Exposure to cigarette smoke induces overexpression of von Hippel-Lindau tumor suppressor in mouse skeletal muscle

Abstract

Cigarette smoke (CS) is a well-established risk factor in the development of chronic obstructive pulmonary disease (COPD). In contrast, the extent to which CS exposure contributes to the development of the systemic manifestations of COPD, such as skeletal muscle dysfunction and wasting, remains largely unknown. Decreased skeletal muscle capillarization has been previously reported in early stages of COPD and might play an important role in the development of COPD-associated skeletal muscle abnormalities. To investigate the effects of chronic CS exposure on skeletal muscle capillarization and exercise tolerance, a mouse model of CS exposure was used. The 129/SvJ mice were exposed to CS for 6 mo, and the expression of putative elements of the hypoxia-angiogenic signaling cascade as well as muscle capillarization were studied. Additionally, functional tests assessing exercise tolerance/endurance were performed in mice. Compared with controls, skeletal muscles from CS-exposed mice exhibited significantly enhanced expression of von Hippel-Lindau tumor suppressor (VHL), ubiquitin-conjugating enzyme E2D1 (UBE2D1), and prolyl hydroxylase-2 (PHD2). In contrast, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression was reduced. Furthermore, reduced muscle fiber cross-sectional area, decreased skeletal muscle capillarization, and reduced exercise tolerance were also observed in CS-exposed animals. Taken together, the current results provide evidence linking chronic CS exposure and induction of VHL expression in skeletal muscles leading toward impaired hypoxia-angiogenesis signal transduction, reduced muscle fiber cross-sectional area, and decreased exercise tolerance.

Fig. 1.

Increased expression levels of von Hippel-Lindau tumor suppressor (VHL) in skeletal muscles of cigarette smoke (CS)-exposed mice. A: qRT PCR analysis of VHL mRNA levels [Control: Median = 3.91, interquartile range (IQR) = 3.31–4.3; N = 8 vs. CS: Median = 5.58, IQR = 4.6–5.9; N = 6; **P = 0.0055]. B: representative Western blot showing VHL protein expression levels. C: quantification of VHL protein expression levels by densitometry. VHL density signals were normalized to α-tubulin signal values (Control: Median = 1.41, IQR = 0.53–2.1 vs. CS: Median = 3.04, IQR = 2.77–3.98; N = 6, *P = 0.016).

Fig. 2.

Chronic CS exposure increases expression levels of ubiquitin-conjugating enzyme E2D1 (UBE2D1) in mice skeletal muscles. A: qRT PCR analysis of UBE2D1 mRNA levels. **P < 0.001 (Control: Median = 3.62, IQR = 2.98–4.5, N = 7 compared with the CS: Median = 5.68, IQR = 5.17–5.95; N = 5; **P = 0.0057). B: representative Western blot showing UBE2D1 protein expression levels. C: quantification of UBE2D1 protein expression levels by densitometry. UBE2D1 density signals were normalized to α-tubulin signal values (Control: Median = 0.86, IQR = 0.66–1.02 compared with the CS: Median = 1.77, IQR = 1.4–1.94; N = 6; *P = 0.03).

Fig. 3.

Increased expression levels of prolyl hydroxylase-2 (PHD2) in skeletal muscle of CS-exposed mice. qRT PCR analysis of PHD2 mRNA levels. *P < 0.05 (Control: Median = 22.87, IQR = 19.13–24.06, N = 7 vs. CS: Median = 30.13, IQR = 28.27–35.9, N = 6; *P = 0.038). B: representative Western blot showing VHL protein expression levels. C: quantification of PHD2 protein expression levels (Control: Median = 0.92, IQR = 0.75–1.1 vs. CS: Median = 2.22, IQR = 1.39–3.1, N = 6; *P = 0.021).

Fig. 4.

CS exposure destabilizes hypoxia-inducible factor-1α (HIF-1α) protein. A: representative Western blot showing HIF-1α protein expression levels. B: quantification of HIF-1α protein expression levels by densitometry. HIF-1α density signals were normalized to α-tubulin signal values. (Control: Median = 1.07, IQR = 0.74–1.64 vs. CS: Median = 0.39, IQR = 0.3–0.47, N = 6; P = 0.027). C: qRT PCR analysis of HIF-1α mRNA values (Control: Median = 11.83, IQR = 11.04–12.91, N = 6 vs. CS: Median = 14.06, IQR = 13.27–15.39, N = 8, *P = 0.045).

Fig. 5.

Decreased vascular endothelial growth factor (VEGF) abundance in skeletal muscles of CS-exposed mice. A: qRT PCR analysis of VEGF mRNA levels (Control: Median = 10.37, IQR = 7.28–13.82, N = 8 vs. CS: Median = 9.16, IQR = 8.31–11.42, N = 6; P = 0.75). B: representative Western blot showing VEGF protein expression levels. C: quantification of VEGF protein expression levels by densitometry. VEGF density signals were normalized to α-tubulin signal values (Control: Median = 14.9, IQR = 12.27–17.05 vs. CS: Median = 9.55, IQR = 8.92–9.82, N = 6; *P = 0.037).

Fig. 6.

Mean skeletal muscle cross-sectional area (CSA) and capillary-to-fiber ratio in skeletal muscles of CS- and air-exposed mice. Representative hematoxylin and eosin staining of gastrocnemius cross-section from 129 SvJ mice Control group (A), CS-exposed group (B), Mean fiber CSA in control animals (C) relative to the CS-exposed animal group (Control: Median = 2,771.16, IQR = 2,462.83–2,914.93 vs. CS-exposed animals: Median = 2,429.3, IQR = 2,360.46–2,454.91; N = 6, *P = 0.025). Representative CD31 staining for control group (D) and mice exposed to cigarette smoke (CS) (E). F: capillary-to-fiber ratio (Control: Median = 1.91, IQR = 1.77–2.13 vs. CS: Median = 1.42, IQR = 1.37–1.55, N = 5; *P = 0.014). Arrows point to endothelial cells positive for CD31 staining.

Fig. 7.

Chronic CS exposure decreases exercise tolerance in mice. A: comparison of run time (min) (Control: Median = 34.2, IQR = 33.35–35.0 vs. CS: Median = 30.9, IQR = 30.35–31.75, N = 4; *P = 0.029). B: run distance (m) before exhauster (Control: Median = 614, IQR = 584–640 vs. CS: Median = 536, IQR = 507–567, N = 4; *P = 0.03).