AbstractsBiology & Animal Science

Effects of mechanical ventilation on autophagy in the mouse diaphragm

by Ilan Azuelos




Institution: McGill University
Department: Department of Medicine
Degree: MS
Year: 2014
Keywords: Biology - Molecular
Record ID: 2045391
Full text PDF: http://digitool.library.mcgill.ca/thesisfile126876.pdf


Abstract

Introduction: Mechanical ventilation (MV) is associated with atrophy and weakness of the diaphragm muscle, a condition termed ventilator-induced diaphragmatic dysfunction (VIDD). At the cellular level, VIDD is characterized by increased oxidative stress and augmented proteolysis in the diaphragm. Autophagy is a lysosomally-mediated proteolytic process that can be activated by oxidative stress, which has the potential to exacerbate VIDD. The major aims of this study were to: 1) determine whether MV modulates autophagic flux in the diaphragm, and 2) ascertain the impact of anti-oxidant therapy upon autophagy as well as the relationship of autophagy to MV-induced force loss in the diaphragm.Methods: Mice (male C57BL/6N; 8-10 weeks old) were assigned to control (CTRL), MV (for 6 hours), Fasting (for 48 hours), and MV + N-acetylcysteine anti-oxidant treatment (NAC; 200mg/kg) groups. Autophagy was monitored by quantifying: 1) autophagic vesicles by transmission electron microscopy (TEM), 2) mRNA levels of autophagy-related genes, and 3) the autophagosome marker protein LC3B-II, including after administration of colchicine to block autophagosome degradation and thus permit evaluation of autophagic flux. Force production of diaphragms from CTRL, MV and MV+NAC mice was determined ex vivo and correlated with autophagic flux measurements. Results: Diaphragms exhibited a 2.1-fold increase in the number of autophagic vesicles visualized by TEM relative to CTRL after 6 hours of MV. This was due to augmented autophagosome formation, which was significantly increased by MV in the diaphragm but not in the limb muscle (extensor digitorum longus, EDL). The Fasting group demonstrated increased autophagosome formation in both muscles, and to a greater extent in the EDL. The anti-oxidant NAC effectively prevented MV-induced diaphragmatic specific force loss, but did not alter autophagy gene expression or suppress autophagy induction in the diaphragm during MV.Conclusions: In a mouse model of VIDD, MV specifically induces increased autophagic flux in the diaphragm but not in limb muscle. The anti-oxidant NAC prevents the loss of diaphragmatic force production associated with MV but without preventing the increase in autophagic flux, thus suggesting that autophagy pathway induction does not underlie the early stages of VIDD pathogenesis but may instead be a beneficial adaptive response. Introduction:La ventilation mécanique (VM) est associée à une faiblesse et à une atrophie du muscle diaphragmatique, une affection pathologique nommée ‘VIDD’ (Ventilator-Induced Diaphragmatic Dysfunction). VIDD est caractérisé par un stress oxydant exclusif au diaphragme, induisant ainsi une augmentation de la protéolyse dans le muscle. L’autophagie est un processus protéolytique agissant à travers une dégradation lysosomale de constituants cellulaires. Les objectifs de cette étude sont premièrement de déterminer si la VM induit l’autophagie dans le diaphragme. De plus, nous cherchons à examiner l’effet de l’antioxydant N-acetylcysteine (NAC) sur le…