Intermediate Filaments & Costamere Structure & Function

肋骨是横纹肌表面的一种结构，它使肌膜与附近的肌原纤维规则地排列在一起，并通过肌膜向细胞外基质横向传递收缩力量。因此，壳体必须与附近的结构牢固地连接起来，以承受收缩的力量。在这里，我们关注的是胞囊和肌原纤维之间的联系。基于结蛋白和基于角蛋白的中间丝(IF)以及肌动蛋白微丝将浅层肌原纤维连接到肌膜上的退化雄蕊。我们推测含有角蛋白8和19的IF在胞囊中起着重要的作用，因为与结蛋白不同，它们存在于所有的胞核结构中。我们已经证明，K8/K19IF通过K19与Dystrophin-糖蛋白复合体相互作用，并且K19的过度表达或同源重组消除K19会破坏雄蕊。在小鼠中，K19零突变还导致线粒体定位错误，血清肌酸激酶水平升高，收缩力量降低，这表明含有K19的IF的缺失会导致轻度的骨骼肌病。我们现在有与K19缺失(FVB)具有相同遗传背景的小鼠，它们缺乏结蛋白，并且同时缺乏K19和结蛋白。在这里，我们提出了5个目的是为了检验这一假设，即基于K19的IF与结蛋白IF一起在健康肌肉的收缩装置和尾壳之间形成不同但互补的联系。我们进一步提出，无论是单独或共同缺乏它们，都会导致肋骨和收缩装置之间的联系减弱，肌肉生理学受损，并增加对延长(“偏心”)收缩所引起的损伤的易感性。目的研究K19基因缺失、结蛋白缺失和双基因敲除(DKO)小鼠的胞浆、肌节和细胞内细胞器(包括线粒体)的形态变化。在目标2中，我们将表征单个突变体和DKO的收缩活性、跑步能力和对偏心损伤的敏感性。目的3通过研究肌原纤维之间以及肌原纤维与肌膜之间的连接强度，研究缺乏K19、结蛋白或两者兼有的肌肉的生物力学特性。因此，我们的结果应该阐明在健康肌肉中传递力量的分子途径，以及IF的缺失如何导致肌肉疾病。

Costameres are structures at the surface of striated muscle that align the sarcolemma regularly with nearby myofibrils and transmit contractile force laterally, through the membrane to the extracellular matrix. Costameres must therefore be linked to nearby structures firmly enough to withstand the forces of contraction. Here we focus on links between costameres and myofibrils. Desmin-based and keratin-based intermediate filaments (IFs), as well as actin microfilaments, link superficial myofibrils to costameres at the sarcolemma. We postulate that IFs containing keratins 8 and 19 play an important role at costameres because, unlike desmin, they are found at all costameric structures. We have shown that K8/K19 IFs interact with the dystrophin- glycoprotein complex via K19, and that over-expression of K19 or elimination of K19 by homologous recombination disrupts costameres. The K19-null mutation in mice also leads to mitochondrial mislocalization, increased serum creatine kinase levels and reduced contractile force, suggesting that the absence of IFs containing K19 causes a mild skeletal myopathy. We now have mice in the same genetic background as the K19-null (FVB) that lack desmin, and that lack both K19 and desmin. Here we propose 5 aims to test the hypothesis that K19-based IFs together with desmin IFs form distinct but complementary links between the contractile apparatus and costameres in healthy muscle. We further propose that their absence either alone or together leads to a weakening of the links between costameres and the contractile apparatus, compromised muscle physiology, and increased susceptibility to injury induced by lengthening ("eccentric") contractions. Aim1 addresses the morphological changes in costameres, sarcomeres and intracellular organelles, including mitochondria, in K19-null, desmin-null, and double knock-out (DKO) mice. In Aim 2, we will characterize the contractile activity, running ability, and susceptibility to eccentric injury of the individual mutants and the DKO. Aim 3 addresses the biomechanical properties of muscles lacking K19, desmin or both IF proteins, by investigating the strength of connections between myofibrils, and between myofibrils and the sarcolemma. Our results should therefore elucidate the molecular pathways that transmit force in healthy muscle, and how the absence of IFs can lead to muscle disease.