Supplementary Table 1. Serum cytokine levels in control and tumor-bearing male and female mice at day 15. Male Female Cytokine Control C-26 Control C-26 IL-1β 2.0 ± 0.8 9.6 ± 1.5* 1.8 ± 0.2 6.8 ± 1.4* IL-2 0.4 ± 0.1 5.6 ± 4.0 0.4 ± 0.1 0.4 ± 0.1 IL-4 0.2 ± 0.1 1.6 ± 1.2 0.3 ± 0.04 ND IL-5 7.9 ± 2.0 12.2 ± 9.5 18.3 ± 0.7 6.4 ± 2.4** IL-6 4.3 ± 2.8 207.9 ± 53 * 3.4 ± 2.6 127.1 ± 85 IL-7 6.4 ± 3.0 11.6 ± 7.1 0.8 ± 0.3 3.9 ± 2.7 IL-10 1.4 ± 1.3 6.0 ± 4.7 0 0.6 ± 0.6 IL-12 5.7 ± 2.7 58.1 ± 51 6.8 ± 4.5 6.1 ± 3.3 IL-13 8.2 ± 0.9 39.3 ± 27 9.3 ± 4.6 6.7 ± 1.3 GM-CSF 3.3 ± 1.5 195.2 ± 130 2.0 ± 0.6 2.7 ± 1.4 IFN-γ 1.3 ± 0.2 10.6 ± 6.1 1.8 ± 0.6 0.7 ± 0.07 TNF-α 2.1 ± 0.8 5.0 ± 1.0 3.4 ± 0.6 1.4 ± 0.4 MCP-1 11.6 ± 5.3 120.3 ± 30* 9.6 ± 4.6 25.1 ± 8.9 A Multiplex ELISA was performed on serum from male and female control and tumorbearing mice 15 days post-tumor cell inoculum. Values are in picograms/milliliter (pg/ml) and results are represented as mean ± SEM. n = 3 per group. ** p < 0.01 and *p < 0.05 versus control. Cytokine abbreviations are; interleukin (IL), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1).
Supplementary Table 2. Transthoracic echocardiography on male and female control and tumor-bearing mice 27 days post-tumor cell inoculum. Male Female Control C-26 Control C-26 Heart rate (bpm) 532.2 ± 11 506 ± 9.9 498.2 ± 13 524.3 ± 4 Anterior wall, diastole (mm) 0.73 ± 0.04 0.66 ± 0.01 0.84 ± 0.03 0.78 ± 0.05 Anterior wall, systole (mm) 1.19 ± 0.05 1.11 ± 0.06 1.22 ± 0.03 1.25 ± 0.07 Posterior wall, diastole (mm) 0.88 ± 0.05 0.79 ± 0.04 0.76 ± 0.04 0.69 ± 0.01 Posterior wall, systole (mm) 1.25 ± 0.07 1.17 ± 0.07 1.23 ± 0.06 1.18 ± 0.07 LVID, diastole (mm) 4.05 ± 0.09 3.94 ± 0.13 4.00 ± 0.11 3.87 ± 0.07 LVID, systole (mm) 2.50 ± 0.13 2.35 ± 0.17 2.42 ± 0.12 2.11 ± 0.13 Fractional shortening (%) 38.43 ± 2.6 40.88 ± 2.7 39.5 ± 2.8 45.6 ± 3 Ejection fraction (%) 70.86 ± 2.6 71.27 ± 3.1 70.1 ± 3.3 76.8 ± 3.3 Aortic velocity (mm/sec) 928.8 ± 47.2 781.1 ± 29.2 * 798.8 ± 21 813.9 ± 46 Aortic pressure (mm Hg) 3.50 ± 0.36 2.46 ± 0.19 * 2.56 ± 0.13 2.69 ± 0.31 Cardiac output (ml/min) 19.4 ± 0.8 20.93 ± 1.17 17.89 ± 0.64 19.73 ± 1.69 Stroke Volume (μl) 36.46 ± 1.3 41.5 ± 2.8 36.02 ± 1.5 37.76 ± 3.1 Male and female mice were subcutaneously injected with saline (control) or C-26 adenocarcinoma cells in the right flank. M-mode echocardiography was performed on each group 27 days post-tumor cell or saline inoculum. Values are represented as mean ± SEM. N = 9 per group *p < 0.05 versus control. Abbreviation is; Left ventricular internal diameter (LVID).
Supplementary Figures Supplementary Figure 1. Supplementary Figure 1. Tumor-bearing males have increased levels of pro- and antiinflammatory cytokines compared to females. A Multiplex ELISA was used to quantify the levels of pro-inflammatory cytokines (A) and anti-inflammatory cytokines (B) in male and female mouse serum at days 15 and 27 post-tumor cell inoculum. n = 3 per group. Male and female differences were not significant due to the variability between mice. Mean ± SEM. ** p < 0.01 and *p < 0.05 versus control. Cytokine abbreviations are; interleukin (IL), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α).
Supplementary Figure 2. (A) Western blots of MyHC and actin extracted in high salt show MyHC and actin levels decrease in parallel in atrophic hearts. (B) High-resolution PAGE gels revealed a significant increase in β-myhc protein in the hearts of male and female tumorbearing mice. Graphs are mean ± SEM. **p < 0.01, ***p < 0.0001.
Supplementary Figure 3. (A) Relative atrogin-1 and MuRF-1 transcript levels in control heart and skeletal muscle. (B) Atrogin-1 and MuRF-1 transcript levels are more than two-fold higher in male than female cardiac muscle. Graphs are mean ± SEM. ***p < 0.0001.
Supplementary Figure 4. (A) Electron micrograph of control cardiac tissue. We did not detect any vacuoles in control tissue. Asterisk denotes elements of the sarcoplasmic reticulum, which are regularly patterned along the sarcomere, as opposed to autophagic vacuoles. (B) Electron micrographs of day 27 atrophic hearts showing numerous autophagic vacuoles and autolysosomes. Autophagosomes have a double membrane, while autolysosomes have a single membrane and have already undergone substantial digestion.
Supplementary Figure 5. Immunohistochemistry for cathepsin D revealed more intense staining in atrophic (C-26) hearts.