Fernando L. Martin

Amino-Terminal Pro-B-Type Natriuretic Peptide and B-Type Natriuretic Peptide Biomarkers for Mortality in a Large Community-Based Cohort Free of Heart Failure

Recent studies report that, in the absence of heart failure and renal failure, plasma B-type natriuretic peptide (BNP) has prognostic value for mortality. We sought to confirm and extend these previous studies to assess BNP, measured by 3 distinct assays, as a biomarker for mortality in a strategy to enhance efforts at primary prevention and to better understand the clinical phenotype of such subjects at risk. We used a community-based cohort of 2042 subjects from Olmsted County, Minn, and individuals with heart or renal failure were excluded. BNP was assessed using 3 assays including Biosite and Shionogi for mature, biologically active BNP and the Roche assay for apparently nonbiologically active amino-terminal pro-BNP (NT-proBNP). Thorough echocardiographic and clinical data were recorded for all of the participants. Median follow-up for mortality was 5.6 years. BNP by all 3 of the assays was predictive of mortality. NT-proBNP and Biosite assays remained significant even after adjustment for traditional clinical risk factors and echocardiographic abnormalities including left ventricular hypertrophy and diastolic dysfunction. Echocardiography documented widespread structural changes in those with increasing BNP levels yet below levels observed in heart failure. We report in a large, well-characterized community-based cohort, free of heart failure, the first study to compare 3 distinct BNP assays as biomarkers for mortality in the same cohort. Our findings confirm the potential use of NT-proBNP and BNP biomarkers for future events and underscore that these peptides may also serve as biomarkers for underlying cardiac remodeling secondary to diverse cardiovascular disease entities.

The prognostic value of N-terminal pro-B-type natriuretic peptide for death and cardiovascular events in healthy normal and stage A/B heart failure subjects.

OBJECTIVES Our objective was to determine the prognostic value of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) for death and cardiovascular events among subjects without risk factors for heart failure (HF), which we term healthy normal. BACKGROUND Previous studies report that plasma NT-proBNP has prognostic value for cardiovascular events in the general population even in the absence of HF. It is unclear if NT-proBNP retains predictive value in healthy normal subjects. METHODS We identified a community-based cohort of 2,042 subjects in Olmsted County, Minnesota. Subjects with symptomatic (stage C/D) HF were excluded. The remaining 1,991 subjects underwent echocardiography and NT-proBNP measurement. We further defined healthy normal (n = 703) and stage A/B HF (n = 1,288) subgroups. Healthy normal was defined as the absence of traditional clinical cardiovascular risk factors and echocardiographic structural cardiac abnormalities. Subjects were followed for death, HF, cerebrovascular accident, and myocardial infarction with median follow-up of 9.1, 8.7, 8.8, and 8.9 years, respectively. RESULTS NT-proBNP was not predictive of death or cardiovascular events in the healthy normal subgroup. Similar to previous reports, in stage A/B HF, plasma NT-proBNP values greater than age-/sex-specific 80th percentiles were associated with increased risk of death, HF, cerebrovascular accident, and myocardial infarction (p < 0.001 for all) even after adjustment for clinical risk factors and structural cardiac abnormalities. CONCLUSIONS These findings do not support the use of NT-proBNP as a cardiovascular biomarker in healthy normal subjects and have important implications for NT-proBNP-based strategies for early detection and primary prevention of cardiovascular disease.

Corin Is Present in the Normal Human Heart, Kidney, and Blood, with Pro–B-Type Natriuretic Peptide Processing in the Circulation

BACKGROUND B-type natriuretic peptide (BNP), which is activated in heart failure (HF), is processed to an active form by corin. The corin gene is expressed in the human heart and kidney, but corin protein expression in the heart, kidney, and circulation, along with whether proBNP is processed by circulating corin, remains unknown. METHODS We examined corin protein expression by immunostaining and Western blot in human heart and kidney, and we assessed the circulating corin concentration by ELISA. We examined histidine-tagged (His-tag) proBNP(1-108) processing in serum and plasma by immunoprecipitation and Western blot and sequenced the processed form. RESULTS Normal human heart and kidney displayed the presence of corin, especially in cells around the vasculature. Both corin and proBNP(1-108) were present in the plasma of healthy human subjects, with circulating corin significantly higher in men than women (P < 0.0001) and a positive correlation of corin to age (P = 0.0497, r = 0.27). In fresh normal plasma and serum, His-tag proBNP(1-108) was processed to a lower molecular weight form confirmed to be BNP. Processed BNP was higher in men than women (P = 0.041) and was positively correlated to plasma corin concentrations (P = 0.041, r = 0.65). CONCLUSIONS Our results support the concept that proBNP(1-108) may be processed outside of the heart in the circulation where the proprotein convertase is present. Moreover, sex may impact this process, since corin concentrations are higher in men. These findings may have important physiologic and pathophysiologic implications for the proBNP/corin system in the human.

The Aging Heart, Myocardial Fibrosis, and its Relationship to Circulating C-Type Natriuretic Peptide

Myocardial aging is characterized by left ventricular (LV) fibrosis leading to diastolic and systolic dysfunction. Studies have established the potent antifibrotic and antiproliferative properties of C-type natriuretic peptide (CNP); however, the relationship between circulating CNP, LV fibrosis, and associated changes in LV function with natural aging are undefined. Accordingly, we characterized the relationship of plasma CNP with LV fibrosis and function in 2-, 11-, and 20-month–old male Fischer rats. Further in vitro, we established the antiproliferative actions of CNP and the participation of the clearance receptor using adult human cardiac fibroblasts. Here we establish for the first time that a progressive decline in circulating CNP characterizes natural aging and is strongly associated with a reciprocal increase in LV fibrosis that precedes impairment of diastolic and systolic function. Additionally, we demonstrate in cultured adult human cardiac fibroblasts that the direct antiproliferative actions of high-dose CNP may involve a non-cGMP pathway via the clearance receptor. Together, these studies provide new insights into myocardial aging and the relationship to the antifibrotic and antiproliferative peptide CNP.

Long-Term Cardiac pro-B-Type Natriuretic Peptide Gene Delivery Prevents the Development of Hypertensive Heart Disease in Spontaneously Hypertensive Rats

Background— Diastolic dysfunction associated with high blood pressure (BP) leads to cardiac remodeling and fibrosis and progression to congestive heart failure. B-type natriuretic peptide (BNP) has BP-lowering, antifibrotic, and antihypertrophic properties, which makes BNP an attractive agent for attenuating the adverse cardiac remodeling associated with hypertension. In the current study, we tested the effects of sustained cardiac proBNP gene delivery on BP, cardiac function, and remodeling in spontaneously hypertensive rats (SHR). Methods and Results— We used the myocardium-tropic adeno-associated virus serotype 9 (AAV9) vector to achieve continuously enhanced cardiac rat proBNP expression. In SHR, a single systemic administration of AAV9 vector allowed long-term cardiac BNP overexpression, resulting in reductions in systolic and diastolic BP for 9 months after injection. Left ventricular (LV) thickness, LV end-systolic dimensions, and LV mass were reduced, whereas ejection fraction was significantly increased, in BNP-treated compared with untreated SHR. Circumferential systolic strain and strain rate of the early phase of diastole were improved in BNP-treated compared with untreated SHR. Noncardiac overexpression of BNP via AAV2 vector was not associated with changes in BP and plasma BNP in SHR. Furthermore, normal Wistar rats injected with AAV9 proBNP vector showed significantly reduced heart weights 4 weeks after injection without BP reduction. Conclusions— AAV9 vector facilitates sustained cardiac proBNP overexpression and improves LV function in hypertensive heart disease. Long-term proBNP delivery improved both systolic and diastolic function. The effects on cardiac structure and function occurred independently of BP-lowering effects in normal Wistar rats.

Experimental mild renal insufficiency mediates early cardiac apoptosis, fibrosis, and diastolic dysfunction: a kidney-heart connection

Impaired renal function with loss of nephron number in chronic renal disease (CKD) is associated with increased cardiovascular morbidity and mortality. However, the structural and functional cardiac response to early and mild reduction in renal mass is poorly defined. We hypothesized that mild renal impairment produced by unilateral nephrectomy (UNX) would result in early cardiac fibrosis and impaired diastolic function, which would progress to a more global left ventricular (LV) dysfunction. Cardiorenal function and structure were assessed in rats at 4 and 16 wk following UNX or sham operation (Sham); (n = 10 per group). At 4 wk, blood pressure (BP), aldosterone, glomerular filtration rate (GFR), proteinuria, and plasma B-type natriuretic peptide (BNP) were not altered by UNX, representing a model of mild early CKD. However, UNX was associated with significantly greater LV myocardial fibrosis compared with Sham. Importantly, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed increased apoptosis in the LV myocardium. Further, diastolic dysfunction, assessed by strain echocardiography, but with preserved LVEF, was observed. Changes in genes related to the TGF-β and apoptosis pathways in the LV myocardium were also observed. At 16 wk post-UNX, we observed persistent LV fibrosis and impairment in LV diastolic function. In addition, LV mass significantly increased, as did LVEDd, while there was a reduction in LVEF. Aldosterone, BNP, and proteinuria were increased, while GFR was decreased. The myocardial, structural, and functional alterations were associated with persistent changes in the TGF-β pathway and even more widespread changes in the LV apoptotic pathway. These studies demonstrate that mild renal insufficiency in the rat results in early cardiac fibrosis and impaired diastolic function, which progresses to more global LV remodeling and dysfunction. Thus, these studies importantly advance the concept of a kidney-heart connection in the control of myocardial structure and function.

CD-NP: a novel engineered dual guanylyl cyclase activator with anti-fibrotic actions in the heart.

Natriuretic peptides (NPs) are cardioprotective through the activation of guanylyl cyclase (GC) receptors A and B. CD-NP, also known as cenderitide, is a novel engineered NP that was designed to uniquely serve as a first-in-class dual GC receptor agonist. Recognizing the aldosterone suppressing actions of GC-A activation and the potent inhibitory actions on collagen synthesis and fibroblast proliferation through GC-B activation, the current study was designed to establish the anti-fibrotic actions of CD-NP, administered subcutaneously, in an experimental rat model of early cardiac fibrosis induced by unilateral nephrectomy (UNX). Our results demonstrate that a two week subcutaneous infusion of CD-NP significantly suppresses left ventricular fibrosis and circulating aldosterone, while preserving both systolic and diastolic function, in UNX rats compared to vehicle treated UNX rats. Additionally we also confirmed, in vitro, that CD-NP significantly generates the second messenger, cGMP, through both the GC-A and GC-B receptors. Taken together, this novel dual GC receptor activator may represent an innovative anti-fibrotic therapeutic agent.

Low-dose nesiritide in human anterior myocardial infarction suppresses aldosterone and preserves ventricular function and structure: a proof of concept study

Background: B-type natriuretic peptide (BNP, nesiritide) has anti-fibrotic, anti-hypertrophic, anti-inflammatory, vasodilating, lusitropic and aldosterone-inhibiting properties but conventional doses of BNP cause hypotension, limiting its use in heart failure. Objective: To determine whether infusion of low-dose BNP within 24 h of successful reperfusion for anterior acute myocardial infarction (AMI) would prevent adverse left ventricular (LV) remodelling and suppress aldosterone. Methods: A translational proof-of-concept study was carried out to determine tolerability and biological activity of intravenous BNP at 0.003 and 0.006 μg/kg/min, without bolus started within 24 h of successful reperfusion for anterior AMI. 24 patients with first anterior wall ST elevation AMI and successful revascularisation were randomly assigned to receive 0.003 (n = 12) or 0.006 (n = 12) μg/kg/min of IV BNP for 72 h in addition to standard care during hospitalisation for anterior AMI. Results: Baseline characteristics, drugs and peak cardiac biomarkers for myocardial damage were similar between both groups. Infusion of BNP at 0.006 μg/kg/min resulted in greater biological activity than infusion at 0.003 μg/kg/min as measured by higher mean (SEM) plasma cGMP levels (8.6 (1) vs 5.5 (1) pmol/ml, p<0.05) and suppression of plasma aldosterone (8.0 (2) to 4.6 (1) ng/dl, p<0.05), which was not seen in the 0.003 μg/kg/min group. LV ejection fraction (LVEF) improved significantly from baseline to 1 month (40 (4)% to 54 (5)%, p<0.05) in the 0.006 group but not in the 0.003 group. Infusion of BNP at 0.006 μg/kg/min was associated with a decrease of LV end-systolic volume index (61 (9) to 43 (8) ml/m2, p<0.05) at 1 month, which was not seen in the 0.003 group. No drug-related serious adverse events occurred in either group. Conclusions: 72 h infusion of low BNP at the time of anterior AMI is well tolerated and biologically active. Patients treated with low-dose BNP had improved LVEF and smaller LV end-systolic volume at 1 month.

Chronic Actions of a Novel Oral B-Type Natriuretic Peptide Conjugate in Normal Dogs and Acute Actions in Angiotensin II–Mediated Hypertension

Background— We previously reported the feasibility of an acute, orally delivered, newly developed, conjugated form of human B-type natriuretic peptide (hBNP) in normal animals. The objective of the present study was to extend our findings and to define the chronic actions of an advanced oral conjugated hBNP (hBNP-054) administered for 6 days on sodium excretion and blood pressure. We also sought to establish the ability of this new conjugate to acutely activate cGMP and to reduce blood pressure in an experimental model of angiotensin II (ANG II) –mediated hypertension. Methods and Results— First, we developed additional novel conjugated forms of oral hBNP that were superior to our previously reported hBNP-021 in reducing blood pressure in 6 normal dogs. We then tested the new conjugate, hBNP-054, chronically in 2 normal dogs to assess its biological actions as a blood pressure–lowering agent and as a natriuretic factor. Second, we investigated the effects of acute oral hBNP-054 or vehicle in 6 dogs that received continuous infusion of ANG II to induce hypertension. After baseline determination of mean blood pressure (MAP) and blood collection for plasma hBNP and cGMP, all dogs received continuous ANG II infusion (20 ng · kg−1 · min−1, 1 mL/min) for 4 hours. After 30 minutes of ANG II, dogs received oral hBNP-054 (400 &mgr;g/kg) or vehicle in a random crossover fashion with a 1-week interval between dosing. Blood sampling and MAP measurements were repeated 30 minutes after ANG II administration and 10, 30, 60, 120, 180, and 240 minutes after oral administration of hBNP-054 or vehicle. In the chronic study in normal dogs, oral hBNP-054 effectively reduced MAP for 6 days and induced a significant increase in 24-hour sodium excretion. hBNP was not present in the plasma at baseline in any dogs, and it was not detected at any time in the vehicle group. However, hBNP was detected throughout the duration of the study after oral hBNP-054, with a peak concentration at 30 minutes of 1060±818 pg/mL. In the acute study, after ANG II administration, plasma cGMP was not activated after vehicle, whereas it was significantly increased after oral hBNP-054 (P=0.01 between the 2 groups). Importantly, MAP was significantly increased after ANG II throughout the acute study protocol. However, although no changes occurred in MAP after vehicle administration, oral hBNP-054 reduced MAP for >2 hours (from 138±1 mm Hg after ANG II to 124±2 mm Hg at 30 minutes, 124±2 mm Hg at 1 hour, and 130±5 mm Hg at 2 hours after oral hBNP-054; P<0.001). Conclusions— This study reports for the first time that a novel conjugated oral hBNP possesses blood pressure–lowering and natriuretic actions over a 6-day period in normal dogs. Furthermore, hBNP-054 activates cGMP and reduces MAP in a model of acute hypertension. These findings advance the concept that orally administered chronic BNP is a potential therapeutic strategy for cardiovascular diseases such as hypertension.

Local renal delivery of a natriuretic peptide a renal-enhancing strategy for B-type natriuretic peptide in overt experimental heart failure.

OBJECTIVES The purpose of this study was to test the hypothesis that local renal delivery of B-type natriuretic peptide (BNP) will overcome renal resistance to BNP without systemic hypotension. BACKGROUND BNP has vasodilating, natriuretic, and renin-inhibiting properties. In overt heart failure (HF), there is development of renal resistance to BNP. METHODS We defined the cardiorenal and humoral effects of systemic (n = 6) or local renal (n = 7) administration of canine BNP (0.01 microg/kg/min) in 2 separate groups of dogs with pacing-induced subacute overt HF complicated by renal dysfunction. We used a commercially available small (3.1-F) bifurcated renal catheter (FlowMedica Inc., Fremont, California) for direct bilateral infusion of BNP into both renal arteries. RESULTS With systemic BNP at this clinically used dose (without the bolus), urine flow increased, but there was only a trend for an increase in urinary sodium excretion and glomerular filtration rate (GFR). In contrast, local renal delivery of BNP resulted in significant diuresis and natriuresis and an increase in GFR. These diuretic and natriuretic responses were greater with local renal BNP compared with systemic BNP, and were associated with increased delivery of BNP to the renal tubules as evident by a greater urinary BNP excretion resulting in a decrease in distal reabsorption of sodium. Importantly, local renal BNP did not result in a significant decrease in mean arterial pressure that was observed with systemic BNP. CONCLUSIONS We conclude that local renal BNP delivery is a novel strategy that may overcome renal assistance to BNP in overt HF by increasing local delivery of BNP to the renal tubules.