Macrophages facilitate post myocardial infarction arrhythmias: roles of gap junction and KCa3.1

Figures

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  Figure 1. Pro-inflammatory mononuclear cells are increased in patients with post-MI arrhythmias at 3 days post-MI. (A). Gating strategy for analyzing monocyte subsets. FSC/SSC was firstly applied to distinguish monocytes from neutrophils and lymphocytes. Monocyte subsets were determined by CD14 and CD16 expression. (B-D). Statistical analysis of monocyte subsets in controls, MI patients without arrhythmias and patients with post-MI arrhythmias (n=8 in each group). **p<0.01. *** p<0.001. NS, not significant.

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  Figure 2. Macrophages form gap junction with cardiomyocytes and accumulate in MI border zones 3 days post-MI. (A-B). Triple immunofluorescence (cardiomyocytes = green, macrophages = red, Cx43 = yellow) in MI border zones of MI patient (A) and MI mouse (B). Arrows indicate gap junction between cardiomyocytes and macrophages. (C-D). Double immunofluorescence of F4/80 (red) and iNOS (yellow) in MI border zone of sham operated (C) and MI mice (D). Arrows indicate pro-inflammatory macrophages. (E). Statistical analysis of the numbers of pro-inflammatory macrophages in sham operated (n=3) and MI (n=4) mice. Scale bars indicate 50 μm. *** p<0.001.

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  Figure 3. KCa3.1 is the most differentially expressed ion channel between MI and sham operated mouse hearts and it is mainly attributed to macrophage accumulation and polarization. (A). The volcano plot of differentially expressed genes between MI and sham operated mice by RNA sequencing (n=3 in each group). Samples were collected 3 days post-MI. The X-axis is the log2 fold-change (MI/sham), the Y-axis is -log10 q value (MI/sham). Up-regulated and down-regulated genes are shown in purple and green, respectively. Several differentially expressed genes encoding ion channels are annotated. (B-C). Western blotting results of KCa3.1 expression in different cells 3 days post-MI (B) and statistical summary (C) (n=3 in each group). (D). Double immunofluorescence of F4/80 (red) and KCa3.1 (green) in MI border zone of MI mice 3 days post-MI. Arrows indicate overlay of F4/80 and KCa3.1. Scale bars indicate 50 μm.

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  Figure 4. KCa3.1 and intracellular Ca2+ are elevated in M1 macrophages. (A). Double immunofluorescence of F4/80 (red) and iNOS (yellow) of M0 and M1 macrophages. (B-C). Representative curves of KCa3.1 before and after TRAM34 perfusion in M0 (B) and M1 (C) macrophages. (D-E). Subtraction of TRAM34 sensitive currents of M0 and M1 macrophages (D) and statistical analysis (n=6 in each group) (E). (F-G). Western blotting results of KCa3.1 expression of M0 and M1 macrophages (F) and statistical analysis (n=3 in each group) (G). (H). Flow cytometry analysis of KCa3.1 expression of M0 and M1 macrophages. (I). Flow cytometry analysis after application of Fluo-3AM to detect intracellular Ca2+. Scale bars indicate 50 μm. * p<0.05.

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  Figure 5. M0 and M1 macrophages electrically modulate cardiomyocytes oppositely via gap junction. (A-B) Representative action potential curves (A) and statistical summary (B) of NMVMs cocultured with M0 (n=14 for solitary NMVMs and connected NMVMs) or M1 macrophages (n=15 for solitary NMVMs and connected NMVMs). (C). Resting membrane potentials of NMVMs cocultured with M0 (n=9 for solitary NMVMs and connected NMVMs) or M1 macrophages (n=10 for solitary NMVMs and connected NMVMs). (D). Cell capacitances of NMVMs cocultured with M0 (n=8 for solitary NMVMs and connected NMVMs) or M1 macrophages (n=7 for solitary NMVMs and connected NMVMs) (E). Summary of APDs of solitary NMVMs cocultured with M1 (n=6) and NMVMs connected with M1 (n=8) before and after GAP26 perfusion. (F). Triple immunofluorescence of cocultured cells (cardiomyocyte = green, macrophage = red, Cx43 = yellow). Arrow indicate Cx43 between cardiomyocyte and macrophage. Scale bars indicate 50 μm.*p<0.05. **p<0.01. *** p<0.001. NS, not significant.

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  Figure 6. KCa3.1 and elevated intracellular Ca2+ of macrophages play crucial roles in APD prolongation of cardiomyocytes. (A-B). Representative action potential curves (A) and statistical summary (B) of solitary NMVMs cocultured with M1 (n=6) and NMVMs connected with M1 (n=6) before and after TRAM34 application. (C-D). KCa3.1 current curves (C) and summary (D) of M1 macrophages treated with siRNA (n=5) or negative control (n=5). (E-F). Western blotting results and statistical analysis of KCa3.1 expression of M1 macrophages treated with siRNA or negative control. (G). APDs of NMVMs connected with M1 after application of siRNA (n=6) or negative control (n=6). (H-I). Representative action potential curves (H) and summary (I) of solitary NMVMs cocultured with M1 (n=5) and NMVMs connected with M1 (n=5) before and after SKF96365 perfusion. **p<0.01. *** p<0.001.

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  Figure 7. KCa3.1 inhibition effectively attenuated post-MI arrhythmias in vivo at 3 days post-MI. (A-B). Heart rates (A) and QTc durations (B) of sham operated mice treated with vehicle (n=5) or TRAM34 (n=6) and MI mice treated with vehicle (n=6) or TRAM34 (n=5). (C-D). Representative surface ECGs and images of intracardiac electrical stimulation of MI mice treated with vehicle (C) or TRAM34 (D). (E-F). VT/VF inducibility (E) and duration (F) of MI mice treated with vehicle (n=6) or TRAM34 (n=6). (G). Representative ambulatory ECGs of MI or sham operated mice treated with TRAM34 or vehicle. (H). Summary of PVC incidence in 60 minutes in MI or sham operated mice treated with TRAM34 or vehicle (n=6 in each group). * p<0.05. NS, not significant.