Sodium calcium dependent arrhythmias – NaCaR

To address the objectives of the project, we implemented a multidisciplinary, multi-scale experimental strategy combining molecular, cellular, tissue, and organ-level approaches. Genetically modified mouse models carrying disease-causing RyR2 mutations associated with CPVT, a Nav1.5 mutation associated with LQTS3, and a desmin mutation were used to investigate arrhythmia mechanisms in an intact physiological context. These models were complemented by studies in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs), cultured as monolayers or as three-dimensional engineered heart tissues (EHTs), ensuring direct relevance to human disease. CRISPR/Cas9-based genome editing was used to generate isogenic control and mutant iPSC lines. As a first line of investigation, the functional consequences of NCX1 mutations were examined using heterologous expression systems, combining patch-clamp electrophysiology with sodium-dependent ⁴⁵Ca uptake assays. For CPVT models, intracellular calcium dynamics were analyzed using advanced confocal and super-resolution imaging techniques, allowing quantitative assessment of calcium sparks, calcium waves, and local recovery of calcium release with high spatial and temporal resolution. Innovative optical approaches, including two-photon calcium uncaging, were employed to probe the refractoriness and sensitivity of calcium-induced calcium release in situ. For the other disease models, calcium transients were recorded using optical mapping techniques. For all models, cellular electrophysiological recordings (patch-clamp and/or sharp microelectrodes) were combined with optical and electrical mapping in isolated hearts to assess action potential properties, conduction patterns, and repolarization dynamics under basal conditions and during adrenergic stress. Computational modeling was used to integrate experimental data and test mechanistic hypotheses linking disturbances in calcium and sodium handling to electrical instability. Finally, targeted molecular interventions, including modulation of FKBP12.6 expression, peptide-based tools, and selected pharmacological agents (some derived from venoms) were applied to identify key determinants of arrhythmia susceptibility and to evaluate potential therapeutic strategies. Depending on the specific project, functional analyses were complemented by molecular biology approaches, including Western blotting, immunostaining, and transcriptomic and proteomic analyses.

The project yielded several major findings that significantly advance the understanding of calcium-dependent ventricular arrhythmias. At the subcellular level, disease-causing RyR2 mutations increased calcium release sensitivity and shortened refractoriness, resulting in more frequent spontaneous calcium release events. These abnormalities were linked not only to altered channel gating but also to nanoscale structural remodeling of calcium release units, including disrupted coupling between RyR2 and junctophilin-2. At the cellular level, enhanced calcium leak promoted both delayed and early afterdepolarizations, particularly under adrenergic stimulation. Importantly, the balance between sarcoplasmic reticulum calcium uptake and leak emerged as a critical determinant of arrhythmogenic calcium signaling. At the tissue and whole-heart levels, calcium abnormalities were shown to act not only as triggers of ectopic activity. Subthreshold calcium-dependent depolarizations induced local conduction slowing and increased activation dispersion, while calcium-mediated action potential prolongation reduced repolarization reserve. Together, these mechanisms create a highly vulnerable electrical substrate favoring re-entrant and triggered arrhythmias. The project also identified modulatory factors within calcium signaling pathways exerting dose-dependent protective or deleterious effects on arrhythmia susceptibility, highlighting the importance of precise regulation rather than global suppression of calcium release.

In LQTS3 mice, a major finding was that Huwentoxin IV, an inhibitor of neuronal sodium channels specifically expressed in the T-tubule–sarcoplasmic reticulum interaction microdomain, prevented arrhythmias without correcting repolarization abnormalities. This demonstrates that these channels play a key role in regulating calcium release and arrhythmogenesis. Maurocalcin, a RyR2 regulator, was much less effective. In this model, abnormal sodium handling due to the SCN5A mutation in ventricular fibroblasts altered calcium homeostasis and activated the calcineurin-NFAT pathway, leading to fibroblast proliferation in the myocardium, a recognized arrhythmogenic factor in addition to altered cardiomyocyte repolarization. The five NCX1 mutations reduced inward NCX1 current, shortening action potentials in iPSC-derived cardiomyocytes. Mathematical modeling showed that decreased NCX1 current in the T-tubule microdomain altered sarcoplasmic reticulum calcium release and early repolarization in subepicardial cardiomyocytes. At the pseudo-ECG level, these combined abnormalities reproduced the typical early repolarization syndrome phenotype observed in patients.

In contrast, despite altered expression of intracellular calcium regulators, our study of the Desmin R406W mutation showed no involvement of calcium dysregulation in arrhythmogenesis, which instead resulted from re-entries favored by short refractory periods and slower conduction.

These results redefine the role of intracellular calcium in ventricular arrhythmogenesis, demonstrating its dual contribution as both a trigger and a substrate for electrical instability. They have important implications for the management of CPVT, LQTS3, and other calcium-related arrhythmias.

By linking molecular and nanostructural defects to whole-heart electrical behavior, this work provides a framework for mutation-specific, mechanism-based therapeutic strategies. The identification of impaired repolarization reserve and conduction abnormalities as calcium-dependent processes opens new avenues for antiarrhythmic interventions beyond conventional sodium channel blockade.

More broadly, the mechanisms uncovered in CPVT and LQTS3 are highly relevant to acquired cardiac diseases such as heart failure, where calcium mishandling and arrhythmias frequently coexist. The approaches and concepts developed here may therefore inform future research and therapeutic innovation in common cardiovascular disorders.

Future studies will aim to translate these mechanistic insights into targeted treatments, refine risk stratification based on calcium signaling profiles, and explore precision medicine approaches tailored to specific molecular defects.

In the LQTS3 model, one perspective is to apply photopharmacology to selectively target neuronal sodium channels in the heart without affecting the brain, as previously demonstrated for another target (hERG channel). In the short term, prior to submitting our manuscript on pharmacological prevention of arrhythmias in the LQTS3 model, we plan to test whether a modified Maurocalcin form is more effective than the E12A variant used previously.

The main objective of this project is to elucidate the mechanisms of life-threatening ventricular arrhythmias involved in sudden cardiac death (SCD), which accounts for about 15% of casualties worldwide. SCD may happen in a diseased heart, but also in structurally normal hearts where a channelopathy, i.e., a genetic disease affecting ion channel, is most often at the origin of the lethal tachyarrhythmia. We will focus on two adrenergically-induced syndromes: Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) and Long QT syndrome type 3 (LQTS3). CPVT is a severe genetic disease manifested by syncope or SCD in children and young adults during physical or emotional stress and in the absence of structural heart disease. Most of the identified CPVT causative mutations are in the gene coding for the intracellular Ca2+ release channel, the ryanodine receptor type 2 (RyR2). Other CPVT-related mutations affect RyR2 regulatory proteins, indicating that CPVT is related to RyR2 malfunctioning. Aberrant openings of the RyR2 during diastole produce an elevation in [Ca2+]i that is rapidly extruded by the electrogenic sodium/calcium exchanger producing delayed after depolarizations, which are at the origin of triggered activity and arrhythmogenesis in CPVT. When ß-adrenergic blockers (first therapeutic option) do not confer enough protection, the Na+ channel blocker flecainide is added with good efficiency, although with a narrow therapeutic window. While a direct effect of flecainide on the RyR2 has been invoked as the mechanism of action, this is controversial. Thus we hypothesize that alteration in Na+ homeostasis contributes to arrhythmogenity in CPVT. This is a completely unexplored mechanism.
LQTS is a severe hereditary disorder of cardiac electrical activity. It is caused by delayed repolarization in ventricular cardiomyocytes, which results in a prolonged QT interval on the ECG and an increased susceptibility to polymorphic ventricular tachycardia and ventricular fibrillation. Mutations in genes encoding ion channels or their accessory subunits are linked to different types of LQT syndrome. Mutations in the Na+ channel Nav1.5, (SCN5A gene), which impair its inactivation, inducing the so called late Na+ current, are involved in LQTS3. Interestingly, our recently published data show that intracellular Ca2+ is enhanced in LQT3 mice (Scn5a+/?QKP) cardiomyocytes probably by the sodium/calcium exchanger activity extruding excess Na+. Thus we formulate the novel hypothesis that Ca2+ handling alteration contributes to arrhythmogenicity in LQTS3.
Gathering a group of international recognized experts, we propose an original and multidisciplinary project focused in the key role of [Ca2+]i and [Na+]i in the genesis of life-threatening arrhythmias, using both knock-in mice and rabbit models of CPVT or LQTS3, and cardiomyocytes differentiated from induced pluripotent stem cells derived from patients exhibiting the same mutations. Beyond the rare diseases CPVT and LQT3, Ca2+ and Na+ mishandling have been evidenced in acquired cardiac diseases like heart failure, where cardiac arrhythmias account for half of the mortality. The new knowledge obtained in these rare diseases may also yield benefits for common diseases in which the affected channel may show altered expression and/or function such as in heart failure, where SCD accounts for about half of the deaths. Thus elucidating CPVT and LQTS fundamental mechanisms will deliver essential knowledge to provide a solid rationale for new and effective antiarrhythmic therapies.