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

• Applicants: Professorin Dr. Theresia Kraft; Privatdozent Dr. Robert Zweigerdt
• Subject Area: Anatomy and Physiology
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 252944158

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.

• DFG Programme: Research Grants