Fat tissue regulates the pathogenesis and severity of cardiomyopathy in murine chagas disease

Figures

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  Fig 1. Fat ablation increases cardiac parasite load, lipid accumulation and cardiac pathology in T. cruzi infected acute (30 DPI) CD murine model (n = 6; minimum 5 images/section were analyzed). A. Hematoxylin and eosin (H&E) staining of hearts in indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab-). Infiltrated immune cells, black arrowhead; amastigote nests, black long arrow, and presence of lipid granules (see S2 Fig). Bar = 100 μm, 20x magnification. Additional images are presented as S3 Fig. B.Magnified (40x) images of H&E stained heart sections of indicated mice showing macro-vesicular (black long pointer) and micro-vesicular (black short pointer) lipid droplets. Bar = 50 um. C.Histologic grading of heart tissue pathology was carried out according to experimental groups and classified in terms of degree of parasite load, infiltrated immune cells, size of lipid droplets (macro lipid (≥20um) and micro lipid (≤20um) droplets) in the H&E sections of hearts in acute T. cruzi infected mice with and without fat ablation. The values plotted are mean ± standard deviation (SD) from n = 5. **, p < 0.01***, p < 0.001.

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  Fig 2. Loss in adipocytes increases cardiac enlargement during acute T. cruzi infection (30DPI). Left ventricle internal diameter (LVID) and right ventricle internal diameter (RVID), measured by ultrasound analysis of the hearts both at diastole (d) and systole (s) conditions at 30 DPI, in infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab-) mice as indicated. The error bars represent SEM. *p≤0.05, **p≤0.01 or ***p≤0.001 compared with uninfected fat-unablated. #p≤0.05 compared with infected fat-unablated.

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  Fig 3. T. cruzi infection alters adipose tissue pathology and fat-ablation further increases infiltration of immune cells into adipose tissue in FAT-ATTAC mice during early chronic stage (90 DPI). A. Hematoxylin and eosin (H&E) staining of adipose tissue in indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab-) n = 5). Infiltration of immune cells, long black arrow; multi-ocular lipid droplets/smaller size lipid droplets, black arrowhead. Bar = 50 μm, 20x magnification. B. Histological grading of adipose tissue pathology was carried out according to experimental groups and classified in terms of degree of infiltrated immune cells, size of adipocytes (micro-lipid droplets ≤20 um, and macro-lipid droplets ≥50 um), and number of dead adipocytes in adipose tissue during chronic T. cruzi infection and/or fat ablation (5 images per section/mouse in each group. Each class was graded on a six-point scale ranging from 0 to 5+ as discussed in Method section and presented as a bar graph. The values plotted are mean ± standard deviation (SD) from n = 5. *p≤0.05, **p≤0.01 or ***p≤0.001 compared with uninfected fat-unablated. #p≤0.05, ##p≤0.01 or ###p≤0.001 compared…

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  Fig 4. T. cruzi infection and fat ablation alter adipogenesis and lipolysis in adipose tissue during chronic stage in the infected mice. A. Immunoblot analyses of (A) adipogenic markers (adiponectin, FABP4 and PPAR) and (B) lipid degradation markers such as lipases (ATGL, HSL and p-HSL), loss of lipid droplets (p-perilipin) and lipid oxidation (PPARα) in adipose tissue of indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab-) n = 8). The change in protein levels were normalized to the levels of Guanosine nucleotide dissociation inhibitor (GDI) and plotted column scatter graph. The error bars represent SEM. A.U. indicates arbitrary unit. *p≤0.05, **p≤0.01 or ***p≤0.001 compared with uninfected fat-unablated. #p≤0.05, ##p≤0.01 or ###p≤0.001 compared with infected fat-unablated.

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  Fig 5. T. cruzi infection induces inflammation and cell death in adipose tissue via necrosis and apoptosis. A. Immunoblot analysis of (A) markers of inflammation (infiltrated macrophages (F4/80), and cytokines (INFᵧ and TNFα)), and (B) cell death (BNIP3 (a marker of necrosis), and cleaved Caspase3 (a marker of apoptosis)) in the adipose tissue of indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab -) n = 8). The change in protein levels were normalized to the levels of Guanosine nucleotide dissociation inhibitor (GDI) and plotted column scatter graph. The error bars represent SEM. A.U. indicates arbitrary unit. *p≤0.05, **p≤0.01 or ***p≤0.001 compared with uninfected fat-unablated. #p≤0.05, ##p≤0.01 or ###p≤0.001 compared with infected fat-unablated.

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  Fig 6. Fat ablation increases adipogenic signaling and elevates lipid levels in the hearts during the early chronic stage in T. cruzi infected mice. Immunoblot analysis of (A) adipogenic markers (FABP4, PPARᵧ and adiponectin), and (B) lipid metabolism (LDL and lipid metabolism markers (p-perilipin, PPARα, acyl-CoA ligase (ACSL) and acetyl Co-A carboxylase, CPT1, ABCA1 and CETP) in the hearts of indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab -) n = 8). The change in protein levels were normalized to the levels of Guanosine nucleotide dissociation inhibitor (GDI) and plotted column scatter graph. The error bars represent SEM. A.U. indicates arbitrary unit. *p≤0.05, **p≤0.01 or ***p≤0.001 compared with uninfected fat-unablated. #p≤0.05, ##p≤0.01 or ###p≤0.001 compared with infected fat-unablated.

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  Fig 7. Fat ablation on mitochondrial dysfunction, ER stress and inflammation in the hearts of chronic CD mice. A. Immunoblot analysis of (A) markers of mitochondrial function (cytochrome C, pyruvate dehydrogenase, SDHA, HSP60 and SOD1), (B) markers of ER stress (BIP and CHOP) and (C) markers of infiltration of macrophage (F4/80) and cytokines (TNFα and IFNᵧ) in the hearts of indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab -) n = 8). The change in protein levels were normalized to the levels of Guanosine nucleotide dissociation inhibitor (GDI) and plotted column scatter graph. The error bars represent SEM. A.U. indicates arbitrary unit. *p≤0.05, **p≤0.01 or ***p≤0.001 compared with uninfected fat-unablated. #p≤0.05, ##p≤0.01 or ###p≤0.001 compared with infected fat-unablated.

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  Fig 8. Loss in fat cells exacerbates cardiac pathology and causes bi-ventricular enlargement in the hearts of chronic CD mice. A. Hematoxylin and eosin (H&E) staining of hearts in indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab-)). Infiltrated immune cells, red arrowhead; vasculitis, black arrowhead; and presence of lipid droplets, black arrow. Bar = 100 μm, 20x magnification. B. Histologic grading of heart tissue pathology was carried out according to experimental groups and classified in terms of degree of infiltrated immune cells, size of adipocytes (macro lipid and micro lipid droplets), and fibrosis in the H&E sections of hearts in chronic T. cruzi infected mice with and without fat ablation (5 images per section/mouse in each group). Each class was graded on a six-point scale ranging from 0 to 5+ as discussed in Method section and presented as a bar graph. The values plotted are mean ± standard deviation (SD) from n = 5. C. Cardiac ultrasound imaging analysis in indicated mice (infected or uninfected mice, fat-ablated (Fab +) or fat-unablated (Fab-) 90DPI). Left ventricle internal diameter (LVID) and right ventricle internal diameter (RVID)…