Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmacology

First Advisor

Roy B. McCauley

Abstract

Autophagy has been implicated in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury; however, it is not clear whether autophagy is beneficial or detrimental to cell survival. We hypothesized that autophagy is beneficial to cardiomyocytes during I/R and that the paucity of ATP may limit its occurrence. To test this hypothesis, we developed a model of ischemia (hypoxia) involving exposure of HL-1 murine atrial cardiomyocytes to two concentrations of oxygen (0.5% and 2.0%) and 2-deoxyglucose.

Hypoxia, irrespective of the oxygen concentration, caused a dramatic drop in cellular ATP and an increase in the phosphorylation of AMPK (an indication of energy starvation),. The activation of the pro-autophagic AMPK coincided with an inactivation of mTOR and Akt in hypoxic myocytes. While these molecular events would be expected to lead to a stimulation of autophagy, we found that the level of autophagy remained at or near control levels under both conditions of ischemia. Autophagy was limited by the shortage in ATP as the presence of glucose instead of 2-deoxyglucose in the ischemia buffer increased ATP content and stimulated autophagy in hypoxic cells. In addition, cardiac cell death was significantly increased by 2 hours of hypoxia, to a higher extent with 0.5% oxygen. Importantly, inhibition of autophagy by 3-methyladenine increased hypoxia-induced cell death. In contrast, the stimulation of autophagy mediated by glucose coincided with a significant reduction in cardiac cell death following hypoxia. These results suggest that autophagy is beneficial to HL-1 cells during ischemia, but that the marked reduction in ATP limits the autophagic response. In turn, reoxygenation of hypoxic cells exposed to 0.5% or 2.0% oxygen caused a partial restoration of ATP levels, which coincided with an activation of apoptosis. Importantly, the rates of these events were dependent on the degree of hypoxia. Interestingly, the levels of phosphorylation/activation of AMPK, Akt and mTOR returned to basal levels in reoxygenated cells. However, autophagy was greatly compromised as cells pre-exposed to 0.5% and 2.0% oxygen exhibited low levels of autophagy after 2 and 4 hours of reoxygenation, respectively. As under ischemic conditions, the presence of 3-methyladenine during reoxygenation enhanced cardiac cell death.

In conclusion, we found that autophagy was differentially affected by the degree of hypoxia during both hypoxia and reoxygenation. The residual level of autophagy was essential to protect cardiomyocytes against the injurious effects of both hypoxia and reoxygenation, further validating our hypothesis.

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