T⁻⁷⁸⁶→C polymorphism of the endothelial nitric oxide synthase gene is associated with insulin resistance in patients with ischemic or non ischemic cardiomyopathy

Insulin resistance (IR) is a common condition in patients with both ischemic and non-ischemic cardiomyopathy. It may be secondary to heart failure (HF) syndrome but also contributes to the disease [1–3]. As IR represents a potentially reversible alteration, its early identification may have a crucial role in HF prevention and treatment [4, 5].

Insulin resistance has often been linked to endothelial dysfunction, defined as paradoxical or inadequate endothelial-mediated vasodilation [6–8]. Specifically, IR has been associated with the decreased synthesis and/or release of nitric oxide (NO), as occurs in many clinical conditions including HF, or with its exaggerated consumption due to high tissue levels of reactive oxygen and nitrogen species in a condition of altered glucose and lipid metabolism [7]. Alternatively, genetically determined deficiency of endothelial-derived NO could predispose to IR. In an experimental study, Duplain et al. showed that knockout mice for the endothelial NO synthase (eNOS) gene were insulin-resistant and hypertensive, suggesting that eNOS is important not only for cardiovascular control but also for glucose homeostasis [9]. In humans, a thymidine-to-cytosine (T to C) transition mutation (T⁻⁷⁸⁶→C) in the promoter region of the gene has been associated with reduced eNOS expression [10] and has been linked to IR both in non-diabetic subjects and Type 2 diabetic patients [11, 12].

Based on these premises, we hypothesized that genetic eNOS impairment could be an independent determinant of IR in patients with HF. Accordingly, in the present study we tested the reciprocal relationships between T⁻⁷⁸⁶→C eNOS polymorphism and IR in patients with ischemic and non-ischemic cardiomyopathy fully characterized for cardiac function and other cardiovascular risk factors.

This study provides evidence that the T⁻⁷⁸⁶→C polymorphism in the promoter region of eNOS gene is the major independent determinant of IR in patients with both ischemic and non- ischemic cardiomyopathy. Interestingly, among all classic cardiovascular risk factors, IR is the only one independently associated with the extent of LV dysfunction and the presence of HF in this population. Taken together, our results suggest that in either ischemic or non- ischemic cardiomyopathy, IR may occur preferentially in patients with a genetic predisposition to endothelial dysfunction (eNOS gene polymorphism) and may worsen the severity of the cardiac disease. Specifically, the mutual order among (genetically determined) endothelial dysfunction, IR and LV dysfunction was found independently of etiology of cardiac disease.

A link between insulin homeostasis and endothelial function has been long documented [6–8]. Endothelial function is modulated by insulin through the stimulatory effects of the hormone on NO production [6, 8]. Nitric oxide is synthesized from L-arginine by a family of enzymes called NO synthases (NOSs). The constitutively expressed eNOS gene, which maps on the 7q35–36 chromosome, is mainly expressed in endothelial cells. It has been shown that eNOS activity is modulated by insulin through a series of phosphorylations triggered by the hormone binding with its receptor on the endothelial cell membrane. Specifically, the insulin signaling cascade leads to a specific phosphorylation of eNOS that increases the enzyme activity [8].

Nevertheless, some experimental data suggest that primary abnormalities of eNOS function may influence insulin homeostasis. In fact, eNOS knockout mice have been reported to be hypertensive and insulin resistant [9, 15, 16]. One elegant study in a mouse model of eNOS partial knockout showed that the partial deletion of the gene does not alter per se insulin sensitivity and blood pressure. However, when challenged with nutritional stress, partial eNOS deficiency facilitates the development of IR and arterial hypertension, providing further evidence for the importance of this gene in predisposing to glycometabolic and vascular abnormalities [17]. Anyway, data showing a clear, exact causal order between eNOS gene expression, hypertension and insulin resistance are unavailable. Likewise, the mechanisms by which the primitive endothelial alteration can affect glucose homeostasis and systemic blood pressure are not fully known.

In keeping with the above experimental findings, in a population of patients with ischemic and non-ischemic cardiac disease, we found a clear correlation between eNOS gene promoter polymorphism, the occurrence of an insulin-resistant phenotype and the presence of arterial systemic hypertension. Given the location of -786T>C in the promoter region of the eNOS gene, it may affect eNOS expression levels. Actually, lower eNOS mRNA and serum nitrite/nitrate levels have been found in individuals with the -786C variant [9], and reporter gene studies have supported this functional role [18, 19].

One possible hypothesis linking the primitive impairment of eNOS function with insulin resistance states that endothelial dysfunction causing systemic NO depletion may affect systemic vascular tone, leading to a decrease in blood flow to the myocardium and muscles. This in turn may reduce myocardial and muscular glucose uptake. As a result, high glucose levels in the blood stimulate insulin secretion and in the long run may cause IR and diabetes. Therefore, a genetic variation that affects NO regulation may contribute to both alteration in vascular tone and to IR. Consistent with this hypothesis, few clinical studies have shown a significant association between that T⁻⁷⁸⁶→C polymorphism in the promoter region of the eNOS gene and IR in both non-diabetic subjects and Type 2 diabetic patients [11, 12]. In this framework, our study confirms and extends previous results in patients without known diabetes but with systolic LV dysfunction.

An additional finding of the present study was that in our population IR, which occurred preferentially in subjects with eNOS gene polymorphism, was also an independent determinant of a more severe cardiac dysfunction with HF, irrespective of etiology. A recent study showed that the T⁻⁷⁸⁶→C promoter polymorphism was specifically associated with a significant reduction in eNOS mRNA expression in myocardial tissue obtained from failing human myocardium, while a different eNOS polymorphism G(894)-->T of exon 7 was not. The authors concluded that the reduced eNOS expression associated with the promoter gene polymorphism might be involved in the pathogenesis of cardiac failure [12]. Our findings did not discover a direct relationship between eNOS polymorphism and the severity of LV dysfunction; however, these conditions were linked by the presence of IR.

Insulin resistance, often manifested clinically through the feature of the metabolic syndrome or type 2 diabetes mellitus, has reached epidemic levels in many nations throughout the world [5, 20]. Furthermore the presence of diabetes mellitus is more than 7 times as potent risk factor for mortality in the non-ischemic and ischemic cardiomyopathy population [21]. Certainly, the eNOS defective gene cannot be the only cause leading to insulin resistance and cardiovascular damage. Rather, our results suggest that this condition might create an individual substrate where the addition of other common factors (such as high fat diet or altered lipid profile) is poorly tolerated and enough to predispose to development of insulin resistance and more severe cardiac damage.

Our study findings should be interpreted bearing in mind some limitations. Firstly, we acknowledge that our sample size may temper statistical estimations in some categories. Secondly, genetic and acquired factors able to condition the presence and the extent of cardiac damage and the development of HF in different individuals are multiple and interactions are complex. Accordingly, adequate experimental models and large longitudinal clinical studies are needed to better elucidate the pathogenetic and prognostic relevance of these observations.

Nevertheless, this study first shows a clear link between a genetic endothelial dysfunction and abnormalities in glycometabolic profile in patients with both ischemic and non-ischemic cardiomyopathy. Confirming the impact of a defective eNOS gene in the development of IR and HF will have major clinical relevance for identifying subjects at higher risk among patients with cardiac dysfunction.