Hereditary cardiomyopathies are a leading cause of cardiovascular morbidity and mortality globally. A considerable proportion of these conditions result from premature termination codons (PTCs), which trigger nonsense-mediated mRNA decay (NMD)—a surveillance pathway that degrades faulty transcripts. Hypertrophic cardiomyopathy (HCM), marked by pathological cardiac hypertrophy and arrhythmias, is often linked to autosomal-dominant PTC mutations in the MYBPC3 gene.
To investigate the role of NMD in MYBPC3-associated HCM, we used induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients and healthy controls. Through CRISPR/Cas9 genome editing, we generated isogenic iPSC lines carrying or lacking MYBPC3 PTC mutations.
In mutant iPSC-CMs, we detected abnormal calcium handling, specifically prolonged calcium decay kinetics, despite preserved contractile function. Although MYBPC3 mRNA levels were significantly reduced, protein levels remained similar to isogenic controls, indicating that haploinsufficiency may not explain the observed phenotype. For further validation, we generated isogenic iPSC with a heterozygous MYBPC3 promotor knockout (MYBPC3-WT/PrKO). While we observed decreased mRNA levels of MYBPC3, protein levels were not decreased here, either. However, MYBPC3-WT/PrKO iPSC-CMs exhibited calcium kinetics comparable to healthy controls. RNA sequencing to evaluate RNA decay (decay-seq) confirmed accelerated degradation of NMD-sensitive mRNAs in MYBPC3-WT/PTC compared to MYBPC3-WT/WT iPSC-CMs.
Furthermore, pharmacological inhibition of NMD via SMG1 inhibition restored normal calcium handling properties in MYBPC3-WT/PTC iPSC-CMs and upregulated several known NMD target transcripts such as HDGF and RPL12. In addition, we observed a significant downregulation of CASQ2—a gene that binds and buffers calcium ions in the sarcoplasmic reticulum—in MYBPC3-WT/PTC. Knocking down CASQ2 in MYBPC3-WT/WT iPSC-CM recapitulated the phenotype observed in MYBPC3-WT/PTC iPSC-CM, indicating a mediating role of CASQ2 in driving the HCM phenotype.
Collectively, our findings demonstrate that NMD activation rather than haploinsufficiency in MYBPC3-mutant iPSC-CMs contributes to HCM pathogenesis which results in disrupted calcium dynamics. Identification of the link between genotype and phenotype will offer a novel therapeutic strategy for PTC-associated HCM.
