Boutjdir, M. | Search bioRxiv

bioRxiv2026

Modeling Sympathetic Neuro-Cardiac Interactions in a hiPSC-Based Microphysiological System

The cardiac autonomic nervous system is a key driver of various cardiac disorders and arrhythmias. However, investigating neuronal regulation of the human heart has proven difficult due to immitted and reliable experimental models. Here, we present a novel microphysiological system utilizing a compartmentalized microfluidic device (MFD) to integrate co-cultured human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). MFD is composed of two wide-open chambers separated by microfluidic microchannels. hiPSC-SNs were characterized by confocal imaging and RT-qPCR for the expression of peripherin, tyrosine hydroxylase, and {beta}-tubulin III, as well as high levels of dopamine {beta}-hydroxylase and nicotinic acetylcholine receptors. Furthermore, patch-clamp techniques confirmed their functional maturity, showing spontaneous action potentials and positive responses to nicotine (1{micro}M). Co-culturing hiPSC-CMs and hiPSC-SNs within the MFD facilitated axonal projection into the cardiomyocyte chamber, establishing a physical connection between the two cell types. After 10 days of co-culture, functional integration was confirmed by a significant increase in the action potential frequency and beating rate of hiPSC-CMs, as recorded by patch-clamp and video motion tracking, respectively. Notably, nicotine application in the neuronal chamber accelerated these rates in hiPSC-CMs chamber, whereas the administration of the {beta}-blocker, propranolol (5{micro}M), effectively decreased the beating rates. Collectively, these data demonstrate the feasibility of differentiating hiPSCs into functional sympathetic neurons and establishing a robust neuro-cardiac interface. This microphysiological system represents a powerful platform for investigating disorders characterized by impaired neuro-cardiac interactions, offering a valuable tool for both disease modeling and pharmacological screening.

physiology↗

bioRxiv2025

Electrophysiological abnormalities associated with a CACNA1D variant are rescued by AAV6-Cav1.3-C-terminus gene therapy in patient-iPSC-CMs

Inherited arrhythmia syndromes are caused by genetic variants that alter cardiac ion channel function. We investigated a complex presentation in a pediatric patient with ventricular tachycardia and conduction abnormalities, harboring a de novo CACNA1D (c.3786G>T) variant, and two inherited variants, the SCN5A (c.2618C>G), and a DSP desmosome (c.1582C>G). The CACNA1D variant, which encodes Cav1.3 L-type calcium channel is the focus of this study, because the C-terminus fragment of Cav1.3 has recently been identified as a transcription auto-enhancer of its own gene and able to prevent arrhythmic events in a mouse model of ischemic heart failure. Leveraging this intrinsic property, we hypothesized that the Cav1.3-C-terminus could reverse the arrhythmic events associated with the CACNA1D variant. Patch-clamp and optical mapping experiments demonstrated a loss of Cav1.3 function, characterized by reduced L-type calcium current densities, and decrease of conduction velocity, leading to inducible re-entrant arrhythmias in human induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs). RNA sequencing confirmed this loss-of-function via the downregulation CACNA1D gene expression. Interestingly, Cav1.3-C-terminus treatment of hiPSC-CMs successfully normalized Cav1.3 gene expression, restored calcium currents, and conduction velocity, and prevented the susceptibility to arrhythmias. These findings highlight the electrophysiological consequences resulting from a de novo Cav1.3 variant and demonstrate an important transcriptional role of Cav1.3-C-terminus as a transcriptional regulator and as a promising therapeutic tool to restore normal electrical properties in patients with calcium channels loss of function.

physiology↗