Contractile imbalance among cardiomyocytes as pathogenic factor for Hypertrophic Cardiomyopathy – investigations on human pluripotent stem-cell derived cardiomyocytes carrying cMyBP-C-mutations.

肥厚型心肌病是最常见的遗传性心脏病，主要由心肌肌球蛋白结合蛋白C(cMyBP-C，MYBPC3)和β-肌球蛋白重链(β-MyHC，MYH7)杂合突变引起。解释为什么不同的突变会导致相似的HCM表型的共同机制尚不清楚。我们以前对MYH7错义突变或MYBPC3截短突变的肥厚型心肌病患者的心肌细胞的研究表明，个体心肌细胞之间的力产生和钙敏感性高度可变，即所谓的收缩失衡。对于MYH7突变，这与个体CMS中突变(MT)和野生型(WT)mRNA的比例不同有关。对于单倍体功能不全诱导的MYBPC3突变，患者CMS中不同水平的WT-cMyBP-C表明细胞间存在镶嵌样表达。有证据表明，对于MYH7和MYBPC3，观察到的细胞间等位基因不平衡是由于各自等位基因的突发式、随机、独立转录所致。我们推测，至少对于MYH7和MYBPC3的突变，导致相邻CMS之间的收缩不平衡是发生HCM的潜在原因-典型的细胞紊乱，并可能导致肥大和纤维化。为了进一步研究HCM的发病机制，在之前的资助期间，我们基于患者来源的带有MYH7错义突变的HiPSC-CMS建立了HCM的细胞模型。我们建立了在大多数细胞质雄性不育系中唯一表达β-MyHC的HiPSC-CMS成熟策略。通过一种新的单细胞定位方法，我们将功能归因于同一CM的mRNA/蛋白表达。MYH7-MT-CMS的单细胞分析显示出典型的HCM特征，如细胞面积较大，收缩参数改变。与HCM患者CMS一样，MYH7-MT-hiPSC-CMS在单个CMS中也显示出MYH7的爆发式转录以及WT与MT MYH7-mRNA的高度可变部分，表明细胞间等位基因失衡。然而，在我们单一的HiPSC-CM方法中，无法研究WT和MT蛋白丰度的镶嵌样差异与促肥大和促纤维化通路的收缩失衡、错乱和激活之间的直接联系。因此，在拟议的研究中，我们将通过研究具有单倍性诱导MYBPC3-突变的HiPSC-CMS的2D单层培养作为功能性CM合胞体的模型来验证我们的假设，这使得可以直接看到嵌合体样WT-cMyBP-C的表达。通过单细胞定位，HIPSC-CMS的功能特性与构成收缩失衡的细胞之间的差异将归因于单个CMS中WT-cMyBP-C蛋白的表达。进一步的分析将解决等位基因/收缩失衡对疾病发展的影响，以及对纤维化和肥大相关标记物表达变化的影响。此外，针对力量产生的选定物质将根据其减少收缩不平衡的潜力进行评估。

Hypertrophic Cardiomyopathy (HCM), the most frequent inherited cardiac disease, is mostly caused by heterozygous mutations in cardiac myosin binding protein C (cMyBP-C, MYBPC3) and β-myosin heavy chain (β-MyHC, MYH7). A common mechanism explaining why different mutations induce a similar HCM phenotype is unclear. Our previous studies on ventricular cardiomyocytes (CMs) from HCM-patients with missense mutations in MYH7 or a truncating mutation in MYBPC3 revealed highly variable force generation and calcium-sensitivity among individual CMs, so-called contractile imbalance. For MYH7 mutations this was associated with unequal fractions of mutated (MT) vs. wildtype (WT) mRNA in individual CMs. For a haploinsufficiency-inducing MYBPC3-mutation varying levels of WT-cMyBP-C in patient-CMs indicated mosaic-like expression from cell to cell. Evidence suggests that for MYH7 and MYBPC3 the observed cell-to-cell allelic imbalance is due to burst-like, stochastic, independent transcription of the respective alleles. We hypothesize that at least for mutations in MYH7 and MYBPC3 the resulting contractile imbalance among neighboring CMs is an underlying cause for development of HCM-typical cellular disarray and may induce hypertrophy and fibrosis.To further investigate HCM-pathomechanisms, in the previous funding period we established cellular models for HCM based on patient-derived hiPSC-CMs with missense mutations in MYH7. We developed a maturation strategy of hiPSC-CMs with exclusive expression of β-MyHC in most CMs. With a new single cell remapping method we attributed function to mRNA/protein expression of the same CM. Single cell analyses of MYH7-MT-CMs showed HCM-typical properties like larger cell area and altered contraction parameters. Like HCM-patient CMs, the MYH7-MT-hiPSC-CMs also showed burst-like transcription of MYH7 and highly variable fractions of WT vs. MT MYH7-mRNA in individual CMs indicating cell-to-cell allelic imbalance. Yet, in our single hiPSC-CM approach, immediate links between mosaic-like differences of WT and MT protein abundance and development of contractile imbalance, disarray, and activation of pro-hypertrophic and pro-fibrotic pathways could not be studied. Therefore, in the proposed study we will test our hypothesis by studying 2D monolayer cultures of hiPSC-CMs with haploinsufficiency-inducing MYBPC3-mutations as models for functional CM syncytia, which allow direct visualization of mosaic like WT-cMyBP-C expression. Functional properties of hiPSC-CMs, with differences between cells constituting contractile imbalance will be attributed to WT-cMyBP-C protein expression in individual CMs by single cell mapping. Further analyses will address the effects of allelic/contractile imbalance on development of disarray, and on changes in expression of fibrosis- and hypertrophy-related markers. Moreover, selected substances that target force generation will be assessed towards their potential for reducing contractile imbalance.