Macrophages modulate cardiac function in lipotoxic cardiomyopathy

Abstract

Diabetes is associated with myocardial lipid accumulation and an increased risk of heart failure. Although cardiac myocyte lipid overload is thought to contribute to the pathogenesis of cardiomyopathy in the setting of diabetes, the mechanism(s) through which this occurs is not well understood. Increasingly, inflammation has been recognized as a key pathogenic feature of lipid excess and diabetes. In this study, we sought to investigate the role of inflammatory activation in the pathogenesis of lipotoxic cardiomyopathy using the α-myosin heavy chain promoter-driven long-chain acylCoA synthetase 1 (MHC-ACS) transgenic mouse model. We found that several inflammatory cytokines were upregulated in the myocardium of MHC-ACS mice before the onset of cardiac dysfunction, and this was accompanied by macrophage infiltration. Depletion of macrophages with liposomal clodrolip reduced the cardiac inflammatory response and improved cardiac function. Thus, in this model of lipotoxic cardiac injury, early induction of inflammation and macrophage recruitment contribute to adverse cardiac remodeling. These findings have implications for our understanding of heart failure in the setting of obesity and diabetes.

Figures

Fig. 1.

Inflammatory cytokines are induced in lipotoxic cardiomyopathy. A: transthoracic echocardiograms were performed in lightly anesthetized MHC-ACS (ACS) and nontransgenic (NTG) littermates at 4 and 8 wk of age. The bars represent the mean fractional shortening. B: hearts from 4-wk-old ACS and NTG littermates were harvested and quantitative RT-PCR (qRT-PCR) was performed on total mRNA to assess the expression of inflammatory cytokines. Graphs display mean expression relative to 36B4 ± SE for n = 3 to 4 per group. *P < 0.05 and **P < 0.005 for ACS vs. NTG. ns, Not significant. C: total protein was isolated from the hearts of 4-wk-old NTG or ACS mice. Phosphorylated and total STAT3 levels were determined by Western blotting. MCP-1, monocyte chemoattractant protein 1; iNOS, inducible nitric oxide synthase.

Fig. 2.

Macrophages are recruited to the myocardium in lipotoxic cardiomyopathy. A: hearts from 4-wk-old ACS and NTG littermates were harvested and qRT-PCR was performed on total mRNA to assess the expression of macrophage markers. Graphs show mean expression relative to 36B4 ± SE for n = 3 to 4 per group. B: formaldehyde-fixed, paraffin-embedded heart tissue from 4-wk-old NTG and ACS mice was stained for the macrophage marker MAC3 with detection using immunoperoxidase. Representative images are shown in low power (top) and high power (bottom). Bars, 100 μm. C: myocardial triglyceride (TAG) levels were determined in ACS and NTG littermates at 3 and 4 wk of age. Each dot represents an individual mouse with the mean shown for n = 4 per group. D: CD68 mRNA expression in heart tissue was determined by qRT-PCR from 3- and 4-wk-old ACS and NTG mice. The graph shows mean expression relative to 36B4 ± SE for n = 3 to 4 per group. *P < 0.05 and **P < 0.005 for ACS vs. NTG. OPN, osteopontin; Arg, l-arginase.

Fig. 3.

F4/80 and CDllc expressing cells accumulate in the myocardium of MHC-ACS mice. A: hearts from 4-wk-old NTG and ACS mice were digested to prepare single cell suspensions, stained with fluorochrome-coupled antibodies, and analyzed by flow cytometry. CD45+ cells (leukocytes) were gated (far left) and further analyzed for the expression of CD11b, CD11c, and F4/80. Representative flow plots are shown with the percentage of cells in each quadrant as indicated. B: quantification of CD11c+ and F4/80+ myeloid cells in NTG vs. ACS hearts. Graphs display means ± SE for n = 3 mice per group. *P < 0.05 and **P < 0.005 for ACS vs. NTG.

Fig. 4.

Macrophage depletion reduces leukocyte influx and modulates the cytokine response in MHC-ACS mice. ACS mice were injected with clodrolip (clp; red bars) or vehicle (veh; black bars) beginning at 22 days of age and continuing every 4 days until heart tissue was harvested at 4 wk of age. A: mRNA was isolated from heart tissue and expression of leukocyte markers and cytokines (relative to 36B4) was determined using qRT-CPR and compared with veh-treated NTG mice (white bars). Graphs display mean expression ± SE for n = 4 per group. B: formaldehyde-fixed, paraffin embedded heart tissue from veh-ACS and clp-ACS mice was stained by hematoxylin and eosin (H&E; left) or for MAC3 (right). Representative images are shown. Bars, 100 μm. C: total and phospho-STAT3 levels were determined by Western blotting using myocardial protein extracts from 4-wk-old ACS and NTG mice treated with clp or veh. D: myocardial TAG content was quantified in clp- or veh-treated ACS and NTG mice at 4 wk of age. *P < 0.05 and **P < 0.005 for ACS (veh or clp) vs. NTG; #P < 0.05 and ##P < 0.005 for ACS-veh vs. ACS-clp.

Fig. 5.

Macrophage depletion improves cardiac function in MHC-ACS mice. NTG (●) and ACS mice (▽) were treated with veh or clp every 4 days, beginning at 22 days of age and continuing until 8 wk of age at which time cardiac function was assessed by transthoracic echocardiography. LV mass index (LVMI; A) and LV fractional shortening (B) are shown for each of the treatment groups. Each symbol represents 1 mouse, and bar indicates mean for each group. *P < 0.05 and **P < 0.005 for comparisons indicated (n = 3 to 8 per group).