Genetic and Functional Sequence Variants of the SIRT3 Gene Promoter in Myocardial Infarction

Coronary artery disease (CAD), including myocardial infarction (MI), is a common complex disease that is caused by atherosclerosis. Although a large number of genetic variants have been associated with CAD, only 10% of CAD cases could be explained. It has been proposed that low frequent and rare genetic variants may be main causes for CAD. SIRT3, a mitochondrial deacetylase, plays important roles in mitochondrial function and metabolism. Lack of SIRT3 in experimental animal leads to several age-related diseases, including cardiovascular diseases. Therefore, SIRT3 gene variants may contribute to the MI development. In this study, SIRT3 gene promoter was genetically and functionally analyzed in large cohorts of MI patients (n = 319) and ethnic-matched controls (n = 322). Total twenty-three DNA sequence variants (DSVs) were identified, including 10 single-nucleotide polymorphisms (SNPs). Six novel heterozygous DSVs, g.237307A>G, g.237270G>A, g.237023_25del, g.236653C>A, g.236628G>C, g.236557T>C, and two SNPs g.237030C>T (rs12293349) and g.237022C>G (rs369344513), were identified in nine MI patients, but in none of controls. Three SNPs, g.236473C>T (rs11246029), g.236380_81ins (rs71019893) and g.236370C>G (rs185277566), were more significantly frequent in MI patients than controls (P<0.05). These DSVs and SNPs, except g.236557T>C, significantly decreased the transcriptional activity of the SIRT3 gene promoter in cultured HEK-293 cells and H9c2 cells. Therefore, these DSVs identified in MI patients may change SIRT3 level by affecting the transcriptional activity of SIRT3 gene promoter, contributing to the MI development as a risk factor.


Introduction
Coronary artery disease (CAD) is a common complex disease, which is a leading cause of human death around the world. Myocardial infarction (MI) is a specific type of CAD.
Atherosclerosis, an inflammatory and metabolic disease, is the main cause for CAD and MI. Known risk factors include hypertension, smoking, diabetes, hyperlipoproteinemia and hypercholesterolemia. Though genome-wide association studies have identified more than 50 genetic loci to CAD, these genetic variants account for only 10% of cases [1][2][3]. To date, genetic causes and underlying molecular mechanisms for CAD remain largely unclear. It has been hypothesized that low frequency and rare variants with large effects may account for some of the missing heritability for CAD. Recently, epigenetic factors have been suggested to contribute to aging and age-associated diseases [4].
Sirtuins (SIRTs), a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, have been involved in a wide range of cellular processes, including aging, calorie restriction, stress resistance, apoptosis, inflammation, mitochondrial function and circadian clock. Dysfunctional SIRTs have been implicated in a variety of age-related diseases, including cardiovascular diseases [5,6]. Seven SIRTs, SIRT1-7, have been identified in mammals. SIRT3 is localized in mitochondria and has been extensively studied. Increased expression of SIRT3 gene has been associated with extended lifespan of humans [7][8][9]. SIRT3 regulates the enzymes involving in the respiratory chain, ATP production, fatty acid oxidation, tricarboxylic acid cycle and urea cycle, and reduces levels of reactive oxygen species (ROS) and oxidative stress [10][11][12]. Tissue-specific SIRT3 deletion studies suggest its non-tissue-autonomous roles [13]. Therefore, SIRT3 plays an important role in regulating mitochondrial function and metabolism. Lack of SIRT3 in experimental animal leads to several age-related diseases, such as cancer, metabolic syndrome, cardiovascular disease, and neurodegenerative diseases [14].
SIRT3 has been demonstrated to protect cardiomyocytes from aging, oxidative stress and dysregulated metabolism, and suppresses cardiac hypertrophy [15,16]. In cultured cardiomyocytes, SIRT3 inhibits myocardial reperfusion injury and reduces oxidative stress-induced apoptosis [17,18]. SIRT3 knockdown increases the susceptibility of cultured cardiomyocytes and adult hearts to ischemia-reperfusion injury [19]. In SIRT3-knockout mice, ROS levels are increased in the cardiomyocytes, indicating that SIRT3 prevents cellular ROS accumulation in the heart [20]. Mitochondrial permeability is also increased in the heart, leading to mitochondrial dysfunction [21]. SIRT3 is necessary for bone marrow cell-mediated cardiac repair in post-myocardial infarction [22]. In human endothelial cells, SIRT3 mediates the cellular response to hypoxia and protects the cells from high glucose-induced cytotoxicity [23,24]. In humans and animals, SIRT3 has been shown to regulate lipid metabolism and reduce lipid accumulation in diverse tissues, including heart [25][26][27]. SIRT3 has recently been reported to be involved in inflammation as well as platelet aging and thrombosis [28,29]. In addition, overexpressed SIRT3 enhances autophagy, which plays an important role in the development of cardiovascular diseases, including atherosclerosis, cardiac ischemia/reperfusion, cardiomyopathy and heart failure [30][31][32]. Therefore, SIRT3 may be involved in development of cardiovascular diseases through pathways of metabolism, inflammation and others.
SIRT3 gene expression has been studied and reported in several human diseases. SIRT3 gene expression is increased in hepatocellular carcinoma cells and human embryonic kidney cells (HEK-293) under hyperglycemic conditions [33]. Decreased SIRT3 expression has been reported in the patients with metabolic syndrome, non-alcoholic fatty liver disease, pulmonary arterial hypertension and type 2 diabetes [34][35][36][37]. Downregulated and upregulated SIRT3 gene expression has also been observed in different cancer tissues, depending on the cell and cancer types [38,39]. However, molecular mechanisms by which SIRT3 gene expression is changed have not been reported. We speculated that the DNA sequence variants (DSVs) within the regulatory regions of the SIRT3 gene may account for the changed SIRT3 gene expression. In this study, we genetically and functionally analyzed the promoter region of the SIRT3 gene in large cohorts of MI patients and healthy controls.

Patients and controls
All MI patients (n = 319, male 236, female 89, age range from 33 to 90 years, median age 60.03 years) were recruited between July, 2012 to November, 2014, from Cardiac Care Unit, Division of Cardiology, Affiliated Hospital of Jining Medical University, Jining Medical University, Jining, Shandong, China. All MI patients were diagnosed according to international guideline criteria: ischemic symptoms, ECG changes (ST-segment elevation or depression), typical rise of biochemical markers of myocardial necrosis (troponin or creatine kinase-MB), or coronary angioplasty. Patients with valvular heart disease, cardiomyopathy, myocarditis, or postrevascularization of the coronary arteries were excluded from this study. Ethnic-matched healthy controls (n = 322, male 172, female 150, age range from 21 to 82 years, median age 48.97 years) were recruited from the same hospital. Controls with familial MI history were excluded. In this study, 342 MI patients and 346 controls were initially recruited. Since DNA sequencing for some samples were failed, total 319 MI patients and 322 controls were finally included. The research was carried out according to the principles of the Declaration of Helsinki. This study was approved by the Human Ethic Committee of Affiliated Hospital of Jining Medical University. Written informed consents were obtained.

DNA Sequence analysis
Peripheral leukocytes were isolated and genomic DNAs were extracted. SIRT3 gene promoter (1200bp upstream to the transcription start site) was sequenced and analyzed. Two overlapped DNA fragments, 764bp (-1200bp~-437bp) and 678bp (-498bp~+180bp), were generated by PCR. PCR primers were designed based on genomic sequence of the human SIRT3 gene (NCBI, NC_000011.10), which were shown in Table 1. PCR products were bi-directionally sequenced with Applied Biosystems 3500xL genetic analyzer. The DNA sequences were then aligned with the wild type sequence of the SIRT3 gene promoter.

Functional analysis of the DSVs by dual-luciferase reporter assays
Expression vectors were constructed by subcloning wild type and variant SIRT3 gene promoters into luciferase reporter vector (pGL3-basic) and dual-luciferase activities were examined. Briefly, DNA fragments of wild type and variant SIRT3 gene promoters (1179bp, from -999bp to +180bp to the transcription start site) were generated by PCR and then subcloned into KpnI and Hind III sites of pGL3-basic to generate expression vectors. The PCR primers with KpnI or HindIII sites were shown in Table 1. Designated expression vectors were transiently transfected into human embryonic kidney cells (HEK-293, from ATCC, CRL-1573) or rat cardiomyocyte line cells (H9c2, from ATCC, CRL-1446). After 48 hours, the luciferases activities of the transfected cells were measured using dual-luciferase reporter assay system on a Promega Glomax 20/20 luminometer. Expression vector pRL-TK expressing renilla luciferase was used as an internal control for transfection. Empty vector pGL3-basic was used as a negative control. The transcriptional activities of the SIRT3 gene promoter were represented as ratios of luciferase activities over renilla luciferase activities. All the experiments were repeated three times independently, in triplicate.

Statistical analysis
The quantitative data were represented as mean ± SEM and compared by a standard Student's t-test. DSV frequencies in MI patients and controls were analyzed and compared with SPSS v13.0. P<0.05 was considered statistically significant.
PCR primers are designed based on the genomic DNA sequence of the SIRT3 gene (NC_000011.10). The transcription start site (TSS) is at the position of 236362 (+1).

Discussion
To date, few studies have associated the SIRT3 gene with human diseases. A SNP rs11246020 (NC_000011.10:g.233067C>T) in the human SIRT3 gene, a nonsynonymous point mutation (V208I) which reduces its enzymatic activity, may enhance the susceptibility to metabolic syndrome [35]. A SNP rs11555236 (NC_000011.10:g.233212C>A) in intron 5 of the SIRT3 gene, which increases expression of SIRT3 gene, has been associated with extended lifespan of humans [7]. In this study, we have identified a total of 23 DSVs within the promoter of the human SIRT3 gene. Six novel heterozygous DSVs and two SNPs were identified in MI patients, but in none of controls (Table 2 and Fig 2). Three SNPs were found in MI patients with significantly higher frequencies compared to controls ( Table 2). These DSVs and SNPs, except the DSV g.236557T>C, significantly decreased the transcriptional activities of the SIRT3 gene promoter in both H9c2 cells and HEK-293 cells (Figs 4 and 5). Therefore, these SIRT3 gene promoter DSVs may reduce SIRT3 levels, contributing to the MI development as risk factors.
The human SIRT3 gene is localized to the chromosome 11p15.5, and encodes an NADdependent mitochondrial deacetylase of 399-amino acids containing an N-terminal mitochondrial targeting signal and a central catalytic domain [40][41][42]. SIRT3 gene is expressed in a variety of tissues with higher expression in adipose tissue, brain and heart in embryos and adults (NCBI Unigene EST Profile Viewer), indicating the tissue-specific regulation of the SIRT3 gene expression. The human SIRT3 gene promoter contains high GC contents and lacks the TATA box sequence and there are binding sites for activator protein 1 (AP1), GATA-binding factor, nuclear Factor κB (NF-κB) and transcription factor ZF5, as well as multiple specificity protein 1 (SP1) binding sites [43]. Nuclear respiratory factor 2, a transcription factor that regulates mitochondrial genes, binds to the promoter of SIRT3 gene and induces its expression [44]. In this study, the DSVs reduced the SIRT3 promoter transcriptional activity in HEK-293 cells and H9c2 cells to different extents, which may be due to the tissue-specificity of the SIRT3 gene expression and related transcription factors. Therefore, expression of the human SIRT3 gene may be manipulated for therapeutic purposes.

Conclusions
In this study, we genetically and functionally analyzed the SIRT3 gene promoter. The DSVs and SNPs of the SIRT3 gene promoter identified in MI patients may alter transcriptional activity of SIRT3 gene promoter and change SIRT3 level, contributing to the MI development as a risk factor. The investigation into the molecular mechanisms by which SIRT3 gene promoter activity are affected are being conducted in our laboratory. Therefore, our findings may provide a genetic basis for further translational and clinical studies for MI patients.