Arrhythmia is the leading cause of cardiac arrest and sudden cardiac death, with 50 to 100 cases per 100,000 people worldwide each year. Even when cardiac arrest occurs in a hospital, survival rates are only 3–10%(Ruthirago et al., 2016; Soros and Hachinski, 2012). Stroke is a common central nervous system disease and is considered a leading cause of death and disability worldwide(Tsao et al., 2023; Tu et al., 2023). Importantly, ischemic stroke comprised 62.4% of all kinds of strokes, affecting approximately millions of people in the world(Collaborators, 2021). Notably, arrhythmia is the most prevalent cardiac complication in ischemic stroke, with a prevalence of 74.4% within 24 h of stroke onset(Kallmunzer et al., 2012; Lin et al., 2021; Ruthirago et al., 2016). However, ischemic stroke-induced arrhythmia is insidious and unstable, and <1% of patients receive continuous electrocardiogram (ECG) monitoring after stroke(Lin et al., 2021). Therefore, post-stroke arrhythmia is not promptly diagnosed and treated, increasing the risk of sudden cardiac death after ischemic stroke.

The sinoatrial node plays an important role in the cardiac conduction system. Sinoatrial node dysfunction can be induced by genetic mutation, inflammation, or autonomic dysfunction, resulting in multiple arrhythmias, such as atrial fibrillation (AF) and sinoatrial block(John and Kumar, 2016; Shaw and Southall, 1984). However, the sinoatrial node is a small, anatomically complex tissue with obvious heterogeneity compared to surrounding cardiomyocytes(Boyett et al., 2000; Xia et al., 2020). The limited dimensions of the tissue and the lack of a distinct anatomical position present difficulties in investigating the pathophysiology of arrhythmia associated with the sinoatrial node(Manoj et al., 2023).

Three important factors influence the propagation of cardiac electrical impulses: myocardial cell excitability, intercellular electrical coupling, and the properties of the cardiac tissue network. Of note, intercellular communication via gap junctions is crucial for effective electrical coupling(van Veen et al., 2001). These intercellular gap junctions selectively permit calcium ions, cyclic nucleotides, and adenosine triphosphate to penetrate adjacent cells. In addition, gap junctions are essential for the propagation of electrical signals between cardiomyocytes to synchronise the cardiac cycle and steer cardiac pump function. The gap junctions between adjacent cells are connected by hemichannels, which are formed by the combination of six connexin proteins(Beyer and Berthoud, 2018; Rodriguez-Sinovas et al., 2021). The connexin protein family is a highly conserved family of transmembrane proteins. Connexin 40 (Cx40) is a widely expressed connexin protein encoded by the Gja5 (also called Cx40) gene. Remarkably, previous studies have reported that alterations in the expression, location, or phosphorylation state of Cx40 are associated with various cardiac arrhythmias, such as AF and heart block(Makita et al., 2012; Santa Cruz et al., 2015). However, the role of Cx40 in the sinoatrial node in ischemic stroke-induced arrhythmia remains unclear.

In this study, we hypothesised that the dysregulated Cx40 in the sinoatrial node plays a dominant role in ischemic stroke-induced arrhythmia. To explore this, we first performed a permanent middle cerebral artery occlusion (pMCAO) mouse model to mimic ischemic stroke and assessed the ECG parameters. Furthermore, to investigate potential pathogenic mechanisms, we localized the position of the sinoatrial node via optical tissue clearing and whole-mount immunofluorescence staining. Subsequently, we collected the sinoatrial node after stroke for RNA sequencing. We found that the expression of Gja5 in sinoatrial node was significantly decreased after stroke. Lastly, the rAAV9-Gja5 virus was injected with ultrasound guidance into the heart to increase Cx40 expression in the sinoatrial node, resulting in ameliorated arrhythmia after ischemic stroke. Our data showed that Cx40 in the sinoatrial node could be a therapeutic target for arrhythmia caused by an ischemic stroke.