East Asian variant of aldehyde dehydrogenase 2 is related to worse cardioprotective results after coronary artery bypass grafting

Abstract

OBJECTIVES

Aldehyde dehydrogenase 2 activity is associated with cardioprotection. Individuals carrying an East Asian variant of the ALDH2 genotype (ALDH2*2) have significantly reduced aldehyde dehydrogenase 2 activity. No previous studies have determined the effect of the ALDH2*2 genotype on cardioprotective results after coronary artery bypass grafting (CABG).

METHODS

In total, 207 patients who underwent selective off-pump CABG were prospectively enrolled. Their baseline characteristics and clinical results were collected. Preoperative and postoperative circulating oxidative stress levels (serum malondialdehyde adducts and hydroxynonenal adducts) were measured. After genotyping, the oxidative stress levels and clinical results were compared between the ALDH2*2 carriers and non-carriers.

RESULTS

ALDH2*2 carriers exhibited higher levels of malondialdehyde (P = 0.02) and hydroxynonenal (P = 0.03) adducts after CABG. ALDH2*2 carriers had higher postoperative troponin I levels (P = 0.01) and 24-h inotropic scores (P = 0.02). The intensive care unit time (P = 0.03) and postoperative length of stay (P = 0.03) were longer in ALDH2*2 carriers. The postoperative pulmonary infection rate was higher (P = 0.03) in ALDH2*2 carriers.

CONCLUSIONS

Patients with the ALDH2*2 genotype had higher postoperative oxidative stress levels and poorer clinical results after CABG. Special cardioprotective techniques should be considered for patients with a history of ‘facial flushing’ when performing CABG.

ALDH2*2 genotype, Coronary artery bypass graft, Oxidative stress, Cardioprotection, Clinical results

INTRODUCTION

Despite advancements in surgical techniques, myocardial protection and perioperative care, the mortality rate after coronary artery bypass grafting (CABG) is still high [1–3]. Up to 50% of these adverse outcomes are mainly related to periprocedural myocardial injury [4, 5]. Coronary artery spasm is a known perioperative complication when performing CABG, and it is associated with myocardial injury, circulatory collapse and death. Refractory coronary spasm reportedly occurs in 0.8–1.3% of patients after CABG [6, 7], and transient coronary spasm has been shown to affect up to 11% of patients after CABG [8]. Thus, novel cardioprotective interventions are required to reduce cardiac injury after CABG.

Human aldehyde dehydrogenase 2 (ALDH2) is a 517-amino-acid polypeptide encoded by a nuclear gene located at chromosome 12q24 [9]. ALDH2 is a tetrameric enzyme present in organs that require high mitochondrial capacity for oxidative ATP generation, such as the heart and brain [10]. In 1 study, ALDH2 activation was sufficient to induce cardioprotection in a rodent myocardial infarction model; treatment with Alda-1, a selective activator for ALDH2, increased ALDH2 activity 2-fold and reduced the myocardial infarct size by 60% [11]. About 8% of the world’s population carries a common single-nucleotide polymorphism (rs671) in the ALDH2 gene, forming an ALDH2 loss-of-function allele (ALDH2*2 allele) [12]. ALDH2*2 carriers display dramatically reduced ALDH2 activity [13]. Mizuno et al. [14] recently found that the East Asian variant ALDH2*2 genotype was associated with coronary spastic angina. Coronary spastic angina is known to be associated with a wide variety of cardiac conditions, including myocardial injury and life-threatening arrhythmic events [15, 16]. However, no studies have been performed to investigate the relationship between ALDH2 genotypes and cardioprotective results after CABG.

PATIENTS AND METHODS

Participants and study design

This prospective observational cohort study was approved by the institutional review board of the Fuwai Hospital, and written informed consent was obtained from all participants (registered at the Chinese Clinical Trial Registry Center, http://www.chictr.org.cn/index.aspx; registration number ChiCTR1800015491). From September 2015 to August 2016, 233 consecutive patients with coronary artery disease were considered as candidates. The exclusion criteria were as follows: selective cardiopulmonary bypass CABG, lack of informed consent, concomitant valvular surgery, end-stage renal disease, prior cardiac surgery, New York Heart Association cardiac function Class ≥III, lung or liver function impairment and a switch to on-pump CABG. Finally, 207 participants who underwent selective off-pump CABG were enrolled in this cohort study. All enrolled patients were in stable condition, and no preoperative respiratory or inotropic support was used. Intraoperative anaesthetic, cardioprotective and operative strategies were standardized and performed by the same team in our centre. All participants were admitted to the same intensive care unit (ICU) after CABG and managed according to the same unit protocol.

Sample size calculation

To estimate the group size, a pilot study was conducted to measure the troponin I (TnI) level after off-pump CABG in 20 patients. After genotyping and grouping, we found 14 ALDH2*2 carriers and 6 ALDH2*2 non-carriers. The pooled standard deviation was 0.21 ng/ml. The difference between the 2 groups was 0.10 ng/ml. The sample proportion of the 2 groups was 7/3. With α = 0.05 (2-tailed) and power of 80%, we needed 170 patients. Considering an exclusion rate of 10%, 189 patients were needed to participate in this study. In reality, 233 candidates who underwent selective off-pump CABG were enrolled.

Blood specimen collection

Preoperative blood samples (3 ml) were obtained on the morning of surgery (6:00 AM). All surgeries were performed during the day. Postoperative blood samples (3 ml) were collected in the morning after surgery (6:00 AM), about 12–18 h after surgery. All blood samples were drawn from the peripheral vein. Specimens were centrifuged and stored in sealed vials at −80°C until analysis.

Clinical data collection

Clinical data were prospectively collected from the medical record system of Fuwai Hospital by trained study personnel. Baseline demographic characteristics, variables that can affect cardioprotection results and cardiac function, and detailed surgical and postoperative information were collected. Perioperative inotrope consumption was quantified by a score developed by Wernovsky et al. [17].

Genotyping and grouping

Genomic DNA was isolated from previously obtained whole-blood specimens with the QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany). Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as previously described [18]. After determination of the single-nucleotide polymorphism loci, genotyping was performed using a mass array system (Sequenom, San Diego, CA, USA) according to the manufacturer’s instructions. DNA samples were dispensed onto a 384-element SpectroCHIP Array using a Nanodispenser, and the SpectroCHIP Arrays were introduced into a MassARRAY Compact mass spectrometer. Automated spectra acquisition was then performed. Data analysis was performed using the MassARRAY Typer software version 4.0 (Sequenom). Repeated analyses were undertaken on 10% of randomly selected samples for quality control. Laboratory staff was blinded to the case status of the study participants. Patients were grouped according to their genotypes after genotyping.

Immunoassays and laboratory tests

Excessive accumulation of oxidative stress is accompanied by cardiomyocyte injury [19]. Serum malondialdehyde (MDA) adducts and hydroxynonenal (HNE) adducts were used to measure the oxidative stress status. MDA (Cell Biolabs, San Diego, CA, USA) and HNE (Cell Biolabs) adducts were measured in serum samples using human-specific enzyme-linked immunosorbent assays according to the manufacturer’s instructions. Other clinical tests were carried out at our hospital laboratory.

Statistical analysis

Statistical analysis was performed to compare the clinical outcomes between patients with different genotypes after genotyping and grouping. Continuous variables are summarized using means and standard deviations (or medians and interquartile ranges if the distributions were skewed). Numbers and percentages are used to summarize categorical data. For categorical variables, the χ² test was used to compare differences between groups. For continuous variables, data with a normal distribution and homogeneity of variance were compared by a t-test. If the distribution of the continuous variable was skewed, Wilcoxon’s rank sum test was used. All reported P-values are 2-sided, and P-values <0.05 were considered statistically significant.

RESULTS

Genotype distribution of ALDH2 and baseline characteristics

We enrolled 233 candidates who underwent selective off-pump CABG. Twenty-six patients were excluded because of a valvular lesion on ultrasonography that required concomitant valvular surgery (n = 5), end-stage renal disease (n = 4), prior cardiac surgery (n = 5) and a switch to on-pump CABG (n = 12). Finally, 207 patients were enrolled, and all were successfully genotyped and analysed. The genotype distribution of the ALDH2 gene did not depart from the Hardy–Weinberg equilibrium (P = 0.99). In total, 31.40% of the enrolled patients carried an ALDH2*2 allele. We found significant differences in the baseline characteristics or intraoperative variables that can affect cardioprotective outcomes between the patients with different genotypes (Table 1).

Table 1:

Baseline characteristics

Characteristics	ALDH2*2 non-carriers (n = 142)	ALDH2*2 carriers (n = 65)	P-value
Age (years), mean ± SD	61.92 ± 8.64	60.00 ± 8.95	0.14
Male gender, n (%)	112 (78.90)	53 (81.50)	0.66
BMI (kg/m2), mean ± SD	25.92 ± 2.81	26.18 ± 2.81	0.53
Diabetes, n (%)	50 (35.20)	32 (49.20)	0.06
Hyperlipidaemia, n (%)	98 (69.00)	43 (66.20)	0.68
Previous myocardial infarction, n (%)	43 (30.30)	18 (27.70)	0.70
Previous stent implantation, n (%)	20 (14.10)	8 (12.30)	0.73
Ventricular aneurysm, n (%)	2 (1.40)	2 (3.10)	0.59
Hs-CRP (mg/l), median (IQR)	2.39 (1.28–5.75)	2.09 (1.25–3.82)	0.60
Total cholesterol (mmol/l), mean ± SD	3.79 ± 1.09	3.82 ± 0.96	0.85
Triglycerides (mmol/l), mean ± SD	1.52 ± 0.77	1.63 ± 0.94	0.34
Albumin (g/dl), mean ± SD	43.24 ± 7.44	42.20 ± 6.48	0.33
Creatinine (μmol/l), mean ± SD	79.67 ± 18.94	81.49 ± 27.05	0.58
Smokers, n (%)	96 (67.60)	39 (60.00)	0.29
Alcohol habit, n (%)	51 (35.90)	19 (29.20)	0.35
AFS alcohol flushing syndrome, n (%)	22 (15.50)	51 (78.50)	<0.01
Left main stem stenosis, n (%)	37 (26.10)	17 (26.20)	0.99
1-vessel disease, n (%)	25 (17.60)	8 (12.30)	0.33
2-vessel disease, n (%)	40 (28.20)	21 (32.30)	0.54
3-vessel disease, n (%)	77 (54.20)	36 (55.40)	0.88
Use of CCB, n (%)	62 (43.70)	27 (41.50)	0.78
Use of aspirin, n (%)	51 (35.90)	23 (35.40)	0.94
Preoperative nitric esters, n (%)	133 (93.70)	60 (92.30)	0.72

AFS: alcohol flushing syndrome; BMI: body mass index; CCB: calcium channel blocker; Hs-CRP: high-sensitivity C-reactive protein; IQR: interquartile range; SD: standard deviation.

ALDH2*2 carriers had higher oxidative stress levels after coronary artery bypass grafting

The highly electrophilic aldehydes react with proteins to generate various adducts after surgery. The levels of circulating HNE and MDA adducts were used to represent oxidative stress states in this study. No significant difference in the baseline oxidative stress parameters was found between the patients with different genotypes (Table 2). ALDH2*2 carriers exhibited obviously higher oxidative stress levels after CABG [postoperative MDA: ALDH2*2 non-carriers, 95.36 (67.81–146.05) pmol/ml; ALDH2*2 carriers, 115.59 (79.22–161.93) pmol/ml; P = 0.02, non-parametric Wilcoxon test; postoperative HNE: ALDH2*2 non-carriers, 3.10 (1.91–4.86) µg/ml; ALDH2*2 carriers, 3.80 (2.23–7.08) µg/ml; P = 0.03, non-parametric Wilcoxon test] (Table 2).

Table 2:

ALDH2*2 carriers had higher levels of aldehyde adducts after coronary artery bypass grafting

Parameters	ALDH2*2 non-carriers (n = 142), median (IQR)	ALDH2*2 carriers (n = 65), median (IQR)	P-value
Preoperative MDA (pmol/ml)	91.63 (59.69–135.02)	85.05 (63.25–139.89)	0.95
Postoperative MDA (pmol/ml)	95.36 (67.81–146.05)	115.59 (79.22–161.93)	0.02
Preoperative HNE (μg/ml)	2.79 (1.75–5.18)	2.50 (1.37–5.36)	0.59
Postoperative HNE (μg/ml)	3.10 (1.91–4.86)	3.80 (2.23–7.08)	0.03

HNE: hydroxynonenal; IQR: interquartile range; MDA: malondialdehyde.

ALDH2*2 carriers had poorer cardioprotective results

ALDH2*2 carriers had significantly higher postoperative TnI levels [0.13 (0.05–0.43) ng/ml vs 0.07 (0.03–0.24) ng/ml, P = 0.01, non-parametric Wilcoxon test] and 24-h inotropic scores [5.00 (3.00–8.00) vs 3.00 (0.00–6.25), P = 0.02, non-parametric Wilcoxon test] (Table 3). The ICU time was longer in ALDH2*2 carriers [ALDH2*2 non-carriers 41.79 (22.08–64.51) h; ALDH2*2 carriers 45.57 (22.36–93.29) h; P = 0.03, non-parametric Wilcoxon test], as was the postoperative length of stay [ALDH2*2 non-carriers 7.00 (7.00–8.25) days; ALDH2*2 carriers 8.00 (7.00–10.50) days; P = 0.03, non-parametric Wilcoxon test]. The ratio of postoperative pulmonary infection was higher in ALDH2*2 carriers (ALDH2*2 non-carriers 2.10%; ALDH2*2 carriers 9.20%; P = 0.03, χ² test) (Table 3).

Table 3:

Clinical outcomes after coronary artery bypass grafting

Parameters	ALDH2*2 non-carriers (n = 142)	ALDH2*2 carriers (n = 65)	P-value
Postoperative TnI (ng/ml), median (IQR)	0.07 (0.03–0.24)	0.13 (0.05–0.43)	0.01
Postoperative 24 h inotrope score,a median (IQR)	3.00 (0.00–6.25)	5.00 (3.00–8.00)	0.02
Mechanical ventilation time (h), median (IQR)	17.00 (14.00–19.00)	16.00 (13.00–19.00)	0.85
ICU time (h), median (IQR)	41.79 (22.08–64.51)	45.57 (22.36–93.29)	0.03
Death, n (%)	0	0
Pulmonary infection, n (%)	3 (2.10)	6 (9.20)	0.03
Renal failure, n (%)	3 (2.10)	4 (6.20)	0.21
Hepatic failure, n (%)	2 (1.40)	2 (3.10)	0.59
ARDS, n (%)	2 (1.40)	4 (6.20)	0.08
Postoperative arrhythmia, n (%)	7 (4.90)	3 (4.60)	1.00
Major bleeding stroke, n (%)	3 (2.10)	5 (7.70)	0.11
Postoperative LOS (days), median (IQR)	7.00 (7.00–8.25)	8.00 (7.00–10.50)	0.03

a

The inotropic score is calculated by obtaining the total amount of inotropic support the patients received at each sampling point. A higher inotrope score indicates poorer cardiac function.

ARDS: acute respiratory distress syndrome; CPB: cardiopulmonary bypass; ICU: intensive care unit; IQR: interquartile range; LOS: length of stay; TnI: troponin I.

DISCUSSION

Coronary artery spasm can lead to perioperative myocardial injury, and it affects up to 11% of patients undergoing CABG [8]. Coronary spasm angina is a common disease affecting East Asians, while it is rare in Western populations [20]. A recent study showed that the ALDH2*2 genotype was associated with coronary spasm angina in East Asians [14]. The relationship between the ALDH2 genotype and cardioprotective results after CABG remains unknown. In the present study, we found that ALDH2*2 carriers had higher postoperative oxidative stress levels and poorer cardioprotective results after CABG.

Excessive reactive oxygen species accumulate in the myocardium during cardiac surgery. Most reactive oxygen species are highly reactive and short-lived, but reactive oxygen species can peroxidate unsaturated lipids to generate more stable aldehydes. The highly electrophilic aldehydes then react with biomolecules to generate various adducts, thus amplifying and propagating reactive oxygen species-initiated damage during cardiac surgery [21]. ALDH2 is the key enzyme in the metabolism of reactive aldehydes and is expressed abundantly in the myocardium [9]. Previous studies have shown that ALDH2 plays a key role in cardioprotection and that ALDH2 activation is correlated with reduced heart damage [11, 21].

ALDH2 is encoded by the ALDH2 gene. The ALDH2*2 allele exerts a dominant-negative effect over the wild-type ALDH2*1 allele. Patients with the heterozygous ALDH2*1*2 genotype have severely reduced ALDH2 activity and those with the homozygous ALDH2*2*2 genotype have negligible ALDH2 activity [22]. In the present study, 142 patients had the ALDH2*1*1 genotype, 59 had the ALDH2*1*2 genotype and 6 had the ALDH2*2*2 genotype. The genotype distribution of the ALDH2 gene in this study did not depart from the Hardy–Weinberg equilibrium (P = 0.99). In total, 31.40% of the enrolled patients carried an ALDH2*2 allele, which is similar to a previous study [12].

When individuals carrying an ALDH2*2 allele consume alcohol, they manifest in ‘facial flushing’ due to the impaired ALDH2 activity and aldehyde accumulation [12]. In line with this phenomenon, ALDH2*2 carriers accumulated more toxic aldehydes after CABG in our study. The levels of aldehyde adducts (HNE adducts and MDA adducts) were much higher in the ALDH2*2 carriers (Table 2). Our previous studies have shown that excessive aldehyde accumulation is associated with myocardial injury [21, 23]. The present study is the first to indicate that ALDH2*2 carriers with higher aldehyde levels had poorer cardioprotective results after CABG. ALDH2*2 carriers had higher cardiac TnI levels, a longer ICU time, a longer postoperative length of stay and a higher pulmonary infection rate. ALDH2*2 carriers also consumed more inotropes (Table 3). Higher cardiac TnI levels and greater inotrope use indicate poorer cardioprotective results and more severe myocardial injury. More severe myocardial damage leads to a longer ICU time and longer postoperative length of stay. The longer ICU time and longer postoperative length of stay increased the pulmonary infection rate in the ALDH2*2 carriers of our study (Table 3).

Previous studies have revealed that an increased oxidative stress level is associated with coronary spasm [19, 24]. Mizuno et al. [14] found that ALDH2*2 carriers are prone to developing coronary spasm. These reports are in high accordance with our results. In our study, we found that ALDH2*2 carriers had higher oxidative stress levels after CABG (Table 2). Coronary artery spasm is associated with poor cardioprotective results after CABG [6, 7], and elevated oxidative stress is related to coronary artery spasm [25]. We thus infer that ALDH2*2 carriers obtained poor cardioprotective results because of their impaired ALDH2 activity, toxic aldehyde accumulation and consequent coronary artery spasm. The detailed mechanism requires further study.

Coronary artery disease is very common. It is of great importance to reduce the myocardial injury caused by coronary stenosis. The present study established the relationship between the ALDH2 genotype and cardioprotective results after CABG. We found that ALDH2*2 allele carriers with impaired ALDH2 activity had higher postoperative oxidative stress levels and poorer cardioprotective results after CABG (Table 3). About 40% of East Asian populations carry an ALDH2*2 allele and thus have impaired ALDH2 activity [12]. Therefore, restoration of ALDH2 activity has great clinical significance. Chen et al. [11] identified a profound ALDH2 agonist, Alda-1, and this small compound can increase the activity of the mutant ALDH2*2*2 11-fold, the heterotetramer ALDH2*2*1 2.2-fold, and the wild-type ALDH2*1/*1 homotetramers 2.1-fold over basal activity. Patients subject to CABG or other events that induce cardiac ischaemia may benefit from the use of an ALDH2 agonist in the future, and ALDH2*2 carriers with negligible ALDH2 activity may gain more from the pharmacological enhancement of ALDH2 activity.

Limitations

Our study has several limitations. First, the sample size of the present cohort was relatively small. We only enrolled patients who underwent CABG performed by a single surgical team to avoid the impact of surgical skill on the outcomes. These findings require confirmation in large multicentre prospective studies. Second, no patient using cardiopulmonary bypass during CABG was enrolled. This is because cardiopulmonary bypass and hypothermia may influence the ALDH2 activity. Therefore, the conclusion of this study should be interpreted with caution when involving patients undergoing cardiopulmonary bypass CABG. A third limitation is that we did not measure myocardial ALDH2 activity. Myocardial tissue cannot be obtained during CABG; thus, we could not measure ALDH2 activity.

CONCLUSIONS

In the present study, patients carrying the East Asian variant of ALDH2 (the ALDH2*2 genotype) had significantly higher postoperative oxidative stress levels and higher TnI levels and consumed more inotropes after off-pump CABG. These patients also had poorer clinical results. These findings may inform surgeons that special cardioprotective techniques should be used when performing surgeries on patients with a history of ‘facial flushing’. This will become a classic example of precision medicine. An ALDH2 activator may also be used in the future to restore ALDH2 activity in ALDH2*2 carriers to improve the cardioprotective result.

ACKNOWLEDGEMENTS

The authors thank Xueli Yang from the Division of Biostatistics, National Center for Cardiovascular Diseases of China, for his assistance in statistical analysis. They also thank all the doctors and nurses at the Adult Cardiac Surgery Center, Fuwai Hospital, for their participation.

Funding

This study was supported by the National Natural Science Foundation of China [NSFC 81500238], the Peking Union Medical College (PUMC) Youth Fund and the Fundamental Research Funds for the Central Universities [3332016017 and 3332016014].

Conflict of interest: none declared.

Author notes

Dingxu Gong and Lin Zhang two authors contributed equally to this work.