Isolation, homology modeling and renal effects of a C-type natriuretic peptide from the venom of the Brazilian yellow scorpion (Tityus serrulatus)

Mammalian natriuretic peptides (NPs) have been extensively investigated for use as therapeutic agents in the treatment of cardiovascular diseases. Here, we describe the isolation, sequencing and tridimensional homology modeling of the first C-type natriuretic peptide isolated from scorpion venom. In addition, its effects on the renal function of rats and on the mRNA expression of natriuretic peptide receptors in the kidneys are delineated. Fractionation of Tityusserrulatus venom using chromatographic techniques yielded a peptide with a molecular mass of 2190.64Da, which exhibited the pattern of disulfide bridges that is characteristic of a C-type NP (TsNP, T. serrulatus Natriuretic Peptide). In the isolated perfused rat kidney assay, treatment with two concentrations of TsNP (0.03 and 0.1μg/mL) increased the perfusion pressure, glomerular filtration rate and urinary flow. After 60min of treatment at both concentrations, the percentages of sodium, potassium and chloride transport were decreased, and the urinary cGMP concentration was elevated. Natriuretic peptide receptor-A (NPR-A) mRNA expression was down regulated in the kidneys treated with both concentrations of TsNP, whereas NPR-B, NPR-C and CG-C mRNAs were up regulated at the 0.1μg/mL concentration. In conclusion, this work describes the isolation and modeling of the first natriuretic peptide isolated from scorpion venom. In addition, examinations of the renal actions of TsNP indicate that its effects may be related to the activation of NPR-B, NPR-C and GC-C. © 2013 Elsevier Ltd.

Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50 %, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70 %). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (n = 10) and HF (n = 6) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts:a possible defense against transforming growth factor-β mediated fibrosis

BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.

METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.

CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Vascular effects and electrolyte homeostasis of the natriuretic peptide isolated from Crotalus oreganus abyssus (North American Grand Canyon rattlesnake) venom

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Utilizações de informações genômicas para o melhoramento genético de características de crescimento em bovinos da raça Nelore

Pós-graduação em Genética e Melhoramento Animal - FCAV

A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy

Guanylyl cyclase-A (NPR-A; GC-A) is the major and possibly the only receptor for atrial natriuretic peptide (ANP) or B-type natriuretic peptide. Although mice deficient in GC-A display an elevated blood pressure, the resultant cardiac hypertrophy is much greater than in other mouse models of hypertension. Here we overproduce GC-A in the cardiac myocytes of wild-type or GC-A null animals. Introduction of the GC-A transgene did not alter blood pressure or heart rate as a function of genotype. Cardiac myocyte size was larger (approximately 20%) in GC-A null than in wild-type animals. However, introduction of the GC-A transgene reduced cardiac myocyte size in both wild-type and null mice. Coincident with the reduction in myocyte size, both ANP mRNA and ANP content were significantly reduced by overexpression of GC-A, and this reduction was independent of genotype. This genetic model, therefore, separates a regulation of cardiac myocyte size by blood pressure from local regulation by a GC-mediated pathway.

Hazardous alcohol consumption is associated with increased levels of B-type natriuretic peptide: evidence from two population-based studies

Russia has very high mortality from cardiovascular disease (CVD), with evidence that heavy drinking may play a role. To throw further light on this association we have studied the association of alcohol with predictors of CVD risk including B-type natriuretic peptide (BNP). Levels of BNP increase primarily in response to abnormal cardiac chamber wall stretch which can occur both as a result of atherosclerosis as well as due to other types of damage to the myocardium. No previous population-based studies have investigated the association with alcohol. We analysed cross-sectional data on drinking behaviour in 993 men aged 25-60 years from the Izhevsk Family Study 2 (IFS2), conducted in the Russian city of Izhevsk in 2008-2009. Relative to non-drinkers, men who drank hazardously had an odds ratio (OR) of being in the top 20 % of the BNP distribution of 4.66 (95 % CI 2.13, 10.19) adjusted for age, obesity, waist-hip ratio, and smoking. Further adjustment for class of hypertension resulted in only slight attenuation of the effect, suggesting that this effect was not secondary to the influence of alcohol on blood pressure. In contrast hazardous drinking was associated with markedly raised ApoA1 and HDL cholesterol levels, but had little impact on levels of ApoB and LDL cholesterol. Similar but less pronounced associations were found in the Belfast (UK) component of the PRIME study conducted in 1991. These findings suggest that the association of heavy drinking with increased risk of cardiovascular disease may be partly due to alcohol-induced non-atherosclerotic damage to the myocardium.

Gene expression profiling of human hibernating myocardium: Increased expression of B-type natriuretic peptide and proenkephalin in hypocontractile vs normally-contracting regions of the heart

A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization. Paired heart biopsies were taken from hypocontractile and normally-contracting myocardium (identified by cardiovascular magnetic resonance) from 6 patients with chronic stable angina scheduled for bypass grafting. Gene expression profiles of hypocontractile and normally-contracting samples were compared using Affymetrix microarrays. The data for patients with confirmed hibernating myocardium were analysed separately and a different, though overlapping, set (up to 380) of genes was identified which may constitute a molecular fingerprint for hibernating myocardium. The expression of B-type natriuretic peptide (BNP) was increased in hypocontractile relative to normally-contracting myocardium. The expression of BNP correlated most closely with the expression of proenkephalin and follistatin 3, which may constitute additional heart failure markers. Our data illustrate differential gene expression in hypocontractile and/hibernating myocardium relative to normally-contracting myocardium within individual human hearts. Changes in expression of these genes, including increased relative expression of natriuretic and other factors, may constitute a molecular signature for hypocontractile and/or hibernating myocardium.

Involvement of the atrial natriuretic peptide in cardiovascular pathophysiology and its relationship with exercise

In this minireview we describe the involvement of the atrial natriuretic peptide (ANP) in cardiovascular pathophysiology and exercise. The ANP has a broad homeostatic role and exerts complex effects on the cardio-circulatory hemodynamics, it is produced by the left atrium and has a key role in regulating sodium and water balance in mammals and humans. The dominant stimulus for its release is atrial wall tension, commonly caused by exercise. The ANP is involved in the process of lipolysis through a cGMP signaling pathway and, as a consequence, reducing blood pressure by decreasing the sensitivity of vascular smooth muscle to the action of vasoconstrictors and regulate fluid balance. The increase of this hormone is associated with better survival in patients with chronic heart failure (CHF). This minireview provides new evidence based on recent studies related to the beneficial effects of exercise in patients with cardiovascular disease, focusing on the ANP. © 2012 de Almeida et al; licensee BioMed Central Ltd.

B-type natriuretic peptide in the evaluation of cardiac function

B type natriuretic peptide (BNP) and its precursor, the inactive form of NT-pro-BNP, are currently the most studied laboratory parameters in the heart disease spectrum. The assessment of their blood concentrations provides invaluable information on the likelihood, severity and prognosis of the disease. The present review aims to describe the biological determinants, the factors that influence these peptide concentrations, the suggested cutoff values for the diagnosis of heart failure and the use of this biomarker in the assessment of cardiac function.

Plasma Pro-B-Type Natriuretic Peptide Testing as a Screening Method for Hypertrophic Cardiomyopathy

Background: Clinical multistage risk assessment associated with electrocardiogram (ECG) and NT-proBNP may be a feasible strategy to screen hypertrophic cardiomyopathy (HCM). We investigated the effectiveness of a screening based on ECG and NT-proBNP in first-degree relatives of patients with HCM. Methods and Results: A total of 106 first-degree relatives were included. All individuals were evaluated by echocardiography, ECG, NT-proBNP, and molecular screening (available for 65 individuals). From the 106 individuals, 36 (34%) had diagnosis confirmed by echocardiography. Using echocardiography as the gold standard, ECG criteria had a sensitivity of 0.71, 0.42, and 0.52 for the Romhilt-Estes, Sokolow-Lyon, and Cornell criteria, respectively. Mean values of NT-ProBNP were higher in affected as compared with nonaffected relatives (26.1 vs. 1290.5, P < .001). The AUC of NT-proBNP was 0.98. Using a cutoff value of 70 pg/mL, we observed a sensitivity of 0.92 and specificity of 0.96. Using molecular genetics as the gold standard, ECG criteria had a sensitivity of 0.67, 0.37, and 0.42 for the Romhilt-Estes, Sokolow-Lyon, and Cornell criteria, respectively. Using a cutoff value of 70 pg/mL, we observed a sensitivity of 0.83 and specificity of 0.98. Conclusion: Values of NT-proBNP above 70 pg/mL can be used to effectively select high-risk first-degree relatives for HCM screening. (J Cardiac Fail 2012;18:564-568)

Accuracy of B-type natriuretic peptide tests to exclude congestive heart failure: systematic review of test accuracy studies

BACKGROUND: Congestive heart failure (CHF) is a major public health problem. The use of B-type natriuretic peptide (BNP) tests shows promising diagnostic accuracy. Herein, we summarize the evidence on the accuracy of BNP tests in the diagnosis of CHF and compare the performance of rapid enzyme-linked immunosorbent assay (ELISA) and standard radioimmunosorbent assay (RIA) tests. METHODS: We searched electronic databases and the reference lists of included studies, and we contacted experts. Data were extracted on the study population, the type of test used, and methods. Receiver operating characteristic (ROC) plots and summary ROC curves were produced and negative likelihood ratios pooled. Random-effect meta-analysis and metaregression were used to combine data and explore sources of between-study heterogeneity. RESULTS: Nineteen studies describing 22 patient populations (9 ELISA and 13 RIA) and 9093 patients were included. The diagnosis of CHF was verified by echocardiography, radionuclide scan, or echocardiography combined with clinical criteria. The pooled negative likelihood ratio overall from random-effect meta-analysis was 0.18 (95% confidence interval [CI], 0.13-0.23). It was lower for the ELISA test (0.12; 95% CI, 0.09-0.16) than for the RIA test (0.23; 95% CI, 0.16-0.32). For a pretest probability of 20%, which is typical for patients with suspected CHF in primary care, a negative result of the ELISA test would produce a posttest probability of 2.9%; a negative RIA test, a posttest probability of 5.4%. CONCLUSIONS: The use of BNP tests to rule out CHF in primary care settings could reduce demand for echocardiography. The advantages of rapid ELISA tests need to be balanced against their higher cost.

Impact of B-type natriuretic peptide on short-term clinical outcomes following transcatheter aortic valve implantation

Aims: We aimed to assess the impact of B-type natriuretic peptide (BNP) on short-term outcomes in patients undergoing transcatheter aortic valve implantation (TAVI). Methods and results: Of 500 consecutive patients with severe aortic stenosis undergoing TAVI at our institution, we studied 340 patients who had a BNP assessment prior to TAVI. Patients were divided into tertiles - low: BNP ≤201 pg/mL (n=114), mid: BNP 202-595 pg/mL (n=113) and high: BNP ≥596 pg/mL (n=113). The primary endpoint was all-cause mortality, cardiac death and major adverse cardiac and cerebrovascular events (MACCE; death, major stroke and myocardial infarction) at 30 days. Compared with low tertile, high tertile patients were at higher baseline surgical risk (STS score 5.5±3.0 vs. 7.4±4.1, p=0.002). On echocardiography, high tertile patients had smaller valve areas (0.74±0.21 vs. 0.66±0.23 cm2, p=0.008), higher left ventricular (LV) mass indices (123.40±33.66 vs. 168.22±47.96 g/m2, p<0.001) and lower LV ejection fractions (61.59±7.18 vs. 42.65±15.41%, p<0.001) as compared with low tertile patients. At 30 days, a significantly higher incidence of death (hazard ratio [HR] 7.41, p=0.001) cardiac death (HR 5.82, p=0.006) and MACCE (HR 9.04, p<0.001) was observed among high as compared to low tertile patients. Conclusions: In TAVI patients, higher BNP values at baseline are associated with an increased risk for an adverse event periprocedurally and after 30 days, respectively.