Burst-like transcription of sarcomeric genes as pathogenic factor in Hypertrophic Cardiomyopathy

肌节基因的爆发式转录作为肥厚型心肌病的致病因素

• 批准号: 266761022
• 负责人: Professorin Dr. Theresia Kraft
• 金额: --
• 依托单位: Institut für Molekular- und Zellphysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/266761022?language=en
• 关键词: Burst; like; transcription; sarcomeric; genes

Most patients with Hypertrophic Cardiomyopathy (HCM) are heterozygous for mutations in sarcomeric genes. These mutations lead to altered force generation of cardiomyocytes (CMs). Yet, our work on HCM-patient's myocardium revealed that calcium-dependent force generation was highly heterogeneous among individual CMs from the same patient. This heterogeneity was significantly larger than among donor CMs. We hypothesize that this contractile imbalance among neighboring CMs disturbs the coordinated function of the cardiac syncytium during systole in HCM patients. Eventually contractile imbalance activates pro-hypertrophic and pro-fibrotic pathways and thereby contributes to HCM-pathogenesis. When analyzing underlying mechanisms of contractile imbalance we observed that mutant (MUT) and wildtype (WT)-alleles of the respective gene are transcribed in stochastic and independent bursts. This leads to unequal ratios of MUT vs. WT mRNA and protein, respectively, among CMs, thus most likely causing contractile imbalance. So far, we analyzed burst-like transcription and transcriptional and functional heterogeneity on CMs from HCM patient’s myocardium at the time of tissue extraction. Aims of the proposed project are to analyze burst kinetics and to modulate bursts and heterogeneity. We will focus on how bursts are linked to transcriptional and functional heterogeneity, and on approaches to reduce heterogeneity.As a model, we will use human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) with MYH7-mutation R723G and WT-controls. We will establish reporter cell lines for real-time visualization of allele-specific transcription and for detection of WT and MUT β-myosin heavy chain protein. These CMs will be used to analyze and modulate burst kinetics. Parallel analysis of contractility of individual CMs and fractions of WT- vs. MUT mRNA and protein, respectively, will enable us to directly associate bursts with heterogeneity of transcriptional and functional parameters. Furthermore, we will analyze the potential of specific inhibition of the MUT-allele to reduce heterogeneity.In human myocardium and hPSC-CMs, we will address the relationship between unequal expression of WT/MUT-mRNA and pro-hypertrophic and pro-fibrotic gene expression in individual CMs and study the effect of contractile imbalance on gene-expression and chromatin modulation using RNA- and ChIP-sequencing techniques. We aim to identify pathways related to the expression of the mutant allele, which could be targets for inhibition of secondary mutation effects. To reduce functional heterogeneity at the sarcomere level we will analyze effects of different modulators of sarcomeric protein function on heterogeneous force generation among CMs.

大多数肥厚性心肌病（HCM）患者的肌瘤基因突变为杂合型。这些突变导致心肌细胞（CMs）产生力的改变。然而，我们对hcm患者心肌的研究表明，来自同一患者的不同cm的钙依赖性力产生是高度异质性的。这种异质性明显大于供体CMs。我们假设这种相邻cm之间的收缩不平衡扰乱了HCM患者收缩期心脏合胞体的协调功能。最终，收缩失衡激活促肥厚和促纤维化途径，从而促进hcm发病。在分析收缩失衡的潜在机制时，我们发现各自基因的突变型（MUT）和野生型（WT）等位基因在随机和独立的爆发中转录。这导致CMs中MUT与WT mRNA和蛋白质的比例不相等，从而很可能导致收缩不平衡。到目前为止，我们分析了HCM患者心肌CMs在组织提取时的爆发样转录和转录和功能异质性。提出的项目的目的是分析爆发动力学和调节爆发和异质性。我们将重点关注爆发如何与转录和功能异质性联系起来，以及减少异质性的方法。作为模型，我们将使用myh7突变R723G和wt对照的人类多能干细胞来源的心肌细胞（hPSC-CMs）。我们将建立报告细胞系，用于实时可视化等位基因特异性转录和检测WT和MUT β-肌球蛋白重链蛋白。这些CMs将用于分析和调节爆发动力学。分别对单个CMs和WT- vs. MUT mRNA和蛋白质组分的收缩性进行平行分析，将使我们能够直接将爆发与转录和功能参数的异质性联系起来。此外，我们将分析mut -等位基因特异性抑制的潜力，以减少异质性。在人类心肌和hPSC-CMs中，我们将研究WT/ mutmrna的不平等表达与个体CMs中促肥厚和促纤维化基因表达之间的关系，并利用RNA和chip测序技术研究收缩不平衡对基因表达和染色质调节的影响。我们的目标是确定与突变等位基因表达相关的途径，这可能是抑制继发性突变效应的目标。为了减少肌节水平上的功能异质性，我们将分析不同的肌节蛋白功能调节剂对肌节间异质力产生的影响。