Inflammatory Macrophage Interleukin-1β Mediates High-Fat Diet-Induced Heart Failure With Preserved Ejection Fraction

Abstract

Diabetes mellitus (DM) is a main risk factor for diastolic dysfunction (DD) and heart failure with preserved ejection fraction. High-fat diet (HFD) mice presented with diabetes mellitus, DD, higher cardiac interleukin (IL)-1β levels, and proinflammatory cardiac macrophage accumulation. DD was significantly ameliorated by suppressing IL-1β signaling or depleting macrophages. Mice with macrophages unable to adopt a proinflammatory phenotype were low in cardiac IL-1β levels and were resistant to HFD-induced DD. IL-1β enhanced mitochondrial reactive oxygen species (mitoROS) in cardiomyocytes, and scavenging mitoROS improved HFD-induced DD. In conclusion, macrophage-mediated inflammation contributed to HFD-associated DD through IL-1β and mitoROS production.

Keywords: CCR2, C-C motif chemokine receptor 2; CM, cardiomyocyte; DD, diastolic dysfunction; DM, diabetes mellitus; EF, ejection fraction; FABP4, fatty acid binding protein 4; HF, heart failure; HFD, high-fat diet; HFpEF; HFpEF, heart failure with preserved ejection fraction; IL, interleukin; IL-1β; IL1RA, interleukin 1 receptor antagonist; KO, knockout; MCP, monocyte chemoattractant protein; MyBP-C, myosin binding protein C; TGF, transforming growth factor; TNF, tumor necrosis factor; Timd4, T cell immunoglobulin and mucin domain containing 4; WT, wild-type; diabetes; diastolic dysfunction; inflammation; macrophage; mitoROS, mitochondrial reactive oxygen species; mitochondria.

Graphical abstract

Figure 1

HFD Causes Cardiac Diastolic Heart Failure in Mice High-fat diet (HFD)-induced (A) hyperglycemia (n = 14 mice per group), (B) an increased ratio of transmitral Doppler early filing velocity to tissue Doppler early diastolic mitral annular velocity (E/E′) (n = 9 to 11 per group), and (D) left ventricular end-diastolic pressure (LVEDP) elevation by hemodynamic test (n = 7 to 18 mice per group) with (C) preserved ejection fraction (EF) value (n = 9 to 11 mice per group). (E) Representative echocardiographic images of tissue Doppler and pulsed wave Doppler from control (Ctrl) and HFD mice. Bars are mean ± SEM. Unpaired t-test was used. ∗P <0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs Ctrl.

Figure 2

IL-1β Mediates HFD-Induced Diastolic Dysfunction (A) Cardiac interleukin (IL)-1β level tested by enzyme-linked immunosorbent assay was increased in the mice with HFD; n = 8 to 9 mice per group. (B) IL-1β antagonist improved E/E′ in HFD mice; n = 11 to 14 mice per group. (C) The EF remained unchanged after IL1RA treatment; n = 11 to 14 mice per group. (D) Other cytokines in hearts were tested by enzyme-linked immunosorbent assay; n = 10 to 12 mice per group. Bars are mean ± SEM. Unpaired t-test (A and D) or 1-way analysis of variance with Bonferroni post hoc tests (B and C) were used. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs Ctrl; §P < 0.05 vs HFD. IL1RA = interleukin 1 receptor antagonist; TGF = transforming growth factor; TNF = tumor necrosis factor; other abbreviations as in Figure 1.

Figure 3

Cardiac Macrophages Are Activated in HFD Hearts (A) Monocyte chemoattractant protein (MCP)-1 level was significantly higher in HFD hearts by Western blot; n = 7 mice per group. (B) Representative flow cytometry images showing the gating strategy and analysis to identify macrophages subsets. Isolated noncardiomyocyte cells were pregated on CD11b⁺F4/80⁺ as cardiac macrophages and were further divided into subsets based on their expression of CCR2, T cell immunoglobulin and mucin domain containing 4 (Timd4), CD206, or CD86. (C) Percentage of cardiac macrophages (CD11b⁺F4/80⁺) among noncardiomyocyte interstitial cells; n = 4 to 21 mice per group. (D) Percentage change of each macrophage subset; n = 8 to 12 mice per group. Bars are mean ± SEM. Unpaired t-test (A and D) or 1-way analysis of variance with Bonferroni post hoc tests (C) were used. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs Ctrl; and &P < 0.01 vs HFD. CCR2 = C-C motif chemokine receptor 2; PI = propidium iodide; other abbreviations as in Figure 1.

Figure 4

Cardiac Macrophages Are Required for HFD-Induced Diastolic Dysfunction (A) Macrophage (Mφ) depletion improved E/E′ ratio (n = 6 to 13 mice per group) and decreased (B) cardiac IL-1β level tested by enzyme linked immunosorbent assay (n = 8 to 12 mice per group) without altering (C) EF (n = 7 to 10 mice per group) or (D) homeostatic model assessment for insulin resistance (HOMA-IR), an indicator of insulin resistance (n = 8 to 13 mice per group). Bars are mean ± SEM. One-way analysis of variance with Bonferroni post hoc tests were used. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs Ctrl; and &P < 0.01; #P < 0.001 vs HFD. Abbreviations as in Figures 1 and 2.

Figure 5

Suppressing Proinflammatory Macrophages Prevented HFD-Induced Diastolic Dysfunction (A) Inflammatory gene expression of fatty acid binding protein 4 (FABP4) knockout (KO) macrophage cell line by microarray. (B) FABP4 KO macrophages cannot secrete IL-1β under lipopolysaccharide stimulation; n = 3 independent experiments per group. (C) With HFD, FABP4 KO mice had lower cardiac IL-1β level; n = 5 to 9 mice per group. (D) Insulin resistance as indicated by HOMA-IR (n = 8 to 9 mice per group) and (E) hyperglycemia (n = 8 to 9 mice per group) were unchanged between wild-type (WT)+HFD and KO+HFD groups. (F) FABP4 KO mice were resistant to HFD-induced diastolic dysfunction; n = 8 to 9 mice per group. (G) EF was unaffected by FABP4 KO; n = 8 to 10 mice per group. Bars are mean ± SEM. Unpaired t-test (C) or 1-way analysis of variance with Bonferroni post hoc tests (D to G) were used. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs WT; §P < 0.05; #P < 0.01 vs WT+HFD. Abbreviations as in Figures 1, 2, and 4.

Figure 6

MitoROS Scavenging Reduced HFD-Induced Diastolic Dysfunction (A) IL-1β incubation up-regulated the cardiomyocyte (CM) mitochondrial reactive oxygen species (mitoROS) level. The cardiomyocytes were isolated from 5 mice, and 48 cardiomyocytes were tested for each group. (B) Mitochondrial antioxidant, mitoTEMPO (MT), improved E/E′ in HFD mice without affecting (C) the EF; n = 11 to 17 mice per group. The HFD group was the same as in Figures 2B and 2C. (D) Representative confocal microscopy images showing mitoROS in cardiomyocytes by mitoSOX red staining; scale bar represents 20 μm. Bars are mean ± SEM. Unpaired t-test were used. ∗∗P < 0.01 vs CM; §P < 0.05 vs HFD. a.u. = arbitrary units; other abbreviations as in Figures 1 and 2.

Figure 7

Innate Immunity Mediates High-Fat Diet–Induced HFpEF Through IL-1β Secretion and Mitochondrial Reactive Oxygen Species Modulation cMyBP-C = cardiac myosin binding protein C; HFpEF = heart failure with preserved ejection fraction; other abbreviations as in Figures 2 and 3.