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β2‐adrenergic receptor Thr164Ile (threonine (Thr) is replaced by an isoleucine (Ile) at codon 164) polymorphism was postulated to contribute to lower exercise tolerance and poor prognosis in patients with congestive heart failure. However, heart failure is associated with several abnormalities of β receptor signalling, and underlying mechanisms are not clear.
To investigate whether Thr164Ile polymorphism negatively modulates myocardial contractile performance and is associated with adverse long‐term prognosis of patients with congestive heart failure.
Among 55 subjects, cardiac contractile response to the β2‐adrenergic receptor agonist terbutaline was assessed from the peak myocardial velocity of systolic shortening (Sm) in 18 subjects with the Ile‐164 variant and 37 matched controls. In total, 24 subjects had normal left ventricular (LV) function and 31 presented with congestive heart failure due to idiopathic dilated cardiomyopathy.
In patients with normal LV function, peak terbutaline‐induced increase (Δ) in Sm was lower in subjects with the Ile‐164 variant than in controls (Δ33% (4%) vs Δ56% (4%), p<0.01). In patients with heart failure, subjects with Ile‐164 showed further severe reduction of β2‐adrenergic‐mediated increase in Sm as compared with controls with heart failure (Δ20% (5%) vs Δ39% (4%), p<0.05). Patients with heart failure with Ile‐164 showed a severely blunted force–frequency relationship in response to agonist stimulation. At 2‐years of follow‐up, patients with heart failure with the Ile‐164 variant showed higher incidence of adverse events than controls with heart failure (75% (6/8)] vs 30% (7/23), p<0.05).
The β2‐adrenergic Thr164Ile polymorphism directly modulates adrenergic‐mediated cardiac responses in patients with normal and failing myocardium. Furthermore, blunted β2 adrenergic‐mediated myocardial contractile response in patients with Ile‐164 variant seems to adversely modulate the course of congestive heart failure.
In the normal myocardium, β2‐adrenergic receptors represent ~20% of the total β‐adrenergic receptor population, and, similar to β1‐adrenergic receptors, they mediate cardiac inotropic and chronotropic responses.1 In congestive heart failure, chronic activation of the cardiac sympathetic nervous system leads to abnormalities at several levels of the adrenergic receptor signal transduction pathway. A reduction in the number of receptors (down regulation) and responsiveness (uncoupling) cause blunted adrenergic‐mediated responses that contribute to the progression of congestive heart failure.1 β2‐adrenergic receptor desensitisation is mainly determined by the uncoupling phenomenon.2 The number of receptors remains relatively stable and this is considered to be one of the compensatory mechanisms preserving adrenergically mediated cardiac responses in heart failure.3,4
The β2‐adrenergic receptor gene polymorphism, in which a threonine (Thr) is replaced by an isoleucine (Ile) at codon 164 (Thr164Ile), leads to “loss of the function” of the receptor.5 Despite the rare incidence,6 individuals with the Ile‐164 allele and normal left ventricular (LV) function show blunted haemodynamic responses to adrenergic stimulation.7 Furthermore, patients with heart failure with Thr164Ile polymorphism seem to be characterised by reduced exercise tolerance and higher mortality.6,8 However, pathophysiological mechanisms contributing to the poor outcome of these patients are not clear—namely, it is unclear whether the poor outcome is related to direct effects of the Ile‐164 polymorphism on the myocardial contractile performance or to systemic haemodynamics. Earlier studies1,3 indicated that β2‐adrenergic receptors may be also hyporesponsive (uncoupled) in heart failure, which is raising doubts about the importance of the Thr164Ile polymorphism in the modulation of myocardial contractility in patients with failing myocardium. Accordingly, we investigated β2‐agonist‐mediated contractile performance in patients with normal and failing myocardium to test the hypothesis that the dysfunctional Ile‐164 β2‐adrenergic receptor directly modulates myocardial contractile performance. Second, we studied the effect of the Ile‐164 variant on long‐term prognosis of patients with congestive heart failure due to idiopathic cardiomyopathy.
A total of 786 Caucasian patients referred to cardiac catheterisation because of chest pain or dyspnoea were screened for β2‐adrenergic receptor polymorphisms in codons 16, 27 and 164. An additional 100 healthy Caucasian blood donors were screened and served as control population. Among screened patients, 55 were eligible for further functional studies. Exclusion criteria were LV hypertrophy, hypertension, valvular heart diseases or coronary artery stenosis with >30%. Informed consent was obtained from all subjects. The study was approved by the ethical committee of the OLV Ziekenhuis, Aalst, Belgium.
Genomic DNA was subjected to a PCR. To amplify the polymorphic site (nucleotide 491), the following primers were used: 5′‐CTT‐TTG‐GCA‐ACT‐TCT‐GGT‐GCG‐AG‐3′ (forward) and 5′‐AGT‐CAC‐AGC‐AGG‐TCT‐CAT‐TG‐3′ (reverse; TIB MOLBIOL, Berlin, Germany). The amplification was performed on the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) using the following hybridisation probes: 5′‐TTG‐TGT‐CAG‐GCC‐TTA‐CCT‐CCT‐TCT‐T‐3′ (donor dye, labelled with fluorescein) and 5′‐LCRed640‐CCC‐ATT‐CAG‐ATG‐CAC‐TGG‐TAC‐AGG‐GC‐3′ (acceptor dye, labelled with LightCycler‐Red640). The reaction conditions consisted of 10 ng DNA, 300 μM of each deoxyribonucleotide triphosphate, 3.25 mM MgCl2, 5% bovine serum albumin, 0.5 μM of each primer, 0.2 μM of each probe, 0.05 U/μl Taq‐polymerase and a ×10 dilution of the supplied buffer in a final volume of 10 μl. PCR conditions were initial denaturation of 95°C for 5 min, then 40 cycles of 95°C for 15 s, 59°C for 5 s and 72°C for 10 s, followed by melting from 95°C for 10 s to 40°C for 20 s and an increase to 85°C with a speed of 0.2°C/s and a final cooling to 40°C. Genotype frequencies in our control population were as follows: Arg16Arg 44%, Arg16Gly 21%, Gly16Gly 35%; Gln27Gln 32%, Gln27Glu 53%, Glu27Glu 15%; and Thr164Thr 98%, Thr164Ile 2%. In patients with low LV ejection fraction (LVEF <45%), genotype frequencies were not statistically different from control population: Arg16Arg 47%, Arg16Gly 15%, Gly16Gly 38%; Gln27Gln 37%, Gln27Glu 47%, Glu27Glu 16%; and Thr164Thr 95%, Thr164Ile 5%.
Pulsed‐wave Doppler recordings of longitudinal myocardial velocities of basal segments were obtained in standard apical views as described previously, 9 using a commercial ultrasound system (Acuson Sequoia, Mountain View, California, USA). During the recording, a sample volume of 5 mm was positioned in the centre of the segment parallel to the long axis and 3–5 consecutive beats were averaged for the analysis.
Patients with LVEF <45% (as assessed by angiography) and LV end‐diastolic diameter >60 mm were considered to have congestive cardiomyopathy. None of these patients had documented coronary artery disease. β Blockers, when present, were stopped 24–36 h before terbutaline protocol. Study population was divided according to genotype and LV function as follows: group 1: controls homozygotic for the Thr164Thr polymorphism, with normal LV function (n=14); group 2: subjects heterozygotic for the Thr164Ile polymorphism, with normal LV function (n=10); group 3: controls homozygotic for the Thr164Thr polymorphism, with congestive cardiomyopathy (n=23); and group 4: subjects heterozygotic for the Thr164Ile polymorphism, with congestive cardiomyopathy (n=8).
Haemodynamic and myocardial contractile performance was studied during the infusion of the selective β2‐adrenergic receptor agonist terbutaline (Bricanyl, Pharmastern, Wedel, Germany). After a 1 h period of supine rest, intravenous terbutaline was increasingly infused at 25, 50, 100 and 150 ng/kg/min, each dose for 15 min.7 Heart rate and blood pressure were continuously monitored throughout the study period.
At tissue Doppler imaging, myocardial contractile response was assessed from the peak myocardial velocity of systolic shortening (Sm) during ejection. This index has been recently introduced as an index of myocardial contractility and correlated well with invasive parameters.10,11,12,13 Tissue Doppler analysis of Sm was performed by an experienced operator blinded to the clinical data and genotype of all subjects. Sm was averaged from four basal segments of the left ventricle (anterior, inferior, lateral and septal) at baseline and 30 s before the end of each dose of terbutaline.
Arterial pulse pressure, calculated as a difference between systolic blood pressure and diastolic blood pressure, was used as a surrogate for cardiac output.14 All haemodynamic data were analysed by the observers blinded to the results of genotyping.
The population of patients with heart failure (groups 3 and 4) were followed from the date of entry into the study up to 2 years. Clinical end points were death, heart transplantation, worsening of heart failure requiring increase in diuretics or hospital admission and new onset of atrial fibrillation.
Data are expressed as mean (SEM). Two‐sided Student's t test, Fisher's exact test and one‐way analysis of variance followed by Newman Keuls post hoc analysis were used for appropriate comparisons. p Value <0.05 was considered significant. In patients with congestive heart failure, event‐free survival at 2 years was calculated from the Kaplan–Meier estimates using the log rank test for comparison between patients with and without Ile‐polymorphisms.
Table 11 shows the clinical characteristics of the patients. Groups were matched by age and LVEF. Female gender was slightly more frequent in patients with homozygotic Thr164Thr. No differences were found in the incidence of diabetes, renal failure and atrial fibrillation. There were no differences in medical treatment between patients with the Ile‐164 variant and their respective control group, regarding the treatment with β blockers, ACE inhibitors or spironolactone.
TablesTables 1 and 22 show haemodynamic data. At baseline, patients with LV dysfunction had higher heart rate than individuals with normal LV function regardless of the genotype. In the subgroup of patients with LV dysfunction, no significant change in heart rate was observed during terbutaline in patients with the Thr164Ile polymorphism. At baseline, systolic and diastolic blood pressures were similar in all groups and did not change in response to terbutaline. No significant changes in LV end‐diastolic dimensions during terbutaline were observed in all patients (data not shown).
Figure 1A1A shows dose‐dependent changes of Sm in response to terbutaline in controls (Thr164Thr) and subjects (Thr164Ile) with normal LV function. Terbutaline induced a significant dose‐dependent increase in Sm in subjects with Thr164Ile polymorphism. However, the increase in Sm was lower than in the matched controls without the polymorphism.
In patients with dilated cardiomyopathy (fig 11 B), controls with the wild‐type Thr164Thr genotype showed a significant dose‐dependent increase in Sm. Yet, as expected, the maximal increase was lower than in controls with the same wild‐type genotype but normal LV function (table 22).). Furthermore, patients with dilated cardiomyopathy and the Ile‐164 variant showed only a minimal increase in Sm response to terbutaline, which was markedly lower than in patients with idiopathic cardiomyopathy and the Thr164Thr genotype.
To further corroborate the myocardial contractile response assessed by the Sm, arterial pulse pressure was measured as an index of cardiac output.14 At baseline, pulse pressure tended to be lower in both groups of patients (groups 3 and 4) with LV dysfunction (table 22).). In individuals with normal LV function, pulse pressure increased in response to terbutaline in controls, whereas no changes were observed in carriers of the Ile variant. In patients with dilated cardiomyopathy, pulse pressure did not change in response to terbutaline in controls or in carriers of the Ile variant.
To investigate whether observed effects on the cardiac performance are directly related to effects of Ile‐164 variant on the myocardial contractility, we analysed the force–frequency relationship at baseline and during peak terbutaline infusion (fig 22).). In subjects with normal LV function, carriers of the Ile‐164 variant showed a blunted increase in the force–frequency relationship at peak terbutaline infusion as compared with individuals without the Ile‐164 variant. Notably, the slope of force–frequency relationship at baseline and at peak terbutaline remained similar, suggesting reduced chronotropic response as the main mechanism underlying blunted increase in contractility in these subjects. As expected, in patients with dilated cardiomyopathy with the Thr164Thr genotype, the force–frequency relationship at baseline and at peak terbutaline showed downward and rightward shift as compared with controls with normal LV function. Patients with the Ile‐164 variant showed a further reduction in the slope of the force–frequency relationship as compared with controls with dilated cardiomyopathy and with carriers of the Ile‐164 with normal LV function. This is consistent with blunted inotropic and chronotropic responses as underlying mechanisms of blunted myocardial performance to terbutaline in patients with the Ile‐164 variant and idiopathic cardiomyopathy.
Only one patient with the Thr164Thr genotype underwent heart transplantation and died 1 month after the surgery. No patient with the Ile‐164 variant died or underwent heart transplantation. New York Heart Association class (2.2 (0.3) vs 1.35 (0.2), p<0.05), number of patients with worsening heart failure (50% vs 4%, p<0.05) or >1 event (50% vs 4%, p<0.05) was higher in patients with the Ile‐164 variant than in patients with the Thr164Thr genotype. As fig 33 shows , the event‐free period was significantly better in patients with heart failure with the wild‐type receptor than in patients with the Ile‐164 variant. In addition, at the 2‐year follow‐up, patients with heart failure with the Thr164Thr genotype showed a significantly higher increase in LVEF whereas no changes were noted in patients with heart failure with the Ile‐164 variant (7.0% (2.3%) vs 0.1% (1.8%), p<0.05).
The present study investigates the effect of the Thr164Ile polymorphism of the β2‐adrenergic receptors on cardiac contractility in normal subjects and in patients with LV dysfunction. Our data demonstrate that (1) subjects with Thr164Ile polymorphism and normal LV function show blunted chronotropic and contractile response to β2‐adrenergic stimulation; (2) the Thr164Ile genetic variant is associated with absence of β2‐adrenergically mediated myocardial contractile reserve in patients with idiopathic dilated cardiomyopathy; and (3) depressed contractile reserve in patients with heart failure with the Thr164Ile variant was associated with worse event‐free survival than in controls with heart failure.
The β‐adrenergic receptors are the main regulators of cardiac inotropy and chronotropy.1 Despite many similarities, β1‐adrenergic and β2‐adrenergic receptors have distinct genetic and pharmacological characteristics.15 β1‐Adrenergic receptors stimulate c‐AMP production by interacting exclusively with G stimulatory proteins, whereas β2‐aderenergic receptors can couple with both stimulatory and inhibitory G proteins. Furthermore, β1‐adrenergic receptor‐mediated responses are mainly related to c‐AMP production, whereas β2‐adrenergic receptor‐mediated signalling is more complex and not entirely defined.16,17,18
Previous studies demonstrated that congestive heart failure is associated with several abnormalities of downstream signalling of β‐adrenergic receptors.3,19,20,21,22 In particular, dysfunctional myocardium is characterised by a down regulation of β1‐adrenergic receptors, whereas the number of β2‐adrenergic receptors remains relatively stable. In addition, in various experimental models, genetic manipulations of β2‐adrenergic receptor‐dependent signalling were shown to protect or even rescue LV function of the failing heart.23,24,25 This corroborates the hypothesis that β2‐adrenergic receptors play a pivotal role in the control of myocardial contractility of the failing heart. Accordingly, it was also hypothesised that genetic changes in receptor function could profoundly affect the course of heart failure. Nevertheless, β2‐adrenergic receptors have been to be reported hyporesponsive in the failing myocardium,1,2,3,4 questioning the significance of genetic variants in β2‐adrenergic receptors in congestive heart failure.
Genetic heterogeneity of the β2‐adrenergic receptor with the replacement of threonine by isoleucine at codon 164 is associated with “loss of function” of the polymorphic receptor due to increased uncoupling.5 The presence of this genetic variant caused blunted cardiac inotropic and chronotropic responses,26 as well as impaired vasomotion.27,28 In humans with normal LV function and Thr164Ile polymorphism, Brodde et al7 reported a blunted increase in the duration of ECG‐derived electromechanical systole, an indirect index of cardiac performance, in response to β2‐adrenergic agonist stimulation.29 Our findings of reduced myocardial contractile response and pulse pressure in similar subjects extend this observation and corroborate the postulate that the Thr164Ile polymorphism directly modulates the β2‐adrenergic‐mediated inotropic reserve. In addition, analysis of the force–frequency relationship suggests that the blunted contractile response in the carriers with normal function is mainly related to reduce chronotropic response.
However, in light of existing abnormalities in β2‐adrenergic receptor signalling, it remains controversial whether similar effects would be observed in humans with failing myocardium. As expected, patients with dilated cardiomyopathy showed reduced myocardial contractile reserve and no change in arterial pulse pressure in response to terbutaline. Nevertheless, the Ile‐variant was associated with further deterioration of contractile response and force–frequency relationship at peak terbutaline infusion. Unlike Ile‐164 carriers with normal LV function, blunted increase in the force–frequency relationship in patients with idiopathic cardiomyopathy was related to reduction of both chronotropic and inotropic responses. Taken together, these observations suggest that the Ile‐164 variant further attenuates β2‐adrenergic receptor‐mediated myocardial contractile response also in patients with failing myocardium.
Only a few studies have addressed the clinical importance of the Ile‐164 variant in heart failure in humans.6,8 They suggested that patients with heart failure with the Thr164Ile polymorphism have lower exercise capacity8 and may have higher mortality or progression to transplantation.6 In our study, we failed to observe higher mortality or higher incidence of heart transplantation. However, it should be noted that our patients were characterised by higher LVEF and the vast majority of patients were treated with β blockers, which may account for the more favourable prognosis of our patient cohort. Nevertheless, the presence of the Ile‐164 variant was associated with higher incidence of clinical events including worsening of heart failure. Thus, these data corroborate the hypothesis that the Ile‐164 polymorphism is associated with attenuated contractile response to β2‐adrenergic‐mediated stimulation that may contribute to the worse clinical outcome in patients with heart failure.
The present study was not powered to investigate the effect of other alleles or haplotype combinations on β2‐adrenergic responses. It should be acknowledged that polymorphisms at codons 16 and 27 may have influenced the observed differences in contractile response to terbutaline.8 The incidence of both polymorphisms was similar between patients with dilated cardiomyopathy with and without the Ile‐164 variant. By contrast, the incidence of both variants was lower in subjects with the Thr164Ile variant and normal LV function than in controls. Nevertheless, these subjects showed reduced contractile and haemodynamic response corroborating the notion that the Ile‐164 variant is the main variant responsible for the blunted β2‐adrenergic responsiveness. This study also has an intrinsic limitation in the number of selected patients owing to the rarity of the Ile‐164 variant and the strict inclusion criteria used. The latter were necessary to ensure the absence of confounding factors, such as cardiac hypertrophy, ischaemic or valvular heart disease that could affect evaluation of cardiac contractile reserve.
The present data demonstrate that the presence of the Ile‐164 variant directly modulates β2‐adrenergic‐mediated myocardial contractile response in normal and in the failing human myocardium. Furthermore, the lower event‐free period in patients with heart failure with this variant suggests that the presence of Ile‐164 may also modulate progression and outcome in patients with congestive heart failure.
We thank Dr Philip MacCarthy for the critical reading of our manuscript. Secretarial help of Mrs Josefa Cano is greatly appreciated. We thank the staff of the Echocardiography laboratory, namely, Eddy Van Schuerbeeck, RN, Linda Mortier, RN, and Frank Staelens, RN, for technical assistance.
LV - left ventricular
LVEF - left ventricular ejection fraction
Sm - peak myocardial velocity of systolic shortening
Thr164Ile - threonine (Thr) is replaced by an isoleucine (Ile) at codon 164
Competing interests: None.