Comparison of genes regulated in P0 Klhl40 WT and KO skeletal muscle

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, and their role in the disease remains unresolved. Here, we demonstrate that loss of a muscle-specific protein, Klhl40, results in a nemaline-like myopathy in mice that closely phenocopies the muscle abnormalities observed KLHL40 deficient patients. We show that Klhl40 dynamically localizes to the sarcomere I-band and A-band and binds to Nebulin (Neb), a protein frequently implicated in NM, as well as a putative thin filament protein, Lmod3. Klhl40 belongs to the BTB-BACK-Kelch (BBK) family of proteins, some of which have been previously shown to promote degradation of their substrates. In contrast, we find that Klhl40 promotes stability of Neb and Lmod3 and blocks Lmod3 ubiquitination. Accordingly, loss of Klhl40 reduces Neb and Lmod3 protein in skeletal muscle of mice and KLHL40 deficient patients. Because loss of sarcomere thin filament proteins is a frequent cause of NM, our data establishes a possible molecular basis for NM in KLHL40 deficient patients by establishing a novel pro-stability function of Klhl40 for Neb and Lmod3. Total RNA was harvested from quadriceps muscle of three Klhl40 WT (control) and three Klhl40 KO mice. Each KO mouse was sacrificed with a corresponding WT littermate. Tissues were also taken at 0 days of age to minimize confounding gene changes occurring due to malnourishment as the phenotype worsens.

杆状体肌病（Nemaline myopathy, NM）是一类可导致致死性肌肉功能障碍的先天性肌病，目前被认为是肌节细丝相关疾病。近期已有多种功能未明的蛋白被证实与NM发病相关，但其在疾病发生发展中的具体作用仍未明确。本研究证实，肌肉特异性蛋白Klhl40的缺失会在小鼠体内引发类杆状体肌病，其表型与KLHL40缺陷患者的肌肉异常高度相似。研究显示，Klhl40可动态定位于肌节I带与A带，并能与伴肌动蛋白（Nebulin, Neb）——一种频繁被报道与NM相关的蛋白——以及潜在肌节细丝蛋白Lmod3结合。Klhl40属于BTB-BACK-Kelch（BBK）蛋白家族，该家族部分成员此前被证实可促进底物的降解。与之相反，本研究发现Klhl40可提升伴肌动蛋白与Lmod3的稳定性，并抑制Lmod3的泛素化修饰。相应地，Klhl40缺失会降低小鼠骨骼肌及KLHL40缺陷患者体内的Neb与Lmod3蛋白水平。由于肌节细丝蛋白缺失是NM的常见致病原因，本研究通过明确Klhl40对伴肌动蛋白与Lmod3的新型促稳定功能，为KLHL40缺陷型NM患者提供了潜在的分子发病机制。本研究从3只Klhl40野生型（对照组）与3只Klhl40敲除型小鼠的股四头肌中提取总RNA；每只敲除型小鼠均选取同窝野生型小鼠作为配对对照。所有组织均采集于小鼠出生当日，以最大程度减少因表型恶化引发营养不良所导致的基因表达变化带来的混杂干扰。