Spontaneous Physical Activity Downregulates Pax7 in Cancer Cachexia

Abstract

Emerging evidence suggests that the muscle microenvironment plays a prominent role in cancer cachexia. We recently showed that NF-kB-induced Pax7 overexpression impairs the myogenic potential of muscle precursors in cachectic mice, suggesting that lowering Pax7 expression may be beneficial in cancer cachexia. We evaluated the muscle regenerative potential after acute injury in C26 colon carcinoma tumor-bearing mice and healthy controls. Our analyses confirmed that the delayed muscle regeneration observed in muscles form tumor-bearing mice was associated with a persistent local inflammation and Pax7 overexpression. Physical activity is known to exert positive effects on cachectic muscles. However, the mechanism by which a moderate voluntary exercise ameliorates muscle wasting is not fully elucidated. To verify if physical activity affects Pax7 expression, we hosted control and C26-bearing mice in wheel-equipped cages and we found that voluntary wheel running downregulated Pax7 expression in muscles from tumor-bearing mice. As expected, downregulation of Pax7 expression was associated with a rescue of muscle mass and fiber size. Our findings shed light on the molecular basis of the beneficial effect exerted by a moderate physical exercise on muscle stem cells in cancer cachexia. Furthermore, we propose voluntary exercise as a physiological tool to counteract the overexpression of Pax7 observed in cancer cachexia.

Figure 1

C26-bearing mice show reduced muscle regeneration after acute injury. Panel (a) depicts representative details of H&E-stained muscle cross-cryosections of the lesion sites from both control and C26-bearing mice. Bar = 100 μm. (b) Morphometric analysis of regenerating fiber size from (a). Histograms show the distribution of the regenerating fiber cross-sectional area in different size classes 6 days after muscle damage (control = black bars; C26-bearing mice = gray bars). Numbers represent the median value. (c) Number of fibers with centrally located nuclei observed in the lesion site of the tibialis from both C26-bearing and control mice after acute damage. The total number of regenerating fibers was monitored at 6, 8, and 10 days after freeze-injury (control = black dots; C26-bearing mice = white dots).

Figure 2

C26-bearing mice show a prolonged inflammation response during muscle regeneration. (a) Left panels: immunofluorescence analysis of IgG expression performed at the lesion site of reconstructed areas of tibialis muscles from both control and C26-bearing mice. (a) Right panels: esterase staining uptake at the same lesion sites highlighting the presence of macrophages in the inflamed muscles. Insects depict magnification of areas defined in the squares. Black arrows show macrophages. White bar = 0.5 mm; black bar = 100 μm. (b) WB analysis of IgG expression on extracts from muscles shown in (a). First lane was loaded with the extract from a healthy, uninjured control muscle. GAPDH was used as loading control.

Figure 3

Early regenerative marker expression pattern in injured muscles. Representative WB images for Pax7, MyoD, and Desmin at 3, 6, and 8 days after freeze-injury. Blots were performed in duplicate for both control and C26-bearing mice. The first lane was loaded with the extract from a healthy, uninjured control muscle. GAPDH was used as loading control.

Figure 4

Exercise effects on myogenic markers. (a) Representative WB of Pax7 expression of muscle extracts from mice treated as indicated. (b) Densitometric analysis of Pax7 quantification. GAPDH was used as loading control. (c) Representative WB of MyoD expression of muscular extract from mice treated as indicated. (d) Densitometric analysis of MyoD quantification. Error bars are shown as means ± SEM of five independent experiments; ^∗ p < 0.005 by one way ANOVA.

Figure 5

Exercise effects on NF-kB. WB of pNF-kB and total NF-kB of muscle extracts from mice treated as indicated. Each group of samples was loaded as triplicate of independent experiments. GAPDH was used as a loading control.

Figure 6

Voluntary wheel running rescues skeletal muscle atrophy in C26-bearing mice. (a) NADH staining in TA muscles. Glycolytic fibers are shown as pale colored while oxidative fibers stain as dark. Bar = 100 μm. Morphometric analysis of glycolytic fibers among healthy controls (black bars) and C26-bearing mice (gray bars) at rest (b) and in the presence of voluntary wheel running (c). Numbers represent the median value ± SEM of three independent experiments (C26 versus C26 wheel: F = 1126; p = 0.0001).