Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis

Abstract

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis.

Keywords: alcoholic cardiomyopathy; cardiac steatosis; heart failure; mitochondrial dysfunction; oxidative stress.

Fig. 1.

Protocol outline and blood alcohol levels in the study groups. A: feeding protocol used in our study. Green arrows represent maltose dextrin [9 g/kg body wt (BW) gavage]; red arrows represent ethanol (5 g/kg BW) gavage in the early morning. Animals were euthanized at the end of the feeding period, 9 h after gavage. Groups that did not receive gavage were euthanized at the end of the feeding period. At the end of the feeding period, echocardiography, invasive hemodynamic examination, and tissue collection were performed. Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). B: time course and results of blood alcohol levels in different study groups. *P < 0.05 vs. baseline (in case of the time course study) or vs. Pair-fed in chronic EtOH-fed groups.

Fig. 2.

Chronic alcohol feeding and binge drinking impairs cardiac performance. A: representative pressure-volume loops in the study groups. B: representative M-mode images taken of the short-axis view at the midpapillary level of the left ventricle in the study groups. Yellow lines represent anterior and posterior wall thickness in end systole (AWs and PWs) and in end diastole (AWd and PWd). Red lines represent internal diameter in end systole (IDs) and in end diastole (IDd). Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B).

Fig. 3.

Chronic alcohol feeding and binge drinking is associated with impaired cardiac function. Systolic indexes were derived from pressure-volume analysis of the study groups. The following indexes are shown: slope of end-systolic pressure-volume relationship (E_es), preload recruitable stroke work (PRSW), maximal slope of systolic pressure increment (dP/dt_max), dP/dt_max-end-diastolic volume (dP/dt_max-EDV), ejection fraction (EF), and efficiency. B: indexes of diastolic function: time constant of left ventricular pressure decay (Tau_w, according to Weiss method) and maximal slope of systolic pressure decrement (dP/dt_min). Stiffness parameters: left ventricular end-diastolic pressure (LVEDP) and slope of end-diastolic pressure-volume relationship (EDPVR). Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). *P < 0.05 vs. Pair-fed; #P < 0.05 vs. corresponding chronic EtOH-fed groups.

Fig. 4.

Chronic alcohol feeding and binge drinking induces vascular dysfunction. Vascular indexes: arterial elastance (Ea), mean arterial pressure (MAP), ventriculo-arterial coupling and total peripheral resistance. Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). *P < 0.05 vs. Pair-fed; #P < 0.05 vs. corresponding chronic EtOH-fed groups.

Fig. 5.

Chronic alcohol intake and binge drinking increases myocardial oxidative/nitrative stress. A: myocardial 3-nitrotyrosine (3-NT) content in the left ventricle. B: gene expression values of gp91phox, p47phox, and angiotensin II receptor, type 1a (AGTR1a) of the LV cardiac tissue are represented. Groups: Pair-fed (isocalorically fed mice with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). *P < 0.05 vs. Pair-fed; #P < 0.05 vs. corresponding chronic EtOH-fed groups.

Fig. 6.

Chronic and binge drinking impairs mitochondrial function and biogenesis. A: mitochondrial complex I, II, and IV activites are shown in the myocardium. B: gene expression values of the following members of mitochondrial biogenesis are shown: PGC1α, peroxisome proliferator-activated receptor alpha (PPARα), estrogen-related receptor α (ERRα), medium-chain acyl-CoA dehydrogenase (MCAD), acetyl-CoA oxidase 1 (ACOX1). Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). *P < 0.05 vs. Pair-fed; #P < 0.05 vs. corresponding chronic EtOH-fed groups.

Fig. 7.

Chronic alcoholism and binge drinking leads to myocardial fat accumulation. A: gene expression values of acetyl-CoA carboxylase 1 and 2 (ACC1, ACC2) of the left ventricle. B: representative images of Oil Red O stained left ventricle sections with lipid droplets in the cardiomyocytes. Magnification: 400×. Scale bar: 50 μm. Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). *P < 0.05 vs. Pair-fed; #P < 0.05 vs. corresponding chronic EtOH-fed groups.

Fig. 8.

Chronic alcoholism and binge drinking induces mild cardiomyocyte hypertrophy. A: cardiomyocyte cross-sectional area and left ventricular gene expression values of atrial natriuretic peptide (ANP) in our study groups. B: representative images of wheat germ agglutinin (WGA; red color)-stained left ventricular sections. Nuclear counterstaining of DRAQ5 is shown as blue. Magnification: 200×. Scale bar: 20 μm. Groups: Pair-fed (isocalorically fed with Lieber-DeCarli control liquid diet), ethanol (EtOH) Lieber-DeCarli liquid diet containing 5% (vol/vol) EtOH for 10 days (EtOH 10d); for 20 days (EtOH 20d); for 40 days (EtOH 40d); liquid diet containing 5% (vol/vol) EtOH for 10 days and binge feeding on day 11 with 5 g/kg BW EtOH solution (EtOH 10d 1B); for 20 days combined with binge feeding on days 11 and 21 with 5 g/kg BW EtOH solution (EtOH 20d 2B); for 40 days combined with binge feeding on days 11, 21, 31, and 41 with 5 g/kg BW EtOH solution (EtOH 40d 4B). *P < 0.05 vs. Pair-fed.