Gene Modulation of Acetylation Modifiers to Reveal Regulatory Links to Human Cardiac Electromechanics

项目摘要 表观遗传调控对心脏电机学和病理学具有重要意义。表观遗传调节子，如 组蛋白脱乙酰酶(HDAC)是已知的基因表达的主要调节者，影响心脏功能 通过染色质重塑，直接作用于转录因子(TF)，以及作用于细胞骨架和 收缩蛋白等。最近，新的药理学试剂，HDAC抑制剂，已经被 被开发用于癌症和免疫疾病的治疗，促使人们对HDAC的强健特征产生兴趣 心功能控制。我们的初步实验集中在RNAi信息的计算建模上 人诱导多能干细胞来源的心肌细胞(hiPSC-CM)的转录数据，但SAW 使用siRNA的击倒效率和仅丧失功能的调制的限制。延伸和完善 在这项工作中，我们提出了一种基于双向扰动(抑制/激活)的实验方法 通过CRISPR干扰和激活在HiPSC-CM中单独表达HDAC基因(CRISPRi/a)。转录体 对这些样本的分析将为模型的计算基因调控网络(GRN)推理提供依据 高密度脂蛋白、转铁蛋白和心脏离子通道的关系。GRN预测的关系将通过以下方式验证 全光机电分析测量HIPSC-CM中的电压、钙和收缩痕迹。一个 迭代方法将允许来自功能实验的反馈来改进我们的计算模型。是这样的 研究将增进我们对某些HDAC如何驱动心脏电生理表型的理解， 这在心脏损伤、心脏治疗学和心脏肿瘤学领域是至关重要的。

PROJECT ABSTRACT Epigenetic regulation is critical for cardiac electromechanics and pathology. Epigenetic modulators, such as histone deacetylases (HDACs), are known master regulators of gene expression and influence cardiac function through chromatin remodeling, direct action on transcription factors (TFs), and action on cytoskeletal and contractile proteins, among others. Recently, novel pharmacological agents, HDAC inhibitors, have been developed as treatments for cancer and immune diseases, driving an interest in robust characterization of HDAC control in cardiac function. Our preliminary experiments focused on computational modeling of RNAi-informed transcriptomic data in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) but saw limitations in knockdown efficiency and loss-of-function-only modulation using siRNAs. To extend and improve this work, we propose an experimental approach based on bidirectional perturbation (repression/activation) of individual HDAC genes in hiPSC-CM by CRISPR interference and activation (CRISPRi/a). Transcriptomic analysis of these samples will inform computational gene regulatory network (GRN) inference to model relationships between HDACs, TFs, and cardiac ion channels. GRN-predicted relationships will be validated by all-optical electromechanical assays measuring voltage, calcium, and contraction traces in hiPSC-CM. An iterative approach will allow feedback from functional experiments to refine our computational models. Such studies will advance our understanding of how certain HDACs drive electrophysiological phenotypes in the heart, which is critical in the fields of cardiac injury, cardiac therapeutics, and cardio-oncology.