Author + information
- Received May 15, 2019
- Revision received June 21, 2019
- Accepted July 15, 2019
- Published online September 11, 2019.
- Melissa A. Lyle, MDa,∗,
- Seethalakshmi R. Iyer, MSa,∗,
- Margaret M. Redfield, MDa,
- Yogesh N.V. Reddy, MD, MSca,
- G. Michael Felker, MD, MHSb,
- Thomas P. Cappola, MDc,
- Adrian F. Hernandez, MDb,
- Christopher G. Scott, MSd,
- John C. Burnett Jr., MDa and
- Naveen L. Pereira, MDa,e,∗ ()
- aDepartment of Cardiovascular Medicine, Mayo Clinic, Rochester, Minnesota
- bDivision of Cardiology, Duke Clinical Research Institute, Durham, North Carolina
- cDivision of Cardiovascular Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
- dDivision of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota
- eDepartment of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota
- ↵∗Address for correspondence:
Dr Naveen L Pereira, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905.
Background In heart failure with reduced ejection fraction (HFrEF), elevated soluble neprilysin (sNEP) levels are associated with an increased risk of cardiovascular death, and its inhibition with sacubitril/valsartan has improved survival.
Objectives This study sought to determine the relevance of sNEP as a biomarker in heart failure with preserved ejection fraction (HFpEF) and to compare circulating sNEP levels in HFpEF patients with normal controls.
Methods A case-control study was performed in 242 symptomatic HFpEF patients previously enrolled in the Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Heart Failure with Preserved Ejection Fraction (RELAX) and Nitrates’s Effect on Activity Tolerance in Heart Failure With Preserved Ejection (NEAT-HFpEF) clinical trials and 891 asymptomatic subjects without HF or diastolic dysfunction (confirmed by NT-proBNP levels <200 pg/ml and echocardiography) who were enrolled in the Prevalence of Asymptomatic Left Ventricular Dysfunction study. sNEP was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) in all subjects.
Results Overall, sNEP levels were lower in HFpEF compared with controls (3.5 ng/ml; confidence interval [CI]: 2.5 to 4.8 vs. 8.5 ng/ml; CI: 7.2 to 10.0; p < 0.001). After adjusting for age, gender, body mass index (BMI), and smoking history, mean sNEP levels were also lower in HFpEF compared with controls (4.0 ng/ml [CI: 2.7 to 5.4] vs. 8.2 ng/ml [CI: 6.8 to 9.7]; p = 0.002). The cohorts were propensity matched based on age, BMI, diabetes, hypertension, smoking history, and renal function, and sNEP levels remained lower in HFpEF compared with controls (median 2.4 ng/ml [interquartile range: 0.6 to 27.7] vs. 4.9 ng/ml [interquartile range: 1.2 to 42.2]; p = 0.02).
Conclusions Patients with HFpEF on average have significantly lower circulating sNEP levels compared with controls. These findings challenge our current understanding of the complex biology of circulating sNEP in HFpEF.
Heart failure with preserved ejection fraction (HFpEF) can result from abnormalities in the process of both active relaxation and passive stiffness of the heart (1). Atrial natriuretic peptide (ANP) activates phospholipase C-b, resulting in intracellular calcium mobilization and active relaxation of the cardiomyocyte. The natriuretic peptides (NPs) via cyclic guanosine monophosphate (cGMP) inhibit smooth muscle and fibroblast proliferation and collagen deposition that can affect passive stiffness (2–4). NPs directly reduce peripheral vascular resistance (5) and affect natriuresis (6–9), therefore playing an important role in maintaining homeostasis in the HFpEF population (10–12).
Neprilysin (NEP) is a cell-membrane–bound metalloendopeptidase that mediates the antifibrotic, antiproliferative, myocardial relaxation, vasodilator, and diuretic properties of NPs by being responsible for removal of at least 50% of circulating NPs (13), and its inhibition therefore results in significant increases in plasma ANP, urinary cGMP and urinary sodium, and reduction in intracardiac filling pressures (14,15). Sacubitril/valsartan, when administered to patients with HFpEF in a phase 2 study, resulted in significantly reduced NT-pro B-type natriuretic peptide (BNP) levels, a surrogate marker of LV wall stress, compared with being treated with valsartan alone (16). Sacubitril/valsartan therefore is being tested as treatment for HFpEF in a phase 3 trial, PARAGON-HF (Prospective Comparison of Angiotensin Receptor-Neprilysin Inhibitor [ARNI] With Angiotensin Receptor Blocker [ARB] Global Outcomes in Heart Failure With Preserved Ejection Fraction), evaluating its efficacy in reducing cardiovascular (CV) death and heart failure (HF) hospitalizations compared with valsartan.
NEP can be released from the cell surface, and, in a recent study, circulating soluble neprilysin (sNEP) level in a linear relationship was significantly associated with CV death or HF hospitalization in patients with HF and reduced ejection fraction (HFrEF) (17), supporting the hypothesis that its inhibition may be beneficial in HFrEF. sNEP levels in HFpEF were evaluated in 144 patients and not found to be associated with New York Heart Association (NYHA) functional class, 6-min walk test, or cardiac hospitalization and death (18). However, to understand its role as a biomarker, sNEP levels should be measured in and compared with control subjects without diastolic dysfunction or HF: a study that has not been previously performed.
The aim of our study was to determine the role of sNEP levels as a biomarker in HFpEF and its concentration in patients with HFpEF compared with normal controls.
This case-control study included 242 patients with definite clinical HFpEF, previously enrolled in the National Institutes of Health funded multicenter Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Heart Failure With Preserved Ejection Fraction (RELAX) and Nitrates's Effect on Activity Tolerance in Heart Failure With Preserved Ejection (NEAT-HFpEF) clinical trials for whom blood samples were available for testing (19,20). The RELAX study was a multicenter, randomized clinical trial of 216 stable outpatients with HFpEF (19). Patients with normal (≥50%) EF and symptomatic HF (NYHA functional class II–IV) with objective exercise limitation (peak oxygen consumption <60% predicted) were recruited. Definite clinical HFpEF was required as defined by previous HF hospitalization, chronic loop diuretic therapy with left atrial enlargement, invasively documented elevation in left-sided filling pressures. In addition, study entry also required an elevated (≥400 pg/ml) NT-proBNP level, or invasive confirmation if NT-proBNP level was >400 pg/ml, further increasing the rigor of HFpEF diagnosis.
NEAT-HFpEF was a 110-subject multicenter randomized study evaluating the effect of oral nitrate therapy on daily physical activity in outpatients with HFpEF (20). Similar to RELAX, patients were required to have an EF ≥50% or more and objective evidence of exacerbation of heart failure by either previous HF hospitalization, elevated invasive filling pressures, elevated level of NT-proBNP (>400 pg per ml), or Doppler echocardiographic evidence of elevated filling pressures.
The control group comprised 891 asymptomatic subjects without HF or diastolic dysfunction and with NT-proBNP <200 pg/ml who were previously enrolled in the Prevalence of Asymptomatic Left Ventricular Dysfunction (PAVD) study from Olmsted County (21,22). Diastolic dysfunction was rigorously defined by comprehensive Doppler techniques and established criteria, as previously described (22). These subjects underwent physical examination, echocardiogram, and phlebotomy for biorepository samples and a follow-up visit that included echocardiography 4 years after the initial evaluation to determine the effects of aging on the development of diastolic dysfunction and HFpEF and were followed for more than 10 years after initial evaluation (21,22). We have previously used the PAVD population in multiple studies to interpret the distribution of BNP concentration and other biomarkers in a normal subset of subjects (23).
Assessment of ventricular structure and function
The echocardiographic methods have been extensively described previously (22,23). Ejection fraction was measured by a semiquantitative method, integrating 2-dimensional (2-D) quantitative measurements and visual assessment. Left-ventricular mass and left-atrial volume were calculated from M-mode and 2D measurements (22). Diastolic dysfunction was recorded as indeterminate, mild (grade 1), moderate (grade 2), or severe (grade 3/4), based on the analysis of transmitral inflow velocities at rest and with Valsalva maneuver, mitral annular-tissue velocity, and pulmonary venous inflow patterns (22). Pulmonary artery systolic pressure was estimated using the modified Bernoulli equation, 4 * (peak tricuspid regurgitation velocity) + 5 mm Hg (24).
Soluble neprilysin assay
A commercially available sandwich enzyme-linked immunosorbent assay (ELISA) for sNEP (R & D Systems, Minneapolis, Minnesota) was used to measure circulating NEP levels in serum. ELISA plates were coated with NEP-capture antibody overnight, and then plates were washed per manufacturer’s instructions and used for analysis of the samples. The samples were incubated in the antibody coated plate for 2 h at room temperature. At the end of the incubation, the plates were washed 4 times and incubated with the NEP-detection antibody for another 2 h. The plates were then washed and incubated with streptavidin-HRP conjugate for 20 min and washed before incubating for another 20 min with the substrate. At the end of the incubation period, the reaction was halted by adding a stop solution, and the plate was corrected for background interference at 570 nm, read at 450 nm. SoftmaxPro 7.2 software was used to extrapolate sNEP concentrations of samples. The standard curve range was from 0.12 ng/ml to 8 ng/ml. The intra-assay and interassay variability was 7.5% and 9.5%, respectively. Based on initial sNEP measurement, any sample exceeding the standard curve range was diluted appropriately and reassayed for accurate measurement. All the measurements were done in duplicate. A freeze-thaw analysis was performed on other control samples and found that sNEP levels were stable for up to 2 freeze thaw cycles. In addition, NEP was obtained from R & D Systems and was dissolved in PBS and diluted to the varying concentrations as described in Online Table S1. Subsequently, NEP concentration and activity in buffer solution was measured as described above and by using a fluorimetric assay, with 5-FAM/QXL 520 Neprilysin substrate (SensoLyte Neprilysin ELISA Kit [AnaSpec, Freemont, California]) respectively.
All the samples from the control and test cohort were analyzed using the same lot of Neprilysin Kit. The analyses of calibration controls from randomly selected plates are shown in Online Table S2. The overall percentage coefficient of variation for the different calibration controls was <10%.
Subjects with HFpEF from both RELAX and NEAT-HFpEF were compared with PAVD subjects without evidence of HF or diastolic dysfunction. Continuous variables were reported as mean (SD) or median (interquartile range) depending on data distribution, and categorical data were reported as frequency and percentage. sNEP was transformed using the natural logarithm prior to analyses to approximate a normal distribution. Linear regression was used to analyze log transformed sNEP with and without adjustment for comorbidities. Results of these analyses are presented using the adjusted mean sNEP and associated 95% confidence interval after back transformation to original scale. A secondary analysis was performed after propensity matching PAVD subjects to HFpEF subjects on age, body mass index (BMI), diabetes, hypertension, estimated glomerular filtration rate (eGFR), and smoking history. Owing to large differences among groups on these important characteristics, only a subset of HFpEF subjects was able to be matched.
To examine the associations between sNEP and baseline characteristics, sNEP was grouped into tertiles, and associations were tested as a trend across tertiles after adjusting for age, gender, BMI, and smoking history. All tests were 2-sided, with a p value < 0.05 considered to be significant. All analyses were performed using SAS version 9.4 (SAS institute, Cary, North Carolina).
The final study cohort included 242 patients with HFpEF from RELAX/NEAT (n = 165 and n = 77), respectively, and 891 from PAVD who had sNEP levels measured and were without HF or diastolic dysfunction at baseline (Table 1). Clinical characteristics were, in general, comparable between the patients with HFpEF from RELAX and NEAT trials with similar sNEP levels and so were combined to form the overall HFpEF cohort (Online Table S3).
The patients with HFpEF represented a typical HFpEF demographic and were elderly (mean age 69 ± 10 years), obese (mean BMI 34 ± 7 kg/m2), with a high prevalence of hypertension (86%) and diabetes (40%). More than one-half of this population (n = 144, 60%) were previous or active smokers. At baseline, the median calculated GFR was decreased at 56 ml/min/1.73 m2 (interquartile range [IQR]: 43 to 73) and median baseline NT-proBNP was elevated at 497 pg/ml (IQR: 149 to 1,075). Baseline ejection fraction was preserved at 64 ± 9%, with an elevated mean E/e’ ratio of 18 ± 10 m/s and pulmonary artery systolic pressure of 41 ± 13 mm Hg.
The control subjects from PAVD (n = 891) were middle aged (mean age 58 ± 8) with a mean BMI in the overweight range (28 ± 5 kg/m2). Approximately one-half of the population (n = 453, 51%) were previous or active smokers, and subjects also had hypertension (18%) or diabetes (5%). The median calculated GFR was preserved at 85 (IQR: 74 to 90), median NT-proBNP was normal 48 (IQR: 20 to 87) as was the ejection fraction (64 ± 5%), and mean E/e’ ratio was 8 ± 2 m/s. As expected, clinical characteristics and echocardiographic parameters were significantly different between HFpEF subjects and controls (Table 1).
Tertiles of sNEP across control and HFpEF subjects
The non-HF PAVD control group was split into tertiles of sNEP (Table 2) to determine variables associated with sNEP in a large community-based cohort. In this group, there was a clear association between smoking and lower levels of sNEP (p < 0.001). There was no association between sNEP tertiles and age, gender, BMI, diabetes, hypertension, NT-proBNP levels, or cardiac structure and function determined by echocardiography in this cohort.
To further investigate nonlinear relationships between low and high sNEP, the HFpEF group was also split into tertiles of sNEP levels (Table 3). The lowest tertile group (n = 85) had sNEP levels <0.6 ng/ml, the highest tertile group (n = 80) had circulating levels of NEP above 9.4 ng/ml, and the intermediate group (n = 77) had levels in between. After adjustment for age, gender, and smoking history, there was a paradoxical association of low sNEP with hyperlipidemia (p = 0.005) and statin use (p = 0.03) in the lowest tertile group compared with the mid and the highest tertile groups. There also was a trend toward an association with a higher prevalence of hypertension (p = 0.09) and angiotensin converting-enzyme inhibitor (ACE-I) use (p = 0.01) in the lowest tertile group. In contrast, there was a higher prevalence of baseline NYHA functional class III/IV symptoms in patients with HFpEF in the highest tertile (51%) versus the lowest tertile (35%); p = 0.08. However, there did not appear to be an association among sNEP tertiles and NT-proBNP levels, echocardiographic measurements, or previous hospitalizations for HF.
Comparison of sNEP levels between HFpEF and controls
Despite age and comorbidity burden being significantly higher in patients with HFpEF, median sNEP levels were lower in these patients (3.5 ng/ml; confidence interval [CI]: 2.5 to 4.8 compared with controls (8.5 ng/ml; CI: 7.2 to 10.0); p < 0.001 (Central Illustration). After adjusting for age, gender, BMI, and smoking history, sNEP levels remained lower in the HFpEF subjects (4.0 ng/ml; CI: 2.6 to 5.0) compared with controls (8.2 ng/ml; CI: 6.8 to 9.7); p = 0.002 (Table 4A). With further adjustment for hypertension as well, this association remained significant with sNEP being lower in HFpEF (4.2 ng/ml; CI: 2.8 to 6.4) compared with controls (8.0 ng/ml; CI: 6.7 to 9.6); p = 0.01. Despite the subset of HFpEF patients with Class III/IV symptoms having higher sNEP levels compared with Class I/II patients, this subgroup had sNEP levels that were lower than controls on average (similar to the overall HFpEF group); p = 0.01 (Table 4B).
As described above, there was no association of comorbidities with sNEP levels in the large control group other than smoking. However, to adjust for more comprehensively imbalances between comorbidities, the 2 groups were propensity matched by age, BMI, diabetes, hypertension, smoking history, and renal function (Table 5). In this subgroup of 111 subjects from each cohort, median sNEP levels remained lower in the HFpEF group—2.4 ng/ml (IQR: 0.6, 27.7)—compared with controls: 4.9 ng/ml (IQR: 1.2, 54.2) (p = 0.02) (Figure 1).
This is the first study to directly compare circulating NEP levels between rigorously defined HFpEF subjects and controls that had no evidence of diastolic dysfunction or HF at the time of evaluation or long-term follow-up. We have previously used the PAVD population to interpret the determinants and concentration of biomarkers in a normal subset of subjects (26). There were lower sNEP levels in HFpEF patients compared with controls despite worse cardiac remodeling, elevated filling pressures, and comorbidity burden in HFpEF. These results remained robust despite both rigorous multivariable adjustment and propensity matching. sNEP levels also failed to correlate with NP levels, cardiovascular remodeling, or HF hospitalization. These data fundamentally question our simplistic assumptions of sNEP as a potential biomarker in HFpEF, contrary to its recent importance in HFrEF.
Previous literature has focused on the importance of NEP as a biomarker in HFrEF, given its significant association with morbidity and mortality and the decrease in mortality associated with NEP inhibition (15). A linear correlation between NEP levels and adverse outcomes in HFrEF has been demonstrated in a previous study, but sNEP levels in ambulatory patients with HFrEF have not been compared with normal controls (17); therefore, inferences regarding sNEP levels in HFrEF compared with controls cannot be made.
It is important to emphasize the complexity of the NEP-NP pathway. NEP is very nonspecific in terms of substrate selection and breaks down both beneficial peptides (25–27) in addition to harmful substrates. Therefore, the pathophysiological role and levels of sNEP may differ, depending on the complex interplay of these various substrates in the pathophysiology of the HF syndrome. HFpEF remains an incompletely understood disease process, with systemic inflammation believed to drive cardiac remodeling. In addition, natriuretic peptide elevation is not universal in HFpEF, given many patients have normal BNP levels despite elevated filling pressures (28), and, on average, BNP levels are lower in HFpEF compared with HFrEF (29). Taken together, our findings of lower sNEP compared with controls without HFpEF question whether sNEP concentration is a reliable theranostic biomarker in HFpEF or whether NEP plays an important role in the pathophysiology of HFpEF.
We recently investigated sNEP levels to determine clinical correlates and relationship to the onset of cardiovascular disease in the general population (n = 1,536) (30). In this large community-based cohort of participants without HF, there was no relationship between sNEP and circulating ANP, BNP, or C-type natriuretic peptide (CNP), a finding that highlights the complexity of the role and contribution of sNEP in the proteolysis of natriuretic peptides. Importantly, this study also found that lower sNEP levels were associated with diastolic dysfunction, dyslipidemia, and hypertension similar to our current findings in HFpEF. Overall, our study extends the concept that lower sNEP levels are associated with not only diastolic dysfunction in the community, as observed in PAVD, but also with clinical HFpEF.
Goliasch et al. (18) measured sNEP in a well-phenotyped single-center HFpEF cohort and, similar to our findings, found its association with smoking but did not find any association with functional or cardiac parameters including symptoms, 6-min walk test, invasive hemodynamics, left-ventricular fibrosis by biopsy or cardiac magnetic resonance. Neprilysin levels also failed to correlate with clinical outcomes (17). The sNEP levels in our HFpEF cohort (3.5 ng/ml; CI: 2.5 to 4.8) were also comparable to that found in the cohort of Goliasch et al of 144 patients with HFpEF (2.9 ng/ml), therefore demonstrating the validity and reproducibility of our sNEP assay (22). Our study builds upon the findings by Goliasch et al. with a larger multicenter HFpEF cohort and, for the first time, compares these patients with controls, clarifying its role as a biomarker in HFpEF.
Limitations of this study include the possibility that circulating sNEP levels may not correlate with biologically active tissue levels, which is a common limitation amongst all serological biomarker research. However, previous investigations have demonstrated low lung tissue NEP expression (both at a protein and transcript level) in smokers (31,32), which is concordant with our independent finding in a previous study of low circulating NEP levels in smokers when analyzed in a large community cohort (33). This trend of lower circulating NEP levels in smokers was also observed in the smaller cohort of Goliasch et al. of patients with HFpEF (71% smokers in the lowest sNEP tertile vs. 52% in the highest tertile) (18). The lower circulating levels in smokers may be related to increased urinary excretion of NEP compared with nonsmokers (34). The significant association between lower levels of circulating sNEP and smoking in the general community and patients with HFpEF, and its independent corroboration in previous observations of lower NEP protein expression in smokers’ lung tissue and higher NEP excretion in smokers, strengthens the validity of our results and affirms the reliability of our assay.
We did not measure sNEP activity and assess its relationship to HFpEF compared with controls. A previous study in 639 patients by Vodovar et al. did not demonstrate a correlation between sNEP levels and NEP activity (35). Also, Nougué et al. demonstrated that switching 73 patients with HFrEF from an ACE-I or angiotensin receptor blocker to sacubitril/valsartan was associated with a dose-dependent decrease in sNEP activity, whereas sNEP levels remained unchanged, which highlights that sNEP levels may not correlate with enzymatic activity (36). These findings are not surprising because enzymatic activity in the blood can vary based on the concentration and potential interference of circulating factors in the blood that may serve as substrates of NEP. We demonstrate this concept by comparing NEP levels with NEP activity in buffer solution, and unlike the findings in plasma as described by Vodovar et al., we illustrate a direct linear correlation between NEP concentration and activity (R2 = 0.99) (Online Figure S1). We have also previously shown a similar linear correlation between NEP cellular expression and enzymatic activity (37). Finally, given the lack of ethnic diversity in the PAVD control group, generalizability to other racial populations may not be valid.
Our study shows, for the first time, that patients with HFpEF have lower levels of sNEP when compared with controls without diastolic dysfunction or HF. In addition, there was no association in HFpEF among baseline sNEP with baseline NPs, cardiac structure, or function. These findings may have important clinical implications on the role of sNEP as a biomarker and its potential importance in predicting clinical efficacy with NEP inhibition in HFpEF. Further investigation is needed to elucidate the complex relationship of sNEP concentration, sNEP activity, and NPs and its feedback loops in subjects with and without HFpEF.
COMPETENCY IN MEDICAL KNOWLEDGE: Circulating sNEP levels have not been measured in HFpEF patients and compared with well-defined controls without diastolic dysfunction or HFpEF. After multivariable adjustment including age- and gender- and propensity-matching, including multiple comorbidities such as diabetes and hypertension, sNEP levels were lower in HFpEF compared with controls.
TRANSLATIONAL OUTLOOK: Higher sNEP levels were associated with increased morbidity and mortality, and inhibition of NEP with sacubitril/valsartan improved survival in HFrEF. Whether sNEP levels can predict efficacy of sacubitril/valsartan in HFpEF remains to be proved.
↵∗ Drs. Lyle and Iyer have contributed equally to this work.
Dr Felker has received research grants from NHLBI, American Heart Association, Amgen, Merck, Cytokinetics, and Roche Diagnostics; served as a consultant to Novartis, Amgen, BMS, Medtronic, Cardionomic, Relypsa, V-Wave, Myokardia, Innolife, EBR Systems, Arena, Abbott, Sphingotec, Roche Diagnostics, Alnylam, LivaNova, and SC Pharma. Dr Pereira is supported by National Institute on Aging Grant R21AG53512. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- body mass index
- ejection fraction
- heart failure
- heart failure with preserved ejection fraction
- heart failure with reduced ejection fraction
- natriuretic peptides
- New York Heart Association
- soluble neprilysin
- Received May 15, 2019.
- Revision received June 21, 2019.
- Accepted July 15, 2019.
- 2019 American College of Cardiology Foundation
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