Methods

Participants included 200 patients at the Veteran’s Affairs San Diego Healthcare System who were referred for an outpatient echocardiogram from April 2010 to September 2010 and who provided consent for the study. Patient referrals were made by either clinic physicians or nurse practitioners for the purpose of evaluating cardiac function. Exclusion criteria consisted of patients who were referred solely for ruling out thrombus or vegetation. Participants were followed for 4 years from the date of their echocardiogram. The study was approved by the University of California Institutional Review Board, and all participants provided written informed consent.

M-mode and 2-dimensional images, spectral and color flow Doppler, and Doppler tissue imaging recordings were performed according to guidelines of the American Society of Echocardiography and obtained with commercially available instruments operating at 2.0 to 3.5 MHz. Two-dimensional imaging examinations were performed in the standard fashion in parasternal long- and short-axis views and apical 4- and 2-chamber views. Pulsed Doppler spectral recordings were obtained in the apical 4-chamber view from a 4 × 4-mm sample volume positioned at the tips of the mitral leaflets and in the right upper paraseptal pulmonary vein and adjusted to yield velocity signals of maximal amplitude. Two-dimensional echocardiograms were subjected to careful visual analysis to detect regional contractile abnormalities. Left ventricular (LV) systolic and diastolic volumes and ejection fraction were derived from biplane apical (2 and 4 chamber) views with a modified Simpson’s rule algorithm. Left atrial and LV dimensions were measured from M-mode images according to recommendations from the American Society of Echocardiography and were used to calculate LV mass, which was indexed to body surface area. The transmitral pulsed Doppler velocity recordings from 3 consecutive cardiac cycles were used to derive measurements as follows: E and A velocities were the peak values reached in early diastole and after atrial contraction, respectively, and deceleration time (DT) was the interval from the E-wave peak to the decrease of the velocity to baseline. In those cases in which velocity did not return to baseline, extrapolation of the deceleration signal was performed. In addition, pulmonary venous systolic and diastolic flow velocities were obtained as the maximal values reached during the respective phase of the cardiac cycle.

Diastolic function was assessed based on published recommendations using Doppler measurements of mitral inflow and Doppler tissue imaging of the mitral annulus using the medial annulus velocity (E′) and was classified as abnormal if it fell into 1 of 3 categories: impaired relaxation, pseudonormal, and restrictive. Impaired relaxation (mild diastolic dysfunction) was defined as an E/A ratio <1 in patients <55 years or E/A ratio <0.8 in patients ≥55 years, DT >220 ms, and E′ <8 cm/s. Pseudonormal (moderate) was defined as E/A ratio 1 to 2 with DT <220 ms; confirming evidence included E′ <8 cm/s or pulmonary vein diastolic flow greater than pulmonary vein systolic flow. Restrictive pattern (severe) was defined as an E/A ratio >2, DT <150 ms, and E′ <8 cm/s. Diastolic function was categorized as indeterminate in the presence of atrial fibrillation, severe mitral stenosis or regurgitation, mitral valve prosthesis, or missing or inconsistent data. All echocardiograms were interpreted by experienced cardiologists who were blinded to the biomarker levels.

Participants were grouped into stages of HF based on the American College of Cardiology/American Heart Association (ACC/AHA) published guidelines. Stage A consisted of patients “at high risk of developing HF because of the presence of conditions that are strongly associated with the development of heart failure,” but without structural heart disease or HF symptoms, and included those with systemic hypertension (HTN), coronary artery disease, diabetes, dyslipidemia, obesity (defined as body mass index [BMI] ≥30 kg/m ² ), a history of alcohol abuse, and cigarette smokers, as recommended in the ACC/AHA Practice Guidelines. Stage B comprised patients with structural heart disease (based on recommendations and cutpoints from the American Society of Echocardiography guidelines ) but without symptoms of HF, including LV hypertrophy (defined as mean LV wall thickness of septum and posterior wall ≥12 mm), LV enlargement (at least moderate in severity, defined as LV end-diastolic diameter ≥64 mm in men or ≥58 mm in women, or LV mass index ≥132 in men or ≥109 in women), LV systolic dysfunction (defined as left ventricular ejection fraction [LVEF] <55% or wall motion abnormality), LV diastolic dysfunction, asymptomatic valvular heart disease of at least moderate severity, or previous myocardial infarction. Patients with a history of symptomatic HF were grouped into a combined stage C/D and were excluded from the study and subsequent analysis. A history of HF was obtained from the patient, and from review of the patient’s medical records, and required confirmation with ≥1 objective findings from the medical record (emergency department visits or hospitalizations documenting HF or treatment for HF, documentation of HF signs and symptoms by the patient’s physician, or regular visits to the outpatient cardiomyopathy clinic for HF).

All samples were collected by venipuncture into EDTA tubes at the time of echocardiography.

ProENK was measured using a chemiluminometric sandwich immunoassay. The assay for stable proENK consisting of amino acids 119 to 159 of proENK A has been previously reported and was recently modified. In brief, 2 mouse monoclonal anti-proENK antibodies were developed by immunizing them with proENK peptide consisting of amino acids 119 to 159 of proENK A. Two micrograms of 1 antibody were used to coat polystyrene tubes. The other antibody labeled with methylacridinium ester served as the detector antibody. Standards, that is, proENK peptide, amino acids 119 to 159 of pro-ENK A, and 50-μl samples, were incubated in tubes with 150 μl of the detector antibody. After equilibration, tubes were washed, and bound chemiluminescence was detected with a luminometer LB952 T/16 (Berthold, Bad Wildbad, Germany). The lower detection limit of the assay was 5.5 pmol/L. Intra-assay and interassay coefficients of variation were 6.4% and 9.5% at 50 pmol/L and 4.0% and 6.5% at 150 pmol/L. Normal ranges are not dependent on age or gender.

Statistical analyses were performed using SPSS, version 22 (IBM SPSS Statistics; IBM Corporation, Armonk, New York) and Stata, version 12.1 (Stata Corp., College Station, Texas).

All biomarker levels were log ₁₀ transformed. Continuous variables are presented as means ± SD, median, and IQR and dichotomous variables as percentages. The primary outcome, major adverse cardiac events (MACE) included cardiovascular-related hospital admission, cardiac death, and all-cause mortality. Participants were followed for 4 years for incident events, which were confirmed by review of medical records. Differences in baseline levels of risk factors and clinical characteristics between participants with and without incident events were analyzed with t tests and chi-square tests. For calculation of number of HF risk factors, the following risk factors were considered: diabetes, HTN, former or current smoking, obesity (defined as BMI ≥30 kg/m ² ), hyperlipidemia, history of coronary artery disease, and renal insufficiency (defined as serum creatinine ≥1.2 mg/dl). The analysis of the mean difference between continuous variables and plasma proENK levels was assessed using the Mann-Whitney and Kruskal-Wallis tests. Between-group differences in categorical variables were assessed using the chi-square test or Fisher’s exact test, as appropriate. Spearman’s correlation analysis was performed for proENK and other clinical parameters.

For survival analyses, participants with >1 event were censored at the time of their first qualifying event. Kaplan-Meier plots were constructed, and the log-rank test was used to compare event-free survival across groups. To assess the prognostic value of the biomarkers, a base model was generated using Cox survival analysis, which included variables that were significantly (p <0.10) associated with any of the study end points on univariable analysis (age, gender, history of ischemic heart disease, HTN or diabetes, estimate glomerular filtration rate [eGFR], and echocardiographic evidence of systolic dysfunction).

Results

The baseline clinical characteristics of 200 participants are displayed by proENK tertiles in Table 1 . Nearly all participants were men (97.5%), with the average age of 63.7 years. Participants with higher proENK levels were older and had higher serum creatinine and lower eGFR, lower LVEF, and higher rates of HTN and diabetes (all p ≤0.009). Patients in the higher tertiles also had more MACE and nonfatal MACE events (p ≤0.003). The log ₁₀ -transformed distribution of proENK levels in our sample is shown in Figure 1 .

Table 1

Characteristics of the 200 patients according to proenkephalin tertiles

	All n = 200	Proenkephalin Tertiles			p Value
		1	2	3
		n = 66 <49.7	n = 67 49.7 – 67.1	n = 67 >67.5
Proenkephalin (pmol/l)	58.5 (46.3, 74.22)	40.2 (36.1, 46.3)	58.2 (53.5, 64.1)	85.5 (74.2, 104.7)	<0.0001
Age (years)	63.7 ± 12.5	58.1 ± 11.6	63.4 ± 11.8	69.22 ± 11.7	<0.0001
Men	195 (98%)	64 (97%)	65 (97%)	66 (99%)	0.811
Heart rate (bpm)	72.2 ± 14.8	72.9 ± 14.5	73.7 ± 15.1	70 ± 14.7	0.329
Systolic blood pressure (mm Hg)	130.8 ± 17.7	132.3 ± 18.8	129.7 ± 17.4	130.4 ± 17	0.682
Diastolic blood pressure (mm Hg)	76.5 ± 12.3	78.1 ± 14.1	77.4 ± 10	74.1 ± 12.2	0.139
Creatinine (mg/dL)	1.04 ± 0.30	0.91 ± 0.17	0.98 ± 0.25	1.24 ± 0.39	<0.0001
Estimated glomerular filtration rate (ml/min/1.73 m 2 )	102 ± 31.8	116.4 ± 25.0	105.5 ± 31.1	84.0 ± 30.4	<0.0001
B-type natriuretic peptide (pg/ml)	74.8 (27.15, 241.0)	56.8 (20, 182.3)	61.7 (22.6, 183.6)	185.4 (60.9, 292.4)	0.051
High-density lipoprotein (mg/dl)	49.5 ± 16.8	45.6 ± 9.5	51.1 ± 15.6	51.9 ± 22.6	0.116
Low-density lipoprotein (mg/dl)	90.9 ± 33.0	95.3 ± 29.9	89.6 ± 34.4	88 ± 34.6	0.551
Triglyceride (mg/dl)	121 (85, 177)	139 (94.5, 197)	108 (77.3, 155.3)	120 (84, 164)	0.073
Left ventricular ejection fraction	61.6 ± 14.2	63.8 ± 11.0	64.8 ± 11.3	56.4 ± 17.8	0.001
Fractional shortening (%)	35.4 ± 11.7	36.9 ± 11.9	36.4 ± 8	33 ± 14.2	0.115
Stage A	0.510	0.52	0.58	0.43	0.223
Hypertension	0.285	44 (67%)	42 (63%)	57 (85%)	0.009
Diabetes	0.293	29 (45%)	10 (15%)	10 (28%)	0.001
Smoker	0.340	25 (38%)	17 (25%)	26 (39%)	0.187
Beta-blocker use	0.555	37 (56%)	33 (49%)	41 (61%)	0.378
ACE Inhibitor use	0.345	23 (35%)	21 (31%)	25 (37%)	0.766
Calcium channel blocker use	0.211	15 (23%)	12 (18%)	15 (22%)	0.775
Statin use	0.500	33 (50%)	32 (48%)	35 (52%)	0.874
Death	0.140	7 (11%)	7 (10%)	14 (21%)	0.137
Major adverse cardiac events	0.215	8 (12%)	11 (16%)	24 (36%)	0.002
Nonfatal major adverse cardiac events	0.100	1 (2%)	6 (9%)	13 (19%)	0.003

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