Role of SNPs of CPTIA and CROT genes in the carnitine-shuttle in coronary artery disease: a case-control study

Introduction

The heart is an organ of high energy demand and requires continuous ATP production in order to maintain its contractile function, basal metabolic processes, and its ionic balance. The main energy fuels used by the heart are carbohydrates and fats. ATP production at large part of the normal adult heart (approximately 95%) is provided by mitochondrial oxidative phosphorylation and the rest is by GTP generated from glycolysis and tricarboxylic acid cycle (TCA) [1]. There is substantial evidence that affecting cardiac mitochondrial energy production by changing long-chain fatty acid oxidation, can influence to heart diseases. In obese Zucher rat, it was observed that the long-chain fatty acyl-CoAs cannot be oxidized in the condition of the fatty acid uptake by the cell exceeds exceeds β-oxidation. The resulting lipid accumulation leads to contractile dysfunction [2]. Sharma et al. observed that lipid accumulation in myocardium cause to increase of toxic lipid intermediates leading to lipotoxicity [3]. Zhou et al. reported that the disruption of cardiac function and energy metabolism by lipotoxicity may contribute to the development of progressive myocardial atrophy and protein breakdown [4]. In cardiac tissue, the acetyl-CoA carboxylase 2 (ACC2) isoform is an important regulator of mitochondrial fatty acid uptake by carnitine palmitoyltransferase-I (CPTI). Essop et al. reported that the Acc2-mutant mice had about decreased 25% left ventricular mass compared with Acc2-wild types [5].

Fatty acid entry into mitochondria via carnitine shuttle in order to produce energy by β-oxidation is carried out by three different proteins [6], [7]. CPTI is localized within intermembrane space of the inner mitochondrial membrane. Acyl-CoA catalyzes separation of acyl which is connected to CoA-SH group and transfer to OH group of carnitine at intermembrane space. Carnitine forms an ester-bond with long chain carboxylic acids through this protein [8]. Carnitine palmitoyltransferase 2 (CPTII) is located on the matrix side of the inner membrane surface and catalyzes retransfer of an acyl group, which is previously transferred by CPTI, from carnitine to CoA [6], [7]. Carnitine-acylcarnitine translocase, which is located on the mitochondrial membrane, provides acyl-carnitine entry into the mitochondria while carnitine is being moved out as antiport reaction [6]. Carnitine octanoyl transferase (CROT) is a kind of acyltransferase which catalyzes the reversible transfer of acyl groups between carnitine and CoA [9].

Carnitine palmitoyltransferase I, which is encoded by CPT1 gene is rate limiting enzyme for β-oxidation of fatty acids. CPT1 is inhibited by malonyl-CoA protein which is expressed by CPTIA gene and it provides rate-control of β-oxidation in all tissues [10]. Three isoforms of CPT1 gene were described. CPTIB is expressed in muscle while CPTIA is found in most of the tissues and especially in the brain [8]. It is lethal when CPTIA and CPTIB are knocked out and is still unknown the exact role of CPT1 in hemostasis of energy [11], [12]. However, several studies have been performed to emerging the function of CPTIA in different metabolic disorders. It was shown that the spontaneous mutations in CPTIA gene result in repeated hepatic insufficiency of CPTAI, hypoketotic hypoglycemia, hepatomegaly, stroke, and coma. Blockage of CPTI activity by selected inhibitors protects the heart from fatty-acid induced ischemic injury and changed the activity of hypothalamic CPTI by molecular and pharmacological approaches effects endogen production of glucose have been declared in previous studies [13].

Carnitine octanoyl transferase which is encoded by CROT gene transforms 4,8-dimethyl-nonanoyl-CoA to adequate carnitine ester. This transesterification process takes place at peroxisome and it is required for transport of medium and long-chain acyl-CoA molecules from outside of peroxisome to cytosol and mitochondria. Thus, the protein product of CROT gene has an important role in lipid metabolism and fatty acid β-oxidation [9].

β-Oxidation of fatty acids is the main energy source used to maintain contractile function in especially heart and skeletal muscle [14]. Therefore, the defect/dysregulation in the β-oxidation in the heart may result in a decrease in energy input of the heart. In many studies, it was investigated the association of CPTIA gene variations with the obesity [15] and lipid disorders [8], [16]. However, the effects of the variations in both CPTIA and CROT genes on coronary artery disease (CAD) risk factors have not been investigated. Martin et al. [17] studied activities of CPTI and CPTII enzymes in the myocardial tissue from patients with congestive heart failure, and from control donor hearts. They reported that CPTI and CPTII activities decreased in patients and they suggested that deficiency of CPT enzyme activity may be related to ventricle function. Therefore, in the present study, we questioned whether the combination of CPTIA rs3019613-G>A and CROT rs2214930-T>C gene variations and risk of CAD and association with lipid parameters in patients with CAD. This is the first study investigating the effect of variations in both CPTIA and CROT genes on lipid/obesity parameters in CAD.

Materials and methods

Results

Discussion

CAD which is the most important cause of mortality in developed countries develops over years. CAD is the leading cause of myocardial ischemia in consequence of vessel narrowing due to an atherosclerotic process which includes inflammatory cytokines and modifications of low density lipoprotein (LDL) such as oxidation, acetylation, glycosylation [18], [19], [20]. The heart needs to produce high energy for its contractile function, basal metabolic processes and its ionic balance by ATP production (approximately 95%) via mitochondrial oxidative phosphorylation and GTP generated from glycolysis and TCA [1].

Recently, it was reported that changes in cardiac mitochondrial energy metabolism contribute to contractile dysfunction and to a decrease in cardiac efficiency [1], [21]. Carnitine palmitoyltransferase IA (CPTIA) provides fatty acid transport into the inner mitochondrial membrane via binding carnitine. Carnitine is removed when fatty acids enter the inner mitochondrial stage and metabolism of fatty acids starts. CPTIA controls fatty acid influx and oxidative pathways via esterification and malonyl-CoA sensitivity, respectively [16]. Binding of malonyl-CoA to the regulatory site of CPTIA allosterically inhibits acylcarnitine formation while it prevents the transition of fatty acids into mitochondria. Inhibitors of CPTI act as regulators of fatty acid biosynthesis via this mechanism. Inhibition of CPTI might be proposed to have a protective effect against cardiac hypertrophy and heart attack via reducing fatty acid oxidation as well [22].

It was proposed in a study with donor and receptors of cardiac transplantation that total CPT activation and carnitine deficiency might be associated with ventricle function and this association might be useful in order to establish a link between CPT enzymes and heart diseases [17].

Homozygous CPTIB knockout (CPTIb^+/+) mice die in the embryonic stage due to cardiac structure and function. In a pressure stimulated aortic contraction study with heterozygous CPTIB knockout (CPT1b^+/-) mice, heart defects, mitochondrial anomalies and myocardial lipid accumulation were observed depend on pressure level. These findings indicate that cardiac pathology increases depend on lipotoxicity under pathologic stress in CPTIb deficiency [22].

Limited numerous studies have been found yet relating to CPT1A polymorphism and obesity/lipid association [8], [16], [23]. However, this is the first study investigating the effect of a polymorphism in CPTIA gene on lipid/obesity parameters in CAD patients. The role of CPTI gene variations in heart diseases has been CPTI is a key enzyme in the fatty acid oxidation process, it has been proposed to have an association with healthy obesity with the contribution of SNPs. No association was reported between CPTIA polymorphisms and obesity/lipid profiles in Japanese type 2 diabetes patients by Hirota et al. [23], while CPTIA A275T (rs17610395) SNP was found related to BMI (p=0.05) and waist circumference (p=0.008) in French-Canadian cohorts by Robitaille et al. [15]. Furthermore, Rajakumar et al. [16] investigated the effects of CPTIA P479L variant on metabolic parameters such as serum lipid profile, blood pressure in Arctic populations. They reported that the L479 allele in the P479L variant was associated with elevated fasting HDL-C and ApoA1 levels in Greenland Inuit. In a recent study from the Center of Alaska Native Health Research (CANHR) which was carried out with Yu’pik Eskimo population, polymorphisms at CPTIA gene has been found to be associated with anti-obesity phenotypes such as decreased body fat and adiposity. It also has been proposed to have a cardioprotective effect due to the association of increased fasting levels of HDL-C [8].

The present study is consistent with suggesting studies CPTIA gene variations are associated with obesity and HDL-C levels. We found that CPTIA rs3019613-A allele associated with the increased BMI (BMI≥27) in CAD group (p=0.03), while HDL-C threshold (HDL-C ≤0.90 mmol/L) was found lower (p=0.015). In addition, the logistic regression analysis confirmed CPTIA rs3019613-A allele has a significant relationship with decreased serum HDL-C levels (p=0.009, Table 6) and increased BMI values (p=0.062, Table 6) in CAD group. No association was observed between CPTIA rs3019613 genotypes and metabolic risk thresholds in the control group. Carnitine-O-octanoyl transferase (CROT) enzyme which is a kind of carnitine/choline acetyltransferase takes part in lipid metabolism and fatty acid β-oxidation by catalyzing transesterification of 4,8-dimethyl nonanoyl-CoA to carnitine ester in peroxisome [13]. It is vital for the transfer of medium and long-chain acyl-CoA molecules. There has been no study yet assessing variations in carnitine-O-octanoyl transferase (CROT) gene which encodes CROT in terms of lipid profile in diabetes and atherosclerotic diseases. CROT rs2214930 T>C polymorphism, CC genotype was found to be associated with low HDL-C (p=0.029) in healthy controls. These findings suggested that the CROT rs2214930 T>C polymorphism might have an effect on HDL-C levels. However, this association was not found in CAD patient group. We think that the effect of rs2214930 CC genotype on HDL-C may be masked due to the patient group is under statin therapy.

The limitations of the study include relatively small sample of the study groups. We believe that our findings will be more meaningful and more concrete in a study group on a wider scale, and will guide the future work in this context.

The present study is a preliminary study to establish whether CPT1A and CROT polymorphisms have individual effects in the pathogenesis of CAD in the Turkish population. Based on our findings, we propose that rs3019613 and rs2214930 variations in CPTIA and CROT genes which encode key proteins in fatty acid metabolism might have important effects on serum lipid profile, obesity, and blood pressures. Therefore, these results suggested that the CROT rs2214930 T>C and CPTIA rs3019613 G>A SNPs may contribute to the increased CAD risk dependently other metabolic parameters.