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

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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.