Alteration of metabolic genes in peripheral blood isolated from patients with acute myocardial infarction

Accumulating evidence suggests that molecular alterations of peripheral leucocytes are possible diagnostic markers of acute myocardial infarction (AMI). Changed lipid/glucose metabolism is a prominent feature of the pathogenesis of AMI. Silent mating type information regulation 2 homolog 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator factor-α (PGC-1α) regulate mitochondrial function and energy metabolism. Thus, the gene expression of SIRT1 and PGC-1α in peripheral leucocytes isolated from AMI patients and their association with dyslipidemia have been evaluated. Fifty-five male subjects were divided into 40 patients with AMI and 15 healthy control subjects. Peripheral blood samples were obtained on the first day of AMI. Lipid profile parameters were assessed spectrophotometrically. Relative SIRT1 and PGC-1α expression were measured by real-time PCR. Compared with the control group, SIRT1 and PGC-1α expression were significantly decreased in the AMI group. SIRT1 expression was significantly negatively correlated with the age of the participants. SIRT1 expression was significantly positively correlated with PGC-1α expression. Both SIRT1 and PGC-1α expression were negatively correlated with markers of dyslipidemia. In conclusion, SIRT1 and PGC-1α expression are reduced in the acute phase of AMI, which addresses their possible role as potential biomarkers for AMI.


INTRODUCTION
Ischemic heart disease (IHD) is a major cause of death and disability worldwide. Acute myocardial infarction (AMI) is responsible to the major percent of IHD deaths due to its accompanied fatal complications 1 . Recently, gene expression studies are gaining attention in revealing potential molecular biomarkers for AMI detection 2, 3 . Peripheral leucocytes were found to exhibit an alteration in their gene expression profile in patients with AMI 4 .
Disrupted lipid/glucose metabolism is associated with the oxidative stress and inflammatory response encountered in the pathophysiology of atherosclerosis and plaque instability 5 . Moreover, AMI is associated with augmented fatty acid (FA) oxidation and increased myocardial oxygen consumption resulting in complications such as arrhythmias or reduced cardiac contractility 6 .
Silent information regulator factor 2-related enzyme 1 (SIRT1) is NAD + dependent deacetylase, which can deacetylate both histone and non-histone proteins to regulate multiple cellular functions including energy metabolism, autophagy, apoptosis, and cellular senescence 7 . SIRT1 controls the cellular energy metabolism through the deacetylation and activation of the master metabolic regulator, the peroxisome proliferator-activated receptor gamma coactivator factor-α (PGC-1α) 8 . PGC-1α upregulates the expression of several genes of the tricarboxylic acid cycle and mitochondrial FA oxidation pathway 9 . Additionally, the SIRT1/PGC-1α pathway plays a role in regulating the activity of antioxidant enzymes 10 .
Previous reports described the role of SIRT1/PGC-1α in protecting against myocardial ischemia in which activation of SIRT1 and/or PGC-1α was associated with smaller infarct size and better post-AMI outcome 11-13 . However, this study was conducted to address the possible diagnostic role of SIRT-1 and PGC-1α expression in peripheral leucocytes isolated from AMI patients and the association between these markers and the dyslipidemic profile observed in AMI patients.

Subjects
A total of 55 men were enrolled in the study, 40 were AMI patients and 15 were healthy control subjects. The blood was collected on the first day of the infarction. Patients were recruited from the Intensive Care Unit, Cardiology Department, Ain Shams University Educational Hospitals, Cairo, Egypt. Exclusion criteria included inflammatory diseases, autoimmune disorders, malignancies, hematological diseases, skeletal muscle diseases, hepatic or renal diseases, acute or chronic infections, or administration of immunosuppressive drugs.
The study was conducted in accordance with the regulations and recommendations of the Declaration of Helsinki and was approved by the Faculty of Pharmacy, Ain Shams University Ethical committee (Ph.D.: no. 22). An informed consent was obtained from all participants.

Sample preparation
Fasting peripheral blood samples were obtained from all participants. Samples were divided into 2 aliquots; the first aliquot was collected on plain vacutainer for lipids profile analysis. The second aliquot was collected on EDTA for immediate total RNA purification from the human whole blood.

Lipid profile analysis
Lipids profile parameters were measured colorimetrically by enzymatic methods 14-18 according to kits provided by Salucea, Netherlands using (Schimatzu 1650 UV/Visible, Japan).

RNA isolation and real-time quantitative PCR
Total RNA was extracted and purified from human whole blood using a QIAamp RNA blood mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and stored at -80 • C until use. The mRNA expression levels of PGC-1α, SIRT1 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control were measured by quantitative one-step QuantiFast Probe QRT-PCR Kit (Qiagen, Hilden, Germany) using Stratagene MX 3000P thermal cycler (Stratagene, USA) according to the manufacturer's protocol. The analysis was performed using the MxPro-Mx 3000P software (Stratagene, USA). The expression levels in an unknown sample were normalized and analyzed by the 2 −ΔΔCt methods, where ΔΔCt = (Ct target gene − Ct GAPDH) sample − (Ct target gene − Ct GAPDH) calibrator.

Statistical Analysis
The IBM statistical package for social sciences (SPSS) statistics (V.23, IBM Corp., USA, 2015) and GraphPad Prism 7 (La Jolla, CA, USA) were used for data analysis. Data were presented as mean±SEM. Comparisons between groups were done using Students t-test. Spearman correlation test was used for correlation analysis. p<0.05 was considered statistically significant.

Clinical characteristics
Demographic and clinical characteristics of all participants are shown in Table (1). Total cholesterol (TC), triacylglycerol (TAG) and lowdensity lipoprotein-cholesterol (LDL-C) were significantly higher in the AMI group when compared with the control group (all p= 0.000). On the other hand, the high-density lipoprotein cholesterol (HDL-C) was significantly lower in AMI patients in comparison with the control group (p= 0.008).

SIRT1 expression
As shown in Fig. 1, SIRT1 relative expression showed a significant decrease in the AMI group (0.49±0.03) compared with the control group (1.07±0.12) at p<0.0001.

DISCUSSION
Early identification of AMI is necessary for early treatment interventions, which can significantly reduce the mortality rate. Many studies have been conducted to seek potential molecular biomarkers for AMI detection 2 . Ischemia reduces cellular NAD + due to the downregulation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD + synthesis, which leads to the decreased expression of SIRT1 19 . This mechanism may probably explain the low blood SIRT1 expression in AMI patients when compared to the control group. Consistently with our results, Chan et al. claimed decreased SIRT1 mRNA and protein expression in monocytes isolated from patients with CAD 20 . Breitenstein et al. showed that peripheral SIRT1 expression was significantly lower in both AMI and CAD patients when compared to the healthy subjects, but with no significant difference between both patients groups 21 .
Interestingly, the expression of PGC-1α in blood was found to be correlated with its myocardial expression. Thus, detection of blood expression of PGC-1α may provide a non-invasive method to monitor metabolic changes in the heart during myocardial ischemia 22 . We found lower blood PGC-1α in AMI patients when compared with the control group. The myocardial tissue of experimental animals had lower PGC-1α expression levels after AMI 23 . In contrary to our study, Fabregat-Andres et al. revealed that PGC-1α expression is induced in the monocytes of patients with AMI 24 .
In this study, a significant negative correlation was observed between SIRT1 expression and the age of the studied groups. Previously, SIRT-1 showed significantly lower expression in vascular smooth muscle cells obtained from old donors when compared with young donors with a strong significant negative correlation with the age of the donors 25 . These age-related changes contribute to plaque instability and trigger AMI 26 .
Dyslipidemia is a major risk factor for AMI, even more, detrimental for AMI risk in men than in women 27 . SIRT1 mRNA was negatively correlated with TC and LDL-C. SIRT1 regulates hepatic lipid metabolism. It inhibits sterol regulatory element binding protein-1c (SREBP-1c) with subsequent inhibition of lipogenic genes which results in inhibition of lipid synthesis and fat storage 28 . Concomitantly, SIRT1 activates liver X receptor (LXR), thus, enhances reverse cholesterol transport 29 . Therefore, low SIRT1 expression may be accompanied by dyslipidemia.
The role of PGC-1α in augmenting FA oxidation is prominently identified. It stimulates FA transporters expression as well as upregulates the expression of enzymes involved in FA oxidation 30 . Therefore, less FA is available for esterification and TAG formation. This fact comes in agreement with the present study in which PGC-1α expression showed a negative correlation with TAG. On the other hand, hepatic cholesterol synthesis was reduced in relation to activation of the PGC-1α 31 . The former study may explain the negative association between blood PGC-1α and TC levels.
In conclusion, peripheral expression of SIRT-1 and PGC-1α may provide diagnostic keys for patients with AMI. Their pathophysiologic role of the SIRT-1/PGC-α pathway in relation to dyslipidemia in AMI patients should be evaluated.