EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 35, No. 5, 336-349, October 2003 Gene expression profiling of oxidative stress on atrial fibrillation in humans Young Hoon Kim1*, Ji Hye Lee2* picture of the interactions between AF and oxi- Do Sun Lim1, Wan Joo Shim1 dative stress. Total RNAs prepared from the re- 33 Young Moo Ro1, Gil Hong Park2 trieved tissues were used to synthesize P-la- Kevin G. Becker3, Yoon S. Cho-Chung4 beled cDNAs by reverse transcription and hybri- and Meyoung-kon Kim2,5 dized to cDNA microarrays. Gene expression pro- files showed that 30 genes were upregulated and 1Department of Internal Medicine 25 were downregulated in AF patients compared 2Department of Biochemistry with control patients. Moreover, comparison rank College of Medicine, Korea University analysis revealed that the expression of five genes Seoul, 136-701 Korea related to reactive oxygen species (ROS)-including 3DNA Array Unit, National Institute on Aging flavin containing monooxygenase 1, monoamine National Institutes of Health oxidase B, ubiquitin specific protease 8, tyro- Baltimore, MD 21224-6820 sinase-related protein 1, and tyrosine 3-monooxy- 4Cellular Biochemistry Section genase-increased by more than 2.0 of the Z-ratio, Basic Research Laboratory, CCR and two genes related to antioxidantsincluding National Cancer Institute glutathione peroxidase 1, and heme oxygenase National Institutes of Health 2-decreased to the Z-ratio levels of ≤ -2.0. Appa- Bethesda, MD 20892-1750 rently, a balanced regulation of pro- and anti-oxi- 5Corresponding Author: Tel, 82-2-920-6184; dation can be shifted toward pro-oxidation and Fax, 82-2-923-0480; E-mail, [email protected] can result in serious damage similar to that of *The first two authors contributed equally to this work. human AF. Western blotting analysis confirmed the upregulation of tyrosinase-related protein 1 Accepted 24 July 2003 and tyrosine 3-monooxygenase and the downre- gulation of heme oxygenase 2. These results sug- Abbreviations: AF, atrial fibrillation; cDNA, complementary deoxyri- gested that the gene expression pattern of myo- - bonucleic acid; O2 , superoxide anion; ROS, reactive oxygen spe- cardial tissues in AF patients can be associated cies with oxidative stress, resulting in a significant increase in ROS. Thus, the cDNA microarray technique was useful for investigating transcrip- tion profiles in AF. It showed that the intracellular Abstract mechanism of oxidative stress plays a pivotal role Atrial Fibrillation (AF) is thought be caused by in the pathologic progression of AF and offers oxidative stress. Oxidative stress at the cellular novel insight into potential treatment with antioxi- level results from many factors, including expo- dants. sure to alcohol, medications, cold, toxins or ra- diation. In this study we investigated gene trans- Keywords: atrial fibrillation; cDNA microarray; gene criptional profiles on the human myocardial tis- expression profile; oxidative stress sues from AF and oxidative stress conditions. Right atrial appendages were obtained from AF Introduction patients (n = 26) undergoing the Maze procedure, and from control patients (n = 26) who were in Atrial fibrillation (AF), the most prevalent sustained normal sinus rhythm and undergoing coronary ar- arrhythmia, affects more than 900,000 Koreans and tery bypass graft operation. To examine the effec- more than 5 million people worldwide. Its incidence ts of oxidative stress on AF, we used radioactive increases with age and it is present in structural heart complementary DNA (cDNA) microarrays to evalu- disease (Kopecky et al., 1987). The prevalence of AF ate changes in the expression of 1,152 known increases strikingly with advancing age; it occurs in genes. This technology, which monitors thou- approximately 2% of men (2.2%) and women (1.7%) sands of genes simultaneously, gives us a better older than age 20, and 5% of those older than age Oxidative stress related genes in atrial fibrillation 337 65 (Prystowsky et al., 1996). Recent data suggest The atrial tissue subjected to rapid atrial pacing show- that AF-related hospital stays are markedly greater ed direct evidence of increased oxidative stress (in- than for any other arrhythmia. Nevertheless, informa- creased 3-nitrotyrosine formation), and ascorbate was tion about its incidence and prevalence in a general able to minimize this effect (Carnes et al., 2001). population is sparse. Although several events that contribute to oxidative AF-associated morbidity is related to excessive stress are well-established consequences of sustained ventricular rate and systemic embolization, and may AF, underlying mechanisms and relationships between cause syncope, fatigue, or cardiomyopathy. AF pa- AF and oxidative stress by high-throughput appro- tients have marked atrial dilatation and atrial myocyte aches are ill-defined. Recent developments in genome hypertrophy with increased interstitial fibrosis and fatty sciences have led to the development of DNA micro- deposition. It may be seen in normal subjects, par- array technology, a tool of unprecedented power for ticularly during emotional stress or following surgery, the study of gene sequence, structure, and expres- exercise, or acute alcoholic intoxication (Mihm et al., sion. Using cDNA microarrays, the expression of 2001). AF is a frequent postoperative complication of thousands of genes can be monitored simultaneously cardiac surgery, with a reported 20% to 50% inci- and expression patterns compared cDNA microarrays dence, increasing the risk of stroke. Patients under- have been developed to reveal the gene expression going coronary atrial bypass graft surgery have patterns of up- or downregulated genes in response increased plasma lipid peroxidation and decreased to various biological stimuli. The new technology cardiac glutathione levels following release of cross allows automated imaging analysis and is well suited clamp, and these changes persist for at least 24 h for the large-scale study of gene expression patterns after cardiac surgery. Similarly, increased free-radical in vitro and in vivo (Park et al., 2002). production in canine heart subjects lead to rapid ven- In this study, we compared gene transcription pro- tricular pacing, and antioxidants can improve cardiac files in postoperative AF patients with non-AF con- function in animals with pacing-induced failure (Carnes trols. Using a cDNA array approach, we assessed a et al., 2001). Free radical is formed via the diffusion causal relationship between oxidative stress and AF rate-limited reaction of nitric oxide and superoxide in accordance with the hypothesis that increased anion, and is known to oxidize cellular lipids, proteins, oxidative stress may underlie AF derived from post- and DNA, and to promote cardiac cell death via operative oxidative stress and electrophysiological re- necrosis and/or apoptosis. modeling such as atrial pacing. In this study, we assessed a causal relationship between reactive oxygen species (ROS) and AF. Because any oxygen-involving free radical can be Materials and Methods referred to as ROS (Goldfarb, 1999). ROS play im- portant roles in many cardiovascular pathologies, Patients including atrial fibrillation, atherosclerosis, and others. Atrial appendages were obtained as surgical speci- These molecules are so reactive that they act in situ mens from patients undergoing cardiac surgery using very close to where they are generated. Therefore, procedures approved by Korea University Medical most cell structures-including membranes, structural Center. All patients gave informed consents. Right proteins, enzymes, and nucleic acids-are vulnerable artrial appendages were obtained from 26 patients in and can become targets for mutation and cell death permanent AF (＞ 1 month at the time of surgery) (Robert K et al., 1996). Major cellular ROS included undergoing the Maze procedure and mitral valve singlet oxygen (O2), nitric oxide (NO), superoxide - repair (mean age 50, range 25-68 years). Control anion (O2 ), hydrogen peroxide (H2O2), hydroxyl ra- data were obtained from the right appendages of 26 ․ ․ dicals (OH ), alkoxyl radicals (RO ), peroxyl radicals patients in normal sinus rhythm with no history of AF ․ (ROO ), and lipid peroxides (LOOH). During periods and undergoing cardiac surgery (mean age 53, 27-65 of high oxidative stress or tissue injury, loss of ROS years). Surgeries were performed between January control can occur and ROS formation is favored 2000 and July 2001. Table 1 details clinical charac- (Beckman and Koppenol, 1996). teristics of AF patients. Several study have tried to identify the origin and characterize the AF associated with oxidative stress that results in a significant ROS increase. Michael et Human cDNA microarray al., found that myofibrillar creatine kinase, an impor- A human cDNA microarray was primarily derived from tant controller of myocyte contractility, is highly sen- a commercially available master set of approximately sitive to oxidative injury, and that increased oxidative 15,000 human verified sequences (Research Genetics, stress and energetic impairment during AF could Inc., Huntsville, AL). The 15,000-human cDNA clone contribute to contractile dysfunction (Mihm et al., 2001). set was sorted for a list of genes (1,152 elements) 338 Exp. Mol. Med. Vol. 35(5), 336-349, 2003 Table 1. Baseline characterisitics of the patient with atrial fibrillation. µl (specific activity: 200,000 U/ml) of Superscript II ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ reverse transcriptase