Low expression of the K280N mutation is sufficient to increase basal myofilament activation in human hypertrophy cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder with patients typically showing heterozygous inheritance of a pathogenic variant in a gene encoding a contractile protein. Here, we study the contractile effects of a rare homozygous mutation using explanted tissue and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to gain insight into how the balance between mutant and WT protein expression affects cardiomyocyte function.

Methods: Force measurements were performed in cardiomyocytes isolated from a HCM patient carrying a homozygous troponin T mutation (cTnT-K280N) and healthy donors. To discriminate between mutation-mediated and phosphorylation-related effects on Ca-sensitivity, cardiomyocytes were treated with alkaline phosphatase (AP) or protein kinase A (PKA). Troponin exchange experiments characterized the relation between mutant levels and myofilament function. To define mutation-mediated effects on Ca-dynamics we used CRISPR/Cas9 to generate hiPSC-CMs harbouring heterozygous and homozygous TnT-K280N mutations. Ca-transient and cell shortening experiments compared these lines against isogenic controls.

Results: Myofilament Ca-sensitivity was higher in homozygous cTnT-K280N cardiomyocytes and was not corrected by AP- and PKA-treatment. In cTnT-K280N cells exchanged with cTnT-WT, a low level (14%) of cTnT-K280N mutation elevated Ca-sensitivity. Similarly, exchange of donor cells with 45 ± 2% cTnT-K280N increased Ca-sensitivity and was not corrected by PKA. cTnT-K280N hiPSC-CMs show elevated diastolic Ca and increases in cell shortening. Impaired cardiomyocyte relaxation was only evident in homozygous cTnT-K280N hiPSC-CMs.

Conclusions: The cTnT-K280N mutation increases myofilament Ca-sensitivity, elevates diastolic Ca, enhances contractility and impairs cellular relaxation. A low level (14%) of the cTnT-K280N sensitizes myofilaments to Ca, a universal finding of human HCM.