Modelling proteostasis in dilated cardiomyopathy - the role of BAG3 and the HSP70 system

研究遗传决定的原发性心肌病是建立基本疾病机制和新的心肌疾病治疗选择的有利途径。其中一个潜在的分子靶点是bcl -2相关的凋亡基因3 (BAG3)，它在原发性扩张型心肌病（DCM）中发生突变。BAG3作为热休克蛋白70家族成员（hsp70）的核苷酸交换因子（NEF），调控蛋白折叠和降解途径。本研究旨在探讨BAG3 BAG结构域的突变如何影响构成型HSPA8的生化功能，扰乱心肌细胞蛋白稳态，最终导致DCM的分子机制。我们假设BAG3结构域内的DCM错义突变特异性地干扰了BAG3的NEF功能。因此，我们将利用体外伴侣蛋白检测来探索BAG3/HSPA8复合物的生物化学活性。具体来说，我们将在等基因诱导的多能干细胞（iPSC）中模拟一个来自大DCM家族的新型错义突变。使用这些iPSCs，我们将分化心肌细胞，进一步分析蛋白质聚集形成、蛋白质静止、自噬和蛋白酶体活性。在心肌细胞中，与HSPA8相关的共伴侣分子网络尚不清楚。我们将使用来自iPSC衍生心肌细胞的蛋白质组数据和体外伴侣分析来表征心脏特异性HSPA8复合物的组成和活性。由于我们观察到该DCM家族患者具有相当大的表型变异性，我们将利用来自患者和对照个体的iPSCs进行蛋白质组学和RNAseq表达研究，以寻找心脏保护因素。最后，我们的目标是在转基因斑马鱼模型中在病理生理和分子水平上表征BAG3相关的心脏表型。分析特异性BAG3错义突变将有助于探索BAG3如何影响有丝分裂后心肌细胞的蛋白质稳态，从而诱导DCM的分子机制。我们的长期目标是将我们的机制发现和技术工具箱转化为针对DCM的治疗方法。

Studying genetically determined, primary cardiomyopathies is an auspicious approach for establishing basic disease mechanisms and novel therapeutic options in myocardial disease. One such potential molecular target is Bcl-2-associated athanogene 3 (BAG3) that is mutated in primary dilated cardiomyopathy (DCM). BAG3 serves as nucleotide exchange factor (NEF) for heat shock protein 70 family members (HSP70s), which regulate protein folding and degradation pathways.In this proposal we aim to explore the molecular mechanism how mutation of the BAG3 BAG domain affect the biochemical function of constitutive HSPA8, perturb protein homeostasis (proteostasis) in cardiomyocytes, and finally lead to DCM. We hypothesize that DCM missense mutations within the BAG3 BAG domain specifically disturb the NEF function of BAG3. Thus, we will explore biochemically the activity of the BAG3/HSPA8 complex using in vitro chaperone assays. Specifically, we will model a novel missense mutation from a large DCM family in isogenic induced pluripotent stem cells (iPSC). Using these iPSCs we will differentiate cardiomyocytes that we further analyze for protein aggregate formation, proteostasis, autophagy, and proteasome activity. The molecular network of co-chaperones associated with HSPA8 is not well understood in cardiomyocytes. We will use proteome data from iPSC derived cardiomyocytes and in vitro chaperone assays to characterize the composition as well as activity of the cardiac specific HSPA8 complex. As we observe a considerable phenotypic variability in patients of this DCM family, we will utilize iPSCs from patients and control individuals in proteome and RNAseq expression studies to search for cardio protective factors. Lastly, we aim to characterize the BAG3 associated heart phenotype on pathophysiological and molecular level in a transgenic zebrafish model. The analysis of specific BAG3 missense mutations will help to explore the molecular mechanism how BAG3 affects proteostasis in post-mitotic cardiomyocytes and consequently induces DCM. Our long-term goal is to translate our mechanistic findings and technical toolbox into therapeutic approaches targeting DCM.