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. 2017 Apr 20;169(3):510-522.e20.
doi: 10.1016/j.cell.2017.03.050.

Macrophages Facilitate Electrical Conduction in the Heart

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Free PMC article

Macrophages Facilitate Electrical Conduction in the Heart

Maarten Hulsmans et al. Cell. .
Free PMC article

Abstract

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Keywords: atrioventricular node; computational modeling; connexin 43; electrical conduction; gap junctions; heart; macrophages; optogenetics; single-cell RNA-sequencing; tissue clearing.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Resident Cardiac Macrophages in the AV Node
(A) Volumetric reconstruction of confocal microscopy after optical clearing of the atrioventricular (AV) node in a Cx3cr1GFP/+ mouse stained with HCN4 (red). The node is orientated along the AV groove extending from the compact node (CN) into the proximal His bundle. Dashed square indicates the lower nodal or AV bundle. CFB, central fibrous body; IAS and IVS, interatrial and interventricular septum. (B) Higher magnification of dashed square in (A). (C) 3D rendering of GFP+ macrophages in the AV bundle. (D) Electron microscopy of a DAB+ macrophage in the AV node of a Cx3cr1GFP/+ mouse stained with a primary antibody for GFP. Arrow indicates nucleus, arrowheads indicate cellular processes. See also Figure S1.
Figure 2
Figure 2. The AV Node Enriches for Macrophages
(A) Flow cytometric macrophage quantification in microdissected AV node and left ventricular (LV) free wall of C57BL/6 mice. (Left) Representative flow cytometry plots; (right) number of macrophages per mg of heart tissue. Data are mean ± SEM, n = 12 mice from 4 independent experiments, **p < 0.01, Student’s t test. (B) (Left) Representative immunofluorescence images of the AV node and LV free wall of a Cx3cr1GFP/+ mouse stained for HCN4 (red) and nuclei (blue), or PDGFRα (red) and nuclei (blue). (Right) Percentage of positive staining per region of interest (ROI). Data are mean ± SEM, n = 3–6 mice from 2 independent experiments, **p < 0.01, Kruskal-Wallis test followed by Dunn’s posttest. (C) Macrophage chimerism in the LV free wall and AV node, and monocyte chimerism in the blood of C57BL/6 mice that had been joined in parabiosis with Cx3cr1GFP/+ mice for 12 weeks (mean ± SEM, n = 3 [AV node] and n = 7 [LV free wall and blood] from 2 independent experiments). (D) (Top) Workflow; (bottom) heat map of expression levels (cpm, counts per million) among top 200 overdispersed genes from RNA-seq data of 76 AV node macrophages. Unsupervised clustering reflects three macrophage subsets according to expression levels of H2 and Ccr2 (orange, MHCIIlowCCR2low; green, MHCIIhighCCR2high; purple, MHCIIhighCCR2low). See also Figure S2
Figure 3
Figure 3. Macrophages in the Human AV Node
(A) Masson’s trichrome stain of human tissue to identify the AV node. IAS and IVS, interatrial and interventricular septum. (B) Immunohistochemical stain for CD68 in human working myocardium and AV node. Data are mean ± SEM, n = 20 to 30 high-power fields per section, ****p < 0.0001, Student’s t test. (C) Volumetric reconstruction of confocal microscopy after optical clearing of a 500 μm section of the human AV bundle stained with CD68 (green). Autofluorescence signal (AF, red) was used for visualization of tissue morphology. Dashed area indicates the AV bundle. CFB, central fibrous body. (D) Maximum projection images of CD68+ and CD163+ macrophages in the human AV bundle. See also Figure S3.
Figure 4
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 Cx3cr1GFP/+ 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 Cx3cr1GFP/+ 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.
Figure 5
Figure 5. Optogenetics Stimulation of AV Node Macrophages Improves Nodal Conduction
(A) Experimental outline. Hearts of Cx3cr1wt/CreER (control) or tamoxifen-treated Cx3cr1wt/CreER ChR2wt/fl (Cx3cr1 ChR2) mice were perfused in a Langendorff setup. Recording and pacing electrodes were connected to the heart and illumination with a fiber optic cannula was focused on the AV node. (B) Images illustrating the optogenetics experimental setup during a light off and on cycle. (C) Representative ECG recordings from a Cx3cr1 ChR2 heart illustrating the number of conducted atrial stimuli between two non-conducted impulses of a Wenckebach period during light off and on cycles. Arrows indicate failure of conduction leading to missing QRS complexes. Stim, stimulation. (D) Representative bar graphs of a control and Cx3cr1 ChR2 heart showing the number of conducted atrial stimuli between two non-conducted impulses of a Wenckebach period during light off and on cycles. Data are mean ± SEM, *p < 0.05, Kruskal-Wallis test followed by Dunn’s posttest. See also Figure S5.
Figure 6
Figure 6. Cx43 Deletion in Macrophages and Congenital Lack of Macrophages Delay AV Conduction
(A) Experimental outline of the electrophysiological (EP) study performed on mice lacking Cx43 in macrophages. (B) AV node effective refractory period at 120 ms pacing frequency, and pacing cycle lengths at which Wenckebach conduction, 2:1 conduction and ventriculo-atrial (VA) Wenckebach conduction occurred in control (n = 5 to 9) and Cx3cr1 Cx43−/− (n = 6 to 8) mice. Data are mean ± SEM, 2 independent experiments, *p < 0.05 and **p < 0.01, Student’s t test and nonparametric Mann-Whitney test. (C) Surface ECG from control and Cx3cr1 Cx43−/− mice illustrating the Wenckebach cycle length. Arrows indicate missing QRS complexes. Stim, stimulation. (D) Flow cytometric quantification of AV node macrophages in control and Cx3cr1 Cx43−/− mice. Data are mean ± SEM, n = 6 mice per group, nonparametric Mann-Whitney test. (E) Immunofluorescence images of control and Cx3cr1 Cx43−/− AV node stained for CD68 (green) and HCN4 (red). (F) Quantification of AV node macrophages in control (n = 5) and Csf1op (n = 4) mice by flow cytometry. Data are mean ± SEM, 3 independent experiments, *p < 0.05, nonparametric Mann-Whitney test. (G) Immunofluorescence image of a Csf1op AV node stained for CD68 (green) and HCN4 (red). (H) AV node effective refractory period at 120 ms pacing frequency, and pacing cycle lengths at which Wenckebach and 2:1 conduction occurred in control (n = 8) and Csf1op (n = 7) mice. Data are mean ± SEM, 4 independent experiments, *p < 0.05, nonparametric Mann-Whitney test. See also Figure S6 and Table S1.
Figure 7
Figure 7. Macrophage Ablation Induces AV Block
(A) Experimental outline. DT, diphtheria toxin. (B) Flow cytometric quantification of AV node macrophages three days after intraperitoneal injection of DT (25 ng/g) into C57BL/6 and Cd11bDTR mice. Data are mean ± SEM, n = 6 mice per group, **p < 0.01, nonparametric Mann-Whitney test. (C) Onset of first degree AV block in Cd11bDTR (n = 6) and C57BL/6 (n = 10) animals after DT injection (DT dose: 25 ng/g, 2 independent experiments, ****p < 0.0001, Mantel-Cox test). (D) Telemetric ECG recordings before and after DT injection (25 ng/g) in Cd11bDTR mice. Arrows indicate non-conducted P waves in second degree AV block. (E) Surface ECG of Cd11bDTR and Cx3cr1GFP/+ parabionts three days after DT injection (DT dose: 25 ng/g, mean ± SEM, n = 3 parabiosis pairs). See also Figure S7 and Tables S2 and S3.

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