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

IF 8.9 1区 医学 Journal of Cachexia, Sarcopenia and Muscle Pub Date : 2024-10-31 DOI:10.1002/jcsm.13617
Makayla S Lancaster, Paul Hafen, Andrew S Law, Catalina Matias, Timothy Meyer, Kathryn Fischer, Marcus Miller, Chunhai Hao, Patrick Gillespie, David McKinzie, Jeffrey J Brault, Brett H Graham
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引用次数: 0

摘要

背景:琥珀酰-CoA合成酶(SCS)亚基的致病变异与人类线粒体脑肌病有关。SCS 催化琥珀酰-CoA 与 TCA 循环中 ADP 或 GDP 底物级磷酸化的结合,将琥珀酰-CoA 转化为琥珀酸。本报告介绍了一种肌肉特异性条件性敲除(KO)Sucla2(SCS的ADP特异性β亚基)的小鼠模型,从而建立了线粒体肌病的新型体内模型:该小鼠模型是利用Cre-Lox系统产生的,人骨骼肌肌动蛋白(HSA)启动子在骨骼肌内驱动Cre-重组CRISPR-Cas9产生的Sucla2等位基因。通过 RT-qPCR 和 Western 印迹验证了 Sucla2 的失活,并通过质谱法对酶活性和血清代谢物进行了定量。为了描述该模型的体内特征,对小鼠进行了全身表型分析,并进行了一系列力量和运动行为测定。此外,还对比目鱼肌(SOL)和伸肌(EDL)进行了体外收缩力实验。还对比目鱼肌和伸拇长肌的冷冻切片进行了成像分析,以评估肌肉纤维特异性表型:分子验证证实,突变体骨骼肌中的 Sucla2 转录本减少了 68%(p 结论:突变体骨骼肌中的 Sucla2 转录本减少了 68%):小鼠骨骼肌中 SUCLA2 的缺失产生了一种 SCS 缺失型线粒体肌病模型,该模型具有体重减轻、肌无力和运动不耐受等症状。后肢肌肉的生理学和形态学分析表明,EDL 和 SOL 肌肉的体内外功能和细胞后果存在显著差异,SOL 肌肉受 Sucla2 失活的影响更大。这种新型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,从而更好地了解SCS缺陷性肌病。
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Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Background: Pathogenic 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.

Methods: The 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.

Results: Molecular 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.

Conclusions: SUCLA2 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.

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来源期刊
Journal of Cachexia, Sarcopenia and Muscle
Journal of Cachexia, Sarcopenia and Muscle Medicine-Orthopedics and Sports Medicine
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期刊介绍: The Journal of Cachexia, Sarcopenia, and Muscle is a prestigious, peer-reviewed international publication committed to disseminating research and clinical insights pertaining to cachexia, sarcopenia, body composition, and the physiological and pathophysiological alterations occurring throughout the lifespan and in various illnesses across the spectrum of life sciences. This journal serves as a valuable resource for physicians, biochemists, biologists, dieticians, pharmacologists, and students alike.
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