In silico Identification of Disrupted Myocardial Calcium Homeostasis as Proarrhythmic Trigger in Arrhythmogenic Cardiomyopathy

Background: Patients with arrhythmogenic cardiomyopathy may suffer from lethal ventricular arrhythmias. Arrhythmogenic cardiomyopathy is predominantly triggered by mutations in plakophilin-2, a key component of cell-to-cell adhesion and calcium cycling regulation in cardiomyocytes. Calcium dysregulation due to plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear.Aim: To unravel the mechanisms by which calcium-handling abnormalities in plakophilin-2 loss-of-function may contribute to proarrhythmic events in arrhythmogenic cardiomyopathy.Methods: We adapted a computer model of mouse ventricular electrophysiology using recent experimental calcium-handling data from plakophilin-2 conditional knock-out (PKP2-cKO) mice. We simulated individual effects of beta-adrenergic stimulation, modifications in connexin43-mediated calcium entry, sodium-calcium exchanger (NCX) activity and ryanodine-receptor 2 (RyR2) calcium affinity on cellular electrophysiology and occurrence of arrhythmogenic events (delayed-afterdepolarizations). A population-of-models approach was used to investigate the generalizability of our findings. Finally, we assessed the potential translation of proposed mechanisms to humans, using a human ventricular cardiomyocyte computational model.Results: The model robustly reproduced the experimental calcium-handling changes in PKP2-cKO cardiomyocytes: an increased calcium transient amplitude (562 vs. 383 nM), increased diastolic calcium (120 vs. 91 nM), reduced L-type calcium current (15.0 vs. 21.4 pA/pF) and an increased free SR calcium (0.69 vs. 0.50 mM). Under beta-adrenergic stimulation, PKP2-cKO models from the population of models (n = 61) showed a higher susceptibility to delayed-afterdepolarizations compared to control (41 vs. 3.3%). Increased connexin43-mediated calcium entry further elevated the number of delayed-afterdepolarizations (78.7%, 2.5-fold increase in background calcium influx). Elevated diastolic cleft calcium appeared responsible for the increased RyR2-mediated calcium leak, promoting delayed-afterdepolarizations occurrence. A reduction in RyR2 calcium affinity prevented delayed-afterdepolarizations in PKP2-cKO models (24.6 vs. 41%). An additional increase in INCX strongly reduced delayed-afterdepolarizations occurrence, by lowering diastolic cleft calcium levels. The human model showed similar outcomes, suggesting a potential translational value of these findings.Conclusion: Beta-adrenergic stimulation and connexin43-mediated calcium entry upon loss of plakophilin-2 function contribute to generation of delayed-afterdepolarizations. RyR2 and NCX dysregulation play a key role in modulating these proarrhythmic events. This work provides insights into potential future antiarrhythmic strategies in arrhythmogenic cardiomyopathy due to plakophilin-2 loss-of-function.

X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.

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

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.

Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants

Background: Pathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well. Methods: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell–derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament–directed calcium reporter to monitor changes in myofilament calcium affinity. Results: Hypertrophic cardiomyopathy–associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy–associated variants decreased contraction. TNNT2 variant–dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, HOPX , and NPPB . We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. On the basis of a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 variants of uncertain significance. Conclusions: Our study found that hypertrophic cardiomyopathy–associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy–associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity.

Molecular Mechanism of the pH-Dependent Calcium Affinity in Langerin

The C-type lectin receptor langerin plays a vital role in the mammalian defense against invading pathogens. Its function hinges on the affinity to its co-factor Ca2+ which in turn is regulated by the pH. We studied the structural consequences of pro-tonating the allosteric pH-sensor histidine H294 by molecular dynamics simulations (total simulation time: about 120 μs) and Markov models. We discovered a mechanism in which the signal that the pH has dropped is transferred to the Ca2+-binding site without transferring the initial proton. Instead, protonation of H294 unlocks a conformation in which a protonated lysine side-chain forms a hydrogen bond with a Ca2+-coordinating aspartic acid. This destabilizes Ca2+ in the binding pocket, which we probed by steered molecular dynamics. After Ca2+-release, the proton is likely transferred to the aspartic acid and stabilized by a dyad with a nearby glutamic acid, triggering a conformational transition and thus preventing Ca2+-rebinding.

Effects of calcium on the kinetics of a model disjunctive ligand exchange reaction: implications for dynamic trace metal ion speciation

Differences in the calcium affinity of exchanging multidentate ligands affect the kinetic behavior of disjunctive ligand exchange reactions.