Protein quality control, cardiomyopathy, cardiotoxicity and human isogenic iPSCs

扩张型心肌病(DCM)通常与错误折叠的蛋白质堆积有关 从异常的蛋白质质量控制。针对蛋白酶体(蛋白酶体的关键成分)的化疗药物 蛋白质质量控制)与心脏毒性和心力衰竭有关。我们假设DCM- 相关的BAG3蛋白调节心脏中的蛋白质质量控制，并在 心肌病和心脏毒性。BAG3作为一个脚手架，绑定和协调两类 分子伴侣：热休克蛋白、热休克蛋白和热休克蛋白70。BAG3复合体是应激诱导的 协调蛋白质折叠、蛋白酶体降解和自噬--所有这些都是蛋白质质量的关键步骤 控制力。在心肌和骨骼肌中，这种伴侣复合体定位于Z盘，在那里它准备 调节特定的肌节客户蛋白。BAG3基因突变导致扩张型心肌梗死和突变特异性临床 表型，表明与不同的细胞过程和疾病途径有关。我们将测试特定的 同基因人诱导多能干细胞中BAG3基因工程点突变的功能模型 细胞(IPSCs)产生心肌细胞(iPS-CMS)。我们在IPSC基因组方面取得了重大进展 生产iPS-CMS和模拟人类心脏病的工程。我们还在开发基因调控 基于CRISPR抑制(CRISPRi)的工具可以快速沉默基因，以验证假定的BAG3相互作用。 我们的目标为实现这些目标提供了一条明确的道路。 目的1：在等基因IPSC系中鉴定与BAG3心肌病相关的细胞过程 携带与疾病相关的BAG3突变。我们正在制造杂合型和纯合型等基因iPS- 内源性BAG3基因座存在临床相关突变的CMS。 目的2：直接确定BAG3结合伙伴在心肌病发生发展中的作用 用CRISPRi沉默候选BAG3互作子在iPS-CMS中的表型。我们假设 特定的BAG3蛋白结合伙伴对DCM表型有贡献。 目的3：确定蛋白酶体抑制剂和其他化疗药物是否会导致心脏毒性 方式取决于蛋白质QC网络的特定组件。我们假设这一点改变了 BAG3伴侣复合体的功能导致蛋白酶体抑制剂的心脏毒性增强。 我们建议全面确定BAG3网络在人类中的机制作用 心肌细胞和扩张型心肌病。对BAG3介导的心肌蛋白质质量控制的基本认识 将提供对疾病机制、药物毒性和潜在治疗选择的见解。我们的研究成果是 预测性基因测试的基础，以了解遗传病和避免心脏毒性。我们是 希望机制洞察力将导致心肌病和异常蛋白疾病的治疗 质量控制。

Dilated cardiomyopathy (DCM) is often associated with accumulation of misfolded proteins thought to result from aberrant protein quality control. Chemotherapeutic drugs that target the proteasome (a key component of protein quality control) are associated with cardiotoxicity and heart failure. We hypothesize that the DCM- associated BAG3 protein regulates protein quality control in the heart and plays a critical role in cardiomyopathy and cardiotoxicity. BAG3 serves as a scaffold that binds and coordinates two classes of molecular chaperones: heat shock proteins, HSPBs and HSP70s. The BAG3 complex is stress-inducible and orchestrates protein folding, proteasomal degradation, and autophagy—all critical steps in protein quality control. In cardiac and skeletal muscle, this chaperone complex is localized in the Z-disk, where it is poised to regulate specific sarcomere client proteins. Mutations in BAG3 cause DCM and mutation-specific clinical phenotypes, suggesting a link with distinct cellular processes and disease pathways. We will test specific models of BAG3 function by engineering point mutations in BAG3 in isogenic human induced pluripotent stem cells (iPSCs) to produce cardiomyocytes (iPS-CMs). We have made significant progress in iPSC genome engineering to produce iPS-CMs and model human cardiac disease. We are also developing gene regulation tools based on CRISPR inhibition (CRISPRi) to rapidly silence genes to validate putative BAG3 interactions. Our aims provide a clear path to these goals. Aim 1: Identify the cellular processes involved in BAG3 cardiomyopathy in isogenic iPSC lines bearing disease-associated BAG3 mutations. We are making heterozygous and homozygous isogenic iPS- CMs that harbor clinically relevant mutations in the endogenous BAG3 locus. Aim 2: Directly define the role of BAG3-binding partners in the development of a cardiomyopathy phenotype in iPS-CMs by silencing candidate BAG3 interactors with CRISPRi. We hypothesize that specific BAG3 protein-binding partners contribute to the DCM phenotype. Aim 3: Determine if proteasome inhibitors and other chemotherapeutics cause cardiotoxicity in a manner dependent on specific components of the protein QC network. We hypothesize that altered function of the BAG3 chaperone complex leads to enhanced cardiotoxicity of proteasome inhibitors. We propose to comprehensively determine the mechanistic role of the BAG3 network in human cardiomyocytes and in DCM. A fundamental understanding of BAG3-mediated cardiac protein quality control will provide insights into disease mechanism, drug toxicity, and potential therapeutic options. Our studies lay the foundation for predictive genetic testing to understand genetic disease and avoid cardiotoxicity. We are hopeful that mechanistic insights will lead to treatments for cardiomyopathy and diseases of aberrant protein quality control.