Intermediate Filaments & Costamere Structure & Function

肋粒是横纹肌表面的结构，其使肌膜与附近的肌原纤维规则地对齐，并通过膜将收缩力侧向传递到细胞外基质。因此，Costameres必须与附近的结构牢固地连接起来，以承受收缩力。在这里，我们重点关注肋肌和肌原纤维之间的联系。结蛋白和角蛋白为基础的中间丝（IF），以及肌动蛋白微丝，表面肌原纤维连接到肌膜中的肋。我们假设，含有角蛋白8和19的IF在costameres中发挥重要作用，因为与结蛋白不同，它们存在于所有costameric结构中。我们已经表明，K8/K19 IFs通过K19与肌营养不良蛋白-糖蛋白复合物相互作用，并且K19的过表达或通过同源重组消除K19会破坏中肠。小鼠中的K19无效突变还导致线粒体错误定位、血清肌酸激酶水平升高和收缩力降低，这表明缺乏含有K19的IFs会导致轻度骨骼肌病变。我们现在有与K19-null（FVB）相同遗传背景的小鼠，它们缺乏结蛋白，并且缺乏K19和结蛋白。在这里，我们提出了5个目的来测试的假设，K19为基础的IF与结蛋白IF形成不同的，但互补的联系之间的收缩装置和costameres在健康的肌肉。我们进一步提出，他们的缺席，无论是单独或一起导致costameres和收缩装置之间的联系减弱，受损的肌肉生理学，并增加对损伤的易感性延长（“偏心”）收缩。Aim 1解决了K19缺失、结蛋白缺失和双基因敲除（DKO）小鼠中的肋节、肌节和细胞内细胞器（包括线粒体）的形态学变化。在目标2中，我们将描述单个突变体和DKO的收缩活动、跑步能力和对离心损伤的敏感性。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IF蛋白的肌肉的生物力学特性。因此，我们的研究结果应该阐明在健康肌肉中传递力的分子途径，以及IFs的缺乏如何导致肌肉疾病。

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.