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Cell Biology and Molecular Medicine Program
M.Sc. (Hons.) Biological Sciences &
B.E. (Hons.) Chemical Engineering
Birla Institute of Technology & Science, Pilani, India
Thesis Advisor: Junichi Sadoshima, M.D., Ph.D.
Department of Cell Biology and Molecular Medicine
Friday, September 17, 2010
MSB G-609, 10:00 a.m.
Autophagy is a bulk degradation process of cytosolic proteins and organelles via lysosomes. It is stimulated in cardiac myocytes at baseline and under stress, playing protective and detrimental roles depending upon the stimulus. Cardiac myocytes transduced with adenovirus harboring tandem fluorescent mRFP-GFP-LC3 were starved to evaluate autophagic flux. Glucose deprivation (GD) increased formation of autophagosomes and autolysosomes accompanied by upregulation of Sirt1 and FoxO1. Overexpression of either Sirt1 or FoxO1 was sufficient for inducing autophagic flux, whereas both Sirt1 and FoxO1 were required for GD-induced autophagy. GD increased deacetylation of FoxO1, and Sirt1 was required for GD-induced deacetylation of FoxO1. Overexpression of FoxO1(3A/LXXAA), which cannot interact with Sirt1, or p300, a histone acetylase, increased acetylation of FoxO1 and inhibited GD-induced autophagy. FoxO1 increased expression of Rab7, a small GTP- binding protein that mediates autophagosome-lysosome fusion, which was both necessary and sufficient for mediating FoxO1-induced increases in autophagic flux. Although cardiac function was maintained in control mice after 48 hours of food starvation, it was significantly deteriorated in mice with cardiac specific overexpression of FoxO1(3A/LXXAA), cardiac specific homozygous deletion of FoxO1 (c-FoxO1-/-), and beclin1+/- mice, in which autophagy is significantly inhibited. These results suggest that Sirt1-mediated deacetylation of FoxO1 and upregulation of Rab7 play an important role in mediating starvation-induced increases in autophagic flux, which in turn maintains left ventricular function during starvation.
To evaluate the role of oxidative stress in mediating autophagy, cardiac myocytes were treated with H2O2 in vitro and subjected to myocardial ischemia/reperfusion (I/R) in vivo. H2O2 and I/R significantly increased autophagic flux. Treatment with antioxidant N-2-mercaptopropionyl glycine (MPG) attenuated I/R-induced increases in oxidative stress, autophagic flux and Beclin1 expression, accompanied by a decrease in the size of myocardial infarction (MI)/area at risk (AAR). MI/AAR after I/R was significantly reduced in beclin1+/- mice, whereas beclin1+/- mice treated with MPG exhibited no additional reduction in MI/AAR after I/R. These results suggest that oxidative stress plays an important role in mediating autophagy during I/R. Oxidative stress dependent activation of autophagy has a detrimental effect and mediates myocardial injury in response to I/R in the mouse heart.