武汉云克隆科技股份有限公司

Abstract

Idiopathic dilated cardiomyopathy (DCM) is a rare disease defined by the presence of left ventricular dilatation and cardiac insufficiency, and is a major indication for transplantation. DCM is a heterogeneous monogenic disease, mainly autosomal dominant, in about 30% of the cases. Mutations in more than 30 genes have been identified to date, yet a majority of cases remain without identified mutations, and molecular and cellular mechanisms leading to the disease remain unclear. We recently identified the BAG3 (Bcl2 athanogene 3) gene as a new DCM gene. Two of the identified mutations are missense located in the BAG domain of BAG3 known to interact with the chaperone Hsp70 (Heat Shock Protein 70). BAG3 is a cytoprotective co-chaperone involved in proteostasis, and plays a role in autophagic clearance of misfolded or degraded proteins. The aim of the study is to confirm that BAG3 mutations are responsible for DCM and to identify underlying molecular mechanisms. We studied biochemical and cellular effects of these mutations in rat neonatal cardiomyocytes (RNC) and HeLa cells, with transient overexpression of GFP-fused BAG3 proteins. Fusion protein location was observed by immunofluorescent microscopy. BAG3-wt/mut overexpression in RNC lead to normal expression at the Z-disc. However 48 hours post-transfection, RNC overexpressing BAG3-mut displayed cellular atrophy and disorganized sarcomeres. Potential perturbation of BAG3 partner interaction was investigated using GST pull-down, which demonstrated a loss of BAG3-mut/Hsp70 interaction. The effect of BAG3 mutations on autophagy was examined via western-blot monitoring LC3-II and on proteostasis using a luciferase based reporter assay. These experiments demonstrated that BAG3-mut overexpression, under stress conditions, seems to alter proteostasis regulation. These results suggest an effect of BAG3 mutants on cardiomyocyte survival, with a faulty proteostasis response to proteotoxic stress, which could be linked to a loss of chaperone interaction. It reveals a new putative pathological mechanism leading to DCM.

摘要

先天扩张性心肌病（DCM）是一种罕见的疾病，表现为左心室扩张和心脏功能不足，是移植的一个主要迹象。先天扩张性心肌病是一种异构的单基因疾病，主要是常染色体显性，大约占30%。迄今为止，已经鉴定了30多个基因的突变，但大多数病例仍然没有发现突变，导致这种疾病的分子和细胞机制仍不清楚。我们最近鉴定了BAG3（Bcl2 athanogene 3）基因可作为一种新的先天扩张性心肌病基因。已知的两种突变被错误的定位在BAG3的BAG功能域，这个功能域与伴侣Hsp70（热休克蛋白70）的相互作用是已知的。在蛋白质稳态中，BAG3是细胞保护的分子伴侣，并在错误折叠的自噬清除或蛋白降解的过程中起作用。这项研究的目的是证实BAG3突变导致了先天扩张性心肌病，并确定潜在的分子机制。通过瞬时过表达GFP-融合的BAG3蛋白，我们研究了这些突变在大鼠新生心肌细胞（RNC）和HeLa细胞中的生化和细胞效应。通过免疫荧光显微镜观察融合蛋白的定位。在大鼠新生心肌细胞中，BAG3-野生型/突变型过表达导致了在Z板的正常表达。然而，在转染48小时后，过表达BAG3突变型的大鼠新生心肌细胞显示了细胞萎缩和紊乱的肌节。利用GST pull-down实验研究了BAG3伙伴交互的潜在干扰，证明了BAG3-突变型/hsp70相互作用的缺失。通过WB实验检测LC3-II证明了BAG3突变对自噬的影响，基于荧光素酶报告基因实验分析了BAG3突变对蛋白质稳态影响。这些实验表明，在压力条件下，过表达BAG3-突变型似乎可以改变蛋白质稳态的调控。这些结果表明了BAG3突变体对心肌细胞存活率的影响，并在应对蛋白质毒性应激时，伴有缺陷的蛋白质稳态反应，这可能与伴侣间的相互作用缺失有关。这些结果揭示了导致先天扩张性心肌病的一种新的假定的病理机制。