Abstract 17052: Cardiac Specific mRFP-GFP-LC3 Transgenic Mice Are a Useful Tool to Evaluate Autophagic Flux in vivo
Autophagy mediates bulk protein degradation by the formation of autophagosomes (APs) followed by lysosomal degradation. Transgenic mice (Tg) expressing GFP-LC3 are useful for evaluating AP formation, but not for autolysosome (AL) formation, as GFP loses its fluorescence at the acidic pH in lysosomes. Since accumulation of GFP-LC3 dots is seen even when their clearance in lysosomes is inhibited, accumulation of GFP-LC3 dots does not necessarily indicate increased autophagy. Although chemicals inhibiting lysosomal degradation are used in order to evaluate autophagic flux, they can be toxic and confound analyses in vivo. Tandem fluorescent mRFP-GFP-LC3 (tf-LC3) is an ideal tool to overcome such challenges. We generated Tg with cardiac specific expression of tf-LC3 (Tg-tf-LC3) and starved the mice for 24 hours. Expression of tf-LC3 did not affect the endogenous LC3 levels either at baseline or after starvation. Starvation increased the level of endogenous LC3-II in both Tg-tf-LC3 (3.8 fold, p<0.01) and non-transgenic (NTg) control mice (3.9 fold, p<0.01), and decreased the level of p62 (−42%, -41.2%, p<0.05), a protein degraded by autophagy. Starvation increased both green (3.1 fold vs fed, p<0.05) and red (5.4 fold vs fed, p<0.01) dots in Tg-tf-LC3 mice. By analyzing the merged images for red dots that overlay with green dots and appear yellow, and free red dots that do not overlay with green dots and appear red, the extent of AP and AL formation can be determined, respectively. Starvation significantly increased both yellow dots (3.1 fold, p<0.05) and red dots (7.7 fold, p<0.01) in the mouse heart. To further demonstrate the usefulness of this model in determining autophagic flux in vivo, Tg-tf-LC3 mice were subjected to ischemia/reperfusion (I/R), which we have shown increases autophagy. I/R increased both yellow dots representing APs and red dots representing ALs (5.7 and 8.1 fold, p<0.01), indicating increased autophagic flux. Expression of endogenous p62 was also decreased after I/R (0.2 vs sham, p<0.01) adding further evidence that I/R increases autophagic flux in the heart. These results suggest that Tg-tf-LC3 mice are useful for evaluating autophagic flux in the heart and the cardiomyocytes therein in vivo.
- © 2010 by American Heart Association, Inc.