β1- and β2-adrenergic Receptor Haplotypes Regulate Therapeutic Responses to Placebo and the Biased Ligand β-blocker Bucindolol

Background ADRB1 and ADRB2, encoding cardiac myocyte β1- and β2-adrenergic receptors (ARs) that mediate pathologic myocardial remodeling in response to chronically increased signaling, contain N-terminus haplotype variants capable of influencing agonist- or biased ligand-induced receptor internalization that uncouples canonical signaling and initiates EGFR/ERK1/2 cardioprotection.

In two heart failure (HF) clinical trial genetic substudies we investigated effects of internalizing vs. internalization-resistant αDRB1/ADRB2 haplotypes on clinical or biomarker responses to the biased ligand β-blocker bucindolol vs. placebo or vs. the nonbiased β1-antagonist metoprolol, and in haplotyped isolated human heart preparations we measured ERK1/2 activation in response to these same interventions.

In subjects with ≥3 internalizing ADRB1+ADRB2 haplotypes (6.7% subcohort) placebo treatment was associated with fewer clinical events compared to subjects with internalization-resistant haplotypes (Odds Ratio (OR) 0.28, 95% CI (0.10, 0.82)). In contrast, placebo treatment in subjects with ≥3 internalization-resistant haplotypes (70% subcohort) was associated with more clinical events in comparison to subjects with internalizing haplotype counterparts (OR 1.64 (1.46, 1.84)). Bucindolol treatment was equal to placebo in the ≥3 internalizing subcohort, but was superior to placebo in the internalization-resistant subcohort (bucindolol vs. placebo OR 0.49 (0.41, 0.58)). In subjects with all 4 haplotypes internalization-resistant (25% subcohort), bucindolol vs. placebo reduced time to first event rates by 62.3±17.5% (P the all-haplotypes parent population and additive to 1.92±0.58 fold when the ADRB1 haplotype contained Arg389 rather than Gly389). The same bucindolol vs. placebo pattern was observed for NT-proBNP or norepinephrine reduction vs. metoprolol. In these comparisons ADRB2 and ADRB1 haplotypes behaved similarly, and although the haplotypes differed in frequency between Black and non-Black subjects, within haplotypes there were no by-race differences in therapeutic effects. Bucindolol but not metoprolol activated ERK1/2 signaling in isolated ventricular preparations with ≥3 internalization-resistant haplotypes.

1) Both β1- and β2-AR haplotypes regulate therapeutic responses in HF; internalizing species confer protection against clinical events in placebo-treated subjects, while in internalization-resistant haplotypes the biased ligand β-blocker bucindolol but not the non-biased ligand metoprolol is associated with favorable effects. 2) The biased ligand cardioprotective effect may be related to internalization-dependent or -independent ERK1/2 activation. Competing Interest Statement MRB IAC and SBL own stock in Genvara Biopharma and is sponsor of bucindolol and are Inventors on a patent covering some of the manuscript content MRB and IAC have partial salary support from Genvara MRB is Chief Science and Medical Officer and Chairman of Genvara MRB and SBL are Principal Investigators on a submitted NIH grant related to this work Clinical Trial NCT00000560 NCT01970501 Funding Statement This work was supported by National Heart Lung and Blood Institute (NHLBI) Grants HL052318 HL48013 HL071609 and HL077101 Additional support is from Genvara Biopharma Westminster CO DNA samples from the BEST trial were provided by the BEST DNA Bank co-sponsored by the NHLBI and the Department of Veterans Affairs Cooperative Studies DNA Samples for the GENETIC-AF trial were provided by ARCA Biopharma (Westminster CO) the trial sponsor Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Each clinical trial site received IRB approval and gave ethical approval for all work. The BEST trial and its pharmacogenomics substudy were approved by ethical committees at the NHLBI, Department of Veterans affairs (Palo Alto VA, location of the statistical analysis center), the DNA Oversight Committee and each investigational site. The GENETIC-AF study and its DNA substudy were approved at each investigational site. Colorado Multiple Institutional Review Board COMIRB University of Colorado I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes 1) Title change to be more descriptive. 2) Abstract enlarged from 150 to 350 words. 3) Data on effects of pre-randomization beta-blockers in GENETIC-AF trial removed to reduce word count. Data Availability Lead contact, MRB materials available, this study did not generate new unique reagents; outputs and code for structural equation modeling and haplotyping data are available on GitHub, Group level data not included in the manuscript are available on request, De-identified subject level data for use in a research context will be available on request