Abstract 23: Intracellular ATP Levels and Occurrence of Autophagy in Hl-1 Cardiomyocytes Exposed to Ischemia Alone
Background: Myocyte autophagy is deemed to be a compensatory, prosurvival response to stress, including ischemia/reperfusion injury. The initiation and progression of the autophagic process seem to require ATP, whose intracellular levels dramatically drop during ischemia.
Objectives: To describe the effect of ATP reduction (during ischemia) on occurrence of autophagy.
Methods: HL-1 cells were incubated in ischemic buffer and exposed to increasing periods of hypoxia (30, 60, and 120 minutes) in presence of 0.5% oxygen. ATP was monitored by means of the firefly luciferase-based method before hypoxia (H) and at the end of each H period. Autophagy was assessed by quantitative immunoblotting of LC3 cleavage, as well as induction and activation of autophagy-related proteins, including Akt, ERK and AMPKα.
Results: H induced a 6-fold decrease in ATP levels, which peaked after 30 minutes and remained low and unchanged throughout the subsequent H time points. The occurrence of autophagy was observed after 30 minutes H, unchanged after 60 minutes and slightly, but significantly, diminished at 120 mins H, as documented by the decrease in steady-state levels of LC3-II and levels of LC3-II accumulation mediated by the autophagy inhibitor, Bafilomycin A1. With respect to the expression and activation of key regulators of autophagy, H had no effect at any time point on the total expression levels of Akt and ERK, two negative modulators of autophagy, but differentially modulated the activation profiles of these kinases. The phosphorylative activation of Akt progressively diminished during H and was barely detectable after 2 hours. In contrast, ERK activation was dramatically reduced after 30 minutes and completely disappeared after 1 and 2 hours of H. On the other hand, H cells exhibited higher expression levels of the pro-autophagic kinase, AMPKα, compared to healthy cells. Moreover, the activation levels of AMPKα were markedly elevated at all time points during H.
Conclusions: Although the changes in the profiles of activation of Akt, ERK and AMPKα are consistent with an induction of autophagy, the magnitude of autophagy did not increase during H, suggesting that the drastic H-induced ATP depletion may prevent both the initiation and progression of the autophagic process.
- © 2010 by American Heart Association, Inc.