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.

大多数患有肌瘤的心肌病的患者是肌瘤基因中的杂合ING CMS中的不平衡在HCM患者的收缩期和纤维性途径中打扰了心脏合成。 MUT与WT mRNA和蛋白质的比率，尊重的蛋白质，因此在HCM患者的HCM患者的心肌上最有可能引起收缩性。我们将集中精力与NEITY相关，并在减少异质性的方法上。作为模型，我们将使用人类多能干细胞衍生的心肌细胞（HPSC-CMS），其MYH7-MONNT RY723G和WT-CONTORS我们将建立记者。用于检测WT和MUT肌球蛋白重链蛋白的等位基因特异性转录D的细胞线将使用这些CMS分析和模块化脉冲动力学。 mut mRNA和蛋白质使我们与转录和功能参数的异质性直接相关。我们使用芯片 - 顺序的技术对基因表达和染色质调制的收缩不平衡，我们的目标是确定与突变等位基因的XPRESSION相关的途径，这可能是减少突变效应的目标。肌节水平。