Exercise Attenuate Diaphragm Atrophy in COPD Mice via Inhibiting the RhoA/ROCK Signaling

Abstract

Background: Exercise is an indispensable component of pulmonary rehabilitation with strong anti-inflammatory effects. However, the mechanisms by which exercise prevents diaphragmatic atrophy in COPD (chronic obstructive pulmonary disease) remain unclear.

Methods: Forty male C57BL/6 mice were assigned to the control (n=16) and smoke (n=24) groups. Mice in the smoke group were exposed to the cigarette smoke (CS) for six months. They were then divided into model and exercise training groups for 2 months. Histological changes were observed in lung and diaphragms. Subsequently, agonist U46639 and antagonist Y27632 of RhoA/ROCK were subjected to mechanical stretching in LPS-treated C2C12 myoblasts. The expression levels of Atrogin-1, MuRF-1, MyoD, Myf5, IL-1β, TNF-α, and RhoA/ROCK were determined by Western blotting.

Results: Diaphragmatic atrophy and increased RhoA/ROCK expression were observed in COPD mice. Exercise training attenuated diaphragmatic atrophy, decreased the expression of MuRF-1, and increased MyoD expression in COPD diaphragms. Exercise also affects the upregulation of RhoA/ROCK and inflammation-related proteins. In in vitro experiments with C2C12 myoblasts, LPS remarkably increased the level of inflammation and protein degradation, whereas Y27632 or combined with mechanical stretching prevented this phenomenon considerably.

Conclusion: RhoA/ROCK plays an important role in the prevention of diaphragmatic atrophy in COPD.

Keywords: RhoA/ROCK signaling; chronic obstructive pulmonary disease; diaphragmatic atrophy; exercise training; inflammation.

Figure 1

Long-term CS establish a mouse model of COPD. (A). hematoxylin and eosin staining of lung sections from mice exposed to air or CS. scale bars, 50 μm; and CSA of alveolus; (B). lung function; (C). inflammation levels of BALF. Significance was calculated by one-way ANOVA with Tukey’s post-hoc analysis for the parameters including CSA of alveolus, FVC, FEV50, FEV50/FVC, IL-6, IL-8, and TNF-α.

Figure 2

Long-term CS induces diaphragmatic atrophy and upregulation of RhoA/ROCK. (A).hematoxylin and eosin staining of diaphragm sections from mice exposed to air or CS. scale bars, 50 μm; and CSA of diaphragm fibers; (B). Western blot analysis of protein expression of RhoA, ROCK1, ROCK2 in diaphragm from mice exposed to air or CS. Significance was calculated by one-way ANOVA with Tukey’s post-hoc analysis for the parameters including CSA of diaphragm fibers, RhoA, and ROCK2.

Figure 3

Treadmill training improves diaphragmatic atrophy in mice with COPD. (A). hematoxylin and eosin staining of diaphragm sections from mice exposed to air or CS. scale bars, 50 μm; CSA of diaphragm fibers; (B-F). Western blot analysis of protein expression of Atrogin-1, MuRF-1, MyoD1 and Myf5. Significance was calculated by one-way ANOVA with Tukey’s post-hoc analysis for the parameters including CSA of diaphragm fibers, Atrogin-1, MyoD, and Myf5.

Figure 4

Treadmill training decreases the expression of RhoA/ROCK and inflammation in COPD diaphragm. (A-E). Western blot analysis of protein expression of RhoA, ROCK, IL-1β and TNF-α. Significance was calculated by one-way ANOVA with Tukey’s post-hoc analysis for the TNF-α.

Figure 5

RhoA/ROCK involves in mechanical stretching downregulating inflammation in LPS-treated C2C12 myoblasts. (A and B). levels of IL-1β and TNF-α; (C-G). Western blot analysis of protein expression of Atrogin-1, MuRF-1, MyoD and Myf5; (H-J). Western blot analysis of protein expression of RhoA and ROCK. Significance was calculated by one-way ANOVA with Tukey’s post-hoc analysis for the parameters including IL −1β, TNF-α, Atrogin-1, MuRF-1, MyoD, Myf5, RhoA, and ROCK1.