Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative β subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and β subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM)-and time (0.5 hour and 24 hours)-dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol.Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or β subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions. (J Nippon Med Sch 2015; 82: 237 245)

Cultured mouse heart-derived myocardial and non-muscle cells were exposed to ethanol, stained with cell-permeant fluorescent vital probes, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide) and oxidation-sensitive dihydrorhodamine 123, and analyzed by flow cytometry to elucidate ethanol-induced time-wise alterations in the mitochondrial membrane potential (DeltaPsim) and the production of reactive oxygen species (ROS). Ethanol (50 and 200 mM) not only hyperpolarized DeltaPsim of both types of cells but also dose-dependently increased ROS production at 24 h, although a 200-mM dose reduced the production until 3 h. These cell pathophysiological reactions suggest the depression of mitochondrial ATPase and mitochondrial respiratory chain. However, differences between these cells appeared after a 24-h exposure to 200 mM ethanol: the increase in ROS production was approximately twice as large for myocardial cells as for non-muscle cells; and the side-scatter parameter of light scattering significantly increased for myocardial cells, but not for non-muscle cells. All these myocyte-specific alterations indicate an increase in the mitochondrial fraction in a cell. This reaction might be a countermeasure against ethanol-induced dysfunction of mitochondrial respiration that is needed to meet the energy requirements of spontaneous myocardial contractions.

Background: There have been no comprehensive investigations that examined the alteration of heart gene expression due to ethanol exposure. Therefore, we attempted to obtain gene expression from cultured neonatal rat cardiomyocytes exposed to ethanol (0, 10, 50, 100 mM) for 24 h. Methods: The total RNA extract of beating cardiomyocytes was evaluated using DNA microarray, and fold changes (FCs) of differential gene expression of ethanol-exposed cardiomyocytes were analyzed against the control using Ingenuity Pathway Analysis (IPA) software.Results: The 1,394 genes with |FC| ≥ 1.8 were uploaded to IPA. IPA predicted 23 canonical pathways working in the ethanol groups. Three canonical pathways related to ethanol degradation, "Ethanol Degradation IV", "Oxidative Ethanol Degradation III", and "Ethanol Degradation II", were inhibited in the ethanol groups. IPA predicted "ethanol" as an upregulated upstream regulator of the network having 22 downstream members for only the 100 mM ethanol group, 3 members, NTRK2, TGFB3, and TLR8, being activated in all groups. Certain cellular functions were predicted to alter dose-dependently; "Myocarditis" was dose-dependently inhibited, whereas "Cell death of heart cells" was dose-dependently activated. Several functions were inhibited only in 50 mM ethanol; "Failure of heart" was enhanced only in 50 mM ethanol. Certain functions were activated only in 100 mM ethanol. "Cardiac fibrosis" was not predicted in any of ethanol groups.Conclusions: IPA predicted ethanol-induced activation or inhibition of canonical pathways and functions of cardiomyocytes depending on the concentrations of ethanol, and 3 networks related to heart functions of cardiomyocytes exposed to 3 concentrations of ethanol.

ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CLT (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its V max⁡/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3.

Maturation of whisky delayed ethanol metabolism to lower the level of blood acetaldehyde and acetate with increasing inhibition of liver ADH activity by nonvolatile congeners. It also prolonged drunkenness by enhancing the neurodepressive effects of ethanol, due to increases in the amount of nonvolatile congeners. These biomedical effects of whisky maturation may reduce aversive reactions and cytotoxicity due to acetaldehyde, and may also limit overdrinking with the larger neurodepression.