Hypertrophic Cardiomyopathy (HCM) is mostly caused by heterozygous mutations in sarcomeric proteins, which alter cardiomyocyte force generation. We previously observed highly variable force generation among individual cardiomyocytes of HCM-patients. It is presumably caused by stochastic, burst-like transcription of mutant and wildtype allele, causing unequal ratios of mutant vs. wildtype mRNA among individual cardiomyocytes. The resulting unequal fractions of mutated protein from cell to cell most likely underlie the variable force generation.

This so-called allelic and contractile imbalance could either already exist prior to clinical disease development, or be a consequence of disease-associated adaptations in symptomatic HCM-patients. To test this, we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as model for an early disease stage with mostly fetal-like sarcomeric protein expression. Patient-derived hiPSC-CMs with mutation R723G in the MYH7-gene showed hypertrophy and increased myofibrillar disarray compared to WT-hiPSC-CMs. Mutation related functional alterations were found like increased calcium sensitivity and higher twitch amplitude with prolonged time-to-peak and half-relaxation time.

To test whether MYH7 in hiPSC-CMs is transcribed in bursts or continuously, we performed RNA-fluorescence in situ hybridization (RNA-FISH). Nuclei without, with one and with two or more active transcription sites were observed, comparable to human cardiac tissue. This indicates stochastic, burst-like and independent transcription of the two MYH7-alleles. To test whether this leads to unequal expression of mutant and wildtype mRNA from cell to cell, we isolated individual cardiomyocytes by laser microdissection and quantified ratios of mRNA from both alleles by allele-specific single cell RT-PCR. We found highly variable ratios of mutated vs. wildtype mRNA among individual cardiomyocytes, similar to HCM-patient’s cardiac tissue. Furthermore, functional measurements on myofibrils from demembranated R723G-hiPSC-CMs revealed substantial variability in force generation at physiological calcium concentration (pCa 5.89), whereas heterogeneity was significantly smaller among WT-hiPSC-CMs at the same calcium concentration. In addition, twitch kinetics of intact hiPSC-CMs showed substantially increased variability among R723G- compared to WT-hiPSC-CMs. Altogether, this suggests that allelic imbalance among individual cardiomyocytes leads to functional heterogeneity.

Our results with HCM-patient derived hiPSC-cardiomyocytes as model for an early disease stage indicate that allelic and contractile imbalance among individual cardiomyocytes are primary to the disease and not secondary adaptational effects. Most likely burst-like transcription of MYH7 leads to the observed cardiomyocyte contractile heterogeneity in HCM.