EFFECTS OF CARVEDILOL TREATMENT ON CLINICAL AND INSTRUMENTAL PARAMETERS IN PATIENTS WITH ESSENTIAL HYPERTENSION IN RELATION TO ADRENERGIC RECEPTOR POLYMORPHISMS

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(1)UNIVERSITY OF PISA CARDIOTHORACIC DEPARTMENT PhD THESIS in “CLINICAL PATHOPHYSIOLOGY OF CARDIOVASCULAR AND RESPIRATORY SYSTEMS” President: Prof. A. Mussi ________________________________________________________. EFFECTS OF CARVEDILOL TREATMENT ON CLINICAL AND INSTRUMENTAL PARAMETERS IN PATIENTS WITH ESSENTIAL HYPERTENSION IN RELATION TO ADRENERGIC RECEPTOR POLYMORPHISMS. Tutor:. Candidate:. Prof. ROBERTO PEDRINELLI. Dr. KATYA V. LUCARELLI. Academic Year 2006-2007.

(2) INDEX. INDEX .............................................................................................................................. 2 ABBREVIATIONS .......................................................................................................... 4 1.0 INTRODUCTION ...................................................................................................... 5 1.1 Pharmacogenomic approach to antihypertensive therapy ....................................... 5 1.2 Genetic polymorphisms in adrenergic receptors ..................................................... 6 1.2a β-adrenergic receptor polymorphisms and their functional role ........................ 7 1.2b α-adrenergic receptor polymorphisms and their functional role ...................... 11 1.3 Adrenergic receptor polymorphisms and β-blocker treatment of hypertension .... 12 2.0 END-POINTS OF THE STUDY.............................................................................. 15 3.0 PATIENTS AND METHODS.................................................................................. 16 3.1 Patient enrolment................................................................................................... 16 3.2 Study protocol ....................................................................................................... 17 3.3 Clinical amd instrumental evaluations .................................................................. 19 4.0 STATISTICAL ANALYSIS .................................................................................... 21 5.0 RESULTS ................................................................................................................. 22 5.1 Baseline characteristics of patients and genotype results...................................... 22 5.2 Case and control groups: baseline characteristics and hemodynamic effects of treatments .................................................................................................................... 23 5.3 Responses to monotherapy by genotype groups ................................................... 23 5.4 Responses to monotherapy stratified by codon 49 and codon 389 genotypes of 1-AR........................................................................................................................... 24 6.0 DISCUSSION........................................................................................................... 26 6.1 Baseline analysis ................................................................................................... 26 6.2 Responses to carvedilol ......................................................................................... 28. 2.

(3) 7.0 CONCLUSION......................................................................................................... 32 8.0 PERSPECTIVES ...................................................................................................... 33 REFERENCES ............................................................................................................... 35 TABLES ......................................................................................................................... 45 FIGURES........................................................................................................................ 60 ACKNOWLEDGEMENTS............................................................................................ 67. 3.

(4) ABBREVIATIONS. A= atrial diastolic peak velocity ABPM= Ambulatory Blood Pressure Monitoring AR= Adrenergic Receptor BMI= Body Mass Index BP= Blood Pressure DAP= Diastolic Arterial Pressure DecT= E velocity Deceleration Time E= Early diastolic peak velocity Em= myocardial Early diastolic velocity FS= Fractional Shortening HF= Heart Failure HR= Heart Rate IVRT= Isovolumic Relaxation Time LV= Left Ventricle or Left Ventricular LVM= Left Ventricular Mass LVMI= Left Ventricular Mass Index RWT= Relative Wall Thickness SAP= Systolic Arterial Pressure Sm= myocardial Systolic velocity TD= Tissue Doppler. 4.

(5) 1.0 INTRODUCTION. 1.1. PHARMACOGENOMIC. APPROACH. TO. ANTIHYPERTENSIVE. THERAPY. Hypertension affects about 25% of the adult population in industrialised countries (1,2); it is a complex disease with both genetic and environmental factors contributing to its pathogenesis (2). Hypertension can be readily detected and treated but, if left untreated, it leads to several and sometimes lethal complications. In fact, optimal control of blood pressure values is essential in preventing risk for cardiovascular disease development, i.e. stroke, coronary artery disease, heart and kidney failure. Improvements in the diagnosis and treatment of hypertension have provided the most significant contribution to the decrease of cardiovascular mortality which has occurred in the last twenty years (1,3). In spite of it all, in up to 95% of the cases its etiology is still unknown, thus hypertension in most cases is treated unspecifically using an empirical approach, resulting in a large number of minor side effects and in a relatively high non compliance rate (40%-50%) (1,3). Three major factors contribute to the complex therapeutic approach to hypertension: 1. There is scanty knowledge of the genetic mechanisms underlying the pathogenesis of essential hypertension; furthermore, similar phenotypes, in terms of blood pressure level and organ complications, may arise through different molecular mechanisms in distinct subsets of patients. 2. The difficulty in matching the different mechanisms of action of so many available antihypertensive drugs with the molecular mechanisms underlying essential hypertension in each patient weakens the rational basis for prescribing a given drug to a given patient. 3. As a consequence, a high interindividual variation in the blood pressure response to drug administration and a variety of unpleasant side effects occur. Even though individual differences in drug response may be due to age, sex, lifestyle, health condition and drug interactions, it is reasonable to assume that genetic factors also affect both the efficacy of a drug and the likelihood of an adverse reaction (4,5). New opportunities now exist for overcoming the above-mentioned limitations of current empirical therapy of essential hypertension based on the development of a pharmacogenomic approach to this therapy (4,6,7,8). The pharmacogenomic approach. 5.

(6) consists in the identification of the genetic–molecular mechanisms underlying hypertension in a given subset of patients and in the development of drugs which are able to interfere with such mechanisms, thus leading to very selective therapeutic interventions with enhanced efficacy and reduced side effects. An individualised therapy of hypertension, based on the knowledge of the genetic background of a single patient, could play a key role in the therapeutic management of hypertension disease since its early phases (9,10,11). For this purpose, over recent years there has been an increase of studies investigating the association between genetic polymorphisms and essential hypertension phenotypes. A polymorphism is a genetic variant in a DNA sequence that appears in at least 1% of a given population (6,7). Nowadays there is a systematic search to identify functionally significant variations in DNA sequences (significant polymorphisms) in genes that influence the effects of various drugs (4); among them, angiotensin-converting enzyme (ACE) (12,13), angiotensinogen (14), angiotensin II receptor 1 (15), adrenergic receptors (16,17), alpha-adducin (18,19), G-protein beta-3 subunit (20), atrial natriuretic peptide (21), eNOS (22) and endothelin 1 gene polymorphisms (23) are of particular relevance. Nevertheless, the results of these studies seem controversial (24) and, even though different responses to antihypertensive therapy are well-documented, there has been limited progress towards identifying individual patient characteristics that predict blood pressure response prior to drug administration (25). So far, candidate genes influencing blood pressure responses to pharmacological therapy are those coding for components of a the drug-targeted system (25), i.e. autonomic nervous system. In particular, the sympathetic nervous system and - and adrenergic receptors have a great relevance in the pathophysiology of essential hypertension (16,26,27). In fact, in response to the cathecolamine activation, adrenergic receptors influence blood pressure homeostasis through the regulation of cellular metabolism, hormone production, cardiac output and peripheral resistance (28).. 1.2 GENETIC POLYMORPHISMS IN ADRENERGIC RECEPTORS. Adrenergic receptors (ARs) are members of the G-protein-coupled receptor superfamily, characterized by 7-transmembrane domains. Multiple subtypes of. 6.

(7) adrenoceptors exist on the basis of their affinity profile with pharmacological agents, their amino-acid sequences and their specific cellular messengers. In particular, there are 3 known receptor types (α1-, α2- and β-AR) and 9 molecular subtypes (α1a, α1b, α1d; α2a, α2b, α2c; β1, β2 e β3) (29). The β1-ARs are located mainly in heart, kidney and adipose tissue whereas the β2-ARs in heart, lung, gastrointestinal tract, liver, pancreas and skeletal muscle. When stimulated by agonists, β1- and β2- cardiac ARs primarily activate guanine nucleotidebinding (Gs)proteins, which in turn transduce intracellular signals via activation of the adenylyl-cyclase pathway; this results in increased intracellular cAMP levels and, consequently, in higher cardiac output through increased cardiac inotropy and chronotropy (30). The β3-ARs are located mainly in adipose tissue, but their role is less well defined (31). The various subtypes of α-ARs can affect the control of the vascular tone in different ways. All the AR genes are polymorphic, and genetic polymorphisms in ARs may contribute to intersubject differences in pharmacological response (28).. 1.2a. β-ADRENERGIC. RECEPTOR. POLYMORPHISMS. AND. THEIR. FUNCTIONAL ROLE β1-ADRENERGIC RECEPTOR POLYMORPHISMS β-ARs located on cardiomyocytes are the most powerful means of enhancing the contractile performance of the heart (31). Normally β1-ARs represent approximately 70% (atrial site) to 80% (ventricular site) of the whole amount of cardiac β-ARs (29); thus, cardiac catecholamines effects and cardiac output are mainly affected by this receptor subtype. In 1987 the β1-AR gene (10q24-26) was cloned (32; 33). Two common polymorphisms resulting in amino-acid substitutions in the gene locus were identified in 1999: Ser49Gly and Arg389Gly (34); other allelic variants of this receptor, including Ala59Ser, Arg399Cys, His402Arg, Thr404Ala and Pro418Ala polymorphisms, are much rarer (35) (Figure1). At position 49 in the extracellular amino-terminus of the receptor, a serine is substituted by a glycine (Ser49Gly) with an allele frequency of 0,87 and 0,13 respectively (34). It is. 7.

(8) not clear if this polymorphic variant of the β1-AR has significant cardiovascular phenotypic consequences in hypertensive patients. In-vitro studies using cell lines transfected with the Ser49/Gly49 variants show that long-term agonist-promoted downregulation is greater for Gly49-β1AR compared to Ser49-β1AR (36); on the other hand, after short-term agonist exposure, isoproterenol potency for adenylyl cyclase activation seems higher on membranes expressing Gly49-β1AR than on those expressing Ser49-β1AR (37). Ranade et al (38) found that in a cohort of Chinese and Japanese individuals the Ser49Gly polymorphism was significantly associated with resting heart rate, independent of other variables (i.e. body-mass index, age, sex, race). Their data showed that Gly49 homozygotes had the lowest heart rate and each Ser49 allele increased the basal heart rate in an additive model (38). At position 389 in the intracellular carboxy-terminus, near the seventh transmembrane region of the receptor (which is a putative Gs-protein binding domain), an arginine is substituted by a glycine (Arg389Gly) with an allele frequency of 0,75 and 0,25 respectively (34,39) (Figure1). In vitro, the Arg389 variant mediates a higher isoproterenol-stimulated adenylyl cyclase activity than the Gly389 variant, through an enhanced receptor-Gs coupling (40). Furthermore, in a study of transgenic mice with cardiac-specific overexpression of Arg389Gly β1AR variants, Mialet Perez and colleagues (41) demonstrated that hearts from young Arg389 mice had enhanced receptor function and contractility compared to Gly389 hearts. In vivo, the Arg389 variant appears to be linked with heart failure in both clinical outcomes and response to therapy (42). Moreover, Bengtsson and colleagues (39) suggest that the Arg389Arg genotype confers an increased risk of developing hypertension; in their study, the Arg389 allele and the Arg389Arg genotype of the β1-AR gene were more common in patients with hypertension than in controls. Investigating the relation between the Arg389Gly β1-AR polymorphism and acute myocardial infarction (AMI), others (43) demonstrated that the prevalence of the Arg389Arg genotype was significantly more frequent in patients with AMI than in controls; since AMI is characterized by augmented sympathetic activity, these authors suggested that the hyperresponsive phenotype of Arg389Arg may play a key role in the pathogenesis of infarction (43). Nevertheless, not all in-vivo data support such statements (44); further studies are needed in order to clear the functional importance of the Gly389Arg β1-AR polymorphism especially in clinical outcomes and response to therapy.. 8.

(9) β2-ADRENERGIC RECEPTOR POLYMORPHISMS In humans, β2-ARs mediates physiologic responses, including vasodilatation, bronchial smooth-muscle relaxation and lipolysis in various tissues. The β2-AR gene is localized to chromosome 5 (33). Several polymorphisms are known both in regulatory and coding region of the β2-AR gene: the most common ones involve the coding region (4 polymorphisms) and the short open reading frame, termed the 5' Leader Cystron (5'LC), which is 102 bp upstream of the β2AR coding block (1 polymorphism) (Figure2 and Figure3). The 5’LC region codes for a 19-aminoacid peptide that regulates the mRNA translation and the expression of the β2-AR (45); at position 19 in this peptide, an arginine (denoted wild-type) is substituted by a cysteine (Arg19Cys). In vitro studies show that levels of the two mRNA transcripts are not different and the upstream peptide regulates receptor expression at the translational level (45). In human cells, receptor expression was approximately twofold higher in those bearing the Cys versus the Arg variant (45) and this polymorphism could therefore influence the susceptibility to hypertension, obesity and type 2 diabetes (45,46). In the coding region of β2-AR, the Val34Met polymorphism is extremely rare. Another polymorphism which is relatively uncommon is a mutation resulting in an Isoleucine to Threonine substitution at position 164 (Thr164Ile), within the proposed ligand binding pocket of the receptor. It seems that Ile164 displays a lower binding affinity for isoproterenol, norepinephrine and epinephrine, as compared to the wild-type β2-AR (47); furthermore, functional coupling to Gs-proteins, as determined in adenylyl cyclase assays, is significantly (approximately 50%) depressed with Ile164 under both basal and agonist-stimulated conditions (47). Studies on transgenic mice demonstrate that the Ile164 variant is substantially dysfunctional, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo (48). Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to a disease such as heart failure, this impairment may have important pathophysiologic consequences (49). The most common polymorphisms in the coding region of β2-AR are Arg16Gly (with an allele frequency of 0,40 for Arg16) and Gln27Glu (with an allele frequency of 0,55 for Gln27). They are functionally relevant polymorphisms; in fact, the Gly16 allele of. 9.

(10) the Arg16Gly polymorphism presents a. greater degree of agonist-induced. downregulation of the β2-AR in both transfected fibroblasts and human smooth muscle (50). The effect of the Gly16 allele dominates when combined with the Glu27 variant of the Gln27Glu polymorphism (47, 50). Interestingly, the Glu27 allele has otherwise been shown to be resistant to agonist-induced downregulation when alone or associated with the Arg16 variant (47, 50). Linkage disequilibrium has been observed between the 5'LC-Cys polymorphism and the β2AR coding block polymorphisms Arg16 and Gln27, although several different haplotypes have been identified (45). These and other studies demonstrated that β2-AR polymorphisms may be involved in impaired vasodilatatory responses to circulating β2-AR agonists; thus, these polymorphisms may play a pivotal role in the pathogenesis of hypertension and several cardiovascular diseases (45-51). β3-ADRENERGIC RECEPTOR POLYMORPHISMS The β3-AR gene is localized to chromosome 8; it contains introns, unlike the genes encoding for β1- and β2-AR (52). The tissue distribution of mRNA for β3-AR shows marked species differences; in humans, β3-AR mRNA was detected in brain, gastrointestinal tract, urinary bladder and especially adipose tissue, where the adrenergic stimulation of these receptors causes a lipolytic effect (52). Human myocardium expresses low amounts of β3-AR mRNA and, so far, there are no data in literature on the mRNA expression in vessels. Several reports proposed that the β3-AR may be coupled to more than one second messenger and interacts with both Gs- and Giprotein. Gauthier and colleagues (53) reported the coupling of β3-AR in the septum of human heart with Gi-protein, resulting in negative inotropic effect. At position 64 in the β3-AR, a Tryptophan is replaced by an Arginine (Trp64Arg); the variant form is found in 8 to 10% of the general populations in the United States and Europe (52) (Figure4). In vitro, compared to the dominant form, no difference was observed in ligand binding or adenylyl cyclase activation constants. In Western obese patients, however, the Arg64 allele is associated with an increased dynamic capacity to gain weight and an increased tendency to develop diabetes (52).. 10.

(11) 1.2b. α-ADRENERGIC. RECEPTOR. POLYMORPHISMS. AND. THEIR. FUNCTIONAL ROLE Subclassification and specific functional role of α1- and α2-ARs remain active areas of research to date (54). Three distinct α1-adrenoceptor proteins have been cloned, best distinguished by assessment of the relative potencies of a series of antagonists: α1a, α1b and α1d; the receptor initially termed α1c has the same tissue localisation and pharmacological profile as the α1a, and these two receptors are now known to be one (54). Likewise, there are three distinct α2-adrenoceptor proteins: α2a, α2b and α2c, but most functional responses produced by α2-AR agonists in intact tissues are mediated by the α2a-adrenoceptors (54). α1-ADRENERGIC RECEPTOR POLYMORPHISMS The α1-adrenoceptor subtypes are involved in the control of vascular tone and peripheral resistances; their stimulation results in peripheral vasoconstriction. For this reason, α1adrenoceptor antagonist activity is a key component in the pharmacological actions of antihypertensive agents, such as carvedilol, an antihypertensive drug which combines antagonist activity at α1- and β-ARs (54). Furthermore, uroselective α1-adrenoceptor antagonists are now available for the treatment of benign prostatic hyperplasia (54). α1a-AR activation in vascular smooth cells affects vascular tone through vasoconstriction, but these receptors are also present on the plasma membrane of myocyte, mainly in seno-atrial node, where they can influence heart rate (55). At position 492 of the α1a-AR, an Arginine can be substituted by a Cysteine (Arg492Cys); this polymorphism is a common variant located in the carbossiterminal tail and is postulated to result in an additional putative palmitoylation site (56,57). In transfected cells, the two variants were not associated with changes in ligand binding, signal transduction or receptor desensitation properties (57). On the other hand, in a study performed in vivo, Cys492Cys genotype was associated with a slightly higher systolic blood pressure and with an attenuated response to adrenaline infusion (58). Moreover, another study showed that Arg492Cys polymorphism is strongly related to autonomic control of heart rate (59). Other α1-AR polymorphisms are known, but they do not seem functionally relevant.. 11.

(12) α2-ADRENERGIC RECEPTOR POLYMORPHISMS The α2-adrenoceptor subtypes are located mainly in central nervous system and their agonists are useful in the treatment of several diseases, such as glaucoma, in general anaesthesia or as analgesics (54). As α1-adrenoceptors, α2b-AR are mainly located in vascular smooth cell and their activation contribute to vascular tone. Some polymorphisms of α2-ARs have been identified (such as α2b-adrenoceptor Del301303 polymorphism), but they are not included in this review, since carvedilol is not an α2-AR antagonist.. 1.3 ADRENERGIC RECEPTOR POLYMORPHISMS AND β-BLOCKER TREATMENT OF HYPERTENSION. Beta-adrenoceptor antagonists play an important role in the management of cardiovascular disease and have been used for three decades in the treatment of hypertension and ischemic heart disease; more recently, they have demonstrated to improve survival in patients with mild to moderate congestive heart failure (60). The beneficial effects of beta-adrenoceptor antagonists stem from their ability to limit the deleterious effects of adrenergic stimulation which is transmitted through adrenergic receptors. However, marked variability in response to beta-blocker exists and adequate blood pressure control failing is achieved in 30% to 60% of patients with beta-blocker monotherapy (61). The factors underlying this interpatient variability are not well understood, but several studies demonstrate that genetic differences and polymorphisms in adrenergic receptors may play a role. To date, β1-AR variants seem to represent the genetic component that more likely affects responsiveness to beta-blockers in hypertension and in heart failure also. Nevertheless, conflicting data have been published regarding the importance of β1-AR polymorphisms in response to selective β1-AR antagonists. O’Shaughnessy and colleagues (62), in a retrospective study, failed to find any significant differences between the Gly389 and Arg389 alleles in blood pressure and heart rate response to long-term β1-AR blockade with atenolol or bisoprolol. More recently, another study concluded that “the Ser49Gly and Arg389Gly β1-AR polymorphisms do not seem to exert a major effect on the changes in heart rate and blood pressure during 12 weeks of. 12.

(13) treatment with atenolol in patients with essential hypertension and left ventricular hypertrophy” (63). On the other hand, Sofowora et al (64) studied resting and exercise hemodynamic responses before and three hours after administration of atenolol in volunteers homozygous for the Arg389 or Gly389 variants; they found that atenolol caused a significantly larger decrease in resting systolic and mean arterial blood pressure (BP) in patient homozygous for Arg389 than in the other group. Similarly, Liu et al (65) studied resting and exercise hemodynamic responses before and after 3 doses of metoprolol in healthy Chinese men homozygous for the Arg389 or Gly389 variants, obtaining similar results as Sofowora and colleagues; in addition, Arg389 homozygous showed greater effect of metoprolol on exercise heart rate than Gly389 subjects. In a prospective study, Johnson et al (66) considered the impact of β1-AR polymorphisms on the antihypertensive effect of metoprolol in 40 patients with uncomplicated hypertension. Using 24-hour ambulatory blood pressure monitoring, they found that patients homozygous for Arg 389 had a significantly greater reduction in 24-hour and daytime diastolic blood pressure than patients with the Gly389 allele (66). Moreover, they also found that the Ser 49Gly polymorphism might contribute to different blood pressure responses to metoprolol and that the codon 49/389 haplotype was more informative than the codon 389 genotype alone (66) (they had previously shown that the codon 49 and 389 polymorphisms are in linkage disequilibrium (67)). In vitro studies confirmed that there are important functional differences among the common haplotypes in the β1-AR (68) and a recent study further demonstrated that β1-AR polymorphisms affect the BP response to metoprolol monotherapy in Chinese hypertensive patients using haplotype analysis (69). So far, there is poor literature concerning the association between β2-AR polymorphisms and beta-blocker treatment in hypertensive patients. For example, Iaccarino et al (70) demonstrated that the Glu27 variant of β2-AR enhances hypertension-induced left ventricular hypertrophy, but in their study the Glu27 patients showed a larger reduction of hypertrophy when treated with an angiotensin-converting enzyme inhibitor (enalapril) than with a beta-blocker (atenolol). Actually, all the studies on adrenergic receptor polymorphisms and beta-blocker treatment of hypertension have examined the effects of selective AR antagonists. Nevertheless, beta-blockers are not a homogeneous class of drugs (71). In particular, beta-adrenoceptor antagonists with additional vasodilatatory activity have been recently. 13.

(14) introduced in the treatment of hypertensive patients. The hemodynamic differences between beta-blocking drugs with and without additional vasodilatatory activity may be explained by the associated 1-adrenoceptor blocking activity (71). This activity may provide the following advantages: 1) greater reduction in BP values; 2) greater efficacy on arterial compliance; 3) furthermore, it is possible to hypothesize that alpha- and betablocking combined activities may positively affect BP variability. Carvedilol is an 1/-adrenoceptor antagonist with antioxidant, neuroprotective, cardioprotective and vascularprotective properties. Recently, Rochais and colleagues (72) demonstrated that the β1-AR undergoes conformational changes upon agonist and antagonist binding, specifically in the region of carboxy-terminus and third intracellular loop. The most clinically relevant data from their study is that, as a consequence of these conformational changes, carvedilol led to a much stronger reduction in basal intracellular cAMP content in the Arg389 β1-AR variant as compared to the Gly389 β1AR variant (72). Thus, carvedilol decreases the contractile activity of cardiac myocytes preferentially in the Arg389 β1-AR and, in general, displays the greatest degree of inverse agonism especially for the Arg389 variant (72). To data, no studies exist which investigate in vivo the correlation between the blood pressure lowering effect of carvedilol and functionally relevant 1- and -AR polymorphisms in hypertensive patients.. 14.

(15) 2.0 END-POINTS OF THE STUDY. The present study aimed to evaluate the following end-points: - the association between 1-, 2-, 3- and 1-adrenergic receptor polymorphisms and the baseline characteristics of hypertensive patients considered in terms of blood pressure, left ventricular mass and remodelling, systolic and diastolic left ventricular function; - the different decrease of blood pressure values between the group of patients with essential hypertension treated with an 1- and -receptor blocker (carvedilol) and the control group, due to 1-, 2-, 3- and 1-adrenergic receptor polymorphisms (PRIMARY END-POINT); - the different response to carvedilol in terms of left ventricular mass and remodelling, systolic and diastolic left ventricular function on the basis of 1-, 2-, 3- and 1adrenergic receptor polymorphisms.. 15.

(16) 3.0 PATIENTS AND METHODS. 3.1 PATIENT ENROLMENT. The present study included randomly selected, unrelated subject with primary mild to moderate essential hypertension never treated for the disease. All the patients were white and coming from the South of Italy. In order to enter the study, they had to meet all the inclusion and none of the exclusion criteria (see below). INCLUSION CRITERIA The following inclusion criteria had to be fulfilled: a. Diagnosis of essential hypertension according to ESH/ESC criteria (73); the last of 3 Diastolic Arterial Pressure (DAP) measurements in sitting position ranged between 90 and 109 mm Hg and/or the last of 3 Systolic Arterial Pressure (SAP) measurements in sitting position ranged between 140 e 179 mm Hg; the diagnosis of hypertension was confirmed at the end of a run-in period (4 weeks); the run-in period was shorted (2 weeks) in case of DAP ≥100 mm Hg and/or SAP ≥160 mm Hg. b. Age 18 years and older. c. Willingness to sign informed consent. EXCLUSION CRITERIA Subjects were excluded from the study if one the following conditions was given or expected to become applicable: a. Secondary hypertension. b. Severe or malignant hypertension. c. Previous antihypertensive treatment. d. History of myocardial infarction or angina pectoris. e. Atrial flutter or fibrillation. f. Second or third degree atrio-ventricular block; pace-maker implantation. g. Diabetes mellitus (74). h. Overt heart failure or a history of heart failure. i. Cardiomyopathies. j. Severe valvular heart disease.. 16.

(17) k. Thyroid disease. l. Gastrointestinal disease including duodenal or gastric ulcer within 3 months or a history of gastrointestinal surgery or disease which could interfere with drug absorption. m. History of dialysis. n. Beta-blocker therapy contra-indication. o. Predictable lack of cooperation. p. Participation in other clinical study within 1 month before screening.. 3.2 STUDY PROTOCOL. The study protocol was approved by the local Ethics Committee (Faculty of Medicine University of Bari, Italy). All study visits took place in the Cardiology Institute of the General Hospital of Bari. Both volunteers and clinical investigators were blinded to the genotype during the study. During the first visit (screening visit) each patient’s eligibility was determined and they entered a run-in period of 4 weeks (or 2 weeks in case of DAP ≥100 mm Hg and/or SAP ≥160 mm Hg); than, patients underwent several clinical and instrumental evaluations before (visit 1) and after (visit 2) a 4 weeks period of drug therapy with carvedilol (case group) or amlodipine (control group). All visits, took place in a quiet air-conditioned room and approximately at the same time of the day to minimize the impact of circadian variation on blood pressure check.. SCREENING VISIT (start of the run-in period) Each patient’s eligibility was assessed by: a. Medical history. b. Physical examination. c. Local measurement of SAP and DAP. d. Inclusion and exclusion criteria validation. e. 12-lead electrocardiography. f. Full routine laboratory tests (haematology, serum chemistries, urinalysis) performed in the last 6 months or in the run-in period.. 17.

(18) The elegible patients were requested to return in 4 weeks (run-in period); this period was shortened to 2 weeks if a DAP ≥100 mmHg and/or SAP ≥160 mmHg was detected. In the run-in period the patients were requested to reduce the daily sodium intake, to do some BP measurements and to abstain from coffee and alcohol drinking for a week before the visit 1 and 2.. VISIT 1 (basal evaluations) After the run-in period the arterial hypertension diagnosis was confirmed and the elegible patients were enrolled. Visit evaluations included: a. Local measurement of SAP and DAP. b. Weight and height assessment. c. 12-lead electrocardiography. d. Blood samples collection for genomic DNA analysis. e. Echo-color-Doppler examination. f. 24 hour ambulatory blood pressure monitoring. Once baseline studies were completed, the patients entered the 4-week treatment period with carvedilol 12,5 mg every 12 hours (at 8 AM and 8 PM, “case group”) or amlodipine 5 mg in the morning (at 8 AM, “control group”). All patients were enrolled and consecutively assigned to treatment in a ratio of 2:1 (2 carvedilol to 1 amlodipine).. VISIT 2 (treatment phase evaluations) Four weeks after the beginning of the treatment a second visit took place. This second visit was performed after morning therapy and included the following procedures: a. Local measurement of SAP and DAP. b. Weight assessment. c. 12-lead electrocardiography. d. Echo-color-Doppler examination. e. 24-hour ambulatory blood pressure monitoring. At the end of the study, on the basis of the treatment response, the patients were invited to continue their therapy or modify it.. 18.

(19) 3.3 CLINICAL AMD INSTRUMENTAL EVALUATIONS. BLOOD PRESSURE MEASUREMENTS A) Arterial pressure ambulatory measurements were performed with patients in a sitting position, after a resting period of at least 10 minutes, by trained nurses and using an automatic blood pressure monitor (Omron, Tokyo, Japan). Monitors were validated against. a. mercury. sphygmomanometer.. Three. consecutive. arterial. pressure. measurements were performed at 3-minute interval, the last value being recorded. B) 24-hour ambulatory blood pressure monitoring (ABPM) was performed in baseline condition and after therapy (visit 1 and 2, respectively). ABPM was obtained by using a Spacelab system and an A&D device. Monitors were validated against a mercury sphygmomanometer and were programmed to inflate at 15-minute intervals during “day-time” (6 AM to 11 PM) and at 20-minute intervals during “night-time” (11 PM to 6 AM). These wake and sleep times largely correlated with patients’ active and resting times and sleeping habits. After placement of the ABPM monitor, patients were instructed to follow their usual daily activities. Mean values and standard deviation of SAP and DAP were calculated by considering 24 hours, daytime and night-time.. ECHOCARDIOGRAPHIC EXAMINATION M-mode and two-dimensional echocardiography, doppler and tissue-doppler recordings were obtained using a phased-array echo-Doppler system (Hewlett Packard Sonos 5500) equipped with a 2,5-Hz transducer. The recordings were analysed off-line to assess the following parameters according to American Society of Echocardiography recommendations (75): - left ventricular (LV) diastolic diameter, diastolic septum and posterior wall thickness; - LV mass index (LVMI) dividing the left ventricular mass (LVM) by body surface area (76); - LV remodelling, considering the relative wall thickness (RWT) and LVMI (77); - systolic LV function through fractional shortening (FS); - diastolic LV function through pulsed transmitralic Doppler, recording mitral inflow velocity with the sample volume at the level of mitral annulus and considering the ratio. 19.

(20) (E/A) between the early (E) and atrial (A) diastolic peak velocity, the isovolumic relaxation time (IVRT), the E velocity deceleration time (Tdec); - systolic and diastolic LV function through pulsed Tissue Doppler (TD) at the level of the mitral annulus, considering respectively the myocardial systolic velocity (Sm) and the myocardial early diastolic velocity (Em) (78).. GENOTYPING Peripheral blood samples were collected and genomic DNA was isolated from peripheral lymphocytes using a Qiagen DNA extraction kit as described in the manufacturer’s protocol (Qiagen, Santa Clara, California, USA). All of the subjects were genotyped for the following polymorphisms involved in the pathophysiology of essential hypertension and related to adrenergic receptors targeted by carvedilol: - the Ser49Gly and Arg389Gly polymorphisms of the 1-adrenergic receptor; - the 5’ leader cistron Arg19Cys polymorphism and the Arg16Gly, Gln27Glu, and Thr164Ile polymorphisms in the coding region of the 2-adrenergic receptor; - the Trp64Arg polymorphism of the 3-adrenergic receptor; - the Arg492Cys polymorphism of the 1-adrenergic receptor. All of the considered polymorphisms were characterised by of polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). The RFLP patterns were confirmed by direct sequencing.. 20.

(21) 4.0 STATISTICAL ANALYSIS. Continuous variables were reported as mean and their standard deviations. Categorical variables were described using frequency tables. Continuous variables were compared using the Student’s t-test (paired and unpaired for intra-group and inter-group comparisons, respectively). Non-parametric tests were used when appropriate. Associations between categorical variables were examined by using Chi-square or Fisher test. Hardy-Weinberg equilibrium was tested by Chi-square test with 1 degree of freedom. Associations between genotypes and clinical characteristics were assessed either by Student’s t test for independent samples or by variance and covariance analysis adjusted for sex, age, BMI and smoking status. All statistical analysis was performed using Statistica 6.1 for Windows (StatSoft Inc). A p value of less than 0.05 was considered statistically significant.. 21.

(22) 5.0 RESULTS. 5.1 BASELINE CHARACTERISTICS OF PATIENTS AND GENOTYPE RESULTS. Eighty-two patients out of 288 screened subjects fulfilled the inclusion and exclusion criteria, thus were enrolled in the study. Their baseline characteristics are shown in table1: males and non smokers were prevalent; patients were 47 ± 11 year-old and normal (BMI= 18,5-24,9 kg/m2) or overweight (BMI= 25-29,9 kg/m2). Table1 shows baseline heart rate (HR), SAP and DAP measurements, performed both in an outpatient setting and with 24hour monitoring, as means and their standard deviations. Echocardiographic examination revealed that none of patients had depressed systolic LV function, since all of them showed a fractional shortening (FS) >25% (80) and that they presented mostly concentric LV remodelling (56%); several diastolic LV function indexes were analyzed and displayed in table1. Table2 shows genotype and allele frequency distribution of 1-, 2-, 3- and 1-AR gene polymorphisms in patients enrolled. Genotype frequencies for all polymorphisms were in Hardy-Weinberg equilibrium (p >0,05). As previously reported (67), we observed significant linkage disequilibrium between codons 49 and 389 polymorphisms of 1AR, and also between the 5'LC-Cys polymorphism and the beta2-AR coding block polymorphisms Arg16 and Gln27, although several different haplotypes were identified (45). Nobody showed homozygosity for the second amino acid of Thr164Ile polymorphism in the 2-AR coding region and for the second amino acid of Trp64Arg polymorphism of the 3-AR, according to the low frequency of these polymorphisms reported in the general population (47, 48, 52), and confirmed by the respective allele frequency distribution in this population (table2). For this reason, we have not included these two polymorphisms in the following analyses of correlations between patients’ characteristics and various ARs genotypes. Tables3 to 8 show patients’ characteristics stratified by genotype of the following polymorphisms: Ser49Gly (table3) and Arg389Gly (table4) of 1-AR; 5’LC-Arg19Cys (table5), Arg16Gly (table6) and Gln27Glu (table7) of 2-AR; Arg492Cys (table8) of 1AR.. 22.

(23) Patients with the Gly49 variant of the Ser49Gly β1-AR polymorphism featured more concentric hypertrophy than Ser49Ser patients, although the two groups had not significant difference in LVMI (table3). Most relevant data are those concerning the significant association between the Arg389Gly β1-AR polymorphism and blood pressure parameters (table4). In particular, Arg389Arg patients showed clinical SAP and DAP, 24hours and daytime SAP and DAP values significantly higher than 389Gly carriers (table4). Moreover, the Gly16 homozygotes for the Arg16Gly β2-AR polymorphism displayed lower fractional shortening (FS) compared to the Arg16 carriers (table6). No differences were found in the other examined parameters stratified by genotype of the -AR polymorphisms. As regards Arg492Cys polymorphism of 1-AR (table8), a few differences of gender and age were randomly found between Arg492 carriers and Cys492 homozygotes; furthermore, Cys492Cys patients showed values significantly higher than Arg492 carriers in 24hours and daytime SAP (table8).. 5.2 CASE AND CONTROL GROUPS: BASELINE CHARACTERISTICS AND HEMODYNAMIC EFFECTS OF TREATMENTS. We examined 79 out of 82 enrolled patients after the four-week treatment (53 with carvedilol and 26 with amlodipine); three patients were lost at follow-up. Baseline characteristics of both carvedilol-treated (beta-blocker, “case group”) and amlodipinetreated (calcium antagonist, “control group”) patients are shown in table9: no significant demographic, clinical and instrumental differences were found between case and control groups. As shown in table10, SAP and DAP value decreases using either carvedilol or amlodipine were very similar; on the other hand, carvedilol treatment caused significant reduction of heart rate values.. 5.3 RESPONSES TO MONOTHERAPY BY GENOTYPE GROUPS. Tables11 to 16 show BP responses to carvedilol or amlodipine and echocardiographic variations of systolic and diastolic function stratified according to β1-, β2- and 1-AR. 23.

(24) genotypes (we did not consider Thr164Ile β2-AR and Trp64Arg β3-AR polymorphisms due to their very low frequency in general population). The study results stratified by the position 389 genotypes of β1-AR are shown in table12. There was a significant effect of Arg389Gly polymorphism on BP response to carvedilol: patients who were Arg389 homozygotes presented a significantly greater reduction in clinical SAP and DAP, and, at ABPM data analysis, in 24hour, daytime SAP and DAP and night-time DAP values compared to Gly389 carriers (table12). There was also a trend toward a greater reduction in night-time SAP in the Arg389 homozygotes, but this difference was not significant (p >0,05). Figure 5 displays the different responses to carvedilol by codon 389 genotype at ABPM data analysis in Arg389Arg and Gly389 carriers groups. At multivariate analysis, adjusted for age, sex, BMI and smoking status, the significant association between Arg389Gly genotype and systolic and diastolic blood pressure values was confirmed (p=0.034 for 24h-SAP and 0.015 for 24h-DAP). On the other hand, patients treated with amlodipine (control group) did not show significant differences in BP reduction stratified according to β1AR Arg389Gly genotypes (table12). Moreover, there were no echocardiographic differences in systolic and diastolic function response to monotherapy on the basis of Arg389Gly genotypes. With regard to the other genotypes, there were no differences in blood pressure response to monotherapy or in echocardiographic characteristics among case and control groups.. 5.4 RESPONSES TO MONOTHERAPY STRATIFIED BY CODON 49 AND CODON 389 GENOTYPES OF  1-AR. We found a significant association between Arg389Gly polymorphism on BP response to carvedilol. On the basis of previous data showing that polymorphisms of β1-AR at codon 49 and 389 are in linkage disequilibrium (67), we also considered whether the combination of Arg389Gly genotypes with Ser49Gly genotypes can better predict BP decrease. Figure6 displays the different responses to carvedilol stratified by codon 49 and codon 389 genotypes of β1-AR at ABPM data analysis. There were no significant. 24.

(25) differences in BP decrease among groups and the effect of Arg389Gly polymorphism on BP response seemed not to be affected by Ser49 or Gly49 alleles.. 25.

(26) 6.0 DISCUSSION. Hypertension is a multifactorial disease with a substantial genetic component. In the pathophysiology of essential hypertension, the sympathetic nervous system and adrenergic receptors are of particular relevance (16,26,27): in response to the cathecolamine activation, adrenergic receptors influence blood pressure homeostasis regulating cellular metabolism, hormone production, cardiac output and peripheral resistance (28). As a consequence, it is reasonable to expect that genetic variants of adrenergic receptors can play a major role in the development of hypertension, thus affecting the antihypertensive response to -blockers; in fact, marked variability in response to beta-blockers exists and adequate blood pressure control failing is achieved in 30% to 60% of patients with beta-blocker monotherapy (61). Over recent years, association studies between genetic polymorphisms and essential hypertension phenotype have rapidly expanded and, to date, β1-AR genetic variants seem to represent the genetic component that more likely affects the development of hypertension and responsiveness to beta-blockers in both hypertension and heart failure. Among all, Arg389Gly polymorphism in the 1-AR gene confers an increased risk of developing hypertension (39) and is strongly associated with the blood pressure response to 1-AR selective antagonists (64-66,68,69), although some authors failed to find any significant association (62,63).. 6.1 BASELINE ANALYSIS. In this work we found that in a population of unrelated white subjects with mild to moderate essential hypertension there was a significant association between the Arg389Gly β1-AR polymorphism and blood pressure parameters (table4): in particular, Arg389Arg patients showed significantly higher arterial pressure ambulatory measurements and 24-hour ambulatory blood pressure monitoring (ABPM) values compared to Gly389 carriers (table4). This results are consistent with the literature data on the functional effects of Arg389Gly β1-AR polymorphism (39-41). The Arg/Gly polymorphism is localized in an intracellular loop of the β1-AR (a putative Gs-protein. 26.

(27) binding domain), which is a highly conserved region among many species; as far as we know, Arg in the analogous human position 389 is invariant in β1-ARs of all species sequenced (81). Polymorphic variation at such a highly conserved residue has been considered one criterion for predicting altered function (81) and, in fact, in vitro studies confirm that the Arg389 variant mediates a higher isoproterenol-stimulated adenylyl cyclase activity than the Gly389 variant, through an enhanced receptor-Gs coupling (40). Consequently, it has been shown that hearts from transgenic mice with cardiacspecific overexpression of Arg389 β1-AR variants, had enhanced receptor function and contractility compared with Gly389 hearts (41); moreover, in humans, it has been shown that the Arg389 allele and the Arg389Arg genotype of the β1-AR gene were more common in patients with hypertension than in healthy controls (39), and that Arg389 homozygous presented significantly higher contractile responsiveness to cathecolamines than Gly389 patients (82). Thus, this polymorphism may play a key role in the sympathetic control of BP. The threshold of statistical significance was not reached for night-time BP values at ABPM analysis in this study (table4), as well as in other works addressing the same subject (66), most likely because sympathetic tone is substantially reduced during the night-time, and also the night-time BP is only minimally regulated by the sympathetic nervous system (83). Although in literature no significant associations between β1-AR polymorphisms and echocardiographic measures have been found in hypertensive patients, in this study Arg389 homozygotes showed a trend toward a higher left ventricular (LV) mass index than Gly carriers (table4), suggesting that Arg389 variant of β1- cardiac ARs could mediate an increased sympathetic activity on hearth and may be an underlying mechanism in LV mass degree. In fact, pathogenesis of LV hypertrophy is multifactorial, but there is substantial evidence that the sympathetic system is involved in hypertension-induced progression of cardiac structural alterations (84). Several studies demonstrated that hypertension and hypertensive LV hypertrophy are associated with increased sympathetic activity (84, 85), and a recent study showed that “arterial plasma noradrenaline at baseline, as an index of sympathetic activity, predicts LV mass at follow-up independently of systolic blood pressure and body build in middle-aged men who developed hypertension over a period of 20 years” (86). As regards other echocardiographic baseline results, we also found that the Gly16 homozygotes for the Arg16Gly β2-AR polymorphism displayed lower fractional. 27.

(28) shortening (FS) compared to the Arg16 group. This is not consistent with the few published data on this polymorphism (87), most likely because of the low number of subjects enrolled in our study; thus, further investigations are needed in order to better evaluate this topic. 1-AR activity is involved in arterial blood pressure control by modifying vascular tone and peripheral resistances. In this work, as well as in a recent in vivo study (58), Cys492Cys genotype of Arg492Cys 1-AR polymorphism was associated with higher SAP compared to genotypes with the Arg492 allele. Nevertheless, in our population differences of gender and age were randomly found between Arg492 carriers and Cys492 homozygotes; moreover, in a previous study Xie et al (57) found no significant association between the Arg492Cys polymorphism of the 1-AR gene and modified ligand binding, signal transduction or receptor desensitation properties. Thus, the correct interpretations of such data remain active areas of research.. 6.2 RESPONSES TO CARVEDILOL. A possible approach to test the impact of polymorphisms on receptors’ sensitivity could be to determine the response to receptors’ antagonists. The wide inter-individual variance of blood pressure responses to beta-blocker treatment is already well known (61), at least partly due to differences in rennin levels (88), race (89) and pharmacokinetics (90). Differences in β1-AR genotype and, in particular, the Gly389Arg β1-AR polymorphism might be an additional factor (91). Although so far conflicting data exist in literature (62-66,69), most studies enrolling both healthy and hypertensive subjects suggest that greater responses to selective -blockers may be associated with the presence of the Arg389 allele of the β1-AR (64-66,69). Nevertheless, beta-blockers are not a homogeneous class of drugs (71); in particular, -adrenoceptor antagonists with additional vasodilatatory activity (i.e. carvedilol, an 1/-AR antagonist) have been recently introduced in the treatment of hypertensive patients. This is the first case-control study to evaluate in vivo the correlations between the blood pressure lowering effect of carvedilol and functionally relevant 1- and -AR polymorphisms in hypertensive patients.. 28.

(29) According to previous data using selective -blockers (64-66,69), the main finding of this study was a significant and independent relationship between the Arg389Gly polymorphism of the β1-AR gene and antihypertensive response to carvedilol (table12, figure5). We found that hypertensive patients who were Arg389 homozygotes presented a significantly greater reduction in clinical SAP and DAP, and, at ABPM data analysis, in 24hour, daytime SAP, daytime DAP and night-time DAP values compared to Gly389 carriers; such results were confirmed at multivariate analysis, adjusted for age, sex, BMI and smoking status (p=0.034 for 24h-SAP and 0.015 for 24h-DAP). On the other hand, there was only a trend toward a greater reduction in night-time SAP in the Arg389 homozygotes. As observed in a study using metoprolol (66), we also think that the association between BP response to -blockers and Arg389Gly polymorphism of the β1-AR gene during the day would not necessary be expected to carry over to the nighttime, because of the physiological reduction of the sympathetic tone during the night (83). Figure7 and 8 show 24hour SAP and DAP values (at ABPM data analysis) with 95% confidence intervals before and after carvedilol therapy, stratified by codon 389 genotypes of β1-AR: Arg389 homozygotes showed significantly higher baseline BP values than Gly389 carriers (p<0.001 for 24h-SAP and p=0,05 for 24h-DAP) and, after the 4-week treatment period with carvedilol, they presented a significantly greater reduction in BP values compared to Gly389 patients, also at multivariate analysis (figure5). How baseline blood pressure affects the response to carvedilol is still uncertain, but these results are of particular relevance because they provide a clinical support to the hypothesis that both the sympathetic nervous system and adrenergic receptors influence blood pressure homeostasis especially in patients who are Arg389 homozygotes for Arg389Gly β1-AR polymorphism and that in these patients carvedilol displays a greater degree of inverse agonism. An in vitro study (40) already demonstrated that the Arg389 variant mediates a higher isoproterenol-stimulated adenylyl cyclase activity and a higher cAMP production than the Gly389 variant, through an enhanced receptor-Gs coupling. In fact, the Arg389Gly polymorphism of β1-AR occurs in a region putatively associated with coupling to the Gs-protein (between the seventh transmembrane region and the intracellular tail of the receptor) and the change of the amino acid residue from the polar and basic arginine (Arg389) to the small and non-polar glycine (Gly389) at position 389 of the β1-AR may result in a. 29.

(30) modified structure that could alter receptor Gs-interaction. Recently, new fluorescence resonance energy transfer-based (FRET-based) techniques have allowed investigators to evaluate dynamic protein-protein interactions and protein conformational changes in real time (92). Using specific β1-AR FRET sensors for both Arg389 and Gly389 variants, Rochais and colleagues (72) were able to directly assess the functional consequences of this polymorphism with respect to receptor conformation in response to several antagonists. They showed that carvedilol induces a conformational change of the β1-AR which causes decreased Gs coupling and cAMP production; both Arg389 and Gly389 variants had inverse agonist properties in response to carvedilol, but the effect was 2,5-fold higher in the Arg389 variant, revealing a supersensitivity of the Arg389 variant to this non-selective β-blocker (72). It is not clear which mechanisms and hemodynamic effects are responsible of different degrees of inverse agonism of carvedilol on β1-AR causing different degrees of BP decrease. In this study Arg389Arg patients and Gly389 carriers did not present any differences in systolic and diastolic function on the basis of Arg389Gly genotypes after the 4-week treatment period with carvedilol, although Rochais et al (72), using cardiac myocytes from neonatal Arg389 or Gly389 rats, showed a carvedilol-induced decrease of the contractile activity mainly in Arg389 β1-AR myocytes, thus suggesting the phenotypic relevance of this polymorphism. On the other hand, no data exist on the effects of carvedilol on plasmarenin activity (PRA) stratified by AR polymorphisms, although it has been shown (93) that codon 389 β1-AR polymorphism may play a role in the regulation of PRA and that bisoprolol markedly suppressed the dobutamine-induced PRA increase in Arg389Arg subjects but only marginally in Gly389 carriers. Furthermore, it is possible to hypothesize a different antihypertensive effect of carvedilol stratified by Arg389Gly β1AR polymorphism through a different modulation of cardiovascular reflex patways, but, so far, there are no studies investigating the association between variations in AR genes and the effects of carvedilol on heart rate variability and reflex regulation of blood pressure in hypertensive patients. However, both previous in vitro studies and such clinical data showed that the antihypertensive effect of carvedilol is dependent on the amino acid residue present at position 389 of the β1-AR. A recent study showed that the codon 49 and 389 polymorphisms of β1-AR are in linkage disequilibrium (67) and some authors (66,69) underlined that haplotype is likely more informative than codon 389 genotype alone. Figure6 displays the different. 30.

(31) responses to carvedilol stratified by codon 49 and codon 389 genotypes of β1-AR at ABPM data analysis in our study. The analysis revealed that the two groups with the largest SAP and DAP reduction were those including the Arg389Arg genotype and the impact of codon 49 genotype on antihypertensive response to carvedilol was not relevant. Nevertheless, there were few Gly49 carriers in this population (table2), as confirmed by the allele frequency of Ser49Gly β1-AR polymorphism reported in the general population (34). Further studies with large cohorts of patients are needed in order to clarify the informative power of codon 49/389 haplotype on antihypertensive response to β-blockers. With regard to the other genotypes, the results of correlations between patients’ response to carvedilol and different ARs genotypes were not relevant; in these analyses, Thr164Ile of the 2-AR and Trp64Arg of the 3-AR polymorphisms were not considered, due to the low frequency of these polymorphisms reported in the general population (47,48,52), as further confirmed by the respective allele frequency distribution in this population (table2). Thus, all these analyses seem to support the statement that only variants of β1-AR gene are predictive of antihypertensive response to carvedilol. Finally, since our study was not designed to test the long-term effects of carvedilol, the longer-duration effects of genetic polymorphism on cardiovascular response to carvedilol deserves additional investigation.. 31.

(32) 7.0 CONCLUSION. In summary, we performed a case-control study on a population of white unrelated subjects with mild to moderate essential hypertension and obtained the following relevant results: - at baseline condition, there was a significant association between the Arg389Gly β1-AR polymorphism and blood pressure parameters: in particular, Arg389Arg patients showed significantly higher arterial pressure ambulatory measurements and 24-hour ambulatory blood pressure monitoring (ABPM) values compared to Gly389 carriers; moreover, Arg389 homozygotes showed a trend toward a higher left ventricular (LV) mass index than Gly carriers; these results were not found in the control group of patients treated with amlodipine; - after the 4-week treatment period with carvedilol, Arg389 homozygotes presented a significantly greater reduction in blood pressure values compared to Gly389 patients, as confirmed at multivariate analysis adjusted for age, sex, BMI and smoking status; no similar results were recorded in the control group; - with regard to the other 1-, 2-, 3- and 1-AR polymorphisms, we found no differences in blood pressure response to carvedilol or in echocardiographic characteristics (in terms of left ventricular mass and remodelling, systolic and diastolic left ventricular function). Altogether, the results of this study provide evidence that the Arg389Gly β1-AR polymorphism may play a key role in the sympathetic control of blood pressure and that a greater response to carvedilol may be associated with the presence of the Arg389 allele of this polymorphism. Thus, if one of the goals of pharmacogenetics is a quest for individualized therapy on the basis of genetic information, the present research could contribute by identifying individual patient characteristics which predict blood pressure response prior to carvedilol administration and leading to selective therapeutic interventions with enhanced efficacy in a given subset of hypertensive patients. Additional adequately powered, multiethnic, multi-drug studies are needed before polymorphism analysis can be applied to clinical practice with the aim to personalize therapy on an individual's genetic makeup.. 32.

(33) 8.0 PERSPECTIVES. To date, β1-AR variants seem to represent the genetic component that more likely has a significant impact on agonist-mediated contractility in non-failing and failing human hearts (81) and also on the responsiveness to beta-blockers in hypertension and heart failure (HF) (31). In HF, the myocardium has a significant loss of contractile function and, in order to compensate this pump failure, the heart must increase its rate and contractility through increases in the activity of the sympathetic nervous system and renin-angiotensin system. However, chronic activation of the adrenergic pathway, and especially of the myocardial β1-AR, leads to increased cardiotoxicity and cardiac pathology (30). This partially explains the clinical benefit achieved by β-AR antagonists, which have been shown to significantly improve patients’ survival and reverse cardiac remodelling (94). In vivo, the Arg389 variant of the β1-AR appears to be linked with HF in both clinical outcomes and response to beta-blocker therapy (42,81); all these results seem to indicate that the Arg389Gly polymorphism is a candidate gene for the development of genomics-based antiadrenergic treatments of both hypertension and chronic HF. On the other side, this work shows some effects of the Gly49 variant of the β1-AR (table3) and the Gly16Gly genotype of the β2-AR (table6) on LV performance, apparently independent of BP, in hypertensive patients. Recently (95) it has been shown that the Gly49 allele of the β1-AR and the Gly16Gly genotype of the β2-AR are significantly and independently associated with the idiopathic dilated cardiomyopathy phenotype, thus suggesting their role in favouring susceptibility to the disease. Such findings are of potentially clinical importance when considering the possibility that these genotypes could promote the transition towards heart failure independently of BP also in hypertensive patients, or alternatively that they could identify hypertensive patients at higher risk. Finally, these data are from an experimental study of a cohort of white patients from the South of Italy; some authors (96) demonstrated a lack of benefit in black patients with advanced chronic heart failure treated with the beta-blocker bucindolol, compared with other patients. Hence, the considerable interindividual and interethnic variability in the response to β-AR agonist and antagonists must be taken into account in the aim to. 33.

(34) extrapolate such results to other regions, and, in our opinion, further multicentric studies could be helpful also in elucidating this point.. 34.

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