Macrophage Mineralocorticoid Receptor Is a Pleiotropic Modulator of Myocardial Infarct Healing

Abstract

Myocardial infarction (MI) is a major cause of death worldwide. Here, we identify the macrophage MR (mineralocorticoid receptor) as a crucial pathogenic player in cardiac wound repair after MI. Seven days after left coronary artery ligation, mice with myeloid cell-restricted MR deficiency compared with WT (wild type) controls displayed improved cardiac function and remodeling associated with enhanced infarct neovascularization and scar maturation. Gene expression profiling of heart-resident and infarct macrophages revealed that MR deletion drives macrophage differentiation in the ischemic microenvironment toward a phenotype outside the M1/M2 paradigm, with regulation of multiple interrelated factors controlling wound healing and tissue repair. Mechanistic and functional data suggest that inactivation of the macrophage MR promotes myocardial infarct healing through enhanced efferocytosis of neutrophils, the suppression of free radical formation, and the modulation of fibroblast activation state. Crucially, targeted delivery of MR antagonists to macrophages, with a single administration of RU28318 or eplerenone-containing liposomes at the onset of MI, improved the healing response and protected against cardiac remodeling and functional deterioration, offering an effective and unique therapeutic strategy for cardiac repair.

Keywords: liposomes; macrophages; myocardial infarction; receptors, mineralocorticoid; wound healing.

Figure 1.

Mice with myeloid cell–restricted MR (mineralocorticoid receptor) deficiency display improved cardiac function and remodeling after myocardial infarction (MI). A, Representative sections from MR^flox and MR^LysMCre infarcted hearts and infarct size. B, Representative left ventricular (LV) pressure-volume loops measured in vivo with conductance catheter in sham-operated MR^flox (gray) and MR^LysMCre (black) mice and in MR^flox (orange) and MR^LysMCre (blue) mice with MI. C, LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), LV end-systolic and end-diastolic volumes; LV ejection fraction, LV maximal rate of pressure rise (LV dP/dt_max), maximal rate of pressure decline (LV dP/dt_min), and LV dP/dt_max divided by instantaneous pressure (IP) and the time constant of LV pressure isovolumic decay (Tau). Mean±SEM (n=14–16). *P<0.01 vs MR^flox.

Figure 2.

Enhanced cardiac neovascularization after ischemic injury in MR^LysMCre mice. A and B, Immunofluorescence double staining (CD [cluster of differentiation] 31, red; α-SMA [α-smooth muscle actin], green) showing capillaries, coated vessels, and (myo)fibroblasts in the healing myocardium of MR^flox and MR^LysMCre mice, 7 d after myocardial infarction. In MR^LysMCre infarcts, the expression of α-SMA was mostly restricted to pericytes and arterioles. C, Capillary density quantification in MR^flox and MR^LysMCre mice. Mean±SEM (n=6). *P<0.01 vs MR^flox.

Figure 3.

Enhanced collagen scar formation in MR^LysMCre mice. Immunofluorescence staining for α-SMA (α-smooth muscle actin; green) showing the presence of myofibroblasts (spindle-shaped α-SMA–positive cells) within the infarct region of (A) MR^flox and (B) MR^LysMCre mice. MR^LysMCre infarcts exhibited a loss of α-SMA expression in (myo)fibroblasts. C–F, Sirius red polarization microscopy of scar sections revealed a matrix with a predominance of thin and loosely assembled collagen fibers in MR^flox mice (C and E) and well-aligned and tightly packed collagen fibers in MR^LysMCre infarcts (D and F), 7 d after myocardial infarction. G, Infarct collagen content, ratio of orange-red (O-R) thick to yellow-green (Y-G) thin collagen fibers, and fiber straightness and alignment. Mean±SEM (n=6). *P<0.05 vs MR^flox.

Figure 4.

MR (mineralocorticoid receptor) inactivation drives macrophage differentiation in the ischemic microenvironment toward a phenotype outside the M1/M2 activation paradigm. A, Flow cytometry and gating strategy identifying macrophages (CD [cluster of differentiation] 45⁺/CD11b⁺/Ly6G⁻/F4/80⁺ cells −Ly6C^low and Ly6C^high) and neutrophils (CD45⁺/CD11b⁺/F4/80⁻/Ly6G⁺ cells) in MR^flox and MR^LysMCre infarcts, 3 d after coronary artery ligation. SSC indicates side scatter. Mean±SEM (n=5). *P<0.05 vs MR^flox. B–E, Comparison of gene expression profiles of infarct macrophages from MR^LysMCre and MR^flox mice (n=3). B, Dot plot showing gene expression of macrophage markers; fold changes of M1/M2-like markers are reported (*P_adj<0.1). C, MA plot showing significantly regulated genes with P_adj <0.1 (red). D, Volcano plot; significantly regulated genes with P<0.05 (gray) and with fold change >2 (orange) are shown; upregulated efferocytic genes are highlighted (blue). E, Heatmap showing efferocytic gene expression.

Figure 5.

Macrophage MR (mineralocorticoid receptor) deficiency is associated with enhanced neutrophil efferocytosis, suppression of free radical formation and the modulation of fibroblast activation. A, Quantitative reverse-transcriptase polymerase chain reaction was used to detect the relative gene expression of Mertk, C1qa, C1qb, Gpnmb, Cat, Pxdr4, ApoE, Vegfb, IGF1, and Lcn2. B, Phagocytic index for apoptotic neutrophils and (C) phagocytic capacity for zymosan particles of macrophages (CD [cluster of differentiation] 45⁺/CD11b⁺/Ly6G⁻/F4/80⁺ cells) isolated by cell sorting from MR^flox and MR^LysMCre infarcts, 3 d after coronary artery ligation. D, Superoxide production by CD45⁺/CD11b⁺/Ly6G⁻/F4/80⁺ macrophages fluorescence-activated cell sorting–isolated from MR^flox and MR^LysMCre infarcts, assessed using a highly sensitive isocratic ion-pair high performance liquid chromatography–electrochemical method. E, Immunocytochemical staining showing fibroblasts and differentiated α-SMA (α-smooth muscle actin)–positive myofibroblasts (vimentin, red; α-SMA, green) and zymography of conditioned media. Macrophages (CD45⁺/CD11b⁺/Ly6G⁻/F4/80⁺ cells) and fibroblasts (CD45⁻/CD11b⁻/CD31⁻/TER-119⁻/NG2⁻/MEFSK4⁺ cells) were isolated by cell sorting from the infarct myocardium of MR^flox and MR^LysMCre mice and cocultured using the Boyden chamber system. Mean±SEM (n=4–5). MMP indicates matrix metalloproteinase. *P<0.05 vs MR^flox. HE indicates hydroethidine; IS, internal standard; NG2, chondroitin sulfate proteoglycan; MEFSK4, anti-feeder antibody, clone mEF-SK4; and SSC, side scatter.

Figure 6.

Targeted delivery of MR (mineralocorticoid receptor) antagonists to macrophages protects against cardiac dysfunction and remodeling after myocardial infarction. A single dose of liposomal RU28318 (Lipo+RU) or liposomal eplerenone (Lipo+Eple) or empty (MR antagonists lacking) liposomes (Lipo) were injected intraperitoneally into mice at the onset of myocardial infarction. A, Sections of infarcted hearts and infarct size. B, Representative left ventricular (LV) pressure-volume loops measured in vivo with conductance catheter, 7 d after myocardial infarction. C, LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), LV maximal rate of pressure rise (LV dP/dt_max), maximal rate of pressure decline (LV dP/dt_min), LV end-systolic and end-diastolic volumes, LV ejection fraction, LV dP/dt_max divided by instantaneous pressure (IP), and the time constant of LV pressure isovolumic decay (Tau). Mean±SEM (n=6). *P<0.05 vs Lipo.