The clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. We determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca²⁺-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca²⁺-sensitivity, incorporation of R278C increased Ca²⁺-sensitivity at low and intermediate dose, while it decreased Ca²⁺-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. Using troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca²⁺-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca²⁺-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.

Mutations in genes encoding sarcomeric proteins are a common cause of cardiomyopathy and sudden cardiac death in humans. We evaluated the hypothesis that myofilament dysfunction is coupled to morphological and functional alterations of cardiomyocyte nuclei in a Tnnc1-targeted knock-in (Tnnc1-p.A8V) mouse model of hypertrophic cardiomyopathy (HCM). Tnnc1 is the gene that codes for the isoform of the Ca²⁺-regulatory protein troponin C (cTnC) that is expressed in cardiomyocytes and slow skeletal muscle fibers and resides on thin filaments of sarcomeres in those muscles. This pathogenic mutation in a sarcomere gene alters many aspects of cardiomyocyte function, including sarcomere contractility, cytoplasmic Ca²⁺ buffering, and gene expression. Analysis of myocardial histological sections and isolated cardiomyocytes from adult Tnnc1-p.A8V mouse hearts revealed significantly smaller (cross-sectional area and volume) and rounder nuclei compared to those from age-matched, wild-type control mice. Changes in nuclear morphology could not be explained by differences in cardiomyocyte size or ploidy. Isolated wild-type and mutant cardiomyocyte nuclei, which are embedded centrally within myofibrils, undergo compression during contraction of the cardiomyocyte, indicating that during each heartbeat cardiomyocyte nuclei would be mechanically deformed as well as being exposed to elevated cytoplasmic Ca²⁺. Immunoblotting analysis indicated decreased nuclear localization of cardiac troponin C and decreased histone H4 expression in Tnnc1-p.A8V mouse hearts. Next, we investigated the influence of nucleocytoplasmic transport by immunofluorescence microscopy, and we could not confirm nuclear localization of cardiac troponin C in fixed myocardial tissue from adult mice. However, cardiac troponin C could be detected in healthy human-induced pluripotent stem cell-derived cardiomyocyte nuclei. We conclude that pathological myofilament dysfunction due to a pathogenic, cardiomyopathy-associated mutation can be linked to altered protein composition of cardiomyocyte nuclei and aberrant nuclear morphology.