Role of autophagy in the development of amiodarone induced pulmonary fibrosis

Introduction: Amiodarone (AD) is a well known anti arrhythmic drug, but is limited by its potential side effects. Severe pulmonary toxicity is reported in patients receiving amiodarone, with the most common histological finding being chronic interstitial pneumonia. Apoptosis of alveolar epithelial cells type II (AECII) was reported in a rat model of amiodarone induced pulmonary toxicity. However, the underlying molecular mechanisms remain unknown. We aimed to establish a mouse model for AD induced pulmonary fibrosis and study the involved molecular mechanisms. Methods: C57BL/6 mice were administered AD or vehicle intratracheally and sacrificed on days 7, 14, 21 and 28. Fibrosis development was shown by trichrome staining & hydroxyproline measurement. (MLE)-12 cells were treated with AD/vehicle and harvested after 8, 16 & 24 hours. Autophagy, apoptosis and lysosomal stress markers were studied by western blotting, immunofluorescence and immunohistochemistry. Results: Pulmonary fibrosis development was observed in AD treated mice from day 7. Accumulation of surfactant proteins -B & -C was observed, paralleled by an induction of autophagy (LC3B-II), lysosomal stress (Cathepsin D) and apoptosis (cleaved caspase 3) markers within the AECII. MLE12 cells treated with AD showed the presence of LC3 positive vacuolar structures and induction of apoptosis (cleaved caspase-3). Conclusions: Mice exposed to amiodarone develop severe pulmonary fibrosis, with AECII specific induction of autophagy, lysosomal stress and apoptosis. In vitro data support these findings. The autophagic pathway might thus be involved in the development of amiodarone induced pulmonary fibrosis, but this needs to be further clarified.