Autophagy in Myocardial Ischemia/Reperfusion

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Autophagy is a physiologic process whereby cytoplasmic components including organelles are engulfed by a double membrane structure and targeted for destruction via lysosomes. Although it is a constitutive process in the myocardium, the level of autophagy is upregulated in response to ischemia/reperfusion (I/R) and possibly other pathophysiologic processes. Autophagy has been implicated in cell death and cytoprotection in different contexts. We hypothesize that autophagy is a protective response in the reperfused heart. We will establish the role of autophagy in the isolated perfused heart subjected to global I/R. and in cultured adult cardiomvocvtes subjected to simulated I/R. utilizing the fluorescent reporter of autophagy, LC3-GFP, and will develop novel reagents including a fluorescent reporter delivered by protein transduction and a cardiac-specific LC3-mCherry transgenic mouse. Mitochondria are important targets of autophagy in the heart, and we hypothesize that elimination of individual damaged or dysfunctional mitochondria may avert programmed cell death by preventing the propagation of reactive oxygen species (ROS) and pro-apoptotic signals to the remainder of the mitochondrial population. We propose to monitor ROS-induced ROS release (RIRR) in adult cardiomvocvtes and to assess whether autophagy can function to limit RIRR. using live-cell imaging (Core B). Because autophagy is closely tied to the metabolic state of the cell, we will examine its regulation by Akt, mTOR, and AMP-dependent kinase. In conjunction with Projects 1, 2, and 3, we will examine whether cytoprotective pathways (Akt, hexokinase, Pim-1, alphaB-crvstallin) stabilize mitochondria directly or through upregulation of autophagy. Autophagy is suppressed during hypertrophy; we will test the hypothesis that the resulting accumulation of damaged mitochondria may contribute to the development of failure after thoracic aortic constriction (Core C). In conjunction with Project 5, we will examine whether autophagy contributes to healthy or pathologic remodeling after permanent ischemia, and will determine the role of sphingosine-1-phosphate signaling in the regulation of autophagy. These studies will delineate the role of autophagy in cardiac health and disease.