Renin-Angiotensin Aldosteron System Genes Polymorphisms and Acute Heart Failure

Purpose: The Renin Angiotensin Aldosteron System (RAAS) is one of the major systems involved in the pathophysiology of heart failure. In this study we investigate the association between the renin angiotensin aldosteron system genes polymorphisms and acute heart failure (AHF) and we evaluate the role of serum ACE activity. Methods: We included 300 patients over 20 years old admitted to the Emergency Department for acute dyspnea. According to the clinical findings and results of the B-type natriuretic peptide (BNP level), patients were divided into two groups: heart failure (HF) and non- heart failure (non-HF) groups. Genotyping was done by PCR-RFLP. The level of serum ACE activity was determined by spectrophotometric method using hippuryl histidyl leucine. We found the diagnosis of AHF in 50% of the population. Results: No association between the renin polymorphism and AHF, but we found an association between ACE ID and CYP11B2 polymorphisms and heart failure. Level of serum ACE activity is significantly higher in the group of AHF. Conclusion: We can conclude that blood ACE activity, ACE ID and CYP11B2 can be a marker of acute heart failure.


Introduction
Heart Failure (HF) is an ever increasing problem worldwide and is a major health problem associated with very high morbidity and mortality [1]. It can be defined as a complex clinical syndrome that results from any structural or functional disorder of the heart to achieve the ability of the ventricles to fill or eject blood [2]. The main events of the HF is dyspnea and fatigue, that limit exercise tolerance and induce water retention that may lead to pulmonary congestion and peripheral edema [3]; these two anomalies hinder the functional capacity and quality of life of those affected [4]. Acute heart failure may result from the development of a variety of arrhythmias; acute coronary syndrome, a rapid increase in the work of a failing heart by conditions such as infection, anemia, and pulmonary embolism [5], discontinuation of treatment of chronic HF and progression of the underlying disease [6]. High blood pressure can also bring on heart failure by causing left ventricular hypertrophy [7]. The Renin-Angiotensin System (RAS) is a central regulator of cardiovascular and renal functions and plays an important role the kidneys, will convert angiotensinogen to angiotensin I, which then will become angiotensin II. Then aldosterone will be released from the adrenal cortex, leading to sodium and water retention and an increase in circulating blood volume. In the persistence of this mechanism without control, it can aggravate heart failure as the heart struggles to pump against the increased volume. Genetic The deleterious effect of this kinase on β adrenergic receptor signaling has been also explored in mice, showing that GRK2 gene deletion could prevent the development of post-MI HF and restore heart function (Am J Cardiol. 2011 Apr 15;107:1125-30). Given the important homeostatic actions of the RAAS on the cardiovascular system, it is not surprising that the synthesis and secretion of (pro-) renin are tightly controlled [10]. To date, few published studies have examined the association between the polymorphism of this system and HF. Renin is the key enzyme at the top of the Angiotensin RAS, it cleaves the N terminus of circulating angiotensinogen to angiotensin I, which is then transformed into Angiotensin II by soluble or endothelial cell-associated Angiotensin-Converting Enzyme (ACE) [11]. The ACE ID polymorphism, on intron 16, results from the insertion (I) or deletion (D) of a 287-base pair Alu sequence [12] and the D allele has been linked to an increased plasma ACE activity [13], and a higher risk for heart failure [14].
The association between the polymorphism of the renin gene C-4063 T and HF has not been investigated previously. On the other hand, the aldosterone synthase gene, CYP11B2, encodes for a cytochrome P450 enzyme, involved in the terminal steps of aldosterone synthesis in the zona glomerulosa cells of human adrenal glands and its expression is regulated by angiotensin II and potassium [15].
The candidacy for this gene is based on its pathogenic role in the syndrome of glucocorticoid-remediable aldosteronism [16].
Aldosterone excess is a well-documented cause of hypertension and there is convincing evidence that mineralocorticoids have adverse effects in heart failure [5].The key enzyme in aldosterone synthesis is aldosterone synthase. The corresponding gene CYP11B2 is located on chromosome 8. A cytosine/thymidine (C/T) substitution in the 5' promoter region at location _344 of the CYP11B2 gene has been identified. The functionality of this variant is unclear [1]. Several common polymorphisms have been described in the CYP11B2 [17]. The C-344 T polymorphism, which is located at a putative binding site for the steroidogenic transcription factor (SF-1), has been associated with hypertension [18]; this locus may be important in blood pressure and cardiovascular regulation [17].
In the present study, we investigated the association, for the first time, between renin, ACE ID and aldosterone polymorphisms in North African patients admitted to the emergency department for dyspnea and AHF and we evaluated the role of serum ACE activity.

Methodology
Subjects and study design: This is a prospective descriptive study in the emergency department of Monastir. We included all patients aged over 20 years consulting in the emergency for acute dyspnea. We excluded all patients with cardio-respiratory arrest, coma and shock. At the time of study entry, family history, physical examination findings, medical therapy, and results of prior cardiac testing were collected. Patients were assigned to two groups: the

ACE I Activity
The spectrophotometric method using Hippuryl Histidyl Leucine (HHL), describes an enzymatic assay and detection by ultraviolet ACE I blood. This is the original method of Cushman and Cheung, 1971. Angiotensin converting enzyme is a glycoprotein peptiylpeptide hydrolase which section the histidyl leucine dipeptide angiotensin I in a relatively inactive decapeptide. The reaction is carried out at 37 °C and pH 8.3. The substrate (HHL) is transformed by the action of ACE histidyl leucine + hippuric acid.

Hippuryl-histidyl-leucine hippurate+ histidyl-leucine
After extraction of hippuric acid with ethyl acetate hippurate assayed by reading the optical density at a wavelength of 228 nm.

Five milliliters of venous blood was collected with Ethylene
Diamine Tetra Acetic Acid (EDTA) as anticoagulant. The genomic DNA was extracted from the peripheral leucocytes using the standard phenol-chloroform extraction method.

Renin C-4063T Polymorphism
Renin C-4063T polymorphism was using the following primers:

Statical Analyses
The SPSS version 18.0 program was used for the statistical analysis, and the significance level was set at 5%. The Pearson chi-square test was used to determine the association between categorical variables. The odds ratio (OR) and their 95% Confidence Interval (CI) were also calculated. Differences between frequencies were assessed using the chi-square test, with a p-value of 0.05 or less was considered as significant.

Study subjects
We included 300 patients, a HF group (n=150; 50%); and a Non HF group (n=150; 50%). The characteristics of study participants are shown in Table 1, with a mean age of 67.5 ±13 years and a mean left ventricular ejection fraction of 59% for the Non HF group and 46% for the HF group. All patients were NYHA (New York Heart Association) class III or IV at the time of admission. Clinical exams findings were similar between the two groups. Compared with the Non-HF patients, HF patients showed a significantly higher prevalence of conventional risk factors for heart failure, including high blood pressure, diabetes and smoking. The HF group also showed significantly higher levels of NT-pro BNP, lipids and troponin.

Angiotensin-Converting Enzyme ID Polymorphism
The ACE polymorphism has a potential role as a modifier gene in heart failure; in the present study, we found an association between ACE ID polymorphisms and a higher risk of HF (p=0.021, OR=1.922, CI 95% [0.269-2.993]), furthermore, the DD genotype is associated with the risk of HF in our study's population. Like our finders, many studies have found that the DD genotype can be associated with the risk of HF in Swedish patients [19], and Caucasian's patients [20] also it can reduce the cardiac function and increased cavity size in South African patients with cardiomyopathy [21]. Another study on Chinese patients, despite they confessed that the study's population was not in Hardy-Weinberg equilibrium, approved the presence of this association [22]. Most studies did not find an association between the ACE polymorphism and heart failure.
Bleumink and colleague did not find an association to ischemic or idiopathic dilated cardiomyopathy with the ACE ID polymorphism [1]. Yamada et al. also did not detect an association between ACE genotypes and hypertrophic cardiomyopathy in Japanese, neither in Caucasian study published by Marian [23] and a second one where Bilsel confirmed the association between the D allele and dilated cardiomyopathy in Caucasian population [24]. These results also were confirmed on a Chinese population [25] and black South African subjects [26,27]. In addition, no association was found between ACE genotypes and echocardiographic measurements [28]. However Schunkert and colleagues described an association between DD genotype and electrocardiographic left ventricular hypertrophy in an European population [29].

Serum ACE Activity
The serum ACE activity measured is higher in the HF group with  [30] and in particular myocardial infarction [31].

Aldosterone Synthase C-344 T Polymorphism
Few studies have described the association between heart failure and C-344 T polymorphism. In our study we found an association between the CYP11B2 polymorphism and the HF group with a significant p=0.025 and an OR=1.267, CI95% [0.478-3.359]. Studies in Caucasian and Japanese patients did not find an association between this variant and idiopathic dilated cardiomyopathy [32].
However, the C allele was associated with increased left ventricular volume in the Japanese patients [33]. But this finding was not confirmed in black South Africans with heart failure [26]. Studies in hypertrophic cardiomyopathy also yielded conflicting results [26,34]. Consequently, there is no firm evidence for the role of this polymorphism in heart failure [1]. In young healthy Finns, the C-344 T variant seems to be associated to the left ventricular hypertrophy [18]. Although one small study confirmed this association [35], other larger studies failed to detect a significant effect of this polymorphism on left ventricular structure [36,37]. Mayosi et al.
investigated the contribution of several markers in the CYP11B2 gene, including the C-344 T polymorphism, as individual variants and haplotypes [38]. No significant association was detected with cardiac mass [1].

Renin C-4063 T Polymorphism
Heart Failure (HF) is often complicated by decreased renal blood flow and a subsequent decrease in glomerular filtration rate. In our study we didn't found any association between the renin C-4063 T polymorphism and HF (p=0,294). Decreased renal function is one of the strongest predictors of mortality in patients with advanced systolic HF [39]. The evidence for a potential effect of renin blockade in diastolic heart failure is rare [10]. Mtiraoui  there was no asso¬ciation between renin -4063T allele or -4063C/ T genotypes and diabetic nephropathy [40]. The RFLP analysis of the study of Shuchi among an Indian Population demonstrated no significant difference between high altitude pulmonary edema and acclimatized control subjects' groups for the Renin C(-4063)T polymorphism [41]. Data related to Renin C-4063T polymorphism and its association with essential hypertension is also available [42].

Limitations
Some potential limitations should be noted to our study. We

Conclusion
Renin C-4063 T polymorphism showed no association with the risk of developing heart failure in this study. ACE ID and CYP11B2 polymorphisms can serve as a marker for early diagnosis of heart failure as previously reported. In addition, blood ACE activity can be a marker of acute heart failure.