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.

大多数患有肥厚性心肌病（HCM）的患者对于肌肉基因的突变都是杂合的。这些突变会导致力量产生心肌细胞（CMS）。然而，我们在HCM-Patient的心肌上的工作表明，在同一患者的单个CMS中，钙依赖性力的产生高度异质。这种异质性明显大于供体CMS。我们假设相邻CMS之间的这种收缩不平衡在HCM患者的收缩期间干扰了心脏合胞体的协调功能。最终，转换性不平衡会激活亲脑和促纤维化途径，从而有助于HCM通过病理发生。当分析收缩不平衡的潜在机制时，我们观察到相应基因的突变体（MUT）和野生型（WT） - 甲表示在随机和独立的爆发中转录。这会导致CMS之间的MUT与WT mRNA和蛋白质的不等之比，因此很可能导致收缩不平衡。到目前为止，我们分析了组织提取时HCM患者心肌的CMS上的爆发样转录以及转录和功能异质性。拟议项目的目的是分析爆发动力学并调节爆发和异质性。我们将重点介绍爆发如何与转录和功能异质性链接，以及降低异质性的方法。作为模型，我们将使用MYH7-MONT的人类多能干细胞衍生的心肌细胞（HPSC-CMS）使用R723G和WT-wt-Controls。我们将建立报告细胞系，以实时可视化等位基因特异性转录以及检测WT和MUTβ-肌球蛋白重链蛋白。这些CMS将用于分析和调节爆发动力学。单个CMS和WT-MUT mRNA和蛋白质分数的收缩力的平行分析将使我们能够将突发与转录和功能参数的异质性直接相关联。 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.我们旨在确定与突变等位基因表达相关的途径，这可能是抑制次级突变效应的靶标。为了降低肌节水平的功能异质性，我们将分析肉瘤蛋白功能不同调节剂对CMS中异质力产生的影响。