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

大多数肥厚型心肌病（HCM）患者的肌节基因突变是杂合子。这些突变导致心肌细胞（CM）的力产生改变。然而，我们对HCM患者心肌的研究表明，同一患者的单个CM之间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CM。我们推测，这种收缩不平衡之间的相邻CM干扰协调功能的心脏合胞体在收缩期HCM患者。最终，收缩失衡激活促肥大和促纤维化途径，从而导致HCM发病机制。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型（MUT）和野生型（WT）等位基因以随机和独立的爆发形式转录。这导致CM中MUT与WT mRNA和蛋白质的比例分别不相等，因此很可能导致收缩失衡。到目前为止，我们分析了在组织提取时来自HCM患者心肌的CM上的突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和调制爆发和异质性。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。作为一个模型，我们将使用MYH 7突变R723 G和WT对照的人多能干细胞衍生的心肌细胞（hPSC-CM）。我们将建立报告细胞系，用于等位基因特异性转录的实时可视化和WT和MUT β-肌球蛋白重链蛋白的检测。这些CM将用于分析和调节爆发动力学。对单个CM的收缩性以及WT-与MUT mRNA和蛋白质的组分的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相关联。此外，我们将分析特异性抑制MUT等位基因以减少异质性的潜力。在人类心肌和hPSC-CM中，我们将解决WT/MUT-mRNA的不平等表达与个体CM中促肥大和促纤维化基因表达之间的关系，并使用RNA和ChIP测序技术研究收缩失衡对基因表达和染色质调节的影响。我们的目的是确定相关的突变等位基因的表达，这可能是抑制二次突变效应的目标途径。为了减少肌节水平的功能异质性，我们将分析肌节蛋白功能的不同调节剂对CM之间异质力产生的影响。

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.