Hove-Madsen, L. | Search bioRxiv

bioRxiv2026

Calsequestrin localization at RyR2 clusters enables calcium wave propagation in ventricular myocytes

Intracellular calcium waves in cardiac myocytes propagate through a fire-diffuse-fire mechanism in which calcium released from one RyR2 cluster diffuses to neighboring clusters and triggers their activation. Yet propagation faces a fundamental physical difficulty: the calcium signal must cross distances of 1-2 {micro}m between Z-planes while being attenuated by cytosolic buffering and diffusion, and at the same time the release site depletes its local sarcoplasmic reticulum calcium store. How waves propagate efficiently despite these constraints has remained unclear. We developed a three-dimensional computational model of mouse ventricular myocytes at 100 nm resolution to address this question. Our central finding is that co-localization of calsequestrin2 (CASQ2) with RyR2 clusters is required for robust wave propagation. In a physiological model, where CASQ2 is concentrated at release sites as observed experimentally, calcium waves propagate reliably across the cell with velocities that match the experimental range. In contrast, when CASQ2 is distributed uniformly throughout the sarcoplasmic reticulum, keeping total CASQ2 unchanged, the wavefront stalls. These results identify CASQ2-RyR2 co-localization as a key structural requirement for effective calcium wave propagation in ventricular myocytes. Author summaryCalcium waves in cardiomyocytes are thought to underlie the onset of malignant cardiac arrhythmias, such as ventricular tachycardia and fibrillation. Yet, the specific conditions that regulate the transition from local calcium sparks to sustained waves remain poorly understood. Using a newly developed computational model of calcium handling, we demonstrate that the spatial distribution of key regulatory proteins is a critical determinant of arrhythmogenicity. Specifically, we found that calsequestrin2, which buffers Ca2+ within the sarcoplasmic reticulum, must be strictly colocalized with Ca2+ release proteins to facilitate sustained wave propagation. This discovery suggests that cardiac stability depends less on the total quantity of protein and more on its precise architectural organization. The consequences of this finding are significant: it implies that "spatial dysregulation"--where proteins are present but mislocalized--may be a hidden driver of arrhythmias even when protein levels appear normal. This shifts the therapeutic focus from simply altering ion channel conductance to preserving or restoring the structural tethering of the junctional SR. By focusing on the nanodomain architecture, we can better understand how cellular remodeling leads to life-threatening electrical instability.

Cell Biology↗

bioRxiv2026

Programmed electrical stimulation in human iPSC-derived cardiomyocytes reveals mechanisms of lethal arrhythmias in Calcium Release Deficiency Syndrome

BackgroundCalcium release deficiency syndrome (CRDS) is a recently described inherited channelopathy caused by loss-of-function variants in RYR2. Clinically, CRDS patients present with lethal ventricular arrhythmias which are not reproduced on exercise stress testing, unlike catecholaminergic polymorphic ventricular tachycardia. A hallmark trigger identified for CRDS mimics a long-burst, long-pause, short-coupled extra-stimulus (LBLPS) programmed electrical stimulation protocol, which was experimentally validated in humans and mouse models. Moreover, application of a long-burst, long-pause (LBLP) protocol alone can induce an abnormal repolarization on the first sinus beat that is unique to CRDS. However, the electrophysiological basis of CRDS in human cardiac tissue, including other triggers, are not fully understood, and whether clinically relevant arrhythmias can be observed in human stem cell models remains unknown. MethodsWe performed electrophysiological and arrhythmia inducibility studies using clinically relevant programmed electrical stimulation protocols in two-dimensional cardiac tissue generated from metabolically matured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRDS variant RyR2-E4146D. High spatiotemporal optical mapping and multielectrode arrays were used for electrophysiological phenotyping. ResultsAt baseline, E4146D+/- monolayers showed no arrhythmias, similar to controls. During rapid pacing, E4146D+/- promoted electrical vulnerability by reducing the threshold for action potential duration (APD) alternans and Ca2+ alternans and increasing the propensity for spatial discordance of alternans. In response to LBLP pacing, E4146D+/- monolayers often demonstrated an abnormal repolarization response characterized by spatially dispersed APD prolongation and large Ca2+ release. Notably, LBLPS pacing produced early-after depolarization (EAD)-driven triggered activity resulting in re-entrant tissue conduction patterns, explaining the short-coupled ectopy driven arrhythmias seen in CRDS patients. Similar arrhythmias were observed when EADs developed during spatially discordant alternans. Lastly, flecainide showed efficacy in suppressing arrhythmia inducibility for the here studied variant. ConclusionsWe developed the first hiPSC model for CRDS which recapitulates clinically observed and inducible arrhythmias. Our model provides novel insights into tissue-level, re-entrant arrhythmias, which are initiated by EADs during electrically vulnerable states in CRDS human cardiac tissue and can be suppressed by flecainide. This model provides the framework for studying other CRDS variants and complex arrhythmias in hiPSC-CMs and establishes a human-based new approach method (NAM) for drug and gene therapy development for CRDS. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABS{blacksquare} We developed the first human stem cell-derived cardiomyocyte (hiPSC-CM) tissue model for calcium release deficiency syndrome (CRDS) which recapitulates its hallmark clinical features, including inducible ventricular arrhythmias with programmed electrical stimulation and post-pacing repolarization abnormalities. {blacksquare}Using genome edited and metabolically matured hiPSC-CMs combined with high spatiotemporal optical mapping, we show that tissue-level arrhythmias are initiated by early-after depolarizations (EADs) which develop during electrically vulnerable states, leading to re-entrant conduction patterns. We comprehensively characterize the features of EAD-induced triggered activity, showing that these ectopic beats promote re-entry through slower conduction velocities and shorter action potential durations. This uncovers how EAD-induced short-coupled ectopy leads to malignant ventricular arrhythmias in CRDS patients, and establishes the phenotype for future hiPSC-CM investigations. {blacksquare}We identified flecainide as an effective agent in suppressing arrhythmias on single cell and tissue levels in hiPSC-CMs for this CRDS variant, reproducing clinical results. What are the clinical implications?{blacksquare} CRDS has only recently been described as a unique channelopathy caused by loss-of-function RYR2 variants, and much of its triggers and mechanisms in human cardiomyocytes remain unclear. The arrhythmias observed are often not related to exercise, and exercise stress testing does not reproduce these abnormalities. No human models exist to date which closely recapitulate the triggers shown to induce tissue-level arrhythmias in patients and mouse models. Our model demonstrates that programmed electrical stimulation, without pharmacological {beta}-adrenergic stimulation, can reliably induce the same arrhythmias seen clinically, enabling accurate disease modeling and drug development. {blacksquare}Combining programmed electrical stimulation in cardiac tissue derived from genome-edited hiPSC-CMs with high spatiotemporal optical mapping is a robust and novel approach to identify the mechanisms of complex, tissue-level arrhythmias which remain underexplored, such as short-coupled ventricular fibrillation, in a patient-specific and translational manner.

cell biology↗