When AMPK is no longer active during reperfusion, Akt phosphorylation is kicked on to activate mTOR, resulting in an Akt-dependent inhibition of autophagy. overtly inhibited autophagy during reperfusion accompanied by the activation of Akt and mTOR. Inhibition and induction of autophagy mitigated ALDH2-induced protection against cell death in hypoxia and reoxygenation, respectively. In addition, levels of the endogenous toxic aldehyde 4-hydroxy-2-nonenal (4-HNE) were elevated by ischaemia and reperfusion, which was abrogated by ALDH2. Furthermore, ALDH2 ablated 4-HNE-induced cardiomyocyte dysfunction and protein damage, whereas 4-HNE directly decreased pan and phosphorylated LKB1 and PTEN expression. == Conclusion == Our data suggest a myocardial protective effect of ALDH2 against I/R injury possibly through detoxification of toxic aldehyde and a differential regulation of autophagy through AMPK- and Akt-mTOR signalling during ischaemia and reperfusion, respectively. Keywords:ALDH2, Myocardial ischaemia/reperfusion, Akt, AMPK, Autophagy, 4-HNE == Introduction == The mitochondrial isoform of aldehyde dehydrogenase (ALDH2) plays a key role in the metabolism of acetaldehyde and other toxic aldehydes.1Evidence from our laboratory and others has revealed a beneficial role of ALDH2 against alcohol, acetaldehyde and toxic aldehyde-induced reactive oxygen species (ROS) formation, and tissue injury.24More recent seminal finding by Chenet al.5has indicated a potential role of ALDH2 activation in the cardioprotection against ischaemia injury. Nonetheless, neither the feasibility of ALDH2 activation nor the mechanisms behind ALDH2-offered cardioprotection against ischaemic injury has been defined. Recent observation has indicated that ethanol treatment prior to ischaemia may enhance ALDH2 activity and retard formation of 4-hydroxy-2-nonenal (4-HNE)-protein adducts.64-HNE, a specific electrophilic reactive aldehyde, is capable of modifying key enzymes by forming protein adducts to inhibit protein function.7Kinase function is often disrupted by 4-HNE due to its reactivity with amino acids including cysteine, histidine, and lysine. Interestingly, 4-HNE may be produced as a result of ischaemia/reperfusion (I/R) insult in various tissues including hearts.84-HNE imposes severe myocardial toxicity encompassing from disturbed coronary flow9to impaired cardiac contractility.10,11Recent findings suggest that elevated HNE content may modify essential cardiac survival signalling molecules including p38-α MAPK-IN-1 AMP-dependent protein kinase (AMPK)12and Akt.13Given the elevated cardiac 4-HNE in ischaemia and reperfusion along with its detrimental role in the heart,8,10,11it is plausible to speculate a possible action of ALDH2 against I/R injury through the detoxification of the toxic aldehyde. Autophagy, an evolutionarily conserved process of lysosome-dependent turnover of damaged KIAA0849 proteins and organelles, is present in normal hearts and may be upregulated in response to stress and cardiovascular anomalies such as heart failure and ischaemic injury.14Autophagy is critical to cell survival, the interruption of which may initiate severe ventricular dysfunction and cardiomyopathy.15Autophagy is believed to exert a beneficial role in I/R as rapamycin, a potent inducer of autophagy, reduces infarct size, and improves functional recovery in ischaemic murine hearts.16,17Paradoxically, autophagy has been shown to be both the cause and consequence of cardiac cell death. Autophagy is upregulated during myocardial ischaemia and is further enhanced during reperfusion.18,19Despite the seemingly cardioprotective role of autophagy during ischaemia, the jury is still out with regard p38-α MAPK-IN-1 to the possible harmful effect of autophagy during reperfusion.14,20Moreover, the signalling mechanism(s) behind autophagy appear(s) to be rather complicated. Several lines of evidence have indicated a pivotal role of the mammalian target of rapamycin (mTOR) in autophagy such that autophagy may be negatively regulated by mTOR through the phosphorylation of its downstream targets such as p70s6k.21,22Other evidence suggested that glucose deprivation during I/R triggers autophagy via the activation of AMPK and the inhibition of mTOR in cardiomyocytes.20In addition, the PI3K-Akt pathway has been indicated to play a critical role in autophagy.23,24Interestingly, mTOR can be activated by Akt although it is inhibited by AMPK via the phosphorylation of tuberous sclerosis complex 2 (TSC2).25,26Hence, the AMPK and Akt pair may quarterback a series of post-transcriptional processes such as cardiac hypertrophy and adaptation to stress at the convergence of mTOR. Nevertheless, the precise role of AMPK/Akt/mTOR pathway in the regulation of p38-α MAPK-IN-1 autophagy in myocardial ischaemia and reperfusion is unclear. Therefore, the aim of this study was to examine (i) whether ALDH2 protects against myocardial I/R injury through detoxification of 4-HNE; (ii) whether autophagy is involved in ALDH2-elicited cardioprotective effect, if any; and (iii) the signalling mechanism(s) behind ALDH2 and/or autophagy-induced myocardial response during I/R with a focus on the AMPK- and Akt-mTOR signalling cascades. == Methods == == Experimental animals and ALDH2 activity == All animal procedures were approved by our Institutional Animal Care and Use Committee (University of Wyoming). Production of ALDH2 transgenic mice using the chicken -actin promoter was described.