Intermediate Filaments & Costamere Structure & Function

筋膜是横纹肌表面的结构，它使肌膜与附近的肌原纤维有规律地排列，并通过膜向细胞外基质横向传递收缩力。因此，建筑物必须与附近的建筑物牢固地连接起来，以承受收缩的力量。在这里，我们关注的是肌原纤维和纤维之间的联系。蛋白基和角蛋白基中间丝（IFs）以及肌动蛋白微丝将浅表肌原纤维与肌膜上的纤维连接起来。我们假设含有角蛋白8和19的if在costameres中起重要作用，因为与desmin不同，它们在所有costameres结构中都存在。我们已经证明，K8/K19干扰素通过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会破坏染色体。小鼠的K19缺失突变还会导致线粒体定位错误、血清肌酸激酶水平升高和收缩力降低，这表明含有K19的IFs缺失会导致轻度骨骼肌病。我们现在有了与K19-null （FVB）具有相同遗传背景的小鼠，它们缺乏desmin，并且同时缺乏K19和desmin。在这里，我们提出5个目标来验证假设，即基于k19的干扰素和desmin干扰素在健康肌肉的收缩装置和肌肉之间形成了不同但互补的联系。我们进一步提出，它们单独或一起的缺失会导致肋肌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”）收缩引起的损伤的易感性。Aim1研究了K19-null、desmin-null和双敲除（DKO）小鼠中脊柱、肌节和胞内细胞器（包括线粒体）的形态学变化。在Aim 2中，我们将描述单个突变体和DKO的收缩活动、跑步能力和对偏心损伤的易感性。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，解决了缺乏K19、desmin或两种IF蛋白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的分子途径，以及if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.