Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of varepsilonPKC and activation of aldehyde dehydrogenase 2

Abstract

The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (varepsilonPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of varepsilonPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 min prior to (but not 15 min prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of varepsilonPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and varepsilonPKC-mediated protection from cardiac ischemia, in vivo.

Figure 1. Ethanol decreases CPK release into the blood in an in vivo model of acute myocardial infarction

Rats were i.p. injected with 0.5 g/kg of ethanol 15 or 60 minutes prior to occlusion of the left anterior descending (LAD) coronary artery (ethanol treated) and compared to animals, which were injected with saline (control treated). Hearts were reperfused for 15 minutes by release of the ligature and humoral creatine phosphokinase (CPK) levels were monitored every five minutes in blood drawn through a cardiac apical punch. CPK released over the 15 minutes of reperfusion was summed together and expressed as % control. CPK values from sham animals that underwent surgery, but did not undergo coronary artery ligation were subtracted from the experimental groups. Comparisons between multiple groups were made using analysis of variance (ANOVA) with a Newman-Keuls multiple comparison test and individual group comparisons were made with a student’s t-test. The number of animals/group with standard error and statistical significance for all data are listed in the figures with a p-value of 0.05 being considered significant. (a) A significant difference in CPK release was seen with ethanol administration 60 minutes prior to ischemia but not in animals injected 15 minutes prior. (b) CPK release from animals injected with ethanol 60 minutes prior to occlusion were monitored every five minutes and expressed as blood CPK units/L. Ethanol significantly decreased CPK release relative to controls at each time point tested.

Figure 2. Ethanol-mediated protection is associated with εPKC translocation to cardiac mitochondria

Mitochondria were isolated from the left ventricles of animals that were treated with 0.5 g/kg of ethanol 15 and 60 minutes prior to LAD occlusion (Et) and compared to animals that were not treated with ethanol (C) and to animals that were not subjected to LAD occlusion (N). Following homogenization, mitochondrial lysates and total cellular lysates were analyzed by Western blot analysis utilizing an anti-εPKC antibody and equal loading was determined by monitoring the levels of mitochondrial ALDH2. (a) Electron micrographs illustrating the membrane integrity of isolated mitochondria. Additionally, the presence of mitochondrial protein (prohibitin), and the lack of cytosolic proteins (enolase), plasma membrane proteins (Na⁺K⁺ ATPase), and ER resident proteins (KDEL) illustrates the purity of our mitochondrial preparation. (b) Data from two separate groups of animals treated with ethanol 60 minutes prior to LAD ligation are shown. Two different molecular weight forms of mitochondrial εPKC are marked with arrows. (c) Quantification of εPKC translocation to the mitochondria from 7 different experiments was done using NIH Image-J software, expressed as % control and significance was determined with a 2 way t-test (*= p<0.05). Significant changes were observed between the ethanol treated and control and normoxic hearts. (1c Insert) Hearts from wildtype and εPKC knock-out mice were isolated and homogenized for Western blot analysis. The lower 87kDa band is present in both wildtype and knock-out animals suggesting non-specific antibody recognition while the upper band represents εPKC. (d) Animals were subjected to ethanol treatment without LAD occlusion and data from two representative experiments are shown. (e) Representative data from 4 separate experiments from animals treated with ethanol 15 minutes prior to LAD ligation are shown.

Figure 2. Ethanol-mediated protection is associated with εPKC translocation to cardiac mitochondria

Mitochondria were isolated from the left ventricles of animals that were treated with 0.5 g/kg of ethanol 15 and 60 minutes prior to LAD occlusion (Et) and compared to animals that were not treated with ethanol (C) and to animals that were not subjected to LAD occlusion (N). Following homogenization, mitochondrial lysates and total cellular lysates were analyzed by Western blot analysis utilizing an anti-εPKC antibody and equal loading was determined by monitoring the levels of mitochondrial ALDH2. (a) Electron micrographs illustrating the membrane integrity of isolated mitochondria. Additionally, the presence of mitochondrial protein (prohibitin), and the lack of cytosolic proteins (enolase), plasma membrane proteins (Na⁺K⁺ ATPase), and ER resident proteins (KDEL) illustrates the purity of our mitochondrial preparation. (b) Data from two separate groups of animals treated with ethanol 60 minutes prior to LAD ligation are shown. Two different molecular weight forms of mitochondrial εPKC are marked with arrows. (c) Quantification of εPKC translocation to the mitochondria from 7 different experiments was done using NIH Image-J software, expressed as % control and significance was determined with a 2 way t-test (*= p<0.05). Significant changes were observed between the ethanol treated and control and normoxic hearts. (1c Insert) Hearts from wildtype and εPKC knock-out mice were isolated and homogenized for Western blot analysis. The lower 87kDa band is present in both wildtype and knock-out animals suggesting non-specific antibody recognition while the upper band represents εPKC. (d) Animals were subjected to ethanol treatment without LAD occlusion and data from two representative experiments are shown. (e) Representative data from 4 separate experiments from animals treated with ethanol 15 minutes prior to LAD ligation are shown.

Figure 3. εPKC associates with mitochondrial ALDH2 following ethanol treatment

Mitochondrial protein was isolated from the left ventricles of animals that were administered 0.5 g/kg of ethanol 60 minutes prior to LAD occlusion (Et) and compared to animals that were not treated with ethanol (C) and animals which did not undergo LAD occlusion (N). (a) Following homogenization 700 μg of mitochondrial protein was immunoprecipitated with an anti-ALDH2 antibody and subjected to Western blot analysis with anti-εPKC. As a control, mitochondrial lysate was used (In). (B) Proteins were subjected to a reverse immunoprecipitation using the antibodies listed in the Figure. (C) ALDH2 activity was measured in mitochondrial fractions isolated from control and animals treated with ethanol 15 and 60 minutes prior to LAD occlusion and the results are expressed as μmole NADH produced/minute/mg of protein of either 7 or 4 independent experiments, respectively. Differences in activity (as determined by a two way t-test) were observed between the control and ethanol groups of the animals injected with ethanol 60 minutes prior to ischemia (*= p<0.05) but not in animals treated 15 minutes prior. (Insert) There was no significant difference in ALDH2 activity between sham and control treated animals.

Figure 4. Ethanol treatment reduces HNE protein-adduct formation following I/R

Mitochondrial protein was isolated from the left ventricles of animals that were administered 0.5 g/kg of ethanol 60 minutes prior to LAD occlusion (Et) and compared to animals that were not treated with ethanol (C) and animals which did not undergo LAD occlusion (N). The right panel shows basal levels of HNE formation in cardiac mitochondria that were not subjected to surgery. Following homogenization, mitochondrial lysate was analyzed by Western blot utilizing antibodies which recognize HNE protein adducts. Two protein bands which showed a reduction upon in HNE-adduct formation upon ethanol treatment are denoted by arrows.

Figure 5. Ethanol treatment decreases activation of MAPK pathway signaling molecules

Mitochondrial protein was isolated from the left ventricles of animals that were administered 0.5 g/kg of ethanol 60 minutes prior to LAD occlusion (Et) and compared to animals that were not treated with ethanol (C) and animals which did not undergo LAD occlusion (N). (a) Following homogenization, cardiac total lysate from the left ventricle was analyzed by Western blot to determine the phosphorylation levels of JNK1/2 (p-p46 and p-p54), and Erk1/2 (p-p44 and p-p42). Data from two separate groups of experiments are shown. (B) Quantification of the data in panel a was done using NIH Image-J software, expressed as % control and significance was determined using a 2 way t-test.