Genetic dysregulation of endothelin-1 is implicated in coronary microvascular dysfunction

Abstract

Aims: Endothelin-1 (ET-1) is a potent vasoconstrictor peptide linked to vascular diseases through a common intronic gene enhancer [(rs9349379-G allele), chromosome 6 (PHACTR1/EDN1)]. We performed a multimodality investigation into the role of ET-1 and this gene variant in the pathogenesis of coronary microvascular dysfunction (CMD) in patients with symptoms and/or signs of ischaemia but no obstructive coronary artery disease (CAD).

Methods and results: Three hundred and ninety-one patients with angina were enrolled. Of these, 206 (53%) with obstructive CAD were excluded leaving 185 (47%) eligible. One hundred and nine (72%) of 151 subjects who underwent invasive testing had objective evidence of CMD (COVADIS criteria). rs9349379-G allele frequency was greater than in contemporary reference genome bank control subjects [allele frequency 46% (129/280 alleles) vs. 39% (5551/14380); P = 0.013]. The G allele was associated with higher plasma serum ET-1 [least squares mean 1.59 pg/mL vs. 1.28 pg/mL; 95% confidence interval (CI) 0.10-0.53; P = 0.005]. Patients with rs9349379-G allele had over double the odds of CMD [odds ratio (OR) 2.33, 95% CI 1.10-4.96; P = 0.027]. Multimodality non-invasive testing confirmed the G allele was associated with linked impairments in myocardial perfusion on stress cardiac magnetic resonance imaging at 1.5 T (N = 107; GG 56%, AG 43%, AA 31%, P = 0.042) and exercise testing (N = 87; -3.0 units in Duke Exercise Treadmill Score; -5.8 to -0.1; P = 0.045). Endothelin-1 related vascular mechanisms were assessed ex vivo using wire myography with endothelin A receptor (ETA) antagonists including zibotentan. Subjects with rs9349379-G allele had preserved peripheral small vessel reactivity to ET-1 with high affinity of ETA antagonists. Zibotentan reversed ET-1-induced vasoconstriction independently of G allele status.

Conclusion: We identify a novel genetic risk locus for CMD. These findings implicate ET-1 dysregulation and support the possibility of precision medicine using genetics to target oral ETA antagonist therapy in patients with microvascular angina.

Trial registration: ClinicalTrials.gov: NCT03193294.

Keywords: Coronary microvascular dysfunction; Endothelin-1; Microvascular angina; Precision medicine; Single-nucleotide polymorphism; Stable angina pectoris.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Figures

Figure 1

Detrimental effects of rs9349379-G allele on coronary microvascular function and endothelin-1. (A) Patients with G allele were over twice as likely to have underlying microvascular dysfunction (odds ratio per G allele 2.33, 95% confidence interval 1.10–4.96; P = 0.027) Even after adjustment for other risk factors the G allele was predictive of microvascular disease (odds ratio 2.31; 95% confidence interval 1.0–4.91). This finding supports a detrimental impact on the coronary microcirculation of a lifetime of increased endothelin gene expression. (B) In a multivariable regression model adjusting for baseline group differences, patients with rs9349379-G allele had higher plasma endothelin-1 (least squares mean 1.59 pg/mL vs. 1.28 pg/mL; 95% confidence interval 0.10–0.53; P = 0.005).

Figure 2

Genotype: phenotype association of G allele with invasive coronary microvascular dysfunction. (A–C) The prevalence of microvascular dysfunction detected during invasive coronary testing was associated with genotype status (AA 60%, AG 75%, GG 83%; P = 0.021). Presence of abnormal coronary flow reserve and microcirculatory resistance were linearly associated with each additional G allele. P-value represents Pearson χ2 test for linear trend (categorical data). (D) Coronary flow reserve was lower amongst subjects with two high-risk G alleles (rs9349379) consistent with detrimental effects of increased endothelin gene expression on the coronary microcirculation (Kruskal–Wallis between groups dotted line, P = 0.046). A priori subgroup analysis (AA vs. GG group—solid line) showed lower CFR in the GG group (P = 0.013). Data are median CFR plus error bars represent 95% confidence intervals for the median. P = 0.021, P = 0.030, P = 0.029 and P = 0.046.

Figure 3

Genotype: phenotype association of G allele with non-invasive ischaemia testing. (A) Cardiovascular stress magnetic resonance imaging at 1.5 T (N = 107). There was a linear relationship between the G allele and presence of an inducible perfusion defect on cardiac magnetic resonance (χ2 test for linear trend P = 0.042). (B) The relationship was more robust when considering with invasive evidence of coronary microvascular dysfunction and/or inducible perfusion defect. Over 90% of GG subjects had at least one abnormality compared with only 65% of AA subjects (P < 0.001). (C and D) Myocardial perfusion reserve was numerically reduced in AG and GG subjects compared with AA subjects; however, this was not statistically significant (P-value represents analysis of variance test for trend). Error bars represent 95% confidence intervals for the mean. (E) Invasive evidence of microvascular dysfunction (defined by abnormal response to intracoronary acetylcholine and/or systemic adenosine) was functionally significant and associated with ischaemic burden on symptom limited exercise treadmill testing (coronary microvascular dysfunction −2.3 vs. no coronary microvascular dysfunction +3.5; difference −5.8 units; −8.2 to −3.3; P < 0.001). (F) Exercise treadmill testing (n = 84). There was a relationship between genotype group and worsening ischaemia on stress testing (analysis of variance P-trend = 0.045). The mean difference in ischaemia by Duke treadmill score between group GG and group AA was −3.0 units (95% confidence interval −5.8 to −0.1; P = 0.045). Error bars represent 95% confidence intervals for the mean.

Figure 4

Endothelin-1 ex vivo vascular biology by genotype. (A) cumulative concentration response curve to endothelin-1 in the three groups in the presence and absence of ETA antagonist BQ123 (n = 44). Similar antagonist potency (rightward curve shift) for each group suggesting firstly that the ETA receptors are the dominant effectors of the endothelin-1 vasoconstrictor response and secondly that the ETA receptor pathway is not down-regulated in spite of the elevated endothelin-1 gene expression and known increase in endothelin-1 activity in the G allele single-nucleotide polymorphism patients. (B) Antagonist potency of novel therapeutic oral ETA receptor antagonist zibotentan [N = 8, mean 7.54 (95% confidence interval 7.27–7.82)] is similar to peptide antagonist BQ123 [N = 27, mean 7.53 (95% confidence interval 7.37–7.69)]. Higher pKB represents a higher antagonist potency. (C) Zibotentan: reversal of established endothelin-1 vasoconstriction. Proof of concept dose-dependent reversal of potent and established endothelin-1-mediated peripheral arteriolar vasoconstriction. Crucially, the highest concentration tested which is also the plasma concentration achieved by a clinically relevant dose of 10 mg/day rapidly and fully reversed the established endothelin-1 constrictor response, indicative of efficacy in conditions of vasospasm. Comparison using ordinary two-way analysis of variance including time and dose both significant factors (P < 0.001 after adjustment for multiple testing).

Figure 5

GG (high-risk endothelin-1 gene enhancer). Illustrative case from a patient with stable angina including representative images from invasive and non-invasive work up are shown in relation to clinical presentation and endothelin-1 enhancer genotype. Maximum ST represents the maximum planar or down sloping ST-segment depression during the exercise treadmill test. Invasive coronary angiography of both subjects is near identical showing only minimal luminal irregularities. White arrows represent subendocardial inducible ischaemic myocardium during adenosine stress magnetic resonance imaging in a patient with severe coronary microvascular dysfunction. Ex vivo vascular biology (bottom panel) shows typical endothelin-1-mediated vessel constriction during wire myography. Increasing vessel tension corresponds to the rising curve at each dose titration. A paired identical vessel experiment is performed after incubation with BQ123, an ETA receptor antagonist. This curve is marked in blue, the curve of endothelin-1 response is shifted to the right indicating that the ETA receptor mediates vasoconstriction. Despite the endothelin-1 gene enhancer, the GG subject does not appear to have ETA receptor down-regulation with similar levels of antagonist potency. This supports that ETA receptor antagonism in this group of patients may have therapeutic benefit. CFR, coronary flow reserve; ETA, endothelin A receptor; FFR, fractional flow reserve; IMR, index of microcirculatory resistance.

Take home figure

Study overview: endothelin-1 gene enhancer in microvascular angina. Three hundred and ninety-one patients with stable angina were prospectively enrolled without prior knowledge of coronary anatomy. One hundred and eighty-five (47%) had no obstructive coronary artery disease and thus eligible for invasive coronary vasoreactivity testing and further sub-studies. One hundred and fifty-one of 185 (82%) were able to undergo adjunctive invasive tests for coronary microvascular dysfunction. One hundred and nine (72%) subjects tested had evidence of coronary microvascular dysfunction. One hundred and forty subjects underwent genetic analysis for rs9349379-G allele with an allele frequency of 46% (129/280 alleles). The frequency of detrimental G alleles was higher than reference genome bank control subjects (46% vs. 39%; P = 0.013). Patients with rs9349379-G allele had higher serum endothelin-1 and over double the odds of coronary microvascular dysfunction (odds ratio 2.33, 95% confidence interval 1.10–4.96; P = 0.027). In addition, subjects were more likely to have impaired myocardial perfusion (P = 0.04) and exercise tolerance (−3.0 units in Duke Exercise Treadmill Score; P = 0.045). Peripheral small artery reactivity to endothelin-1 and affinity of ETA receptor antagonists were preserved in the rs9349379-G allele group (P = 0.209). Crucially, zibotentan tested at clinically relevant concentrations, fully reversed an established endothelin-1 vasoconstriction, indicative of efficacy in conditions associated with vasospasm. This suggests that ETA receptor antagonism in this group of patients may have therapeutic benefit.