Alcohol-induced myocardial fibrosis in metallothionein-null mice: prevention by zinc supplementation

Abstract

Alcohol-induced cardiomyopathy including fibrosis has been recognized clinically for a long time, but its pathogenesis is incompletely understood. Studies using experimental animals have not fully duplicated the pathological changes in humans, and animal models of alcoholic cardiac fibrosis are not available. In the present study, we have developed a mouse model in which cardiac hypertrophy and fibrosis were produced in metallothionein-knockout (MT-KO) mice fed an alcohol-containing liquid diet for 2 months. The same alcohol feeding did not produce cardiac fibrosis in the wild-type (WT) control mice, although there was no difference in the alcohol-induced heart hypertrophy between the WT controls and the MT-KO mice. Zinc supplementation prevented cardiac fibrosis but did not affect heart hypertrophy in the alcohol-fed MT-KO mice, suggesting a specific link between zinc homeostasis and cardiac fibrosis. Serum creatine phosphokinase activity was significantly higher in the alcohol-administered MT-KO mice than in the WT mice, and zinc supplementation decreased serum creatine phosphokinase activities and eliminated the difference between the groups. Thus, disturbance in zinc homeostasis due to the lack of MT associates with alcohol-induced cardiac fibrosis and more severe cardiac injury, making the MT-KO mouse model of alcohol-induced cardiac fibrosis a useful tool to investigate specific factors involved in the alcoholic cardiomyopathy.

Figure 1

MT concentrations in the hearts of WT and MT-KO mice treated with alcohol, zinc, or alcohol plus zinc. The hearts were obtained 2 months after the alcohol-containing liquid diet feeding with or without zinc supplementation. Data were obtained from four animals in each group and the values that do not share the same letter are significantly (P < 0.05) different from each other.

Figure 2

Alcohol-induced increase in the heart weight normalized to body weight in WT and MT-KO mice. Each value was obtained from four to six mice and the values that do not share the same letter differ significantly (P < 0.05) from each other.

Figure 3

Alcohol-induced histopathological changes in the myocardium and the effect of zinc supplementation on the changes. Alcohol (EtOH) feeding markedly increased the size of cardiomyocytes in comparison with the controls (Cont) in both WT and MT-KO mice. Zinc (Zn) supplementation did not alter the morphology of the myocardium or affect the alcohol-induced hypertrophy of the cardiomyocytes (Zn/EtOH). Degenerative morphological changes including myocyte necrosis were identified in the alcohol-treated myocardium.

Figure 4

Alcohol-induced ultrastructural changes in the myocardium and the effect of zinc supplementation. Alcohol (EtOH) feeding induced marked organelle changes in both WT and MT-KO myocardium. More damages, in particular mitochondrial swelling and cristae disorganization, were found in MT-KO mice. Zinc (Zn) supplementation greatly improved the ultrastructural changes in both WT and MT-KO mice (Zn/EtOH).

Figure 5

Effects of alcohol feeding on serum CPK activities in WT and MT-KO mice with or without zinc supplementation. Each value was obtained from four to eight mice and the values that do not share the same letter differ significantly (P < 0.05) from each other.

Figure 6

Top: Alcohol-induced myocardial fibrosis and the effect of zinc supplementation. Alcohol (EtOH) feeding induced collagen deposition in MT-KO myocardium and zinc supplementation prevented the fibrotic effect (Zn/EtOH). Alcohol did not cause the same fibrotic change in WT mice and zinc supplementation per se did not induce fibrotic response in either WT or MT-KO myocardium. Bottom: Perivascular inflammatory infiltration (A), patchy interstitial neutrophil infiltration in fibrotic area (B), and hypertrophic cardiomyocytes surrounding fibrotic area (C) in alcoholic MT-KO myocardium.

Figure 7

Semiquantitative analysis of the proportion of fibrotic area in the whole heart, expressed as percentage of the heart. Each data point was obtained from five mice, and five slides from each mouse and seven random fields (1.2 mm² per field) per slide were examined and digitalized using a computer-assisted morphometric imaging analyzer system. The data presented are mean ± SEM and an asterisk indicates a significant difference from the rest of the groups (P < 0.05).