Published ahead of print on August 17, 2006, doi:10.1164/rccm.200605-721OC Am. J. Respir. Crit. Care Med., Volume 174, Number 9, November 2006, 997-1002 A more recent version of this article appeared on November 1, 2006
Submitted on May 30, 2006 Activation of Ubiquitin-Proteasome Pathway in the Diaphragm in Chronic Obstructive Pulmonary DiseaseCoen A.C. Ottenheijm1*,1 Department of Pulmonary Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Institute for Fundamental and Clinical Human Movement Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington, USA, 2 Department of Pulmonary Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Department of Intensive Care, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Institute for Fundamental and Clinical Human Movement Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 3 Department of Medicine, Baylor College of Medicine, Houston, Texas, USA, 4 Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Movement Sciences, Maastricht University, Maastricht, The Netherlands, 5 Department of Pulmonary Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Institute for Fundamental and Clinical Human Movement Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands * To whom correspondence should be addressed. E-mail: c.ottenheijm{at}long.umcn.nl.
RATIONALE Recent studies show that myosin content of the diaphragm in patients with mild to moderate 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 PCR, and caspase-3 activity by determining the hydrolysis of fluorogenic substrates. RESULTS 20S proteasome activity was ~3-fold 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 COPD patients compared non-COPD patients. Caspase-3-mediated cleavage of actomyosin complexes is considered an initial step in muscle wasting, yielding fragments which 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 (GOLD I/II). Key words: COPD, proteolysis, caspase-3, diaphragm function, myosin
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