Figure 4. AV Node Macrophages Couple to Conducting Cardiomyocytes and Alter Their Electrophysiological Properties.

(A) Relative connexin (Cx) expression levels in FACS-purified AV node macrophages by qPCR (n = 4 to 6 from 2 independent experiments).

(B) Cx43 levels by qPCR in macrophages FACS-sorted from AV node, and LV and RV free wall. n = 6 to 9 from 2 independent experiments.

(C) Whole-mount immunofluorescence microscopy of AV lower nodal area from a Cx₃cr1^GFP/+ mouse stained with Cx43 (red) and HCN4 (white). Arrowheads indicate Cx43 colocalization with GFP⁺ macrophages (green).

(D) Electron microscopy image of a direct membrane contact (arrow) between a DAB⁺ macrophage and a cardiomyocyte in AV node tissue of a Cx₃cr1^GFP/+ mouse stained for GFP. The nodal cardiomyocyte is characterized by its typical high mitochondrial content and junctional contact with the neighboring myocyte (arrowhead).

(E) Immunofluorescence image of a co-cultured desmin⁺ neonatal mouse cardiomyocyte (white) and GFP⁺ cardiac macrophage (green) stained with Cx43 (red, arrow), illustrating setup for patch clamp experiments (F-I). The cells are grown on cover slips coated with fibronectin in a line pattern.

(F) Immunofluorescence images of dextran diffusion during whole-cell patch clamp with a dextran-loaded pipette. (Top) Arrowhead indicates GFP⁺ cardiac macrophage (green); (bottom) Texas Red⁺ dextran (red) diffusion into macrophage.

(G and H) Spontaneous recordings (G) and resting membrane potential (H) of solitary cardiac macrophages (n = 20) and macrophages attached to cardiomyocytes (n = 43) by whole-cell patch clamp. Data are mean ± SEM from 13 independent experiments, ^**p < 0.01, nonparametric Mann-Whitney test. Rhythmic depolarization was observed in 10/43 macrophages attached to cardiomyocytes.

(I) Resting membrane potential of solitary cardiomyocytes (n = 13) and cardiomyocytes coupled to macrophages before (n = 14) and after (n = 7) addition of the Cx43 inhibitor Gap26. Data are mean ± SEM from 3 independent experiments, ^*p < 0.05 and ^**p < 0.01, Kruskal-Wallis test followed by Dunn’s posttest.

(J) Mathematical modeling of ‘single-sided coupling’ between one AV bundle cardiomyocyte and an increasing number of cardiac macrophages. The graph shows the AV bundle cardiomyocyte membrane potential uncoupled or coupled to one, two or four cardiac macrophages at a junctional conductance of 1 nS.

(K and L) Computational modeling of resting membrane potential (K) and action potential duration (L) of an AV bundle cardiomyocyte coupled to an increasing number of cardiac macrophages.

See also Figure S4 and Movie S1.