Transgenic overexpression of aldehyde dehydrogenase-2 rescues chronic alcohol intake-induced myocardial hypertrophy and contractile dysfunction

Abstract

Background: Chronic alcoholism leads to the onset and progression of alcoholic cardiomyopathy through toxic mechanisms of ethanol and its metabolite, acetaldehyde. This study examined the impact of altered acetaldehyde metabolism through systemic transgenic overexpression of aldehyde dehydrogenase-2 (ALDH2) on chronic alcohol ingestion-induced myocardial damage.

Methods and results: ALDH2 transgenic mice were produced with the chicken beta-actin promoter. Wild-type FVB and ALDH2 mice were placed on a 4% alcohol diet or a control diet for 14 weeks. Myocardial and cardiomyocyte contraction, intracellular Ca(2+) handling, histology (hematoxylin and eosin, Masson trichrome), protein damage, and apoptosis were determined. Western blot was used to monitor the expression of NADPH oxidase, calcineurin, apoptosis-stimulated kinase (ASK-1), glycogen synthase kinase-3beta (GSK-3beta), GATA4, and cAMP-response element binding (CREB) protein. ALDH2 reduced the chronic alcohol ingestion-induced elevation in plasma and tissue acetaldehyde levels. Chronic alcohol consumption led to cardiac hypertrophy, reduced fractional shortening, cell shortening, and impaired intracellular Ca(2+) homeostasis, the effect of which was alleviated by ALDH2. In addition, the ALDH2 transgene significantly attenuated chronic alcohol intake-induced myocardial fibrosis, protein carbonyl formation, apoptosis, enhanced NADPH oxidase p47(phox) and calcineurin expression, as well as phosphorylation of ASK-1, GSK-3beta, GATA4, and CREB.

Conclusions: The present results suggest that transgenic overexpression of ALDH2 effectively antagonizes chronic alcohol intake-elicited myocardial hypertrophy and contractile defect through a mechanism that is associated, at least in part, with phosphorylation of ASK-1, GSK-3beta, GATA4, and CREB. These data strongly support the notion that acetaldehyde may be an essential contributor to the chronic development of alcoholic cardiomyopathy.

Fig. 1

(A): Identification of ALDH2 transgenic mice. Genomic DNA was isolated from 2-cm tail clips from 1-month-old mice and ALDH2 gene was identified by PCR. Lanes 1 and 4 are negative and the rest are positive for ALDH2 gene. M: marker; (B). Myocardial tissue acetaldehyde levels from FVB and ALDH2 transgenic mice consuming ethanol or control diets for 14 weeks. (C). ALDH2 expression in the liver, kidney, brain and heart from FVB and ALDH2 transgenic mice. Inset: Representative gel blots depicting ALDH2 and β-actin protein expression using specific antibodies. Mean ± SEM, n = 6-7 mice per group, * p < 0.05 vs. FVB, # p < 0.05 vs. FVB+ETOH.

Fig. 2

Effect of ALDH2 transgene on chronic ethanol (ETOH) intake-induced cardiomyocyte contractile defects. A: Representative traces depicting cell shortening; B: Cross-sectional area (longitudinal); C: Peak shortening (PS); D: Maximal velocity of shortening/relengthening (± dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR₉₀). Mean ± SEM, n = 160 – 161 cells from 8 mice per group, * p < 0.05 vs. FVB, # p < 0.05 vs. FVB+ETOH.

Fig. 3

Effect of ALDH2 transgene on chronic ethanol (ETOH) intake-induced intracellular Ca²⁺ homeostasis, SERCA activity and SR Ca²⁺ store evaluated by frequency (0.1 – 5.0 Hz)-dependent shortening response in murine cardiomyocytes. A: Resting fura-2 fluorescence intensity (FFI); B: Electrically-stimulated rise in FFI (ΔFFI); C: Intracellular Ca²⁺ decay rate; D: SERCA activity evaluated by ⁴⁵Ca²⁺ uptake; and E: Frequency response. Each point represents PS normalized to that of baseline value at 0.1 Hz from the same cell. Mean ± SEM, n = 86 – 87 cells (A-C) and 6 mice (D) or given in parenthesis, * p < 0.05 vs. FVB, # p < 0.05 vs. FVB+ETOH.

Fig. 4

Histological analyses hearts from FVB and ALDH2 mice with or without chronic alcohol intake for 14 weeks. A: Representative H&E staining micrographs showing transverse sections of left ventricular myocardium (x 400); B: Quantitative analysis of cardiomyocyte cross-sectional (transverse) area using measurements of ∼ 200 cardiomyocytes from 3-5 mice per group; C: Representative Masson trichrome staining micrographs showing longitudinal sections of left ventricular myocardium (x 200); and D: Quantitative analysis of fibrotic area (Masson trichrome stained area in light blue color normalized to the total myocardial area). Data were obtained from 3-5 mice per group, *p < 0.05 vs. FVB, # p < 0.05 vs. FVB+ETOH.

Fig. 5

Caspase 3 assay (panel A) and protein carbonyl formation (panel B) in myocardium from FVB and ALDH2 mice administrated with control or ethanol (ETOH) liquid diet for 14 weeks. Mean ± SEM, n = 5 – 7 mice per group, * p < 0.05 vs. FVB, # p < 0.05 vs. FVB+ETOH.

Fig. 6

Effect of ALDH2 on chronic alcohol (ETOH) intake-induced change in p47^phox NADPH oxidase (panel A), calcineurin A (panel B), ASK-1 phosphorylation (normalized to total ASK-1, panel C), GSK-3β phosphorylation (normalized to total GSK-3β, panel D), GATA4 phosphorylation (normalized to total GATA4, panel E) and CREB phosphorylation (normalized to total CREB, panel F). Inset: representative gels using specific antibodies. β-Actin was used as the loading control. Mean ± SEM, n = 5 – 7 mice per group, * p < 0.05 vs. FVB, # p < 0.05 vs. FVB+ETOH.