Original article
Inhibition of aldehyde dehydrogenase 2 activity enhances antimycin-induced rat cardiomyocytes apoptosis through activation of MAPK signaling pathway

https://doi.org/10.1016/j.biopha.2009.12.001Get rights and content

Abstract

Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial-specific enzyme, has been proved to be involved in oxidative stress-induced cell apoptosis, while little is known in cardiomyocytes. This study was aimed at investigating the role of ALDH2 in antimycin A-induced cardiomyocytes apoptosis by suppressing ALDH2 activity with a specific ALDH2 inhibitor Daidzin. Antimycin A (40 μg/ml) was used to induce neonatal cardiomyocytes apoptosis. Daidzin (60 μM) effectively inhibited ALDH2 activity by 50% without own effect on cell apoptosis, and significantly enhanced antimycin A-induced cardiomyocytes apoptosis from 33.5 ± 4.4 to 56.5 ± 6.4% (Hochest method, p < 0.05), and from 57.9 ± 1.9 to 74.0 ± 11.9% (FACS, p < 0.05). Phosphorylation of activated MAPK signaling pathway, including extracellular signal-regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38 was also increased in antimycin A and daidzin treated cardiomyocytes compared to the cells treated with antimycin A alone. These findings indicated that modifying mitochondrial ALDH2 activity/expression might be a potential therapeutic option on reducing oxidative insults induced cardiomyocytes apoptosis.

Introduction

Oxidative stress-induced myocardial apoptosis is one major culprit in the pathogenesis of cardiac failure [1]. During oxidative stress, intracellular redox balance is disturbed with the increased production and accumulations of ROS, which may cause mitochondrial dysfunction and consequent ATP reduction [2]. Recent findings suggested that ALDH2, a mitochondrial-specific enzyme, could restore mitochondrial function by transferring cytotoxic acetaldehyde into non-toxic acetate [3]. In contrast, ALDH2-deficient PC12 cells were observed to be highly vulnerable to antimycin A, an inhibitor of complex III and 4-HNE, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid in mitochondrial membranes [4], [5]. Decreased expression of ALDH2 was also found to enhance human umbilical vein endothelial cells injuries during acetaldehyde attacks [6]. Therefore, mitochondrial ALDH2 deficiency itself might be viewed as a kind of oxidative stress [7]. A mutant allele, ALDH2*2, a single point mutation (G/A) in exon 12 of the active ALDH2*1 gene, could result in glutamate to lysine substitution at position 487 followed by reduced enzymatic activity in vivo [8]. Since nearly 40% of the East Asian population were found to carry a semi-dominant polymorphism of the ALDH2 gene, ALDH2*2 [9] and individuals with the ALDH2*2 allele could be more vulnerable to oxidative insults and late-onset of Alzheimer's disease and other vascular syndromes, possibly due to increased apoptosis [6], it is therefore essential to clarify the association between ALDH2 deficiency and oxidative stress-induced apoptosis and underlying mechanisms. In this study, we tested the hypothesis that suppressing ALDH2 activity with a specific ALDH2 inhibitor Daidzin [10] may further aggravate antimycin A-induced myocardial apoptosis.

Section snippets

Cardiomyocyte culture

Cardiomyocyte culture was prepared as previously described [11] with modifications from neonatal rat hearts obtained from the Sprague-Dawley breeding colony at the Cardiovascular Center of Fudan University. The breeding animals were maintained according to the guidelines of the Guide for the Care and Use of Laboratory Animals and the culture protocol was approved by the Animal Care and Use Committee of the University. Under aseptic conditions, the ventricular apices were surgically removed and

Daidzin inhibited ALDH2 activity in a concentration-dependent manner

ALDH2 activity was inhibited by daidzin in a concentration-dependent manner (Fig. 1a). Daidzin at a concentration of 60 μmol/L reduced about 50% ALDH2 activity without significantly affecting cell vitality (Fig. 1a and b). Therefore, daidzin at this concentration was chosen for inhibiting ALDH2 enzymatic activity. Quantitative RT-PCR showed that ALDH2 mRNA expression was significantly reduced in A + D group compared to that in control group and A group (Fig. 1c).

Suppression of ALDH2 activity increased intracellular ROS levels in antimycin A-treated cardiomyocytes

To determine whether decreased

Discussion

Antimycin A, the inhibitor of mitochondrial electron transport chain, could promote ROS accumulation in mitochondria membranes followed by the generation of cytotoxic aldehyde and its derivatives such as 4-HNE [13]. In the present study, we found that suppressed ALDH2 activity by 60 μmol/L daidzin could significantly enhance antimycin A-induced cardiomyocytes apoptosis and ROS generation through activation of MAPK signaling pathway. In line with previous findings showing ALDH2 could partly

Conclusion

Taken together, our study suggested ALDH2 could protect cardiomyocytes against antimycin A-induced excess ROS production and apoptosis through downregulating the MAPK signaling pathway. Natural or synthetic ALDH2 analogues might be used as potential therapeutics for treating oxidative stress induced cardiac dysfunction.

Conflicts of interest statement

The authors have declared no conflicts of interest.

Acknowledgements

This study was supported by the Grant from the National Natural Science Foundation of China (30700317, 30725036 and 30971250), 863 program (2006AA02A406), Shanghai Basic Research Program (09QH1400500), and 973 program (2005CB523302 and 2006CB503803).

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