In the present study we have demonstrated that direct activation of ALDH2 is sufficient to protect ischemic/reperfused myocardium in the absence of PKCε. These findings are in agreement with previous studies that ALDH2 plays a central role in mediating cytoprotection from cardiac ischemia/reperfusion9, 11–13
. These data also suggest that ALDH2 is downstream of PKCε in ethanol-induced cardioprotection and that this cytoprotective mechanism can be harnessed using ALDH2 activators, such as Alda-44. The study also provides further insight into the cytoprotective mechanism of Alda-44, demonstrating that Alda-44 can prevent 4-HNE-induced inactivation of ALDH2 by preventing the formation of 4HNE-ALDH2 protein adducts (summarized in ).
Schematic diagram of cardiprotection conferred by ALDH2
Much of the cellular damage occurring during IR is predicted to be due to reactive oxygen species (ROS) production28
. IR-induced ROS accumulation leads to lipid peroxidation and the generation of reactive aldehydes including acetaldehyde29
and 4-HNE16, 17, 30
which form protein adducts with cysteine, histidine and lysine residues via
inhibiting protein function. 4-HNE, for example, inhibits key metabolic proteins such as GAPDH31
, the Na/KATPase32
and the 20S proteosome33
and is also an inducer of mitochondrial permeability transition34
. 4-HNE accumulates rapidly in the ischemic heart16
, and has a considerably longer half-life than free radical species16
, therefore, the efficient removal of cytotoxic aldehydes is likely to be of crucial importance for cell survival against ischemia/reperfusion injury. Glutathione (GSH) can scavenge 4-HNE in a reaction catalysed by glutathione-S-transferase (GST)15
, however GSH levels fall rapidly during ischemia35
. 4-HNE is also metabolized by mitochondrial ALDH2 to the non-reactive metabolite, 4-HNA15, 30
. Thus, detoxification of reactive aldehydes, such as 4-HNE is a likely cytoprotective mechanism mediated by ALDH2 activation.
4-HNE also inactivates ALDH2 at high concentrations9, 21
, interacting covalently with Cys302, located at the ALDH2 active site21
and resulting in almost complete inhibition of the enzyme, in vitro
(as shown in ). It is therefore likely that the impaired 4-HNE metabolism30
and accumulation of 4-HNE adducts16
observed during cardiac ischemia/reperfusion occurs as a dual consequence of increased generation of aldehydes from lipid peroxidation and impaired metabolism of these aldehydes due to 4-HNE-induced inactivation of ALDH2. Here, we demonstrate, that in addition to activation of ALDH2, Alda-44 prevents 4-HNE induced inactivation of ALDH2 in vitro
, by reducing the formation of 4-HNE protein adducts on ALDH2 (). Furthermore, Alda-44 prevented IR-induced inhibition of ALDH2 in the heart, with a concomitant decrease in 4-HNE protein adduct formation. Thus, the mechanism of protection mediated by Alda-44 is likely to be due to a combination of increased ALDH2 activity and prevention of 4-HNE-mediated ALDH2 inactivation, resulting in reduced accumulation of cytotoxic aldehydes and enhanced preservation of ischemic myocardium.
Chronic, moderate alcohol consumption results in increased expression and activation of PKCε36
and confers enhanced protection against myocardial ischemia37
. Acute ethanol treatment is also cardioprotective and is abolished by selective inhibition of PKCε3
. Our data that ethanol is not protective in PKCε knockout mice confirms the role of PKCε in ethanol preconditioning. Recent studies by Slater et al
have identified an ethanol binding site on the C1 domain of PKCε, suggesting that ethanol mediates its action on PKCε by direct binding to the enzyme 38
. The direct cytoprotective effect of ethanol on the myocardium has been observed in a variety of species, however, other studies have shown a lack of ethanol-induced protection39, 40
. Furthermore, ethanol can abolish cytoprotection conferred by ischemic preconditioning if it is not sufficiently metabolized or washed out prior to the onset of ischemia6, 41
. Thus, the acute cardioprotective effect of ethanol depends critically on the dose of ethanol used and whether ethanol remains present during the ischemic period. The observation that the continued presence of ethanol blocks its own protection5, 6
and that of ischemic preconditioning5, 41
may perhaps be explained, at least in part, by the identification of ALDH2 as a key cytoprotective enzyme. Acetaldehyde is the primary product of ethanol metabolism and impairs cardiac excitation-contraction coupling, preturbs Ca2+
signaling and forms protein adducts42
. Acetaldehyde is metabolized by ALDH2, and transgenic overexpression of ALDH2 rescues acetaldehyde-induced myocardial dysfuntion43, 44
. It is likely that if ethanol is not sufficiently metabolized prior to ischemia, ethanol-derived acetaldehyde results in competitive inhibition of ALDH2 for 4-HNE metabolism. The dichotomous effect of ethanol in acute cardioprotection may therefore be a consequence of the beneficial effects of ethanol (mediated by PKCε and ALDH2) being countered by the cytotoxic effects of acetaldehyde accumulation (resulting from ethanol metabolism). Direct activation of ALDH2 with Alda-44 offers a means of harnessing the cytoprotective effect, without the associated cytotoxicity caused by acetaldehyde accumulation.
While 4-HNE induces necrosis through inhibition of key metabolic proteins, evidence indicates that 4-HNE can also induce programmed cell death through activation of SAPK/JNK45–49
. It is well established that SAPK/JNK becomes phosphorylated and activated immediately on reperfusion of ischemic myocardium24–26
. SAPK/JNK activation initiates apoptosis through the phosphorylation and activation of pro-apoptotic Bad50
, inactivation of pro-survival BCl-251
and induction of cytochrome c
release from cardiac mitochondria52
. As 4-HNE can also increase SAPK/JNK phosphorylation and activity45–47
, IR-induced apoptosis may, at least in part, be mediated through 4-HNE. Our data that Alda-44 reduced IR-induced formation of 4-HNE protein adducts with a concomitant reduction in SAPK/JNK phosphorylation suggest that ALDH2 activators may reduce IR-induced apoptotic cell death, by reducing 4-HNE mediated activation of SAPK/JNK.
In summary, our data demonstrate that cardioprotection conferred by ethanol preconditioning is abolished in PKCε KO mice, whereas direct activation of ALDH2 with Alda-44 can confer cardioprotection in the absence of PKCε. Our data also provide mechanistic insight into the cytoprotective mechanism of Alda-44. We demonstrate that in addition to ALDH2 activation, Alda-44 prevents 4-HNE mediated inactivation of ALDH2. We provide evidence, in vitro, that the molecular basis for Alda-44 induced protection of ALDH2 is likely due to prevention of 4-HNE protein adducts on ALDH2. Thus, by preventing inactivation of ALDH2, Alda-44 maintains the detoxification of oxidative-stress induced reactive aldehydes, reducing ischemic injury (shown in ). In support of this hypothesis, we show that Alda-44 prevents ALDH2 inactivation, reduces accumulation of 4-HNE adducts and reduces SAPK/JNK phosphorylation induced by ischemia/reperfusion. The evidence supporting the beneficial effects of ethanol on the heart is abundant, however, because the cardioprotective effect of ethanol consumption must be countered by the cytotoxic effects of acetaldehyde, and the associated health risks (such as liver cirrhosis, cancer, alcohol-induced cardiomyopathy) ALDH2 activators, such as Alda-44, may therefore offer a means of exploiting this endogenous cytoprotective mechanism without the unwanted side effects of ethanol consumption.