Therapeutic Targets for Heart Failure Identified Using Proteomics and Mendelian Randomization

Background: Heart failure (HF) is a highly prevalent disorder for which disease mechanisms are incompletely understood. The discovery of disease-associated proteins with causal genetic evidence provides an opportunity to identify new therapeutic targets. Methods: We investigated the observational and causal associations of 90 cardiovascular proteins, which were measured using affinity-based proteomic assays. First, we estimated the associations of 90 cardiovascular proteins with incident heart failure by means of a fixed-effect meta-analysis of 4 population-based studies, composed of a total of 3019 participants with 732 HF events. The causal effects of HF-associated proteins were then investigated by Mendelian randomization, using cis-protein quantitative loci genetic instruments identified from genomewide association studies in more than 30 000 individuals. To improve the precision of causal estimates, we implemented an Mendelian randomization model that accounted for linkage disequilibrium between instruments and tested the robustness of causal estimates through a multiverse sensitivity analysis that included up to 120 combinations of instrument selection parameters and Mendelian randomization models per protein. The druggability of candidate proteins was surveyed, and mechanism of action and potential on-target side effects were explored with cross-trait Mendelian randomization analysis. Results: Forty-four of ninety proteins were positively associated with risk of incident HF (P<6.0×10–4). Among these, 8 proteins had evidence of a causal association with HF that was robust to multiverse sensitivity analysis: higher CSF-1 (macrophage colony-stimulating factor 1), Gal-3 (galectin-3) and KIM-1 (kidney injury molecule 1) were positively associated with risk of HF, whereas higher ADM (adrenomedullin), CHI3L1 (chitinase-3-like protein 1), CTSL1 (cathepsin L1), FGF-23 (fibroblast growth factor 23), and MMP-12 (matrix metalloproteinase-12) were protective. Therapeutics targeting ADM and Gal-3 are currently under evaluation in clinical trials, and all the remaining proteins were considered druggable, except KIM-1. Conclusions: We identified 44 circulating proteins that were associated with incident HF, of which 8 showed evidence of a causal relationship and 7 were druggable, including adrenomedullin, which represents a particularly promising drug target. Our approach demonstrates a tractable roadmap for the triangulation of population genomic and proteomic data for the prioritization of therapeutic targets for complex human diseases.

• PROSPER PROSPER is a randomized, double-blind, placebo-controlled trial designed to test the hypothesis that pravastatin will diminish risk of subsequent major vascular events. Participants include 2000 women and 2804 men aged 70-82 years with a history of vascular disease or at high risk for developing vascular disease recruited between December 1997 and May 1999 from Scotland, Ireland, and the Netherlands. Patients with HF were excluded at baseline. Incident HF was defined as presence of a hospital admission record of HF diagnosed from a combination of symptoms and signs, chest radiograph, and echocardiograph. [27][28][29] PIVUS and ULSAM PIVUS (Prospective Investigation of the Vasculature in Uppsala Seniors) is a population-based cohort which recruited 1,016 subjects aged 70 years living in the community of Uppsala, Sweden. Subjects were chosen from a community register and were invited in a randomized order, with a participation rate of 50.1%. 22 The ULSAM study included 838 men living in Uppsala at age 77 between 1998 and 2001. 23 In the analyses included in the present study, participants from the two cohorts were followed up to 11 years from baseline until heart failure diagnosis, death, or end of follow-up (10 June 2014 in PIVUS and 31 December 2008 in ULSAM). Diagnosis of heart failure was derived from medical records based on the presence of International Classification of Diseases, Ninth Briefly, the PEA technology uses a pair of oligonucleotide-labeled antibodies which bind to the target protein in the sample. On target binding, the oligonucleotide sequences come in close proximity, hybridize, and then are extended by a DNA polymerase to form a polymerase chain reaction (PCR) amplicon. The resulting amplicon is detected and quantified using real-time quantitative PCR method to estimate the circulating protein abundance. 7  Heart failure case status was assigned based on ICD-9 or ICD-10 codes for discharge diagnoses (ICD-10: I50 and subcodes, ICD-9: 428 and subcodes). ✓

EGCUT
Population-based prospective cohort study

Incident + prevalent
Heart failure status was assigned based on ICD-10: I50 and subcodes. ✓

EPHESUS
Heart failure randomised controlled trial Prevalent Patients were recruited 3 to 14 days after acute myocardial infarction according to the following critera: acute myocardial infarction as documented according to standard criteria; left ventricular dysfunction as documented by a left ventricular ejection fraction of 40 percent or lower on echocardiography, radionuclide angiography, or angiography of the left ventricle after the index acute myocardial infarction and before randomization; and heart failure as documented by the presence of pulmonary rales, chest radiography showing pulmonary ✓ ✓ venous congestion, or the presence of a third heart sound. In patients with diabetes who met the criteria for left ventricular dysfunction after acute myocardial infarction, symptoms of heart failure did not have to be demonstrated, since such patients have an increased risk of cardiovascular events similar to that of nondiabetic patients with symptoms of heart failure.

EPIC-Norfolk
Population-based prospective cohort study

Incident + prevalent
Heart failure definition was based on hospital admission or death record listing Heart failure code -ICD-10: I50 and subcodes ✓ ✓

FHS
Population-based prospective cohort study Incident Criteria for defining heart failure in the FHS have been described previously (PMID: 5122894, 16837677). In brief, heart failure was considered to be present if two major or one major plus two minor criteria were present in the absence of an alternative explanation for the symptoms and signs. Major criteria are defined as paroxysmal nocturnal dyspnea, orthopnea, jugular venous distention, hepatojugular reflux, pulmonary rales, radiographic evidence of cardiomegaly, acute pulmonary edema, third heart sound, central venous pressure >16 cm of water, and weight loss >4.5 kg during first 5 days of treatment for suspected heart failure. Minor criteria are defined as bilateral ankle edema, nocturnal cough, dyspnea on ordinary exertion, hepatomegaly, pleural effusion, and heart rate >120 beats per minute. The medical records for all individuals with heart failure diagnosis in any position in the Swedish hospital discharge register were reviewed by two physicians who were blinded to the baseline data. They classified the cases as definite, questionable, or miscoded according to the European Society of Cardiology recommendations. They considered ICD heart failure codes 427.00, 427.10, 428 (ICD-9), I50 (ICD-10) and hypertensive heart disease with heart failure, I11.0 (ICD-10) as possible diagnosis of heart failure. For further details see PMID:15916919.

PREVEND
Population-based prospective cohort study

Incident + prevalent
Heart failure cases were ascertained using criteria in accordance with the Heart Failure Guidelines of the European Society of Cardiology (ESC). In-and outpatient files were inspected for the presence of heart failure at baseline and for new onset heart failure, by recording signs, symptoms, and objective evidence of heart failure. In total, 586 individual cases were identified as suspected heart failure. An endpoint adjudication committee of seven independent experts evaluated all suspected cases of new onset heart failure. Each case was validated by two different experts by reviewing anonymized clinical charts, hospitalization, and physician office records in order to ascertain the incidence of heart failure. In case of consensus, patients were classified as 'definite new onset heart failure', 'definite no new onset heart failure', or 'definite heart failure, with date of onset before time of recruitment. In case of difference of opinion about an individual case, the committee made a joint decision.

PROSPER
Randomised-cont rolled trial

Incident + prevalent
Cases were defined by hospitalization for heart failure with a definition based on a combination of symptoms (e.g. shortness of breath) and signs, including chest radiograph with fluid congestion or echocardiogram with severely diminished LV function. All outcomes were adjudicated by an expert committee blinded to randomized study medication and using pre-defined criteria.

Regeneron/Geis inger
Population-based prospective cohort study

Incident + prevalent
Heart failure status was assigned based on ICD-10: I50 and subcodes. ✓

Rotterdam study 1
Population-based prospective cohort study Incident Prevalent heart failure at baseline was assessed using a validated score based on the European Society of Cardiology recommendation, identified from hospital discharge diagnoses, and restropective medical records screening. Cases of incident heart failure were obtained by continuously monitoring participants for the occurrence of heart failure during follow-up through general practitioners records and hospital discharge diagnoses. The date of incident heart failure was defined as the day of the first occurrence of symptoms suggestive of heart failure, obtained from the medical records, or the day of receipt of a first prescription for a loop diuretic or an ACEinhibitor indicated for treatment of heart failure, whichever came first. The diagnosis of heart failure was classified as definite, probable, possible, or unlikely in ✓ ✓ ✓ accordance with the criteria from the European Society of Cardiology. Potential cases were ascertained by two research physicians and verified by a cardiologist. Only definite and probable cases were considered in the analyses.

SHIP
Population-based prospective cohort study

Incident + prevalent
For these analyses heart failure was defnied according to a modified Rotterdam definition (PMID: 10213348). Prevalent HF cases in SHIP were defined as having history of HF (either chest pain during exercise, bypass, heart transplant, atrial flutter or fibrillation, LV hypertophy in individuals aged 45 or older, known MI) and HF symptoms (dyspnoe at exercise or swollen legs at evening) that were not related to bronchitis (bronchitis that occured recently or during the last 12 months).

SOLID
Randomised-cont rolled trial

Incident + prevalent
Heart failure status at enrolment was identified from medical record with no specific definition. I HF hospitalizations adjudicated during follow up were defined as admission to hospital or attendance at an acute health care facility for administration of intravenous diuretic treatment, escalation of diuretic doses, and/or inotropes. Confirmation of heart failure diagnosis was obtained by chest imaging demonstrating pulmonary congestion or edema, or, in patients without available chest imaging, at least one of the following: Pulmonary edema, (i.e. rales >1/3 up the lung fields thought to be of cardiac causes), pulmonary capillary wedge pressure >18 mmHg or BNP >500 pg/ml (or NT-terminal prohormone BNP >2500 pg/ml).

TwinGene
Population-based prospective cohort study

UK Biobank
Population-based prospective cohort study

ULSAM
Population-based prospective cohort study

Incident + prevalent
The medical records for all individuals with heart failure diagnosis in any position in the Swedish hospital discharge register were reviewed by two physicians who were blinded to the baseline data. They classified the cases as definite, questionable, or miscoded according to the European Society of Cardiology recommendations. They considered ICD heart failure codes 427.00, 427.10, 428 (ICD-9), I50 (ICD-10) and hypertensive heart disease with heart failure, I11.0 (ICD-10) as possible diagnosis of heart failure.