Background Cardiac activities are under the control of the autonomic nervous system. Evidence suggests that cardiac glial cells associated with autonomic innervation participate in the regulation of cardiac functions. We found network-like structures of glia in the human and murine heart ventricular epicardium (Fig.1 A); however, it remains unknown if the cells are connected via gap junctions and form a functional network. Therefore, we aim to study the epicardial glial network by tracer injections and investigate the gene expression of connexins (Cxs).
Methods and Results PLP-CreERT2 LSL-tdTomato mice were used. For patch clamp-based tracer coupling experiments, tangential slices from the epicardium digested by collagenase B and trypsin were used (Fig.1 B). The tracer, biocytin, was loaded via the backfill of the patch pipette. One single glial cell per slice was patched and loaded. Biocytin-coupled glial cells were visualized by post-staining with Alexa-conjugated streptavidin 488. Quantification and analysis of the fluorescence of tracer-coupled areas and compared to tdTomato-positive area reveals that the tracer coupling ratios (biocytin vs glia) is 69.8 ± 4.8% (n = 3). To examine whether the tracer coupling is via gap junction, we applied the non-selective gap-junction channel blocker carbenoxolone (CBX). CBX suppressed trace coupling ratios to 29.4 ± 9.8% (n = 4) (p = 0.057). For quantitative real-time PCR, cardiac glia were isolated and sorted by fluorescence-activated cell sorting (FACS). Preliminary analysis reveals the expression of Cx43, Cx29, Cx40, Cx45, Cx47, Cx23 and Cx36 abundance in descending order.
Conclusion Our data indicate that epicardial glia are connected via gap junctions. This suggests that they form a functional syncytium, which could allow ionic buffering, energy or metabolic support for cardiac nerves directly in the myocardium.
Fig. 1. Cardiac glia network-like strecture and patch clamp-based tracer coupling experiment. A delicate network-like structure of glia was observed on the hearts of transgenic PLP-CreERT2 LSL-tdTomato mice. B. A single glial cell from epicardial slice was patched and loaded with the tracer. Figure created by BioRender.
