Sex Differences in Cardiac Pathology of SARS-CoV2 Infected and Trypanosoma cruzi Co-infected Mice

Figures

• 

  Figure 1. T. cruzi infection modulates pulmonary pathology in CoV2 infected huACE2 mice. (A) H&E stained lung sections (n = 4/sex, a minimum of five images/section were analyzed) of both male and female uninfected, CoV2 infected, T. cruzi infected, and coinfected huACE2 mice showing infiltrated immune cells (black arrowhead), accumulated lipid droplets (black line), fibrosis (red arrowhead), and granulomas (red arrow) (20x magnification, bar-200μm). (B) Histological grading of lung pathology was carried out according to experimental groups and classified in terms of granulomas (numbers), infiltrated immune cells, and accumulated lipid droplets. Each class was graded on a 6-point scale ranging from 0 to 5 as discussed in Materials and Methods. Values plotted are mean ± standard error (SE) from n = 5. The error bars represent the standard error of the mean. **P < 0.01 and ***P < 0.001, between the indicated groups and uninfected mice. #p ≤ 0.05 and ###p ≤ 0.001 for comparisons between CoV2 infected and co-infected mice.

• 

  Figure 2. Changes in ACE2 levels and viral load in the lungs and hearts of CoV2 infected, T. cruzi infected, and coinfected hACE2 mice. (A) Immunoblot analysis (upper panel) of ACE2 in the lungs (left) and hearts (right). GDI was used as loading control. Fold changes in the protein levels of ACE2 were normalized to GDI expression and are shown as a bar graph (A dot plot displaying individual data point is shown in Supplementary Figure 4). The error bars represent standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to uninfected sex matched mice (n = 4/sex/group) (###p < 0.001 for comparisons between CoV2 infected and coinfected mice). (B) Number of viral copies/μg of RNA in the lungs (left) and hearts (right) quantitated by qPCR in male and female CoV2 and coinfected mice. The error bars represent standard error of the mean (**p ≤ 0.01 and ***p ≤ 0.001) (M, male; F, female).

• 

  Figure 3. Histology of the myocardium of uninfected, CoV2 infected, T. cruzi infected, and coinfected huACE2 mice (n = 4/sex, a minimum of five images/section were analyzed). (A) H&E stained sections of left ventricles showing the accumulation of lipid droplets in the capillaries (red arrow) and presence of enlarged cardiomyocyte nuclei (black arrowhead) in infected mice (20x magnification). (B) Masson-trichrome stained sections of right ventricles showing fibrosis (blue and purple) and the presence of immune cells (yellow arrows) in the right ventricles (RV) of infected/coinfected mice (20x magnification; bar – 100 μm) (Additional images are shown in Supplementary Figures 2, 3). (C) Histological grading of heart sections showing the levels of immune infiltrates, lipid droplet accumulation, and fibrosis among CoV2 infected, T. cruzi infected, and coinfected mice compared to uninfected mice in both sexes. The error bars represent standard error of the mean. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with uninfected sex matched mice (M, male; F, female).

• 

  Figure 4. Immunohistochemistry staining (top panel) of huACE2 mouse myocardium using anti-perilipin (left) and anti-phospho-perilipin (right) antibody. The stained sections show altered levels of perilipin and phospho-perilipin during indeterminate T. cruzi infection and coinfection in both male and female mice compared to uninfected and CoV2 infected mice. IHC staining intensity (bottom panel) was analyzed using ImageJ software. Positive staining intensities of perilipin and phospho-perilipin were calculated and plotted separately (n = 4 mice, a minimum of five images/section were analyzed). The error bars represent standard error of the mean. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with uninfected sex matched mice (M, male; F, female).

• 

  Figure 5. Immunoblot analysis of adipogenic markers in the hearts of Cov2 infected, T. cruzi infected, and coinfected huACE2 mice. Western blot images of (A) cardiac high-molecular weight adiponectin (HMW ApN) and (B) ApN receptors (Adipo R1, R2 and T-cadherin). β-Actin was used as loading control. Fold-changes in the protein levels of adipogenic markers normalized to β-Actin are shown as bar graphs. (A dot plot displaying individual data point is shown in Supplementary Figure 4). The error bars represent standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to uninfected sex matched mice (n = 4/sex/group) (##p ≤ 0.01 for comparisons between CoV2 infected and coinfected mice) (M, male; F, female).

• 

  Figure 6. Immunoblot analysis of inflammatory markers in the hearts of CoV2 infected, T. cruzi infected, and coinfected huACE2 mice. Western blot images (A) of macrophage marker (F4/80) and proinflammatory cytokine TNFα. β-Actin was used as loading control. Fold changes in the protein levels of F4/80 (B) and TNFα (C) were normalized to β-Actin expression and are shown as bar graphs (A dot plot displaying individual data point presented in Supplementary Figure 4). The error bars represent standard error of the mean. **p < 0.01 and ****p < 0.0001 compared to uninfected sex matched mice (n = 4/sex/group). (##p ≤ 0.01, and ###p ≤ 0.001 for comparisons between CoV2 infected and coinfected mice) (M, male; F, female).

• 

  Figure 7. Immunoblot analysis of markers of glucose and lipid metabolism in the hearts of CoV2 infected, T. cruzi infected, and coinfected huACE2 mice. Immunoblot images of (A) markers of glucose metabolism (pAMPK and hexokinase II) and (B) markers of lipid metabolism (PPARα and PPARγ). β-Actin was used as loading control. Fold changes in the protein levels normalized to β-Actin are shown as bar graphs (A dot plot displaying individual data points is presented in Supplementary Figure 4). The error bars represent standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to uninfected sex matched mice (n = 4/sex/group) (###p ≤ 0.001 for comparisons between CoV2 infected and coinfected mice) (M, male; F, female).

• 

  Figure 8. Immunoblot analysis of β-Adrenergic Receptor and markers of mitochondrial β-oxidation in the hearts of CoV2 infected, T. cruzi infected, and coinfected huACE2 mice. Western blot images (A) of β-Adrenergic Receptor (β-AR), cytochrome C (Cyto C), and superoxide dismutase (SOD). β-Actin was used as loading control. Fold changes in the protein levels of β-AR (B), Cyto C (C) and SOD (D) were normalized to β-Actin expression and are shown as bar graphs (A dot plot displaying individual data points is presented in Supplementary Figure 4). The error bars represent standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to uninfected sex matched mice (n = 4/sex/group) (#p ≤ 0.05 and ###p ≤ 0.001 for comparisons between CoV2 infected and coinfected mice) (M, male; F, female).

• 

  Figure 9. The proposed models of signaling pathways that differentiate the pathogenesis of dilated cardiomyopathy (indicated by blue arrows) and cachexia (indicated by red arrows) in male and female coinfected mice, respectively. The adiponectin/AMPK/glycolysis pathway dominates in the hearts of male coinfected mice, whereas β-adrenergic receptor signaling activates lipid metabolic pathways in female coinfected mice.