ADRB2 polymorphism Arg16Gly modifies the natural outcome of heart failure and dictates therapeutic response to β-blockers in patients with heart failure

Abstract

We sought to investigate the association of single nucleotide polymorphisms (SNPs) of the genes involved in βAR signaling with the response of patients to βAR blockers. A total of 2403 hospitalized patients with chronic heart failure (HF) were enrolled in a multicenter observational study as the first cohort and followed up for a mean period of 20 months. Genes for β1AR, β2AR, and the major cardiac G-protein-coupled receptor kinases (GRKs) GRK2 and GRK5 were analyzed to identify SNPs, and patients were stratified according to genotypes. A second independent cohort enrolling 919 patients with chronic HF was applied to validate the observed associations. The signaling properties of the key identified SNPs were assessed in vitro. Our data showed that HF patients harboring the Gly16 allele in the gene for β2AR (ADRB2) had an increased risk of the composite end point relative to patients who were homozygous for Arg16. Notably, these patients showed a beneficial response to βAR-blocker treatment in a G allele-dose-dependent manner, whereas Arg16 homozygotes had no response to βAR-blocker therapy. This Arg16Gly genotype-dependent heterogeneity in clinical outcomes of HF was successfully validated in the second independent population. Besides, the in vitro experiments revealed that G allele carriers were defective in β2AR-coupled inhibitory adenylate cyclase g (G_i) protein signaling.

Fig. 1. Enrollment and follow-up of patients with HF in the first stage.

At study termination on July 2015 in the first discovery stage, 2615 patients with HF had been screened for eligibility to be included in the study in three centers in China and 2403 patients were finally enrolled and genotyped. Of these patients, 149 (5.7%) were excluded for repeat admittance, and 63 (2.4%) were excluded because valvular heart disease was considered the main reason for HF. Of the study participants, 17 were lost from follow-up because incorrect contact information had been provided on enrollment. Of the 2386 patients included in the outcome analysis: all were included in the analysis of the prognostic utility of the ADRB2 Arg16Gly, ADRB2 Gln27Glu, and GRK5 Arg304His variants; 2377 patients were included in the analysis of the prognostic utility of the ADRB1 Arg389Gly variants; and 2384 patients were included in the analysis of the prognostic utility of the GRK5 Gln41Leu variants

Fig. 2. Clinical outcomes of heart failure patients and responses to β-blockers.

a Kaplan−Meier curves of the primary composite end point showing that the clinical outcomes significantly varied among the groups of patients with the different genotypes at ADRB2 amino acid site 16 (P < 0.001 by stratified log-rank test). G allele carriers had worse outcomes compared with those who were homozygous for AA (HR, 1.49; 95% CI, 1.21−1.83; P < 0.001 for AG/GG versus AA). b Kaplan−Meier curves of cardiac death demonstrating a similar association with the genotype of this SNP to the primary end point. The G allele was associated with increased risk of this individual end point (HR, 1.44; 95% CI, 1.16−1.79; P = 0.001 for AG/GG versus AA). c Among the entire cohort, β-blocker treatment was significantly associated with a reduced risk of the composite end point of cardiovascular death or heart transplantation (adjusted HR, 0.59; 95% CI, 0.45−0.76; P < 0.001). d For the patients who were not using BB therapy, Kaplan−Meier curves for the primary end point showed that the ADRB2 Arg16Gly genotype was significantly associated with reduced transplantation-free survival (P < 0.001 by log-rank test). e For the patients who received BB therapy for ≥6 months during the study, the genotype-based heterogeneity was not significantly different (log-rank P = 0.732). The probability of the composite primary end point (cardiovascular death or heart transplantation) was significantly decreased with β-blocker use among patients with f the GG genotype (adjusted HR, 0.38; 95% CI, 0.23−0.64; P < 0.001) and g the AG genotype (adjusted HR, 0.64; 95% CI, 0.42−0.96; P = 0.03), but not with h the AA genotype (adjusted HR, 0.70; 95% CI, 0.45−1.10, P = 0.121). The extent of benefit of β-blocker treatment seemed to be G allele-dose-dependent. i Adjusted HRs are shown for the composite primary end point and for the individual end point of death from a cardiovascular cause for patients in the five specified missense variant subgroups. To assess the impact of both of the ADRB2 missense polymorphisms together, we also examined outcomes for patients stratified according to three genotype combinations: homozygous for both Arg16 and Gln27 (that is, patients with only the major alleles; designated as group A), homozygous for both Gly16 and Glu27 (that is, patients with only the minor alleles; designated as group C), and other genotypes (designated as group B). The blue squares and black lines represent the HRs and 95% CIs. The size of the blue square corresponds to the number of patients in the subgroup. The P values were calculated by Cox proportional hazard models, with a two-sided P value of 0.05 indicating statistical significance after adjustment for the clinical covariants, with an unbalanced distribution between groups. BB β-blocker, CI confidence interval, HR hazard ratio

Fig. 3. Functional analysis of the ADRB2 Arg16Gly variants in cell culture systems.

a The western blots of β2AR and GAPDH for lysates of adenovirus-infected rat cardiomyocytes showing that the transfection method (multiplicity of infection of 100 for 24 h) produced equal expression of ADRB2-Arg16 and ADRB2-Gly16, which is true irrespective of deglycosylation treatment of the cell lysates with PNGase F. b PTX treatment (0.75 μg/ml overnight) enhanced the zinterol (1×10⁻⁵ M)-induced contractile response in adult rat cardiac myocytes expressing ADRB2-Arg16, but not in those expressing ADRB2-Gly16 (multiplicity of infection of 100 for 24 h; N = 8 for each group). c ADRB2 knockout mouse cardiomyocytes were infected with Adeno-ADRB2-Arg16 or Adeno-ADRB2-Gly16 viruses as described in Materials and Methods and then subjected to stimulation with zinterol (3×10⁻⁷ M). The zinterol-induced contractile responses were 200−300% in the absence of PTX. PTX enhanced these responses in cells expressing ADRB2-Arg16, but not in cells expressing ADRB2-Gly16. (N = 10–16 cells from four mouse hearts for each data point). d Peripheral lymphocytes were isolated from HF patients with different genotypes at the ADRB2 Arg16Gly locus. The inhibition of G_i by PTX enhanced β2AR-mediated cAMP accumulation in cells with the AA genotype (N = 10; P < 0.05), whereas cAMP accumulation in cells with either the AG (N = 10) or GG (N = 6) genotype was insensitive to PTX treatment. Therefore, peripheral lymphocytes harboring the G allele of the ADRB2 Arg16Gly A > G polymorphism showed deficient coupling of G_i to the β2AR. All the data were shown as the mean ± SEM. β2AR β2 adrenergic receptor, HF heart failure, PTX pertussis toxin. *p<0.05, **p<0.01, ***p<0.001