Pharmacological targeting of mitochondrial function and reactive oxygen species production prevents colon 26 cancer-induced cardiorespiratory muscle weakness

Abstract

Cancer cachexia is a syndrome characterized by profound cardiac and diaphragm muscle wasting, which increase the risk of morbidity in cancer patients due to failure of the cardiorespiratory system. In this regard, muscle relies greatly on mitochondria to meet energy requirements for contraction and mitochondrial dysfunction can result in muscle weakness and fatigue. In addition, mitochondria are a major source of reactive oxygen species (ROS) production, which can stimulate increased rates of muscle protein degradation. Therefore, it has been suggested that mitochondrial dysfunction may be an underlying factor that contributes to the pathology of cancer cachexia. To determine if pharmacologically targeting mitochondrial dysfunction via treatment with the mitochondria-targeting peptide SS-31 would prevent cardiorespiratory muscle dysfunction, colon 26 (C26) adenocarcinoma tumor-bearing mice were administered either saline or SS-31 daily (3 mg/kg/day) following inoculation. C26 mice treated with saline demonstrated greater ROS production and mitochondrial uncoupling compared to C26 mice receiving SS-31 in both the heart and diaphragm muscle. In addition, saline-treated C26 mice exhibited a decline in left ventricular function which was significantly rescued in C26 mice treated with SS-31. In the diaphragm, muscle fiber cross-sectional area of C26 mice treated with saline was significantly reduced and force production was impaired compared to C26, SS-31-treated animals. Finally, ventilatory deficits were also attenuated in C26 mice treated with SS-31, compared to saline treatment. These data demonstrate that C26 tumors promote severe cardiac and respiratory myopathy, and that prevention of mitochondrial dysfunction is sufficient to preclude cancer cachexia-induced cardiorespiratory dysfunction.

Keywords: SS-31; cachexia; diaphragm; elamipretide; heart.

Figure 1. Cardiac function.

Heart left ventricular function for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31 assessed by: (A) fractional shortening percentage and (B) myocardial performance index. Values are presented as means ± SEM. Representative echocardiographic images are shown to the right of the graphs. ^*significantly different versus all groups (p < 0.05); ^#significantly different versus CON-SALINE and CON-SS-31 (p < 0.05).

Figure 2. Diaphragm muscle function and fiber size.

Diaphragm muscle (A) force-frequency response and (B) cross-sectional area for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31. Values are presented as means ± SEM. ^*significantly different versus all groups (p < 0.05); ^{^}significantly different versus CON-SALINE (p < 0.05).

Figure 3. Respiratory function.

Ventilatory pattern for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31 during baseline (normoxic) and challenge (hypoxia) conditions. (A) breathing frequency; (B) tidal volume; and (C) minute ventilation. Values are represented as means ± SEM. ^*significantly different versus all groups (p < 0.05); ^{^}significantly different versus CON-SALINE (p < 0.05).

Figure 4. Mitochondrial reactive oxygen species (ROS) emission and protein expression.

Mitochondrial ROS production from (A) heart and (B) diaphragm permeabilized muscle fiber bundles for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31; and (C) heart and (D) diaphragm citrate synthase and VDAC protein expression for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31. Values are represented as means ± SEM. Representative western blot images are shown to the right of the graphs. ^*significantly different versus all groups (p < 0.05).

Figure 5. Circulating cytokines.

Plasma cytokine levels of TNFα, IL-1b and IL-6 for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31. Values are represented as means ± SEM. ^# significantly different versus CON-SALINE and CON-SS-31 (p < 0.05).

Figure 6. Markers of proteolytic activity.

Western blot analysis of the calpain (145 kDa) and caspase-3 (120 kDa)-specific spectrin breakdown product in (A) heart and (B) diaphragm for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31. Western blot analysis of LC3II/LC3I in (C) heart and (D) diaphragm for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31. Gene expression of MuRF1 and BNIP3 in (E) heart and (F) diaphragm for control (CON) and tumor-bearing (TB) mice treated with saline (SALINE) or SS-31. Values are represented as means ± SEM. Representative western blot images are shown below the graphs. ^*significantly different versus all groups (p < 0.05); ^#significantly different versus CON-SALINE and CON-SS-31 (p < 0.05).