Human-based computational and experimental investigation of disease mechanisms in mutation-specific hypertrophic cardiomyopathy

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 764738. British Heart Foundation Intermediate Basic Science Fellowship (FS/17/22/32644). Background The pathogenic TNNI3R21C/+ variant causes malignant hypertrophic cardiomyopathy (HCM) with high incidence of sudden cardiac death, even in individuals absent of hypertrophy. There is evidence to support a known biophysical defect in the protein, yet the cellular mechanisms that precipitate adverse clinical outcomes remain unclear. Purpose We aim to computationally model and map the TNNI3R21C/+ cellular phenotype observed in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) to human disease, thereby explaining the key mechanisms driving HCM in TNNI3R21C/+ variant carriers. Methods Wild-type (WT) and TNNI3R21C/+ iPSC-CMs were characterised by calcium transient analysis and direct sarcomere tracking to assess cellular contraction and relaxation. In-vitro data was used to inform the in-silico modelling of human cardiomyocytes. We constructed an in-silico population of WT adult cardiomyocytes and used it to transform the in-vitro data into corresponding adult phenotypes by means of a novel iPSC-to-adult data mapping. We tested the hypothesis that the abnormal TNNI3R21C/+ phenotype observed in iPSC-CMs would be explained by alterations in calcium affinity of troponin and increased myofilament calcium sensitivity. Results Analysis of in-vitro iPSC-CM data showed that TNNI3R21C/+ cells exhibit increased contractility with slowed relaxation when compared to WT. They also exhibited a faster rise in the calcium transient with a slowed calcium decay in comparison to WT. The in-silico adult TNNI3R21C/+ phenotype from the iPSC-to-adult mapping replicated the abnormalities observed in iPSC-CMs. The WT in-silico population accurately covered the ranges of electromechanical biomarkers providing a representative cohort of physiological variability. The TNNI3R21C/+ calcium phenotype could be recovered by our in-silico mutant models. Simulation results suggest that calcium abnormalities in TNNI3R21C/+ are a direct consequence of abnormal calcium buffering by thin filaments, mediated by increases in calcium affinity of troponin and myofilament calcium sensitivity. The TNNI3R21C/+ phenotype could not be recovered if these two factors were considered in isolation. Corresponding contractility analyses of in-silico models showed that the calcium level changes caused by the TNNI3R21C/+ phenotype are associated with hypercontractility and diastolic dysfunction, well-known hallmarks of HCM, which were also observed in the iPSC-CM model of disease. Conclusions This study showcases human-based computational and experimental methodologies that unearth direct mechanistic explanations of phenotypes driven by the TNNI3R21C/+ HCM variant. We show that the TNNI3R21C/+ HCM-causing mutation exhibits multifactorial remodelling of troponin calcium affinity and myofilament calcium sensitivity. Unearthing mechanistic pathways in mutation-specific HCM will be key to develop effective pharmacological interventions for a wide variety of understudied variants.