Sonne Alexander, Peverelli Lorenzo, Hernandez-Lain Aurelio, Domínguez-González Cristina, Andersen Jesper L, Milone Margherita, Beggs Alan H, Ochala Julien
Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Neuromuscular and Rare Diseases Unit, Department of Neuroscience, Fondazione, IRCCS Ca' Granda Ospedale Maggiore, Policlinico, Milan, Italy.
Am J Physiol Cell Physiol. 2023 Mar 1;324(3):C769-C776. doi: 10.1152/ajpcell.00002.2023. Epub 2023 Feb 6.
Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with truncating mutations and from five human healthy controls. We then assessed ) MyHCs presence/post-translational modifications using LC/MS; ) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; ) myosin activation with an unloaded in vitro motility assay; and ) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by ) a higher ATP demand of myosin heads in the disordered-relaxed conformation; ) faster actomyosin kinetics; and ) reduced muscle fiber force. Overall, our findings indicate that truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients.
先天性肌病是一大类遗传性肌肉疾病。其病因包括基因中的突变，这些突变导致截短的IIa型肌球蛋白重链（MyHCs）。这些突变诱发骨骼肌症状的确切细胞和分子机制仍不清楚。因此，在本研究中，我们旨在探讨此类基因缺陷是否会改变MyHCs的存在情况、翻译后修饰以及肌球蛋白分子的功能。为此，我们从四名患有截短突变的肌病患者和五名健康对照者身上分离出肌纤维。然后，我们使用液相色谱/质谱法评估MyHCs的存在情况/翻译后修饰；使用加载了Mant-ATP追踪装置评估松弛的肌球蛋白构象和伴随的ATP消耗；使用无负载的体外运动测定法评估肌球蛋白激活情况；并使用肌纤维力学装置评估细胞力产生情况。有趣的是，患者体内存在一种带有一个额外乙酰化赖氨酸（Lys35-Ac）的IIa型MyHC。这伴随着：处于无序松弛构象的肌球蛋白头部对ATP的需求更高；肌动球蛋白动力学更快；以及肌纤维力降低。总体而言。我们的研究结果表明，截短突变通过破坏ATP酶活性和肌动球蛋白复合物，影响人类成年成熟肌纤维中肌球蛋白的存在情况/功能。这些可能是导致患者出现肌病表型的重要分子病理干扰因素。
