Abstract 1779: Autophagy is an Important Survival Response to Bnip3-Mediated Mitochondrial Injury
Myocardial ischemia and reperfusion (I/R) is associated with increased autophagy and cell death. We previously found that the mitochondrial death protein Bnip3 contributes to I/R injury and induces autophagy in cardiac cells. However, the role of Bnip3-mediated autophagy is not clear and has been reported to be a protective, as well as a detrimental process. In this study, we investigated the functional role of Bnip3-mediated autophagy by utilizing adenoviral infections of cells coupled with fluorescence deconvolution microscopy for the analysis of autophagy and Bnip3 function. Imaging of cells infected with adenoviruses expressing GFP-LC3 and Bnip3 or beta-galactosidase (beta-gal) allowed us to monitor the formation of autophagosomes in live cells. Cell death was measured by assessing LDH activity in the media. Monitoring of GFP-LC3 incorporation into autophagosomes confirmed that overexpression of Bnip3 induced upregulation of autophagy in HL-1 myocytes and neonatal myocytes. We previously found that Bnip3 mediates cell death via Bax/Bak and that mouse embryonic fibroblasts (MEFs) lacking Bax and Bak are resistant to Bnip3-mediated cell death. Bnip3 still caused significant upregulation of autophagy in Bax/Bak−/− MEFs and imaging using high resolution fluorescence microscopy showed that a majority of the cells mitochondria were sequestered in autophagosomes. These results were confirmed by electron microscopy which also revealed the presence of a number of giant mitochondria in the cytosol, suggesting stimulation of mitochondrial biogenesis. Although Bnip3 alone did not cause cell death in Bax/Bak−/− MEFs, addition of 3-methyladenine (3-MA), an inhibitor of autophagy induced significant cell death in cells infected with Bnip3 but had no effect on cells overexpressing beta-gal. Overexpression of Bnip3 caused significant cell death in wild type MEFs, which was enhanced when inhibiting autophagy with 3-MA. These results suggest that Bnip3 stimulates autophagy to remove damaged mitochondria and mitochondrial biogenesis to synthesize new functional mitochondria. Since Bnip3 is upregulated in heart failure, stimulation of autophagy and mitochondrial biogenesis might represent potential therapeutic targets to treat heart disease.
This research has received full or partial funding support from the American Heart Association, AHA National Center.