Key result
The cTnT-K280N mutation increases myofilament Ca2+ sensitivity and impairs cellular relaxation at ~14% mutant levels.
Why the study?
HCM patients typically display heterozygous inheritance, but how the balance between mutant and WT protein expression affects cardiomyocyte function remained to be characterized.
Does the cTnT-K280N mutation alter myofilament Ca2+-sensitivity and cardiomyocyte function in human hypertrophic cardiomyopathy models?
Does the cTnT-K280N mutation alter myofilament Ca2+-sensitivity and cardiomyocyte function in human hypertrophic cardiomyopathy models?
Even low levels of the cTnT-K280N mutation are sufficient to increase myofilament Ca2+ sensitivity and alter contractility in human hypertrophic cardiomyopathy models.
Hypothesis-generating in HCM models; leaves open clinical relevance pending human validation.
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. 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 Ca2+-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 Ca2+-dynamics we used CRISPR/Cas9 to generate hiPSC-CMs harbouring heterozygous and homozygous TnT-K280N mutations. Ca2+-transient and cell shortening experiments compared these lines against isogenic controls. Myofilament Ca2+-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 Ca2+-sensitivity. Similarly, exchange of donor cells with 45 ± 2% cTnT-K280N increased Ca2+-sensitivity and was not corrected by PKA. cTnT-K280N hiPSC-CMs show elevated diastolic Ca2+ and increases in cell shortening. Impaired cardiomyocyte relaxation was only evident in homozygous cTnT-K280N hiPSC-CMs. The cTnT-K280N mutation increases myofilament Ca2+-sensitivity, elevates diastolic Ca2+, enhances contractility and impairs cellular relaxation. A low level (14%) of the cTnT-K280N sensitizes myofilaments to Ca2+, a universal finding of human HCM.
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Sequeira et al. (2022) studied Hypertrophic cardiomyopathy (HCM). cTnT-K280N mutation vs. cTnT-WT (healthy donors and isogenic controls) was evaluated on Myofilament Ca2+-sensitivity, Ca2+-dynamics, and cell shortening/relaxation. The cTnT-K280N mutation increases myofilament Ca2+-sensitivity, elevates diastolic Ca2+, enhances contractility, and impairs cellular relaxation, with even a 14% mutant level sufficient for sensitization.
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