Abstract Body: Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmia causing sudden cardiac death in children and young adults, often presenting during exercise or emotional stress as the first clinical manifestation. Ryanodine receptor type 2 gain-of-function mutations trigger CPVT upon sympathetic activation, yet no curative therapy exists. Current human disease modeling is predominantly based on cardiomyocytes alone, without incorporating autonomic innervation. Therefore, arrhythmogenic phenotypes require exogenous stimulation and may not accurately model complex autonomic cardiac interactions, fundamentally limiting physiological relevance and translational utility.

Hypothesis: We tested the hypothesis that a 3D neuro-cardiac model with intrinsic autonomic and cardiac responses recapitulates CPVT arrhythmogenic phenotypes in vitro.

Methods: CPVT patient and wild-type (H9 and WTC lines) stem cell-derived cardiac organoids were assembled with channelrhodopsin-2-expressing spinal cord organoids to generate neuro-cardiac assembloids (NeuCarS). Heart rate variability (HRV) was quantified by sharp microelectrode extracellular recordings at spontaneous baseline and during optogenetic sympathetic-like stimulation (n=6-16).

Results: Spontaneous HRV was significantly elevated in CPVT NeuCarS versus wild-type NeuCarS (p=0.0021) and non-innervated CPVT organoids (p<0.0001, two-way ANOVA genotype×preparation interaction p=0.0008, n=14–16). Isoproterenol did not further elevate HRV (p=0.83, n=8), confirming endogenous sympathetic tone saturates the CPVT arrhythmogenic substrate. Ruxolitinib, a previously FDA-approved Janus kinase 1/2 inhibitor, recently identified as a potential treatment for CPVT, fully rescued spontaneous HRV to baseline levels (p=0.0145, n=8). Optogenetically evoked HRV was similarly elevated in CPVT versus wild-type NeuCarS (p<0.0001, n=14-16), and ruxolitinib partially rescued this innervation-evoked arrhythmogenic signature (p=0.0396, n=6).

Conclusion: The novel 3D neuro-cardiac model, NeuCarS, accurately recapitulates CPVT arrhythmogenic phenotypes through the incorporation of autonomic innervation without pharmacological provocation. Initial therapeutic evidence has been shown with ruxolitinib, fully rescuing innervation-driven and partially rescuing optogenetically evoked arrhythmia in NeuCarS. These data establish NeuCarS as a faithful platform for cardiovascular research and drug discovery.