Introduction

Hypertrophic cardiomyopathy (HCM) is the most common genetically transmitted cardiac disorder, affecting about 1/500 of individuals of the population.¹ While patients may be symptom free in the early stages of the disease, most patients suffer from exercise intolerance and shortness of breath as the disease progresses. Impaired left ventricular filling and dynamic left ventricular outflow obstruction are important mechanisms underlying symptoms in HCM and both can be augmented by myocardial hypercontractility. Therefore, adrenergic activity plays a central role in the clinical course of HCM by increasing cardiac contractility and the heart rate through B1 adrenoceptor activation. Moreover, adrenergic activity can also cause structural changes in the myocardium; previous studies showed accelerated myocyte necrosis and cardiac fibrosis by isoproterenol through beta adrenergic stimulation.² Similarly, renin synthesis in response to B1 adrenoceptor activity activates the renin–angiotensin–aldosterone system, which causes remodeling of the heart chambers along with myocardial fibrosis. Considering the aforementioned potential effects of the adrenergic activation in the pathophysiology of HCM, blockage of the adrenergic activity is an essential therapeutic target in patients with HCM and the current guidelines recommend beta-blocker drugs as the first-line medical therapy in symptomatic patients with HCM, which mainly affect via reducing myocardial contractility and the heart rate.^1,3 However, there may be substantial variability in symptom relief in response to beta blocker therapy among HCM patients and some patients remain unresponsive to beta-blockers.⁴ While the causes of this variability of response to beta blockers are not clearly understood, one potential mechanism may reside in the polymorphism of beta-adrenoceptors. There are previously demonstrated single nucleotide polymorphisms (SNP) of the beta-adrenoceptor coding genes (ADRB-1) which alter the activation of beta-receptors via adrenergic stimulus.⁵ The most commonly studied SNPs at the adrenoceptor gene beta-1 (ADRB-1) are serine–glycine polymorphism at 49. position and arginine–glycine polymorphism at 389. position. Several studies demonstrated that these SNPs have clinical impact on drug responsiveness and disease progression in hypertension and dilated cardiomyopathy patients.^6-9 To the best of our knowledge, there is no study in the literature which investigated the impact of ADRB-1 SNPs on the clinical course of patients with HCM. In this study, our primary aim was to investigate the impact of ADRB-1 gene polymorphism on clinical and structural features of hypertrophic cardiomyopathy. We also searched for the association between ADRB-1 polymorphism and response to beta-blocker therapy in patients with HCM.

Methods

Study Design

The study was conducted in a university hospital cardiology clinic, which is a tertiary reference center for HCM. Patients who were diagnosed with HCM but naive to medical therapy were prospectively enrolled after informed consent was obtained. The study protocol was following the Declaration of Helsinki and was approved by the Local Ethics Board of our institution (date: August 10, 2018; number: 30330229-6040102-42266). The diagnosis of HCM was made according to current guidelines; at least 15 mm of thickness at any region of the left ventricle which was not related to the increased afterload.^1,3 Inclusion criteria were as follows: (i) patients with a clear diagnosis of HCM, (ii) patients over 18 years, (iii) patients naive to medical therapy including beta-blockers and calcium channel blockers. Patients were excluded from the study if one of the following were present: (i) already under medical treatment for HCM, (ii) previous septal reduction therapy either via alcohol ablation or surgical myectomy, (iii) refusal for informant consent, (iv) unable to attend regular visits, and (v) having resistant hypertension. Patients’ demographic features, family history, functional capacity, heart rate and blood pressures were recorded. The 5-year risk of sudden cardiac death was calculated using the ESC risk calculator during the enrolment and ICD implantation was performed according to current guideline recommendations.³

Beta-Blocker Therapy

Following enrolment, metoprolol 25 mg once a day was prescribed to each patient, and dose adjustment was made according to blood pressure and mean heart rate which were derived from 24-hour ambulatory Holter monitoring. The dose of metoprolol was up titrated to achieve symptom free status with a maximum dose of daily 200 mg. Pre-treatment and post-treatment blood pressures, heart rate and functional capacity of the patients were recorded. Functional capacity was defined according to the New York Heart Association (NYHA) classification. Beta-blocker intolerance was defined as symptomatic bradycardia, hypotension, or deterioration of exercise capacity following beta-blocker initiation that required discontinuation of beta-blocker therapy.

Echocardiography

All patients underwent comprehensive echocardiographic evaluation with Philips EPIQ echocardiography machine (Philips, Andover, MA, USA). One of the 2 board-certified cardiologists performed echocardiography and the other one analyzed the data. In case of disagreement, a third senior cardiologist reviewed the data, and a final decision was reached. Maximum wall thickness was measured at the particular left ventricular (LV) segment where the wall thickness is highest. LV ejection fraction was calculated using biplane modified Simpson’s method. Left atrial volume was also measured using biplane Simpson’s method and indexed to the body surface area. LV longitudinal strain assessment was performed by an automatic border detection program, and manual correction was made when necessary. Systolic anterior motion (SAM) was defined as the motion of the anterior mitral valve leaflet towards the interventricular septum during systole. LV apical aneurysm was diagnosed when obvious akinesia/aneurysm was observed at the LV apex. LV outflow gradient was measured in apical 5-chamber view using pulsed wave Doppler at rest and following Valsalva maneuver.

ECG Evaluation

Patients’ initial heart rhythm and rate were determined on a 12 lead ECG. Presence of fragmented QRS which is defined as the presence of notching within the R wave or S wave was recorded. A 24-hour ambulatory ECG Holter monitor was used to monitor heart rate and rhythm and maximum, minimum, and mean heart rate in all the patients. ECG Holter monitoring was performed on every patient during enrolment and at sixth-month visit, and a comparison between these 2 recordings was made to assess the effect of beta-blocker therapy. Ventricular tachycardia was defined as a cardiac rhythm that is originating from the ventricles with a rate of >100 beats/min. Atrial fibrillation was diagnosed when an irregular rhythm with the absence of obvious p waves was seen either on 12-ECG or Holter monitoring. Ventricular extra systole count in 24 hours was recorded.

Genetic Analysis

Single nucleotide polymorphism (SNP) protocol was used for the genetic analyses. To assess polymorphisms in adrenoceptor beta-1 gene rs1801252 (Ser49Gly) and rs1801253 (Arg389Gly), TaqMan System (Applied Biosystems, Life Technologies) Primer ProbMix and qPCR Probes Master (Jena Bioscience, Germany, PCR-360) were used. Two SNPs were evaluated which cause Arginine/Glycine polymorphism at the 389. position and Serine/Glycine polymorphism at the 49. position of adrenoceptor beta-1 gene. Patients were divided into 3 groups according to Arg389Gly polymorphism; Arg389Arg homozygotes, Arg389Gly heterozygotes and Gly389Gly homozygotes. However, due to the lower number Gly49Gly homozygotes, patients were divided into 2 groups according to Ser49Gly polymorphism; Ser49Ser homozygotes constituted group 1, and Ser49Gly and Gly49Gly constituted group 2 (Glycine carriers).

Follow-up

Patients were followed up in a dedicated outpatient clinic. The follow-up visits were done at the end of the first week, first month, and then 3 months after the second visit. Patients’ heart rates, blood pressure and response to beta-blocker therapy were recorded to optimize the therapy. However, only the data from the initial encounter and final visits with the patients were included in the statistical analysis.

Statistical Analysis

All statistical analyses were conducted by Statistical Package for the Social Sciences Statistics, version 25.0, for Windows (SPSS Inc., Chicago, Ill, USA) Program. The sample size is defined by effect size, type 1 error, and power of the study with the G-Power Program. Type 1 error was 5%, power of the study was 80%, effect size was calculated by other studies in the literature. The Kolmogorov–Smirnov test was used to analyze the normality of the data. Normally distributed variables were expressed as mean ± SD, while non-normally distributed variables were expressed as median with minimum–maximum value. The categorical variables are presented as percentages. A Chi-square test was used to assess differences in categorical variables between groups. Student’s t-test or Mann Whitney U-test was used to compare unpaired samples as needed. The primary analysis used ANOVA to compare all reported data for parametric variables, whereas the Kruskal–Wallis test was used to compare nonparametric variables between the median value of the survival days. According to the values distribution, paired t-test or/and Wilcoxon test is used for the comparison of the same subjects’ values before and after treatment with beta-blockers of HC patients. Significance was assumed at a 2-sided P < .05.

Results

The study was conducted from September 2018 to May 2021. One hundred ninety-four patients with a working diagnosis of HCM were screened. Patients with resistant hypertension (36 patients), aortic sclerosis (4 patients), Amyloidosis (6 patients), and Fabry disease (1 patient) were excluded during the study and finally, 147 patients were enrolled. The mean follow-up duration was 23 months (6-32 months). Table 1 demonstrates the baseline demographic variables and echocardiographic features of the study population and group statistics according to gender distinctions. The most common presenting symptoms were shortness of breath and palpitations during physical activity (65% and 50% of the study population respectively). Previous episodes of syncope (20.4%) and exercise angina (17%) were the other common symptoms of the patients. The vast majority of the study population demonstrated septal hypertrophic cardiomyopathy (83%). The distribution of HCM variants in the study population was; 83% septal, 11% apical, 3% global, and 3% mid-ventricle. Systolic anterior motion of the mitral valve was detected in 40 patients (27%).

The target dose beta-blocker therapy 200 mg/day was achieved in 92.5% of the study patients. Beta-blocker therapy was not up-titrated or discontinued in 10 patients due to increased exercise intolerance or hypotension.

The risk of sudden cardiac death which is determined using the HCM sudden cardiac death calculator was low (<4%) in 55.8%, intermediate (4%-6%) in 24.5%, and high (>6%) in 19.7% of the study population.

Arginin389Glycine Polymorphism

Patients were divided into 3 groups according to Arginin389Glycine gene polymorphism; Arg389 homozygotes (n= 43; 29%), Arg389Glycine heterozygotes (n= 42; 29%) and Glycine389 Homozygotes (n= 62; 42%).

Basal heart rate, rhythm, and blood pressures: The mean heart rate was 79.1 ± 15.5 beats/min in Arg389 homozygotes, 81.7 ± 17.2 beats/min in Arg389Gly heterozygotes, and 83.2 ± 15.7 beats/min in Gly389 homozygotes and there was no significant difference between the groups (Table 2). The incidence of atrial fibrillation and non-sustained ventricular tachycardia were similar between the groups (P = .495 and P = .553, respectively). There was also no significant difference between groups with respect to 24 hours VES count (P = .693). Systolic blood pressures during the enrolment were 145.7 mm Hg for Arg389 homozygotes, 148.4 mm Hg for Arg389Gly heterozygotes and 147.1 mm Hg for Gly389 homozygotes. There was no significant difference between the groups (P = .905).

Response to beta-blocker therapy: Following beta-blocker therapy mean and maximum heart rate decreased significantly (P < .001 and P = .001 for arginine homozygotes, P < .001 for glycine homozygotes, P = .006 and P = .002 for heterozygotes). Moreover, total premature ventricular contractions decreased significantly (P = .001 for arginine homozygotes, P < .001 for glycine homozygotes and P = .002 for heterozygotes) (Table 3).

Impact of beta-blocker therapy on NT-proBNP and LVOT gradient: Baseline NT-proBNP and LVOT gradients were similar between groups (P = .934 and P = .516, respectively). Following treatment, a decrease in NT-proBNP values was observed in all patients. This decrease was statistically significant in both the arginine homozygous and heterozygous groups (P = .010 and P < .001, respectively) but did not reach significance in the glycine homozygous group (P = .066). When comparing LVOT gradients after treatment, a significant gradient reduction was observed in all groups (P < .001) (Table 4).

Structural features: The mean maximum wall thickness was 20 ± 5 mm in Gly389 homozygote, 20 ± 5 mm in Arg389 homozygote, and 22 ± 5 mm in heterozygote individuals. There was no significant difference between the groups (P = .230). Left ventricular outflow tract obstruction was also similar between the groups (P = .827). Ventricular strain analyses demonstrated that GLS mildly decreased in the vast majority of the patients and there was no significant difference between the groups (P = .241) (Table 5).

Ser49Gly Polymorphism

Patients were divided into 2 groups according to Ser49Gly gene polymorphism; there were 106 patients with Ser49 homozygote (group 1). There were only 2 patients with Gly49 homozygote and hence rest of the patients were included in group 2 (Gly49 carriers).

Basal heart rate, rhythm and blood pressure: The mean and maximum heart rates were similar in patients with homozygote Ser49 gene and Gly carriers (P = .105 and P = .527, respectively). The incidence of atrial fibrillation and non-sustained VT were also similar between groups. Ser49 homozygotes had significantly more VES count compared to Gly carriers (P = .011) (Table 6). Systolic blood pressure at the first visit was comparable between the groups (148 mm Hg vs. 143 mm Hg, P = .581).

Response to beta-blocker therapy: Mean and maximum heart rate decreased significantly in both Ser49 homozygote and Gly carrier patients (P < .001 and P < .001 for serine homozygotes and P = .054 and P = .012 for glycine carriers, respectively) following beta-blocker therapy. However, the count of daily premature ventricular contractions decreased significantly only in Ser49 patients (P < .001) but not in Gly carriers (P = .297) (Table 7).

Impact of beta-blocker therapy on NT-proBNP and LVOT gradient: There was no significant difference in baseline NT-proBNP levels and LVOT gradients among the groups (P = .947 and P = .191, respectively). Following treatment, a decrease in NT-proBNP values was observed in all patients. This decrease was statistically significant in serine homozygotes (P < .001). After treatment, significant reductions in LVOT gradients were observed in all groups (P < .001) (Table 8).

Structural features: The mean maximum wall thickness was comparable between the groups (20 ± 4 mm vs. 21 ± 5 mm, P = .138). Left ventricular outflow tract obstruction was detected in 24% of Ser49 homozygotes and 17% of Gly carriers (P = .267). Ventricular strain analyses demonstrated that GLS decreased mildly in both groups and there was no significant difference between the groups (−15.6% vs. −15.4%, P = .833) (Table 9).

Discussion

In this study, we searched for the potential effect of 2 common polymorphisms in adrenoceptor beta-1 gene polymorphism on the heart rate and rhythm and response to beta-blocker therapy in patients with hypertrophic cardiomyopathy. Our findings show that the Arg389Gly polymorphism does not have significant effect on the baseline heart rate and response to beta blocker therapy. However, Ser49Gly polymorphism appears to have significant effect on the VES count and suppressibility with beta blockers. In addition, following beta-blocker therapy, natriuretic peptide levels and left ventricular outflow obstruction decreased significantly.

The adrenergic system plays a central role during the course of HCM via increasing heart rate and myocardial contractility which further deteriorates LV diastolic functions and hence beta-blocker therapy has become the cornerstone of medical treatment of symptomatic HCM patients. Our results indicated that beta-blocker therapy reduces mean and maximum heart rate in hypertrophic cardiomyopathy patients regardless of ADRB-1 gene polymorphisms. Moreover, apart from Gly49 carrier subgroup, the total number of daily VES decreases significantly underlying the efficacy of beta-blocker therapy in HCM patients. Previous studies demonstrated that polymorphisms of the genes encoding beta-1 adrenergic receptors, particularly those resulting in amino acid substitution at the 49^th and 389^th positions, may have effects on the response to the adrenergic system and response to beta-blocker therapy.^9-12 However, the results of the studies are inconsistent. In vitro studies by Rathz et al⁸ and Levin et al,⁹ chronic exposure to a beta-receptor agonist resulted in the downregulation of beta-receptors in Gly49 carriers which did not observe in Ser49 homozygous.^8,9 They concluded that Gly49 carrier heart failure patients may have better outcomes. In our study, the mean heart rate prior to beta-blocker therapy was higher in Ser49 homozygous and following beta-blocker therapy, the mean heart rate decreased significantly in Ser49 homozygote and Ser49Gly heterozygote patients. This finding indicates that Ser49 homozygote patients are more susceptible to adrenergic stimulation and beta-blocker therapy is more efficacious in these patient groups which is concordant with Rathz et al⁸ findings. Lanfear et al¹³ have studied the effect of beta-blocker in patients with heart failure and concluded that beta-blocker therapy is 5 times more effective in Ser49 homozygous which is in line with our findings. Talameh et al¹⁴ also studied a similar patient subset and concluded that the survival benefit of beta-blocker therapy in heart failure patients is detected in Ser49 homozygous, not heterozygous. In our study, there was no significant impact of Arg389Gly polymorphism on heart rate and response to beta-blocker therapy. Although the literature includes several studies which have reported that Arg389Gly polymorphism has a significant effect on left ventricular remodeling and response to beta-blocker therapy, there are also several studies reporting contradicting data. In light of these conflicting results, the effect of Arg389Gly polymorphism needs to be clarified.

The principal goal of beta-blocker therapy is to reduce the symptoms particularly those associated with increased myocardial contractility. In this study, we search for the impact of beta-blocker therapy on the quantitative parameters including natriuretic peptides and outflow gradient. Our results demonstrated that following beta-blocker therapy natriuretic peptide levels decreased in all HCM patients particularly in patients with Ser49 homozygotes. Concordant with natriuretic peptide levels, left ventricular outflow obstruction was also decreased in all patients. Recently Dybro et al. investigated the impact of beta-blocker therapy on haemodynamic and structural variables among obstructive HCM patients and demonstrated lower LVOT gradient following beta-blocker therapy and increased LV end-diastolic diameter.¹⁵ Our results are in line with aforementioned study. Geske et al¹⁶ search for the potential utility of natriuretic peptides in HCM patients and concluded that septal reduction therapy which resolves LVOT obstruction significantly is associated with decreased natriuretic peptides. Hamada et al¹⁷ demonstrated that chronic use of cibenzoline, a class 1A antiarrhytmic drug with negative inotropic properties, resulted in lower natriuretic peptide levels among HCM patients. Increased left ventricular end-diastolic volume due to prolonged diastole, decreased myocardial contractility and LVOT obstruction might be associated with decrease in natriuretic peptide levels. In this regard, further studies are needed to clarify our findings.

Study Limitations

Although this is a prospective study, there are several limitations. The major limitation of our study is the under-representation of high-risk HCM patients. The reason for this was patients admitted with palpitations/syncope were hospitalized in the intensive unit to detect VAs and ICD implantation was performed without cardiac MRI confirmation. The second limitation was being unable to report clinical outcomes. Although we recorded clinical outcomes, due to the lower number of cardiovascular events, the comparison would likely be misleading and hence we did not report the outcomes. Additionally, the limited number of participants in our study is another notable limitation, which may impact the generalizability of our findings.

Conclusion

In conclusion, we have demonstrated that polymorphism in the adrenoceptor beta-1 gene, particularly Ser49Gly polymorphism, is associated with structural and clinical features among patients with hypertrophic cardiomyopathy. Moreover, Ser49Gly polymorphism has a significant impact on response to beta-blocker therapy. Further studies with higher populations and longer follow-ups are needed to determine the role of adrenoceptor beta-1 gene polymorphism for the risk assessment and treatment strategy in patients with hypertrophic cardiomyopathy.

Footnotes

Ethics Committee Approval: The ethics committee approval for the study was provided by the İstanbul University-Cerrahpaşa Ethics Committee (date: August 10, 2018; number: 30330229-6040102-42266).

Informed Consent: Verbal informed consent was obtained from the patients who agreed to take part in the study.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept – H.K., B.K.; Design – B.İ., D.R.; Supervision – E.D., R.E.; Resources – A.U.S., N.M.; Materials – M.G., O.A.K.; Data Collection and/or Processing – D.R., U.R.; Analysis and/or Interpretation – D.R., E.Ç.; Literature Search – E.Y., D.R.; Writing – C.i., D.R.; Critical Review – R.E., E.D.

Declaration of Interests: The authors have no conflict of interest to declare.

References

1. Ommen SR, Mital S, Burke MA. 2020 AHA/ACC guideline for the diagnosis and treatment of patients with hypertrophic cardiomyopathy. Circulation. 2020;142(25):558-631. https://doi.org/10.1161/CIR.0000000000000937
2. Ayala P, Montenegro J, Vivar R. Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproterenol. Exp Mol Pathol. 2012;92(1):97-104. https://doi.org/10.1016/J.YEXMP.2011.10.012
3. Zamorano JL, Anastasakis A, Borger MA. ESC guidelines on diagnosis and management of hypertrophic cardiomyopathy: the task force for the diagnosis and management of hypertrophic cardiomyopathy of the European Society of Cardiology (ESC). Eur Heart J. 2014;35(39):2733-2779. https://doi.org/10.1093/eurheartj/ehu284
4. Dybro AM, Rasmussen TB, Nielsen RR, Andersen MJ, Jensen MK, Poulsen SH. Randomized trial of metoprolol in patients with obstructive hypertrophic cardiomyopathy. J Am Coll Cardiol. 2021;78(25):2505-2517. https://doi.org/10.1016/j.jacc.2021.07.065
5. Muthumala A, Drenos F, Elliott PM, Humphries SE. Role of β adrenergic receptor polymorphisms in heart failure: systematic review and meta-analysis. Eur J Heart Fail. 2008;10(1):3-13. https://doi.org/10.1016/j.ejheart.2007.11.008
6. Gjesing AP, Andersen G, Albrechtsen A. Studies of associations between the Arg389Gly polymorphism of the β1-adrenergic receptor gene (ADRB1) and hypertension and obesity in 7677 Danish white subjects. Diabet Med. 2007;24(4):392-397. https://doi.org/10.1111/J.1464-5491.2006.02031.X
7. Wang H, Liu J, Liu K. β1-adrenoceptor gene Arg389Gly polymorphism and essential hypertension risk in general population: a meta-analysis. Mol Biol Rep. 2013;40(6):4055-4063. https://doi.org/10.1007/s11033-012-2483-1
8. Rathz DA, Brown KM, Kramer LA, Liggett SB. Amino acid 49 polymorphisms of the human beta1-adrenergic receptor affect agonist-promoted trafficking. J Cardiovasc Pharmacol. 2002;39(2):155-160. https://doi.org/10.1097/00005344-200202000-00001
9. Levin MC, Marullo S, Muntaner O, Andersson B, Magnusson Y. The myocardium-protective Gly-49 variant of the beta 1-adrenergic receptor exhibits constitutive activity and increased desensitization and down-regulation. J Biol Chem. 2002;277(34):30429-30435. https://doi.org/10.1074/jbc.M200681200
10. White HL, De Boer RA, Maqbool A. An evaluation of the beta-1 adrenergic receptor Arg389Gly polymorphism in individuals with heart failure: a MERIT-HF sub-study. Eur J Heart Fail. 2003;5(4):463-468. https://doi.org/10.1016/S1388-9842(03)00044-8
11. Peng Y, Xue H, Luo L, Yao W, Li R. Polymorphisms of the β1-adrenergic receptor gene are associated with essential hypertension in Chinese. Clin Chem Lab Med. 2009;47(10):1227-1231. https://doi.org/10.1515/CCLM.2009.276
12. Luzum JA, English JD, Ahmad US. Association of genetic polymorphisms in the beta-1 adrenergic receptor with recovery of left ventricular ejection fraction in patients with heart failure. J Cardiovasc Transl Res. 2019;12(4):280-289. https://doi.org/10.1007/s12265-019-09866-5
13. Lanfear DE, Peterson EL, Zeld N, Wells K, Sabbah HN, Williams K. Beta blocker survival benefit in heart failure is associated with ADRB1 Ser49Gly genotype. J Card Fail. 2015;21(8):-. https://doi.org/10.1016/J.CARDFAIL.2015.06.169
14. Talameh J, Garrand A, Ghali J. Beta-1 adrenergic receptor genotype SER49GLY is associated with beta-blocker survival benefit in patients with heart failure. J Am Coll Cardiol. 2012;59(13):E861-. https://doi.org/10.1016/S0735-1097(12)60862-6
15. Dybro AM, Rasmussen TB, Nielsen RR. Effects of metoprolol on exercise hemodynamics in patients with obstructive hypertrophic cardiomyopathy. J Am Coll Cardiol. 2022;79(16):1565-1575. https://doi.org/10.1016/j.jacc.2022.02.024
16. Geske JB, McKie PM, Ommen SR, Sorajja P. B-type natriuretic peptide and survival in hypertrophic cardiomyopathy. J Am Coll Cardiol. 2013;61(24):2456-2460. https://doi.org/10.1016/j.jacc.2013.04.004
17. Hamada M, Ikeda S, Ohshima K. Impact of chronic use of cibenzoline on left ventricular pressure gradient and left ventricular remodeling in patients with hypertrophic obstructive cardiomyopathy. J Cardiol. 2016;67(3):279-286. https://doi.org/10.1016/j.jjcc.2015.05.014