Activation of the ubiquitin-proteasome pathway in the diaphragm in chronic obstructive pulmonary disease

Abstract

Rationale: Studies show that the myosin content of the diaphragm in patients with mild to moderate chronic obstructive pulmonary disease (COPD) is reduced, compromising diaphragm contractile performance. The mechanisms for reduced contractile protein content are unknown. In the present study we hypothesized that the loss of contractile protein content is associated with activation of the ubiquitin-proteasome pathway in the diaphragm of patients with mild to moderate COPD.

Methods: Proteolytic activity of isolated 20S proteasomes was determined in diaphragm biopsies from patients with and without COPD (predicted mean FEV1, 66 and 93%, respectively). In addition, we determined 20S proteasome subunit C8 protein levels by means of Western blotting, ubiquitin-ligase mRNA levels by means of real-time polymerase chain reaction, and caspase-3 activity by determining the hydrolysis of fluorogenic substrates.

Results: The 20S proteasome activity was about threefold increased in the diaphragm of patients with COPD. C8 protein levels were not significantly different between COPD and non-COPD diaphragm, indicating increased specific activity of individual proteasomes, rather than an increased number of proteasomes. mRNA levels of the muscle-specific ubiquitin-ligase MAFbx were significantly higher in diaphragm from patients with COPD compared with patients without COPD. Caspase-3-mediated cleavage of actomyosin complexes is considered an initial step in muscle wasting, yielding fragments that can be degraded by the ubiquitin-proteasome pathway. In line with the increased ubiquitin-proteasome activity, caspase-3 activity was higher in diaphragm homogenates from patients with COPD.

Conclusions: The present study is the first to demonstrate increased activity of the ubiquitin-proteasome pathway in COPD diaphragm. Importantly, these changes occur in patients with only mild to moderate COPD (Global Initiative for Chronic Obstructive Lung Disease stage I/II).

Figure 1.

Myosin heavy chain content in chronic obstructive pulmonary disease (COPD) and non-COPD diaphragm. Myosin heavy chain content was determined in diaphragm homogenates by Western blotting and subsequent densitometric quantification of protein bands. Top: Myosin heavy chain content in diaphragm homogenates from patients with COPD (solid column) versus patients without COPD (open column). Bottom: Representative myosin heavy chain immunoblot from diaphragm homogenates from a patient with COPD and a patient without COPD. For the positive control purified myosin heavy chain was used. For the negative control a homogenate from a patient without COPD was stained without addition of the primary (anti–myosin heavy chain) antibody. Data are presented as means ± SEM. * Significant difference (p < 0.05) from non-COPD.

Figure 2.

Proteasome activity in diaphragm of patients with COPD and patients without COPD. Proteasome activity against fluorogenic substrates succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY) and N-carbenzoxy-Leu-Leu-Glu-7-amido-4-methylcoumarin (LLE) was higher in patients with COPD than in patients without COPD. AMC = amido-4-methylcoumarin. Data are presented as means ± SEM. * Significant difference (p < 0.05) from non-COPD.

Figure 3.

20S proteasome subunit C8 protein content in diaphragm of patients with COPD and patients without COPD. Subunit C8 protein content was determined in diaphragm homogenates by Western blotting and subsequent densitometric quantification of protein bands. Top: Our analyses revealed comparable contents of subunit C8 in the diaphragm of patients with COPD (solid column) and patients without COPD (open column). Bottom: Representative C8 subunit immunoblot from diaphragm homogenates from a patient with COPD and a patient without COPD. For the positive control we used an isolated proteasome fraction from a patient without COPD (the same fraction as was used for the proteasome activity assays; see Figure 2). For the negative control a homogenate from a patient without COPD was stained without addition of the primary (anti-C8) antibody. Data are presented as means ± SEM.

Figure 4.

E3-ligase mRNA levels in the diaphragm of patients with COPD and patients without COPD. MAFbx mRNA levels are higher in COPD versus non-COPD diaphragm. MURF-1 mRNA levels were not significantly different between COPD and non-COPD diaphragm. Data are presented as means ± SEM. * Significant difference (p < 0.05) from non-COPD.

Figure 5.

Caspase-3 activity in the diaphragm of patients with COPD and patients without COPD. Caspase-3 activity against the fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) was higher in diaphragm homogenates from patients with COPD (solid column) versus patients without COPD (open column). Data are presented as means ± SEM. * Significant difference (p < 0.05) from non-COPD.