Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA
Open Access
- 26 February 2009
- journal article
- research article
- Published by Springer Science and Business Media LLC in Basic Research in Cardiology
- Vol. 104 (2), 157-167
- https://doi.org/10.1007/s00395-009-0006-6
Abstract
We have shown that the cellular process of macroautophagy plays a protective role in HL-1 cardiomyocytes subjected to simulated ischemia/reperfusion (sI/R) (Hamacher-Brady et al. in J Biol Chem 281(40):29776–29787). Since the nucleoside adenosine has been shown to mimic both early and late phase ischemic preconditioning, a potent cardioprotective phenomenon, the purpose of this study was to determine the effect of adenosine on autophagosome formation. Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles, and can be monitored by imaging the incorporation of microtubule-associated light chain 3 (LC3) fused to a fluorescent protein (GFP or mCherry) into nascent autophagosomes. We investigated the effect of adenosine receptor agonists on autophagy and cell survival following sI/R in GFP-LC3 infected HL-1 cells and neonatal rat cardiomyocytes. The A1 adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) (100 nM) caused an increase in the number of autophagosomes within 10 min of treatment; the effect persisted for at least 300 min. A significant inhibition of autophagy and loss of protection against sI/R measured by release of lactate dehydrogenase (LDH), was demonstrated in CCPA-pretreated cells treated with an A1 receptor antagonist, a phospholipase C inhibitor, or an intracellular Ca(+2) chelator. To determine whether autophagy was required for the protective effect of CCPA, autophagy was blocked with a dominant negative inhibitor (Atg5K130R) delivered by transient transfection (in HL-1 cells) or protein transduction (in adult rat cardiomyocytes). CCPA attenuated LDH release after sI/R, but protection was lost when autophagy was blocked. To assess autophagy in vivo, transgenic mice expressing the red fluorescent autophagy marker mCherry-LC3 under the control of the alpha myosin heavy chain promoter were treated with CCPA 1 mg/kg i.p. Fluorescence microscopy of cryosections taken from the left ventricle 30 min after CCPA injection revealed a large increase in the number of mCherry-LC3-labeled structures, indicating the induction of autophagy by CCPA in vivo. Taken together, these results indicate that autophagy plays an important role in mediating the cardioprotective effects conferred by adenosine pretreatment.This publication has 35 references indexed in Scilit:
- TAT Protein Transduction Into Isolated Perfused HeartsCirculation, 2002
- Does adenosine protect the heart by acting on the sarcoplasmic reticulum?Cardiovascular Research, 2002
- TAT-Mediated Protein Transduction into Mammalian CellsMethods, 2001
- LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processingThe EMBO Journal, 2000
- A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.2000
- Characterization of adenosine receptors in intact cultured heart cellsBiochemical Pharmacology, 1994
- A comparison of adenosine-induced cardioprotection and ischemic preconditioning in dogs. Efficacy, time course, and role of KATP channels.Circulation, 1994
- A novel cytotoxicity screening assay using a multiwell fluorescence scannerToxicology and Applied Pharmacology, 1992
- 3‐Methyladenine, an inhibitor of autophagy, has multiple effects on metabolismJBIC Journal of Biological Inorganic Chemistry, 1988
- Lysosomal alterations in hypoxic and reoxygenated hearts. I. Ultrastructural and cytochemical changes.1980