Congenital forms of arrhythmia could arise from mutations in genes encoding ion channels and proteins involved in their regulation [1]. The most commonly inherited cardiac arrhythmias are long QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT could be provoked by emotional stress, exercise, or certain drugs causing sudden cardiac death with an overall mortality rate of 30-40% [2,3]. Mutations associated with CPVT have been identified in genes Ryr2 encoding ryanodine receptor calcium release channel, Trdn encoding triadin, Calm1-3 encoding calmodulin proteins, and calsequestrin-2 (Casq2) [4]. Casq2 is a high-capacity Ca2+-binding protein, located in the sarcoplasmic reticulum of cardiomyocytes (CM) [[3], [4], [5]]. Impaired intracellular Ca2+ handling was associated with a rare but severe form of CPVT in transgenic models and humans [1,4,6]. The Casq2-null (Casq2–/–) transgenic mice were reported to display increased diastolic Ca2+ leak and gain of Ca2+ release [4,6].

In the healthy state, tissue macrophages (MΦ) reside in various organs, including the heart, brain, liver, and lung. In addition to the conventional immunomodulatory functions, cardiac MΦs (cMΦ) were shown to participate in cardiac homeostasis [[7], [8], [9]], including angiogenesis, electrophysiological regulation, and proliferation [[10], [11], [12]]. Resident cMΦ originated from the primitive yolk sac maintain a homeostatic population within the heart [11,13]. In ischemia-induced cardiac damage, monocyte-derived MΦ from bone marrow repopulate to repair and remodel the heart [14,15]. Moreover, cMΦ were shown to form physical contact with endothelial cells [16] and CMs [12] modulating their function.

Recent in vitro culture models and optogenetic studies provided intriguing evidence for cMΦ in electrophysiological and contractile regulation of the myocardium [12,17]. In the last decade, resident cMΦ, monocyte-derived MΦ and secreted inflammatory cytokines were implicated in ventricular arrhythmia [13,18,19]. Moreover, the depletion of cMΦ in mice was shown to result in atrioventricular (AV) block and arrhythmia [12]. The parabiosis experiments demonstrated that only about 1% of circulating monocytes were routed toward the AV node, whereas the remaining population was the resident cMΦ [12]. These results indicated that cMΦ located at the AV node were mostly from the tissue-resident population.

The chemokine C-X3-C motif receptor-1 (Cx3cr1) is abundantly expressed in tissue-resident MΦs during the fetal period, and its expression has been shown to be maintained into adulthood [9,14,20]. The Cx3cr1-mediated signaling regulates the immune cell chemotaxis depending on the ligand gradient, thereby contributing to tissue homeostasis [21]. The Cx3cr1GFP/+ reporter mouse strain has been widely utilized for in vivo tracing of tissue-resident MΦs in developmental and transplantation studies [13,14,18,[21], [22], [23], [24], [25]]. Recently, depletion of Cx3cr1+ MΦ were shown to result in cardiac dysfunction, indicative of their irreplaceable role in the heart and designating them as a major cMΦ population [12,26,27].

As the recent reports showed involvement of cMΦ in the regulation of contractility at the electrophysiological level, and the depletion of cMΦ leading to AV block and arrhythmia in WT murine hearts [12,19], we hypothesized that the population and distribution of cMΦ may be altered in the case of a congenital form of tachyarrhythmia. For this, we comparatively explored the localization and abundance of Cx3cr1+ cMΦ population in the AV node regions of the heart from WT and Casq2–/– transgenic mice, which serve as a model for CPVT. First, we immunostained heart sections for Cx3cr1-expressing cMΦ. In parallel, we identified the compact AV node regions by subsequent Masson’s Trichrome staining, Hematoxylin-Eosin (H&E) staining, and coimmunostaining for the Hcn4 nodal marker protein. The Cx3cr1+ cMΦ density were detected to be higher at the AV node regions compared to other cardiac regions in WT murine hearts. Moreover, Cx3cr1+ cMΦ were in close proximity with cTnT+ and cTnI+ CMs in the 3D reconstruction model. Importantly, quantitative intensity analysis of our data revealed that Cx3cr1+ cMΦ immunoreactivity in AV node regions of Casq2–/– mice was statistically lower compared to that in WT mice. Our results demonstrate lower densities of Cx3cr1+ cMΦ interacting with nodal Hcn4+ CMs in Casq2–/– mice, which may indicate a potential contribution of cMΦ to CPVT pathogenesis. Future studies await detailed molecular and functional investigations of cMΦ in arrhythmia pathology.