In the current study, we have identified a mitochondrial target of εPKC and have elucidated a protective mechanism of ethanol-mediated preconditioning in vivo. Our results demonstrate for the first time that acute administration of ethanol 60 minutes prior to coronary occlusion results in εPKC translocation to cardiac mitochondria, ALDH2 and εPKC mitochondrial association, increased ALDH2 activity, diminished HNE-protein adduct formation and decreased pro-death signaling.
Many epidemiological studies in the past decade have demonstrated that chronic moderate consumption of ethanol decreases the risk of myocardial infarction [1
]. We have previously found that acute ethanol exposure before ischemia is sufficient to protect the heart from damage [8
]. Using an isozyme-selective inhibitor, we demonstrated that εPKC mediates the protective effects of ethanol in an adult rat heart ex vivo
model of I/R and in isolated cardiac myocytes in vitro
]. Several groups have shown in ex vivo
models of I/R that ethanol-mediated protection is dependent upon the sustained activation of εPKC [9
]. While it is clear that ethanol can induce the translocation of εPKC, it was not clear where the enzyme translocates to within the cell. Other cardioprotective phenomena utilizing different models have shown that protection is dependent upon εPKC activation [45
]. Ohnuma et al.
demonstrated that this cardioprotection coincided with εPKC translocation to the mitochondria [33
] and we show here that similarly, ethanol administration 60 minutes prior to ischemia was sufficient to induce translocation of εPKC to cardiac mitochondria during I/R. We also show that the protective effects of ethanol and translocation of εPKC did not occur if ethanol was administered only 15 minutes prior to LAD ligation. These data further support our conclusion that the mitochondrial association of εPKC plays a critical role in cardioprotection.
Our findings further support the body of work suggesting that ethanol administration immediately prior to the ischemic period does not protect the heart from I/R-mediated injury [15
]. Krenz et al
. demonstrated that ethanol administrated immediately prior to ischemia did not reduce infarct size induced by I/R in a rabbit MI model [9
]. In the same model, when ethanol was infused one hour prior to ischemia, infarct size was reduced. Ethanol is converted to acetaldehyde via
alcohol dehydrogenase, which in turn is metabolized to acetic acid mainly by ALDH2 [50
]. It is possible that the accumulating acetaldehyde competes for 4-HNE metabolism by ALDH2 allowing the accumulation of reactive 4-HNE thereby increasing 4-HNE-protein-adduct formation, and causing subsequent cellular injury.
We show here that εPKC translocation to the mitochondria was not induced by either ethanol or ischemia alone, supporting the hypothesis of a delayed two-part mechanism of εPKC activation and suggesting that a priming step is necessary for εPKC translocation and protection of the ischemic heart; here this priming was induced by ethanol. We further propose that in addition to this priming step, a second activation event occurring during ischemia is necessary for εPKC translocation to the mitochondria; ethanol treatment without a subsequent ischemic event did not induce mitochondrial translocation. Recently, εPKC has been shown to be activated by mild reactive oxygen species (ROS) [51
]. We therefore propose that ROS, which is generated during ischemia [52
] may oxidatively modify εPKC [53
], resulting in its entry into the mitochondria in a mechanism that has yet to be determined. If the initial priming step does not occur and if sufficient time is not allowed for this second step to occur, εPKC cannot translocate into the mitochondria, and cardiac protection is not afforded.
Translocation of εPKC to cardiac mitochondria suggests that these organelles may play a role in ethanol-mediated cardioprotection. It is well established that mitochondria can regulate cardiac injury during ischemia and reperfusion through increased ROS generation [52
], the release of pro-apoptotic molecules [56
] and alterations in energy utilization [57
]. Additionally, there are several mitochondrial targets of εPKC which protect the heart from I/R injury through mechanisms including ROS generation [59
], opening of the mitochondrial permeability transition pore (MPTP) [61
], regulation of the mitochondrial kATP channels (reviewed in [63
]) and activation of mitochondrial ALDH2 [29
]. One key mediator of cellular injury that accumulates during I/R is the toxic aldehyde 4-HNE [40
]. Under physiological conditions, 4-HNE may act as a signaling molecule [65
]. However, under conditions of oxidative stress, accumulated 4-HNE modifies and regulates enzymes involved in mitochondrial energy production [39
], resulting in increased ROS generation[66
], diminished protein degradation [35
] and increased pro-apoptotic signaling [42
]. Because of the reactivity of this and other aliphatic and aromatic aldehydes, the cell has developed mechanisms to detoxify these molecules [65
]. In addition to glutathione-S transferase, aldehyde dehydrognesase-2 (ALDH2) is a mitochondrial enzyme that detoxifies 4-HNE and other toxic aldehydes, thereby diminishing cellular oxidative stress. Therefore, since 4-HNE accumulation during ischemia [37
] can damage the heart through oxidative mechanisms and ethanol protects the heart through activation and translocation of εPKC to cardiac mitochondria, we hypothesize that εPKC might regulate ALDH2 activity to increase detoxification of 4-HNE, thereby conferring protection by diminishing oxidative stress. Interestingly, human cardiomyocytes in which ALDH2 is overexpressed are significantly protected from acetaldehyde-induced ROS accumulation and apoptosis [67
], suggesting that activation of ALDH2 may be a therapeutic target to reduce ischemic damage to the heart. In a recent study, we identified a small molecule activator of ALDH2 (alda-1) that induces 60% reduction in infarct size, in vivo
, an effect that is similar to the cardioprotection seen with ethanol here (). These data demonstrate that ALDH2 activation is required and sufficient to produce cardioprotection from ischemic injury. Together with the current study, these data suggest that εPKC translocates to cardiac mitochondria where it interacts with ALDH2, and phosphorylates and activates that enzyme to protect the heart from the injury induced by ischemia and reperfusion, in vivo
. Ethanol-induced and εPKC-mediated activation of ALDH2 increase the metabolism of the reactive aldehyde, HNE, to HNA. This prevents HNE-protein adduct formation and diminishes pro-apoptotic signaling, thus improving cardiac function.