Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA

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 A 1 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 A 1 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 (Atg5 K130R ) 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.

Restoration of autophagic flux in myocardial tissues is required for cardioprotection of sevoflurane postconditioning in rats

“…A previous study showed that ischemic preconditioning induces autophagy formation and initiates cardioprotective effects against I/R injuries [15] . It has also been reported that pharmacological pretreatment with the adenosine A1 receptor selective agonist CCPA induces autophagy and confers cardioprotection against I/R injury [36] . In this previous study, the preconditioning was performed prior to the ischemic period and less damage occurred during the ischemic period [36] .…”

“…It has also been reported that pharmacological pretreatment with the adenosine A1 receptor selective agonist CCPA induces autophagy and confers cardioprotection against I/R injury [36] . In this previous study, the preconditioning was performed prior to the ischemic period and less damage occurred during the ischemic period [36] . After CCPA pre-treatment, diminished autophagy during simulated ischemia and reperfusion occurred because the cells experienced less stress, and less autophagy occurred as a response to reperfusion injury.…”

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

“…Adult cardiac myocytes were prepared from 200 to 250 g male Sprague Dawley rats as previously described. 41 All animal procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee. Briefly, the rat was anesthetized with sodium pentobarbital, the heart excised and rapidly cannulated via the aorta.…”

The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning

“…Autophagy is known to be activated during ischemic insult, but its contribution to neuronal death/survival is still being debated [16][17][18] . Recent reports also suggested an association between preconditioning and autophagy in both the heart and brain [19][20][21][22] . In an earlier study, we demonstrated that the neuroprotection induced by IPC is mediated by autophagy in a rat model of IPC, and the pharmacological induction of autophagy mimics the neuroprotection of IPC [23] .…”