolated MuSCs from aged K320Emsc mice were completely devoid of mtDNA alterations. However, after passaging, mtDNA copy number as well as respiratory chain subunits and p62 levels gradually decreased.
Taken together, accumulation of large-scale mtDNA alterations in
myofibres alone is not sufficient to cause sarcopenia. Expression of K320E-Twinkle is tolerated in quiescent MuSCs, but progressively leads to mtDNA and respiratory chain depletion upon activation, in vivo and in vitro, possibly caused by an increased mitochondrial removal. Altogether, our results suggest that the accumulation of mtDNA alterations in
myofibres activates regeneration during aging, which leads to sarcopenia if such alterations have expanded in MuSCs as well.