Leukocyte iNOS is required for inflammation and pathological remodeling in ischemic heart failure

Abstract

In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS^-/- mice (iNOS^-/-c) or WT mice (WTc). Chimeric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ~twofold increase in circulating Ly6C^hi pro-inflammatory monocytes, and ~sevenfold higher cardiac M1 macrophages, which were primarily CCR2^- cells. In contrast, as compared with WTc HF mice, iNOS^-/-c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS^-/-c HF mice exhibited lower circulating Ly6C^hi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2⁺ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomyocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS^-/- HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.

Keywords: Heart failure; Inducible NOS; Inflammation; LV remodeling; Macrophage.

Fig. 1

a. Kaplan–Meier survival curves over 28 days following coronary ligation [to induce heart failure (HF)] or sham operation in wild-type chimeric (WTc) and iNOS^−/− chimeric (iNOS^−/−c) mice. *p = 0.0184 vs. WTc sham; ^#p = 0.0469 vs. WTc HF (log-rank test); n = 8–19/group. b Group data for scar size 4 weeks after coronary ligation in WTc and iNOS^−/−c mice (n = 9/group, unpaired t test). c Representative short-axis LV sections, end-diastolic 2D images, and M-mode echocardiograms from WTc and iNOS^−/−c sham and HF mice 4 weeks after operation. d Example LV pressure and dP/dt tracings from WTc and iNOS^−/−c sham and HF mice 4 weeks after operation

Fig. 2

Representative Masson's trichrome stains (a) and quantitation of interstitial fibrosis and connective tissue growth factor (CTGF) gene expression by real-time PCR (b) in myocardium of WTc and iNOS^−/−c sham and HF hearts. c Representative wheat-germ agglutinin staining (green) of myocyte cell membranes and DAPI staining of nuclei (blue) in WTc and iNOS^−/−c sham and HF hearts, and quantitation of myocyte cross-sectional area and atrial natriuretic factor (ANF) gene expression by real-time PCR. *p < 0.05 vs. respective sham, ^#p < 0.05 vs. WTc HF (two-way ANOVA with Bonferroni post-test); n = 5–7/group

Fig. 3

a Western immunoblotting for iNOS protein in WTc and iNOS^−/−c sham and HF hearts. +C, recombinant iNOS-positive control. Myocardial nitrate + nitrite (NO_x) levels (b) and proteinmalondialdehyde (protein-MDA) adducts (c) corresponding to the same experimental groups. d Representative protein-nitrotyrosine immunostaining (green) of WTc and iNOS^−/−c sham and HF hearts and corresponding quantitative group data for fluorescence intensity. e Protein-nitrotyrosine slot blots from the same experimental groups. *p < 0.05 vs. respective sham, ^#p < 0.05 vs. WTc HF, ^@p < 0.05 vs. WTc sham (two-way ANOVA with Bonferroni post-test); n = 3–6/group

Fig. 4

a Myocardial gene expression of tumor necrosis factor-α (TNF), interleukin (IL)-6, and IL-10 in WTc and iNOS^−/−c hearts as indicated (n = 5–6/group). b EMSA for nuclear factor(NF)-κB DNA binding in WTc and iNOS^−/−c sham and HF hearts and corresponding quantitation of binding intensity (n = 3/group). c Serum levels of various cytokines, measured using cytometric bead array (n = 4–6/group). IFN-γ interferon-γ, MCP-1 monocyte chemoattrac-tant protein-1. *p < 0.05 vs. respective sham, ^#p < 0.05 vs. WTc HF, ^@p < 0.05 vs. WTc sham (two-way ANOVA with Bonferroni post-test)

Fig. 5

a Flow cytometry gating strategy used to identify circulating CD45⁺CD11b⁺F4/80^lowLy6C^hi and CD45⁺CD11b⁺F4/80^lowLy6C^low monocytes using the lymphocyte–monocyte gate from initial side vs. forward scatter plots (SSC vs. FSC, respectively). b Quantitative group data for total blood CD11b⁺F4/80⁺ cells, and Ly6C^hi and Ly6^low subsets, in WTc and iNOS^−/−c sham and HF mice. *p < 0.05, **p < 0.005, ***p < 0.0005 (two-way ANOVA with Bonferroni post-test); n = 4–6/group

Fig. 6

a Confocal microscopic images of immunofluorescent staining for F4/80 (red), CD206 (green), and nuclei (DAPI, blue) from representative WTc and iNOS^−/−c sham and HF hearts (border zone), along with magnified images from the failing heart sections and quantitative data for F4/80⁺CD206⁻ (M1) and F4/80⁺CD206⁺ M2 macrophages (n = 3–7/group). Red arrows M1 cells, and yellow arrows M2 cells. *p < 0.05 vs. WTc sham; **p <0.005 vs. WTc and iNOS^−/−c sham, ^#p < 0.05 vs. WTc HF (two-way ANOVA with Bonferroni post-test). b Myocardial CD206 mRNA expression by real-time PCR for the same experimental groups. *p < 0.05 vs. iNOS^−/−c sham and WTc HF. n = 4–6/group

Fig. 7

a High-power confocal microscopic images of immunofluo-rescent staining for CD68 (red), CCR2 (green), and nuclei (DAPI, blue) from representative WTc and iNOS^−/−c sham and HF hearts (near scar area), and b quantitative group data for CD68⁺CCR2⁻ (locally sourced) and CD68⁺CCR2⁺ infiltrating macrophages (n = 3–5/group). *p < 0.05, **p < 0.005 (two-way ANOVA with Tukey's post-test)

Fig. 8

a Group data for sarcomere shortening from cardiomyocyte– macrophage cell interaction studies in the presence or absence of attachment to macrophages derived from WT or iNOS^−/− HF mice. In some experiments, iNOS^−/− HF-derived macrophages were also pre-stimulated with lipopolysaccharide (LPS). *p < 0.05 vs. baseline and WT unattached, ^#p < 0.05 vs. iNOS^−/− attached, without and with LPS; n = 4–6/group. b Group data for number of WT and iNOS^−/− HF-derived macrophages attached to cardiomyocytes in these studies. NS not significant; n = 5/group. c Top representative fluorescent images of DCFH-loaded cardiomyocytes (green) exposed for 15 min to macrophages derived from either WT or iNOS^−/− HF mice, with or without cell attachment as indicated. Corresponding phase-contrast images are also shown. Bottom quantitative group data for cardiomyocyte fluorescent intensity after 15 min of exposure to macrophages derived from WT and iNOS^−/− (sham and HF) mice with or without attachment. *p < 0.05; n = 4–6/group. d Top representative fluorescent images of cardiomyocytes loaded with peroxynitrite indicator HK-Green2 exposed for 15 min to either positive control SIN-1 (peroxynitrite generator) or attached macro-phages derived from WT and iNOS^−/− HF mice. Bottom quantitative group data for cardiomyocyte fluorescence intensity. *p < 0.05; n = 3–4/group. (a–c two-way ANOVA, d one-way ANOVA; Bon-ferroni post-test)