BackgroundPathogenic variants in subunits of succinyl‐CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl‐CoA to succinate coupled with substrate‐level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle‐specific conditional knock‐out (KO) mouse model of Sucla2, the ADP‐specific beta subunit of SCS, generating a novel in vivo model of mitochondrial myopathy.MethodsThe mouse model was generated using the Cre‐Lox system, with the human skeletal actin (HSA) promoter driving Cre‐recombination of a CRISPR‐Cas9–generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT‐qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model in vivo, whole‐body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, ex vivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre‐specific phenotypes.ResultsMolecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%–40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle‐specific manner; although no genotype‐specific deficiencies were seen in EDL function, SUCLA2‐deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL‐specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls.ConclusionsSUCLA2 loss within murine skeletal muscle yields a model of SCS‐deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in ex vivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle‐specific and fibre type–specific pathogenic mechanisms to better understand SCS‐deficient myopathy.

中文翻译：

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骨骼肌中的 Sucla2 敲除产生具有肌肉类型特异性表型的线粒体肌病小鼠模型


背景琥珀酰辅酶 A 合成酶 （SCS） 亚基的致病性变异与人类线粒体脑肌病有关。SCS 催化琥珀酰辅酶 A 转化为琥珀酸盐，并在 TCA 循环中偶联 ADP 或 GDP 的底物水平磷酸化。本报告介绍了 Sucla2 的肌肉特异性条件敲除 （KO） 小鼠模型，Sucla2 是 SCS 的 ADP 特异性 β 亚基，生成了一种新的线粒体肌病体内模型。方法使用 Cre-Lox 系统生成小鼠模型，人骨骼肌动蛋白 （HSA） 启动子驱动骨骼肌内 CRISPR-Cas9 生成的 Sucla2 絮凝等位基因的 Cre 重组。使用 RT-qPCR 和 western blot 验证 Sucla2 的失活，并通过质谱定量酶活性和血清代谢物。为了在体内表征模型，进行了全身表型分析，小鼠接受了一组力量和运动行为测定。此外，对比目鱼肌 （SOL） 和趾长伸肌 （EDL） 进行了离体收缩实验。SOL 和 EDL 冷冻切片也接受影像学分析以评估肌肉纤维特异性表型。结果分子验证证实，突变骨骼肌内 Sucla2 转录物减少 68% （p < 0.001），功能减少 95% 的 SUCLA2 蛋白 （p < 0.0001）。到 3 周龄时，Sucla2 KO 小鼠的体重是对照组的 44% （p < 0.0001）。突变小鼠的握力也降低 34%-40% （p < 0.01） 和自发运动减少，在跑轮上花费的累积时间减少约 88% （p < 0.0001）。 收缩功能也以肌肉特异性方式受到干扰;虽然在 EDL 功能中没有观察到基因型特异性缺陷，但 SUCLA2 缺陷的 SOL 肌肉产生的特异性强直力降低了 40% （p < 0.0001），同时收缩和松弛速率较慢 （p < 0.001）。同样，观察到线粒体的 SOL 特异性增加 3 倍 （p < 0.0001），COX 和 SDH 的染色定性增加，并且 SOL 中 1 型肌球蛋白重链表达纤维的比例几乎翻了一番（增加 95%，p < 0.0001）与对照组相比，Sucla2 KO 小鼠。结论小鼠骨骼肌中 SUCLA2 缺失产生 SCS 缺陷型线粒体肌病模型，表现为体重减轻、肌无力和运动不耐受。后肢肌肉的生理和形态学分析显示，EDL 和 SOL 肌肉之间的离体功能和细胞后果存在显着差异，其中 SOL 肌肉受 Sucla2 失活的影响明显更大。这种新颖的模型将为研究肌肉特异性和纤维类型特异性致病机制提供宝贵的工具，以更好地了解 SCS 缺陷型肌病。