Resident cardiac macrophages mediate adaptive myocardial remodeling

Abstract

Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2⁺) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2^-) macrophages. Herein, we identified an essential role for CCR2^- macrophages in the chronically failing heart. Depletion of CCR2^- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2^- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.

Keywords: C-C chemokine receptor 2; CCR2; TRPV4; angiogenesis; cardiac remodeling; dilated cardiomyopathy; heart failure; macrophages; transient receptor potentialvanilloid 4.

Figure 1.. Cardiac macrophages in a mouse model of dilated cardiomyopathy.

A, End-diastolic echocardiographic images of 8-week-old control and Tnnt2^{ΔK210/ΔK210} mice. B, Quantification of ejection fraction, LV diastolic dimension, LV mass index, and stroke volume. * denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to controls, ** p<0.05 compared to Tnnt2^{ΔK210/ΔK210} at 8 weeks. n=10–11 per group. C, H&E (top) and immunostaining images (bottom, CD68-white, cardiac actin-green) of 8-week-old control and Tnnt2^{ΔK210/ΔK210} mice. n=7 per group. Scale bar: 20μm. D, Flow cytometry plots of CD45⁺Ly6G⁻CD64⁺ macrophages in control and Tnnt2^{ΔK210/ΔK210} mice at 8 weeks of age. n=4 per group. E-F, Quantification of CD68 immunostaining and flow cytometry. * denotes p<0.05 (Mann-Whitney test) compared to controls. G, CCR2 and CCR5 PET/CT of control and Tnnt2^{ΔK210/ΔK210} mice (6–10 weeks of age). n=4–17 per group. PET: positron emission tomography, CT: computed tomography. H, Quantification of CCR2 and CCR5 tracer uptake within the heart. * denotes p<0.05 (Mann-Whitney test) compared to controls. I, MA plot and pathway analysis of RNA sequencing data comparing control to Tnnt2^{ΔK210/ΔK210} hearts. n=5–6 per group. See also Figure S1–3, Table S1, Data S1.

Figure 2.. CCR2⁻ macrophages influence survival and LV remodeling in dilated cardiomyopathy.

A, Schematic of experimental groups, CCR2⁻ macrophage depletion strategy, and endpoints. B, CD68 immunostaining of control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts after 3 weeks of DT treatment. n=4 per group. Scale bar: 20μm. C, Quantification of CD68 immunostaining. ** denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to all other groups. D, Flow cytometry of CD45⁺Ly6G⁻CD64⁺ macrophages showing specific depletion of CCR2⁻ macrophages in Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts. n=4 per group. E, Kaplan-Meier analysis of survival in control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR mice after DT treatment. n=12–15 per group. F-I, LV ejection fraction, relative wall thickness, LV volumes (μl), and stroke volume in control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR mice 3 weeks after DT treatment. n=4–8 per group. * denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to controls. # denotes p<0.05 compared to Tnnt2^{ΔK210/ΔK210} mice. J, Pressure volume loops showing reduced stroke volume in Tnnt2^{ΔK210/ΔK210} CD169-DTR compared to Tnnt2^{ΔK210/ΔK210} mice. n=4 per group. K, Invasive hemodynamics: LV dP/dt max, heart rate (HR), and LV end systolic pressure (LVESP) at baseline and during peak infusion of dobutamine (64 ng/min) in control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{0394K210/ΔK210} CD169-DTR mice 3 weeks after DT treatment. n=5 per group. * denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to controls. # denotes p<0.05 compared to Tnnt2^{ΔK210/ΔK210} mice. See also Figure S4–6, Tables S2–S3.

Figure 3.. CCR2⁻ macrophages orchestrate myocardial tissue adaptations in dilated cardiomyopathy.

A, Low magnification H&E images of control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts after 3 weeks of DT treatment. n=4–9 per group. Scale bar: 200μm. B, Quantification of the ratio of compact to trabecular myocardium in control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts. C, Wheat germ agglutinin (WGA, red) staining showing alignment of trabecular cardiomyocytes in control, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts. n=4–9 per group. Scale bar: 40μm. D, X-ray microscopy and virtual histology images comparing myocardial architecture of Tnnt2^{ΔK210/ΔK210} and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts following 3 weeks of DT injection. n=4 per group. Scale bar: 200μm. E, Images of cardiomyocytes digested from control, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts. Hearts were relaxed in potassium prior to fixation. Scale bar: 20μm. F, Quantification of cardiomyocyte area, length, and width. * denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to controls. ** denotes p<0.05 compared to control and Tnnt2^{ΔK210/ΔK210} mice. See also Figure S4.

Figure 4.. CCR2⁻ macrophages are essential for coronary angiogenesis in dilated cardiomyopathy.

A, Microfil vascular casting of control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts after 3 weeks of DT treatment. n=4 per group. Scale bar: 200μm. B, Perfused lectin staining of control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts after 3 weeks of DT treatment.. n=4–9 per group. Scale bar: 20μm. C, Quantification of the coronary microvascular in control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts. * denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to controls. D, CCR2⁻ macrophages from Tnnt2^{ΔK210/ΔK210} hearts display increased Igf1, Pdgfc, Cyr61, and Hbegf mRNA expression compared to controls. n=5 per group. * denotes p<0.05 (Mann-Whitney test) compared to controls. E-F, IGF1 (E, white) and CYR61 (F, white) immunostaining of control, CD169-DTR, Tnnt2^{ΔK210/ΔK210}, and Tnnt2^{ΔK210/ΔK210} CD169-DTR hearts after 3 weeks of DT treatment. Red: CD68. n=5 per group. Scale bars: 10μm (E) and 20μm (F).

Figure 5.. CCR2⁻ macrophages interact with neighboring cardiomyocytes through focal adhesion complexes.

A-B, Compressed Z-stack images of CCR2⁻ (A) and CCR2⁺ (B) cardiac macrophages of a 8-week-old control Ccr2^GFP/+ and Tnnt2^{ΔK210/ΔK210} Ccr2^GFP/+ hearts. n=4–6 per group. CD68: white, GFP: green, α-actinin, red, DAPI: blue. Scale bar: 10μm. C-D, Electron microscopy of CCR2⁻ (C) and CCR2⁺ (D) cardiac macrophages in Tnnt2^{ΔK210/ΔK210} Ccr2^GFP/+ hearts. CCR2⁻ macrophage are adjacent to endothelial cells (*) and contact cardiomyocytes (CM). n=4 per group. Scale bars: 2μm (C, left), 500nm (C, center; C, right, D). E, Measurement of projection length in CCR2⁻ and CCR2⁺ macrophages of 8-week-old Ccr2^GFP/+ and Tnnt2^{ΔK210/ΔK210} Ccr2^GFP/+ hearts. * denotes p<0.05 (Mann-Whitney test) compared to controls. F, 2-photon microscopy of live Cx3cr1^GFP/+ Ccr2^RFP/+ papillary muscle cell preparations (n=4) over 90 minutes. CCR2⁻ macrophages (green). Scale bar: 25μm. G, Quantification of the percent of CCR2⁻ (black data points) and CCR2⁺ (red data points) macrophages interacting with cardiomyocytes in control and Tnnt2^{ΔK210/ΔK210} hearts. n=4 per group. * denotes p<0.05 (Mann-Whitney test) comparing CCR2⁻ to CCR2⁺ macrophages. H-I, Immunostaining for CD68 (green), FAK (white), and troponin (red). Scale bar: 10μm. n=12 per group. * denotes p<0.05 (Mann-Whitney test) compared to controls. J, Electron microscopy of bone marrow-derived macrophages (MΦ) and HL1 cardiomyocytes (CM) after 4 hours of co-culture. Physical interactions (white arrows) between macrophages and cardiomyocytes. Right center right panels: high magnification of boxed areas shown in left center panel. Scale bars: 50μm, 2μm, 500nm, 200nm. K, Immunostaining of cell junction markers. Blue-DAPI, red-CD68, white-cell junction markers: Paxillin (focal adhesion complex) Cx43 (gap junction), pan-Cadherin (adherins junction), Claudin (tight junction), Desmoplakin I/II (desmosome). Representative images from 4 independent experiments. Scale bar: 10μm. See also Figure S6, Supplemental movie 1–2.

Figure 6.. TRPV4 regulates growth factor expression in macrophages.

A, mRNA expression of TRP channels in CCR2⁻ macrophages. n=20 samples. B, Ratiometric calcium assay demonstrating that cardiac macrophages have active TRVP4 channels. GSK101: TRPV4 agonist, GSK219: TRPV4 antagonist. * denotes p<0.05 (ANOVA, Post-hoc Tukey) comparing GSK101 treated cells with GSK101 and GSK219 treated cells. C, Immunostaining of Trpv4-GFP BAC transgenic mice showing GFP (green) expression in CD68⁺ macrophages (red). Scale bar: 20μm. D, Flow cytometry of cardiac CD45⁺GFP⁺ leukocytes isolated from Trpv4-GFP hearts. n=4 per group. E, 2-photon imaging of GFP (green) and tdTomato (red) in papillary muscle preparations harvested from Cx3cr1-ertCre; Rosa26-GCaMP6/tdTomato mice treated with either vehicle or the TRPV4 inhibitor GSK219 (TRPV4i). Scale bar: 10μm. F, Quantification of GCaMP6 signal. n=6 per group. * denotes p<0.05 (paired t-test) compared to vehicle. G, Ratiometric calcium assay showing that bone marrow derived macrophages express active TRPV4. GSK101: TRPV4 agonist, Ionomycin: calcium ionophore. H, Cyclic uniaxial stretch (1 Hz, 10% deformation, 24 hours) promotes elongation of bone marrow derived macrophages. n=4 independent experiments. Scale bar: 50μm. * denotes p<0.05 (ANOVA, Post-hoc Tukey) compared to vehicle unstretched. I, Quantitative RT-PCR measuring Igf1, Hbegf, and Cyr61 mRNA expression in stretched macrophages treated with vehicle or TRPV4 inhibitor. n=4 independent experiments. * denotes p<0.05 compared to vehicle treated unstretched cells (ANOVA post-hoc Tukey). J, Quantitative RT-PCR measuring Igf1, Hbegf, and Cyr61 mRNA expression in stretched control, Myd88^−/−, and Trif^−/− macrophages. n=4 independent experiments. * denotes p<0.05 compared to vehicle treated unstretched cells (ANOVA post-hoc Tukey) (F-H). K, Quantitative RT-PCR of macrophages stimulated with vehicle control, LPS, or polyIC. * denote p<0.05 (Mann-Whitney). n=4 independent experiments. See also Figure S6, Data S2.

Figure 7.. TRPV4 regulates IGF1 expression in CCR2⁻ macrophages and is required for coronary angiogenesis.

A, Immunostaining for IGF1 (white) and CD68 (red) inf control and Tnnt2^{ΔK210/ΔK210} hearts treated with vehicle, TRPV4 inhibitor, or TRPV4 agonist. Scale bar: 20μm. B, Quantification of IGF1 protein expression (% IGF1+ macrophages, IGF1 MFI) in control and Tnnt2^{ΔK210/ΔK210} hearts treated with vehicle, TRPV4 inhibitor, or TRPV4 agonist. MFI: mean florescent intensity. n=5 per group. * denotes p<0.05 compared to vehicle treated control hearts. ** denotes p<0.05 compared to vehicle treated Tnnt2^{ΔK210/ΔK210} hearts. (ANOVA, post-hoc Tukey). C, Low magnification H&E images of control and Tnnt2^{ΔK210/ΔK210} hearts treated with vehicle or TRPV4 inhibitor for 2 weeks beginning at 6 weeks of age. n=4 per group. Scale bar: 200μm. D, Quantification of the ratio of compact to trabecular myocardium in control and Tnnt2^{ΔK210/ΔK210} hearts treated with vehicle or TRPV4 inhibitor. * denotes p<0.05 compared to vehicle treated control hearts (ANOVA, post-hoc Tukey). E, LV ejection fraction and LV chamber dimensions in control and Tnnt2^{ΔK210/ΔK210} mice treated with vehicle or TRPV4 inhibitor for 2 weeks beginning at 6 weeks of age. n=5 per group. * denotes p<0.05 compared to vehicle treated control hearts (ANOVA, post-hoc Tukey). F, CD34 (red) immunostaining of control and Tnnt2^{ΔK210/ΔK210} hearts treated with vehicle or TRPV4 inhibitor. Scale bar: 40μm. G, Microfil vascular casting showing that TRPV4 is necessary for expansion of coronary microvasculature in Tnnt2^{ΔK210/ΔK210} hearts. Scale bar: 200μm. H-I, Quantification of coronary microvasculature (G) and coronary microvasculature (H). n=5 per egroup. * denotes p<0.05 compared to vehicle treated control. ** denotes p<0.05 compared to all other groups (ANOVA, Post-hoc Tukey).