Comprehensive Analysis of Allelic, Cellular and Molecular Heterogeneity in Human 3-Dimensional Cardiac Microtissues with MYH7 Mutations

项目摘要/摘要 肥厚型心肌病(HCM)患者有心脏性猝死和进展性心脏病的风险 失败(HF)，而且没有有效的治疗方法，部分原因是我们对HCM的遗传理解有限 发病机制。我们目前对连接特定基因的分子机制的认识存在着严重的空白 β肌球蛋白重链基因(MYH7)突变导致心肌病理性增厚 与HCM相关。我们的长期目标是利用基因组工具结合三维心脏 人诱导多能干细胞来源的微组织探讨人巨细胞瘤继发性发病机制 特定的MYH7变异，并利用这些洞察力来识别新的疾病生物标志物和治疗靶点 适用于特定的肥厚型心肌炎患者。 我们之前的研究发现了两个与HCM相关的MYH7变异体，精氨酸403到谷氨酰胺和 位于肌球蛋白重链蛋白(MHC-MHC)的肌动蛋白结合区。 β)，产生更大的微组织收缩力量，并伴随收缩动力学异常。 其他人的研究表明，MYH7变异位于MHC-β的不同结构域中，导致 不同的表型。这些结果导致了我们的中心假设，即HCM是一种异质性疾病，其中 患者的症状和治疗反应取决于引起MYH7变异的位置(S) 在基因内和在细胞类型的特定转录和表观遗传程序上，这些程序始于 收缩功能异常。在我们全面的初步数据指导下，我们建议继续进行三项 确定HCM发病机制的多尺度洞察力的特定目标：(1)表征功能性 MYH7变异定位于MHC-肌动蛋白结合、ATP结合和转换结构域的后果 β，(2)鉴定微组织中hCM的细胞类型特异性转录和表观遗传机制 单细胞RNA-seq和ATAC-seq以及(3)询问C1ORF105的功能 与HCM相关的线粒体蛋白，在人类心肌细胞中特异表达。 总而言之，执行这些目标将提供对职能作用的更准确的理解 MYH7变异的定位，产生新的细胞类型特异性和分子机制，并鉴定 肌节和线粒体功能之间的关键分子联系将广泛影响 肥厚性心肌病和心力衰竭。

PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) patients are at risk for sudden cardiac death and progressive heart failure (HF), and there are no effective therapeutics, due in part to our limited genetic understanding of HCM pathogenesis. There are critical gaps in our current knowledge of the molecular mechanisms that link specific mutations in the beta myosin heavy chain gene (MYH7) to pathological thickening of the heart muscle that is associated with HCM. Our long-term goals are to utilize genomic tools combined with 3-dimensional cardiac microtissues derived from human induced pluripotent stem cells to interrogate mechanisms of HCM secondary to specific MYH7 variants, and to utilize these insights to identify new disease biomarkers and therapeutic targets for specific HCM patients. Our previous studies identified that two HCM-associated MYH7 variants, arginine 403 to glutamine and valine 606 to methionine that are located in the actin-binding domain of beta myosin heavy chain protein (MHC- β), generate increased microtissue contraction force with associated abnormalities in contraction kinetics. Studies by others have indicated that MYH7 variants located in distinct structural domains of MHC-β cause distinct phenotypes. These results lead to our central hypothesis that HCM is a heterogeneous disorder, in which patient symptoms and therapeutic responses are dependent on the location of the causative MYH7 variant(s) within the gene and on cell-type specific transcriptional and epigenetic programs, which initiate from abnormalities in contractile function. Guided by our comprehensive preliminary data, we propose to pursue three Specific Aims to determine multi-scale insights into HCM pathogenesis: (1) to characterize functional consequences of MYH7 variants localized to the actin-binding, ATP-binding and converter domains of MHC- β, (2) to identify cell type-specific transcriptional and epigenetic mechanisms of HCM in microtissues using paired single-cell RNA-seq and ATAC-seq and (3) to interrogate the function of C1ORF105, a nuclear-encoded mitochondrial protein that is associated with HCM and is specifically expressed in human cardiomyocytes. In summary, the execution of these aims will provide a more precise understanding of the functional role of MYH7 variant localization, generate novel cell-type specific and molecular mechanisms of HCM and identify critical molecular linkages between sarcomere and mitochondrial function that will broadly impact the field of HCM and heart failure.