Nebivolol Prevents Up-Regulation of Nox2/NADPH Oxidase and Lipoperoxidation in the Early Stages of Ethanol-Induced Cardiac Toxicity.

Changes in redox state are described in the early stages of ethanol-induced cardiac toxicity. Here, we evaluated whether nebivolol would abrogate ethanol-induced redox imbalance in the heart. Male Wistar rats were treated with a solution of ethanol (20% v/v) for 3 weeks. Treatment with nebivolol (10 mg/kg/day; p.o. gavage) prevented the increase of both superoxide (O2•-) and thiobarbituric acid reactive substances (TBARS) in the left ventricle of rats chronically treated with ethanol. Neither ethanol nor nebivolol affected the expression of Nox4, p47phox, or Rac-1. Nebivolol prevented ethanol-induced increase of Nox2 expression in the left ventricle. Superoxide dismutase (SOD) activity as well as the concentration of reduced glutathione (GSH) was not altered by ethanol or nebivolol. Augmented catalase activity was detected in the left ventricle of both ethanol- and nebivolol-treated rats. Treatment with nebivolol, but not ethanol increased eNOS expression in the left ventricle. No changes in the activity of matrix metalloproteinase (MMP)2 or in the expressions of MMP2, MMP9, and tissue inhibitor metalloproteinase (TIMP)1 were detected after treatment with ethanol or nebivolol. However, ethanol increased the expression of TIMP2, and this response was prevented by nebivolol. Our results provided novel insights into the mechanisms underlying the early stages of the cardiac injury induced by ethanol consumption. We demonstrated that Nox2/NADPH oxidase-derived ROS play a role in ethanol-induced lipoperoxidation and that this response was prevented by nebivolol. In addition, we provided evidence that MMPs are not activated in the early stages of ethanol-induced cardiac toxicity.

Acute ethanol exposure-induced autophagy-mediated cardiac injury via activation of the ROS-JNK-Bcl-2 pathway.

Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway.

From the Cover: Alcohol Inhibition of the Enzymatic Activity of Glyceraldehyde 3-Phosphate Dehydrogenase Impairs Cardiac Glucose Utilization, Contributing to Alcoholic Cardiomyopathy.

Heavy consumption of alcohol induces cardiomyopathy and is associated with metabolic changes in the heart. The role of altered metabolism in the development of alcoholic cardiomyopathy remains largely unknown but is examined in the present study. The effect of chronic alcohol consumption on cardiac damage was examined in mice fed an alcohol or isocaloric control diet for 2 months. Signaling pathways of alcohol-induced metabolic alteration and pathologic changes were examined in both animal hearts and H9c2 cell cultures. Compared with controls, the hearts from the alcohol-fed mice exhibited cardiac oxidative stress, cell death, a fibrotic response, hypertrophic remodeling, and the eventual development of cardiac dysfunction. All these detrimental effects could be ameliorated by superoxide dismutase mimic Mn (111) tetrakis 1-methyl 4-pyridylporphyrin pentachloride (MnTMPyP) therapy. A mechanistic study showed that chronic alcohol exposure enhanced the expression of proteins regulating fatty acid uptake but impaired the expression of proteins involved in mitochondrial fatty acid oxidation, which compensatively geared the heart to the suboptimal energy source, glucose. However, chronic alcohol exposure also impaired the glycolytic energy production step regulated by glyceraldehyde-3-phosphate dehydrogenase, which further feeds back to enhance glucose uptake signaling and the accumulation of glycolytic intermediate product fructose, resulting in aggravation of alcohol-induced cardiac oxidative stress, cell death, and remodeling. All these dysmetabolic alterations could be normalized by MnTMPyP treatment, along with significant improvement in cardiac cell death and remodeling. These results demonstrate that alcohol-induced oxidative stress and altered glucose metabolism are causal factors for the development of alcoholic cardiomyopathy.

Chronic Alcohol Intoxication Is Not Accompanied by an Increase in Calpain Proteolytic Activity in Cardiac Muscle of Rats.

Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of µ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of µ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of µ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in µ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

Enzymes and signal pathways in the pathogenesis of alcoholic cardiomyopathy.

Owing to its acute psychotropic effects, ethanol is the most frequently consumed toxic agent worldwide. However, excessive alcohol intake results in an array of health, social, and economic consequences, which are related to its property as an addictive substance. It has been well established that exposure to high levels of alcohol for a long period leads to the onset and progression of nonischemic cardiomyopathy through direct toxic mechanisms of ethanol and its metabolite, acetaldehyde. Excessive alcohol ingestion causes myocardial damage including disruptions of the myofibrillar architecture and is associated with reduced myocardial contractility and decreased ejection volumes. Key features of alcoholic cardiomyopathy are cardiac hypertrophy and ventricular dilatation, and the disease is manifested mainly as cardiomegaly, congestive heart failure, and even cardiac death. Mechanisms that have been postulated to underlie the pathogenesis of alcoholic cardiomyopathy include apoptosis, mitochondrial alterations, acetaldehyde protein adduct formation, oxidative stress, and imbalances in fatty acid metabolism. In the following, we give a brief overview of the molecular effects of ethanol-metabolizing enzymes and their impact on myocardial signal transduction pathways.

High-dose alcohol induces reactive oxygen species-mediated apoptosis via PKC-ß/p66Shc in mouse primary cardiomyocytes.

Cardiac dysfunction caused by excessive alcohol consumption is a specific disease, alcoholic cardiomyopathy (ACM). High-dose alcohol has been found to induce oxidation stress and apoptosis in cardiomyocytes, but the signaling link between alcohol-induced oxidation stress and apoptosis in cardiomyocytes remains to be elucidated. To address the issue, we exposed primary cardiomyocytes from neonatal mouse hearts to high doses of alcohol (50mM, 100mM, and 200 mM). We found that alcohol induced dose-dependent phosphorylation of p66shc, and reactive oxygen species (ROS) production increased in parallel with phosphorylation levels of p66shc. Exposure to alcohol also led to loss of mitochondrial membrane potential and cytochrome c release. Depletion of p66Shc and inhibition of protein kinase C-ß (PKC-ß) successfully reversed all the effects and suppressed alcohol-induced apoptosis in cardiomyocytes. Collectively, our study provides a molecular basis for signaling transduction of alcohol-induced oxidation stress and apoptosis of cardiomyocytes, which may facilitate the prevention and treatment of ACM.

[Anti-heat shock protein 70 (anti - Hsp 70) antibodies in alcohol use disorder patients]. / Przeciwciala przeciw bialku wstrzasu termicznego 70 kDa (anty- Hsp 70) u osób z zespolem zaleznosci alkoholowej.

The expression of the most important chaperone protein - Hsp70 and autoimmunity directed against it is a risk factor of cardiovascular diseases, increased in subjects with alcohol use disorder (AUD). The aim of the study was to evaluate the level of anti-Hsp 70 protein antibodies (anti-Hsp 70) in sera of AUD patients during abstinence period. Material and methods. The study included 54 subjects with AUD diagnosed basing on DSM IV criteria. In the studied group clinimetric evaluation was performed, plasma lipids, basic transketolase activity in erythrocytes (TK), thiamine pyrophosphate (TPP) activation of transketolase and the level of anti-Hsp 70 antibodies were evaluated as well. Results. In AUD subjects anti-Hsp 70 level was decreased during abstinence period. During first month of abstinency it correlated negatively with total cholesterol concentration (rS=-0.8857, p=0.0188) and the percentage of TPP stimulation (rS=-0.5960, p<0.05), and during 6 months of abstinence with HDL cholesterol (rS=-0.6848, p=0.0289). After 1 year of abstinence anti-Hsp 70 correlated positively with basic TK activity (rS=0.9550, p=0.0008). Sex is an independent factor influencing anti-Hsp 70 level in AUD subjects (B=60.9469, p=0.0435). In multiple regression model including results of clinimetric evaluation and its effect on the level of anti-Hsp 70 antibodies in AUD patients during 1 month of abstinency anti-Hsp 70 correlated with TWEAK scale score (BETA=-1.4543, p=0.0144) and AUDIT score (BETA-=1.2255, p=0.0224). In 2-6 months of abstinency anti-Hsp 70 correlated with TWEAK score (BETA=1.1110, p=0.0418). After 1 year of abstinency anti-Hsp 70 correlated with AUDIT score (BETA=-1.2161, p=0.0210). Conclusion. The autoimmune reaction against Hsp 70 is decreased during abstinency in AUD patients. Its relation with plasma lipids and thiamine deficiency may lead to increased risk of cardiovascular disorders. TWEAK and AUDIT scoring seem to be most useful for clinimetric evaluation in the context of the role of anti-Hsp 70 antibodies.

ALDH2 in alcoholic heart diseases: molecular mechanism and clinical implications.

Alcoholic cardiomyopathy is manifested as cardiac hypertrophy, disrupted contractile function and myofibrillary architecture. An ample amount of clinical and experimental evidence has depicted a pivotal role for alcohol metabolism especially the main alcohol metabolic product acetaldehyde, in the pathogenesis of this myopathic state. Findings from our group and others have revealed that the mitochondrial isoform of aldehyde dehydrogenase (ALDH2), which metabolizes acetaldehyde, governs the detoxification of acetaldehyde formed following alcohol consumption and the ultimate elimination of alcohol from the body. The ALDH2 enzymatic cascade may evolve as a unique detoxification mechanism for environmental alcohols and aldehydes to alleviate the undesired cardiac anomalies in ischemia-reperfusion and alcoholism. Polymorphic variants of the ALDH2 gene encode enzymes with altered pharmacokinetic properties and a significantly higher prevalence of cardiovascular diseases associated with alcoholism. The pathophysiological effects of ALDH2 polymorphism may be mediated by accumulation of acetaldehyde and other reactive aldehydes. Inheritance of the inactive ALDH2*2 gene product is associated with a decreased risk of alcoholism but an increased risk of alcoholic complications. This association is influenced by gene-environment interactions such as those associated with religion and national origin. The purpose of this review is to recapitulate the pathogenesis of alcoholic cardiomyopathy with a special focus on ALDH2 enzymatic metabolism. It will be important to dissect the links between ALDH2 polymorphism and prevalence of alcoholic cardiomyopathy, in order to determine the mechanisms underlying such associations. The therapeutic value of ALDH2 as both target and tool in the management of alcoholic tissue damage will be discussed.

[The importance of biochemical studies for practical forensic-medical expert examination].

Sudden death along with differential diagnosis of deaths from coronary heart disease and alcoholic cardiomyopathy is a challenging problem in practical forensic medicine. Relevant investigations demonstrated the possibility to address it using a battery of biochemical methods, such as measurement of blood glucose level in heart cavities, lactate dehydrogenase activity in myocardium, etc.

Cardiac overexpression of alcohol dehydrogenase exacerbates chronic ethanol ingestion-induced myocardial dysfunction and hypertrophy: role of insulin signaling and ER stress.

Chronic alcohol intake leads to alcoholic cardiomyopathy characterized by cardiac hypertrophy and contractile dysfunction possibly related to the toxicity of the ethanol metabolite acetaldehyde. This study examined the impact of augmented acetaldehyde exposure on myocardial function, geometry, and insulin signaling via cardiac-specific overexpression of alcohol dehydrogenase (ADH). ADH transgenic and wild-type FVB mice were placed on a 4% alcohol diet for 12 weeks. Echocardiographic, glucose tolerance, glucose uptake, insulin signaling, and ER stress indices were evaluated. Mice consuming alcohol exhibited glucose intolerance, dampened cardiac glucose uptake, cardiac hypertrophy and contractile dysfunction, all of which with the exception of whole body glucose tolerance were exaggerated by the ADH transgene. Cardiomyocytes from ethanol-fed mice exhibited depressed insulin-stimulated phosphorylation insulin receptor (tyr1146) and IRS-1 (tyrosine) as well as enhanced serine phosphorylation of IRS-1. ADH-augmented alcohol-induced effect of IRS-1 phosphorylation (tyrosine/serine). Neither alcohol nor adh affected expression of insulin receptor and IRS-1. Alcohol reduced phosphorylation of Akt and GSK-3beta as well as GSK-3beta expression and the effect was exaggerated by ADH. The transcriptional factors GATA4, c-jun and c-jun phosphorylation were upregulated by alcohol, which was amplified by ADH. The ratios of phospho-c-Jun/c-Jun and phospho-GATA4/GATA4 remained unchanged. Chronic alcohol intake upregulated expression of the endoplasmic reticulum stress markers eIF2alpha, IRE-1alpha, GRP78 and gadd153, the effect of which was exaggerated by ADH. These data suggest that elevated cardiac acetaldehyde exposure via ADH may exacerbate alcohol-induced myocardial dysfunction, hypertrophy, insulin insensitivity and ER stress, indicating a key role of ADH gene in alcohol-induced cardiac dysfunction and insulin resistance.

Study of oxidative stress and trace element levels in patients with alcoholic and non-alcoholic coronary artery disease.

Alcohol consumption induces oxidative stress, and leads to lipid peroxidation. These effects have been linked to alcohol-related toxicity and diseases are considered relevant to alcohol-atherosclerosis interrelationship. Deficiency of many antioxidants and trace elements may impair the antioxidant defense leading to ethanol induced oxidative stress. In the present study, our aim was to investigate the lipid peroxidation, lipid profile, antioxidant enzymes and trace elements in patients with and without alcoholic coronary artery disease (CAD). Our study included 61 patients suffering from CAD, 124 patients suffering from alcoholic CAD with high to moderate alcohol intake, 75 controls were randomly selected for our study. Increased serum lipid peroxidation, total cholesterol, triglycerides, LDL cholesterol and copper levels were high while levels of HDL cholesterol, glutathione peroxdiase, superoxide dismutase, trace elements like Selenium and Zinc were low in high alcoholic CAD patients compared with moderate and non alcoholic CAD patients. The results obtained from present study indicate that high alcohol intake predicts low antioxidant enzyme and that trace element may contribute to the increased susceptibility for the development of CAD.

Myocardial antioxidant status in chronic alcoholism.

BACKGROUND: Excessive ethanol intake is one of the most frequent causes of acquired dilated cardiomyopathy in developed countries. The pathogenesis is multifactorial, with the antioxidant imbalance of cardiac muscle being a potential factor. The current study evaluates myocardial antioxidant status in ethanol consumers and its relation to cardiac damage. METHODS: The authors assessed superoxide dismutase, glutathione peroxidase, and glutathione reductase enzyme activities as well as the total antioxidant status capacity in myocardial samples obtained from organ donors with sudden death of traumatic or neurological origin. They studied 23 high-dose chronic alcohol consumers, 27 individuals with long-standing hypertension, and 11 healthy controls. Cardiomyopathy was defined according to standard functional and histological criteria. RESULTS: Patients with dilated cardiomyopathy, either of alcoholic or hypertensive origin, showed increased myocardial superoxide dismutase activities compared with patients without cardiomyopathy (p < 0.001, both) and controls (p < 0.05, both). Total antioxidant status capacity and the activity of glutathione peroxidase and glutathione reductase enzymes were similar in all groups. Superoxide dismutase activity was related to the presence of cardiac enlargement and the degree of cardiac histological damage. The amount and type of alcoholic beverages as well as the nutritional status of the patients were not related to myocardial antioxidant activity. CONCLUSIONS: The presence of dilated cardiomyopathy, of either alcoholic or hypertensive origin, is related to an increase in myocardial superoxide dismutase activity.

[Enzymatic markers in the alcoholic cardiomyopathy]. / Markeri enzimatici în cardiomiopatia alcoolica.

Alcoholic cardiomyopathy can ensue from heavy consumption of alcohol over a long period of time. The clinical features include dilatation of the left ventricle, poor myocardial contractility with reduced left ventricular ejection volume, raised tissue enzymes. In numerous experimental data has been observed increased generation of oxygen and ethanol free radicals, indicate that free radicals are implied in myocardial and hepatic damage. Ethanol administration also elicits hepatic disturbances in the availability of antioxidant defense. The resulting antioxidative stress leads to enhanced lipid peroxidation and can also affect other important cellular component.

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: a gene for alcohol-induced cardiomyopathy.

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.

[Changes in the enzymatic activity in the myocardium of patients with idiopathic and secondary dilated cardiomyopathy]. / Izmeneniia aktivnosti fermentov v miokarde bol'nykh s idiopaticheskoi i vtorichnoi dilatatsionnoi kardiomiopatiei.

Myocardial biopsies from patients with dilated cardiomyopathy (DCMP) were studied histochemically. The patients were divided into the following groups: 1) idiopathic DCMP; 2) patients with postmyocarditis DCMP (an increase of lymphocytic-macrophagal elements in the myocardium); 3) secondary DCMP against alcoholic myocardial damage. Idiopathic and secondary DCMP are characterized by the following enzymatic changes: a decrease in the activity of the majority of oxidation-reduction mitochondrial enzymes, normal or increased activity of malate dehydrogenase, increased activity of lysosomal and microsomal enzymes. An increased activity of succinate dehydrogenase was observed only in patients who had previous myocarditis. Alterations in idiopathic and alcoholic DCMP were practically identical.

The functional state of the creatine kinase system of myocardial mitochondria in alcoholic cardiomyopathy.

The authors studied the chronic effect of ethanol on the functional state of creatine kinase system in myocardial mitochondria of adult rats. Mitochondrial functions were determined in skinned fibres prepared according to Veksler without mitochondria isolation. Compared with control values, ethanol was found to decrease statistically significantly the velocity of creatine-stimulated mitochondrial respiration (Vcr) and that of maximum ADP-induced stimulation of mitochondrial respiration (Vmax). The chronic action of alcohol also reduces the respiratory activation by creatine (%Cr) which ranks among the most sensitive indicators of mitochondrial respiration regulation by creatine kinase. The authors have demonstrated that the function of the creatine kinase system of myocardial mitochondria is impaired in alcoholic cardiomyopathy in the adult rat.

[Enzymatic diagnosis of alcoholism-induced damage of internal organs]. / Enzymatyczna diagnostyka poalkoholowego uszkodzenia narzadów.

++Post-alcoholic lesion of liver, pancreas, and heart muscle was estimated by measurement of some enzymes activity. Alcoholic in-patients were divided into two groups in regard to the age and the length of the disease. The activity of enzymes in the blood was measured by kinetic methods using the RA-1000/Technicon analyser. It was shown that the increase of activity of alanine aminotransferase (AlAT), gamma-glutamyltransferase (GGTP), and alcohol dehydrogenase (ADH) may indicate the ++post-alcoholic liver damage, while increase of activity of alpha-+-amylase and ++leucine aminopeptidase (LAP) may be useful for the diagnosis of pancreas lesion, and creatine kinase (CK) as well as lactate dehydrogenase (LDH) for the evaluation of postalcoholic lesion of the heart muscle.

Quantitative histoenzymological characteristics of the myocardium in sudden cardiac death.

Enzymes in the human myocardium following sudden death were examined for activity in a quantitative histoenzymological study, these were NAD-dependent dehadrogenases of succinate (SDG), lactate (LDG), beta-hydroxybutyrate (beta-HOBDG), alpha-glycerophosphate (alpha-GPDG), alcohol (ADG), glucoso-6-phosphate (G-6-PDG), and NAD-diaphorase (NADse), and catalase. Autopsies were performed within 3 h after death. beta-HOBDG and LDG were found to show an increase in activity in the cardiomyocytes of sudden death subjects with coronary heart disease without apparent changes. In the myocardium from death subjects with coronary heart disease and large postinfarct cardiosclerosis, the activity of the enzymes was directly related to the severity of myocardial hypertrophy and signs of chronic heart failure. As myocardial hypertrophy developed, the enzyme activity increased; when there appeared signs of chronic heart failure it decreased. The myocardium from sudden death subjects with alcoholic cardiomyopathy showed diminished redox enzyme activity and higher activity of the enzyme utilizing alcohol (ADG and catalase). The findings suggest that changes in the enzyme activity in the myocardium are of various type and depend on previous cardiac abnormalities.