Rationale: Calcium transient analysis is central to understanding inherited and acquired cardiac physiology and disease. While the development of novel calcium reporters enables assays of CRISPR/Cas-9 genome edited pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and primary adult cardiomyocytes, existing calcium-detection technologies are often proprietary and require specialist equipment, while open source workflows necessitate considerable user expertise and manual input. Objective: We aimed to develop an easy to use open source, adaptable, and automated analysis pipeline for measuring cellular calcium transients, from image stack to data output, inclusive of cellular identification, background subtraction, photobleaching correction, calcium transient averaging, calcium transient fitting, data collation and aberrant behavior recognition. Methods and Results: We developed CalTrack, a MatLab based algorithm, to monitor fluorescent calcium transients in living cardiomyocytes, including isolated single cells or those contained in 3-dimensional tissues or organoids and to analyze data acquired using photomultiplier tubes or employing line scans. CalTrack uses masks to segment cells allowing multiple cardiomyocyte transients to be measured from a single field of view. After automatically correcting for photobleaching, CalTrack averages and fits a string of transients and provides parameters that measure time to peak, time of decay, tau, F max /F 0 and others. We demonstrate the utility of CalTrack in primary and iPSC-derived cell lines in response to pharmacological compounds and in phenotyping cells carrying hypertrophic cardiomyopathy variants. Conclusions: CalTrack, an open source tool that runs on a local computer, provides automated high-throughput analysis of calcium transients in response to development, genetic or pharmacological manipulations, and pathological conditions. We expect that CalTrack analyses will accelerate insights into physiologic and abnormal calcium homeostasis that influence diverse aspects of cardiomyocyte biology.

Cardiomyopathies have unresolved genotype–phenotype relationships and lack disease-specific treatments. Here we provide a framework to identify genotype-specific pathomechanisms and therapeutic targets to accelerate the development of precision medicine. We use human cardiac electromechanical in-silico modelling and simulation which we validate with experimental hiPSC-CM data and modelling in combination with clinical biomarkers. We select hypertrophic cardiomyopathy as a challenge for this approach and study genetic variations that mutate proteins of the thick (MYH7R403Q/+) and thin filaments (TNNT2R92Q/+, TNNI3R21C/+) of the cardiac sarcomere. Using in-silico techniques we show that the destabilisation of myosin super relaxation observed in hiPSC-CMs drives disease in virtual cells and ventricles carrying the MYH7R403Q/+ variant, and that secondary effects on thin filament activation are necessary to precipitate slowed relaxation of the cell and diastolic insufficiency in the chamber. In-silico modelling shows that Mavacamten corrects the MYH7R403Q/+ phenotype in agreement with hiPSC-CM experiments. Our in-silico model predicts that the thin filament variants TNNT2R92Q/+ and TNNI3R21C/+ display altered calcium regulation as central pathomechanism, for which Mavacamten provides incomplete salvage, which we have corroborated in TNNT2R92Q/+ and TNNI3R21C/+ hiPSC-CMs. We define the ideal characteristics of a novel thin filament-targeting compound and show its efficacy in-silico. We demonstrate that hybrid human-based hiPSC-CM and in-silico studies accelerate pathomechanism discovery and classification testing, improving clinical interpretation of genetic variants, and directing rational therapeutic targeting and design.

Hypertrophic cardiomyopathy (HCM) affects as many as ~1 in 500 individuals, and is often typified by hyperdynamic contraction and poor cellular relaxation. HCM can be caused by mutations in a variety of key contractile proteins of the sarcomere. A large proportion of these variants are found in MYBPC3, MYH7, TNNT2, and TNNI3. These genes encode proteins that control cardiac muscle contraction at the thick (MYBPC3 and MYH7) and thin filaments (TNNT2 and TNNI3) of the sarcomere. In this study we use human induced pluripotent stem cell derived cardiomyocytes to model HCM across all of these genes. We do this to define key mechanistic differences between thick and thin filament HCM. We define sarcomeric contractility (SarcTrack) calcium transients (CalTrack) and myosin states using the mant-ATP assay. We use the parametric data from these experimental studies in iPSC-CMs to model possible disease mechanisms in silico. Our experimental analysis highlights that both thick and thin filament HCM variants cause cellular hypercontractility, with slowed cellular relaxation. We find that thick filament HCM variants drive cellular HCM phenotypes by destabilising the myosin interacting heads motif (IHM), showing a marked reduction in the super relaxed state of myosin. Counterintuitively thin filament based HCM variants show a reduction in DRX myosin. When applying Mavacamten the allosteric myosin ATPase inhibitor to our thin and thick filament HCM variant iPSC-CMs we find a dichotomy of cellular responses. The thick filament variants studied all show a clear resolution of cellular HCM. However, not all cellular phenotypes of thin filament HCM are corrected by Mavacamten treatment, although there is benefit. We conclude that causal mechanisms of thick filament HCM are well corrected at the molecular and cellular level by Mavacamten, but these causal mechanisms in thin filament based HCM are not suitably corrected. We highlight key mechanistic pharmacological targets for thin filament variants that could add cellular benefit to HCM phenotype resolution.

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.

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