# GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model

## TL;DR

This study identifies a new mechanism for atrial fibrillation caused by inflammation, showing how specific gene changes lead to irregular heartbeats in a lab-grown heart tissue model.

## Contribution

The novel contribution is the discovery that inflammation-induced downregulation of GJA5 and ATP1A1 causes arrhythmias through disrupted connectivity and ion flux in atrial tissue.

## Key findings

- M1 macrophage activation reduced contraction amplitude and increased beat irregularity in atrial tissue.
- Combined knock-down of GJA5 and ATP1A1 induced significant arrhythmia and reduced conduction velocity.
- In silico modeling confirmed that reduced sodium-potassium pump and conductivity can trigger AF under tachycardia.

## Abstract

Atrial fibrillation (AF) is the most common cardiac arrhythmia, linked to greater risk of heart failure, stroke and death. Inflammation has been connected to AF emergence, however mechanisms of inflammation-caused AF remain thus far elusive, leading to a lack of mechanism-based treatments. An isogenic, 3D tissue model containing hiPSC-derived atrial-like cardiomyocytes (aCM), cardiac fibroblasts (cfb), and cardiac macrophages was engineered using custom injection-molded pillar devices. Electrophysiological changes were examined via sharp electrode recordings, calcium imaging, and multi-electrode assays. Gene function was interrogated using siRNA knock-down, lentiviral overexpression, and pharmacological modulation. In silico tissue and whole-heart models validated findings under simulated stress and heterogeneous conditions. Activation of M1 macrophages led to a 50% reduction in contraction amplitude, action potential spike amplitude (aCM+cfb+M1: 61.3 mV ±13.9 vs control: 71.6 mV ±14.5, p < 0.01) and increased beat irregularity (M1: 150.7% ± 388.9 vs control, p < 0.001). Calcium transient amplitude was reduced (12.3 a.u. ± 14.7, p < 0.05) and upstroke velocity slowed. SCN5A knock-down reduced contraction amplitude (−51.9% ± 37.2, p < 0.01) without inducing arrhythmias, whereas combined GJA5 and ATP1A1 knock-down induced significant irregularity (403% ± 371.3, p < 0.001), increased conduction heterogeneity (+18%), and reduced velocity (−52.4%). In silico modeling confirmed that paired 50% downregulation of sodium-potassium pump and tissue conductivity induced AF under tachycardia even without ectopic activity. This work reveals a novel, inflammation-driven mechanism for AF initiation. Combined downregulation of GJA5 (connexin 40) and ATP1A1 (NaK ATPase) disrupted intercellular connectivity and ion flux, establishing a substrate for arrhythmogenesis. These results were robust across in vitro, genetic/pharmacological, and in silico models, defining new avenues for translational intervention.