NADPH Oxidase-4 Driven Cardiac Macrophage Polarization Protects Against Myocardial Infarction–Induced Remodeling

Figures

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  Figure 1. Cardiac Structure and Function in Nox4 TG Mice and Controls 1 Week After Reperfused MI (A to D) Changes in left ventricular end-systolic volume (ESV), end-diastolic volume (EDV), ejection fraction (EF), and heart rate (HR) assessed by echocardiography. (E) Heart weight/body weight ratio (HW/BW). (F) Cardiomyocyte cross-sectional area quantified in left ventricular sections. n = 5 to 6 per group. I/R = ischemia/reperfusion; MI = myocardial infarction; Nox4 = NADPH oxidase 4; TG = transgenic; WT = wild-type.

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  Figure 2. Nox4 Overexpression Alters Basal Inflammatory Profile in the Heart (A and B) Staining and quantification of macrophages (F4/80+ cells, arrows) in cardiac sections from TG and WT mice. Scale bar: 100 μm. (C to G) Messenger ribonucleic acid (mRNA) expression levels of macrophage-specific markers and chemo-attractant molecules in TG and WT heart. n = 5 to 6 per group. EMR1 = EGF-like module-containing mucin-like hormone receptor-like 1; CCR2 = C-C chemokine receptor type 2; CCL2 = C-C motif chemokine ligand 2; CCL5 = C-C motif chemokine ligand 5; VCAM = vascular cell adhesion molecule; other abbreviations as in Figure 1.

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  Figure 3. Nox4 Modulates Cardiac Macrophage Polarization After I/R (A to D) Flow cytometric quantification of total leukocytes, macrophages, and macrophage subsets according to Ly6c or MRC1 expression in the TG and WT left ventricle, 4 days’ post-MI. (E and F) Representative pseudocolor plots for macrophages based on Ly6c or MRC1 expression. (G and H) mRNA levels of the M2 markers MRC1 and CX3CR1 in whole left ventricle. (I and J) Ly6c- and MRC1+ macrophages as a proportion of total macrophages in the remote and ischemic areas of the left ventricle. n = 5 to 6 per group. #Significant interaction according to 2-way analysis of variance. Abbreviations as in Figures 1 and 2.

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  Figure 4. Nox4 Modulates Cardiac Macrophage Polarization After Permanent LAD Ligation Cardiomyocyte Nox4 drives polarization in a region-specific manner after permanent left anterior descending coronary artery (LAD) ligation. (A and B) Representative pseudocolor plots for macrophages based on Ly6c or MRC1 expression. Nox4 drives an inversion of the Ly6c expression pattern in the remote myocardium of TG. (C and D) Ly6c- and MRC1+ macrophage proportions in the infarct and remote area. n = 5 to 6 per group. Abbreviations as in Figure 1.

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  Figure 5. Cardiomyocyte-Targeted Nox4 Overexpression Improves Post-MI Survival and Protects Against Adverse Remodeling (A) Kaplan-Meier survival curves after MI in TG and WT (mouse numbers at 0, 7, 14, 21, and 28 days after MI: TG, 27, 26, 26, 26, and 26; WT, 37, 28, 28, 27, and 27). (B) Infarct size 24 h and 4 weeks after MI. n = 6 per group. (C and D) Echocardiographically quantified left ventricular EDV and EF after MI. n ≥ 15 per group. (E and F) Invasive cardiac contractile function assessed by maximum derivative of left ventricular pressure (dP/dtmax) and minimum derivative of left ventricular pressure (dP/dtmin). n = 8 per group. (G) Postmortem HW/BW. (H) Interstitial fibrosis in remote myocardium of TG and WT hearts after MI. n = 6 per group. #Significant interaction according to 2-way analysis of variance. LV = left ventricle; other abbreviations as in Figure 1.

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  Figure 6. Cardiomyocyte-Targeted Nox4 Alters MMP-2 Activity Profile After I/R Left ventricular matrix metalloproteinase (MMP)-2 activity measured by using gelatin zymography. (A) Representative zymogram. (B) Mean data. (C) mRNA levels of MMP-2 in the left ventricle of WT and TG hearts after ischemia/reperfusion (I/R). n = 5 to 6 per group. #Significant interaction according to 2-way analysis of variance. Abbreviations as in Figures 1, 2, and 5.