Variable cMyBP-C distribution and transition from fast to slow contraction phenotype in patient-specific MYBPC3c.927-2A>G hiPSC cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disorder often associated with mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C). Studies on single cardiomyocytes (CMs) from heterozygous MYBPC3 HCM patients have revealed marked variability in calcium sensitivity and force generation, as well as haploinsufficiency and mosaic-like cMyBP-C distribution from CM to CM. We hypothesized that variable cMyBP-C distribution and functional heterogeneity among individual CMs contribute to HCM progression by destabilizing the functional syncytium of the myocardium.

To investigate this hypothesis, we developed a HCM disease model by using patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a heterozygous truncating MYBPC3 mutation (c.927-2A>G). This mutation is identical to that identified in a patient, who was diagnosed with stage IV heart failure and underwent heart transplantation at age 45 years. Mutant MYBPC3^c.927-2A>G and corrected isogenic control hiPSC-CMs were cultured on laminin-coated coverslips and micropatterned slides for over 70 days. Subsequently, they were analysed for protein expression, cellular morphology, contractile function, and mRNA expression in a time-dependent manner.

Immunofluorescence staining targeting cMyBP-C protein indicated a mosaic-like distribution of cMyBP-C in MYBPC3^c.927-2A>G hiPSC-CMs, becoming increasingly pronounced after day 56 of cultivation. Functionally, twitch parameters, such as time to peak (ttp) and half relaxation time (hrt), were initially shorter in the MYBPC3^c.927-2A>G   hiPSC-CMs, suggesting faster contraction. However, ttp and hrt became longer over time, accompanied by a reduction in contraction velocity. Additionally, we observed altered Ca²⁺-handling. While both the time to peak of Ca²⁺-transient (Ca²⁺-ttp) and Ca²⁺ half decay time (Ca²⁺-hrt) were shorter early on, this phenotype diminished in prolonged culture. The Ca²⁺-transient amplitude was initially elevated, but decreased over time. Furthermore, mRNA sequencing indicated upregulation of HCM related pathways, including Ca²⁺ handling, cardiac muscle contraction, MAPK and TGF signaling.

This study suggests that the variable cMyBP-C protein distribution from CM to CM could be a critical factor in HCM development. This implies that therapeutic strategies tailored to different disease stages might be beneficial.