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Activation of the Ubiquitin–Proteasome Pathway in the Diaphragm in Chronic Obstructive Pulmonary Disease

Departments of Pulmonary Diseases and Intensive Care, and Institute for Fundamental and Clinical Human Movement Sciences, Radboud University Nijmegen Medical Center, Nijmegen; Nutrition and Toxicology Research Institute Maastricht, and Department of Movement Sciences, Maastricht University, Maastricht, The Netherlands; Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington; and Department of Medicine, Baylor College of Medicine, Houston, Texas

Correspondence and requests for reprints should be addressed to C. A. C. Ottenheijm, Ph.D., Department of Pulmonary Diseases, 454, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: c.ottenheijm{at}long.umcn.nl

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).

Key Words: caspase-3 • chronic obstructive pulmonary disease • diaphragm function • myosin • proteolysis

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