Figure 1.
Akt and ubiquitin-proteasome signaling pathways.
On the left side, upon stimulation of their respective receptor, insulin or insulin-growth factor 1 stimulates phosphorylation of Akt. Protein synthesis is then promoted through activation of p70S6K and inhibition of GSK-3β. On the right side, following stimulation by the appropriate stimuli (pro-inflammatory cytokines, oxidative stress, lipopolysaccharide, etc) NF-κB becomes activated and transcribes, among others, MuRF1. In pro-atrophic conditions, FoxO also transcribes MuRF1 and Atrogin-1. These E3-ligases increase total polyubiquitination and therefore promote protein degradation through the proteasome. Mitogen activated protein kinase p38 is also known to induce muscle protein degradation. In addition to promoting synthesis, activated Akt is an inhibitor of protein degradation by restraining nuclear translocation of FoxO. Activation = Filled arrows Inhibition = Hammer head lines Translocation = Dashed arrows.
Table 1.
Primer sequences used for PCR analyses.
Figure 2.
Cigarette smoke exposure decreases whole body weight and skeletal muscle mass.
BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 8 or 24 weeks. Body weight was measured prior to sacrifice (A). Tibia bones were collected from mice exposed 24 weeks to smoke or room air and measured (B). Gastrocnemius (C) and soleus (D) muscles were excised and weighted. Results are mean ± SEM of 9 or 10 mice. *p<0.05. Room air control mice = Light gray Smoke-exposed mice = Black.
Figure 3.
Cigarette smoke exposure time-dependently activates pathways associated to protein degradation in skeletal muscle.
BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week for 8 or 24 weeks. MuRF1 (A), Atrogin-1 (B) and FoxO3 (C) mRNA expression levels were measured by real-time PCR. Protein levels of MuRF1 (D), polyubiquitin linked to lysine 48 (E), total form of p38 (F) and the ratio of phosphorylated p38/total p38 (G) were assessed by Western Blot and normalized to tubulin signal. All results were obtained with gastrocnemius muscle. Results are mean ± SEM of 9 or 10 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black.
Figure 4.
Cigarette smoke exposure time-dependently inhibits pathways associated to protein synthesis in skeletal muscle.
BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week for 8 or 24 weeks. Protein levels of Akt (A), phosphorylated Akt/total Akt ratio (B), GSK-3β (C), phosphorylated GSK-3β/total GSK-3β ratio (D), p70S6K (E) and phosphorylated p70S6K/total p70S6K ratio (F) were assessed by Western Blot and normalized to tubulin signal. All results were obtained with gastrocnemius muscle. Results are mean ± SEM of 9 or 10 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black.
Figure 5.
Soleus muscle weight but not gastrocnemius is normalized following smoking cessation.
BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 24 weeks. All mice were then exposed to room air for an additional 60 days, without smoke exposure. Gastrocnemius (A) and soleus (B) muscles were excised and weighted. Results are mean ± SEM of 5 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black.
Figure 6.
Pro-degradation and anti-synthesis changes induced by chronic cigarette smoke exposure are reversible.
BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 24 weeks. All mice were then exposed to room air for an additional 60 days, without smoke exposure. MuRF1 (A), Atrogin-1 (B) and FoxO3 (C) mRNA levels were measured by real-time PCR. Protein levels of MuRF1 (D), polyubiquitin linked to lysine 48 (E), total p38 (F), phosphorylated Akt/total Akt ratio (G) and total GSK-3β (H) were assessed by Western Blot and normalized to tubulin signal. Results are mean ± SEM of 5 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black.
Figure 7.
Cigarette smoke exposure induces inflammatory, oxidative and pro-angiogenic states in the skeletal muscle that are partially reversible.
BALB/c mice were either exposed to room air or cigarette smoke twice a day, five days a week, for 8 or 24 weeks (9–10 mice per group). Following this protocol, a sub-group (5 mice per group) of control mice and 24-week smoke exposed mice were exposed to room air for an additional 60 days. TNF-α (A), IL-1β (B), IL-6 (C) and VEGF (E) mRNA levels were measured by real-time PCR. Total protein oxidation (D) were assessed by Western Blot and normalized to tubulin signal. Results are mean ± SEM of 5–10 mice. *p<0.05 Room air control mice = Light gray Smoke-exposed mice = Black Ex-smoking mice = Dark gray.