NPPB Gene Research Articles

Gene variability of natriuretic peptides in patients with rheumatic heart disease and atrial fibrillation

Introduction: Atrial fibrillation (AF) is one of the most common arrhythmias in rheumatic heart disease (RHD) patients. The pathophysiological mechanisms of AF development against the background of RHD are still understudied. Similarly to most cardiovascular pathologies, AF is a multifactorial disease with a significant genetic predeterminacy.Objective: The study was aimed at searching for associations of polymorphic gene variants of the natriuretic peptides (NPPA, NPPB and NPPC) with the AF risk in RHD patients. Methods: The study included 251 patients diagnosed with RHD, who were divided into 2 groups: patients with RHD and AF (n = 191) and patients with RHD without AF (n = 60). Genotyping of 7 allelic variants of genes encoding natriuretic peptides was performed using real-time polymerase chain reaction.Results: Polymorphic variants rs198388 and rs198389 of the NPPB gene were found to have a protective effect against the AF in RHD patients on the model of a codominant inheritance. The rs198358 allelic variant of the NPPA-AS1 gene was associated with the two-fold increased AF risk in RHD patients (OR 1.96, 95% CI 1.02-3.75, p = 0.037). Two models of gene-gene interactions characterized by the high efficacy and sensitivity have been identified in this study. The most significant combinations of genotypes associated with an increased AF risk in RHD patients were rs198388 C/T × rs198389 A/G × rs198358 T/C × rs5063 C/C × rs632793 A/G and rs198388 C/T × rs198358 T/ C × rs5063 C/C.Conclusion: Polymorphic gene variants of the natriuretic peptides (NPPB rs198388, rs198389 and NPPA-AS1 rs198358), associated with AF risk, were identified in RHD patients. Three-locus (NPPB rs198388, NPPA-AS1 rs198358 and NPPA rs5063) and five-locus (NPPB rs198388, NPPB rs198389, NPPA-AS1 rs198358, NPPA rs5063 and NPPA rs632793) models of gene-gene interactions were associated with the studied phenotype. Received 16 April 2024. Revised 10 October 2024. Accepted 2 November 2024. FundingThis research was supported by the Complex Program of Fundamental Research of the Siberian Branch of the Russian Academy of Sciences within the framework of the fundamental research project of the Research Institute for Complex Issues of Cardiovascular Diseases No. 0419-2022-0001. Conflict of interestThe authors declare no conflict of interest. Contribution of the authorsConception and study design: A.V. Sinitskaya, M.V. KhutornayaData collection and analysis: O.N. Hryachkova, A.O. Poddubnyak, M.V. Khutornaya, M.A. Asanov Statistical analysis: A.V. SinitskayaDrafting the article: A.V. SinitskayaCritical revision of the article: M.Yu. SinitskyFinal approval of the version to be published: A.V. Sinitskaya, M.V. Khutornaya, O.N. Hryachkova, A.O. Poddubnyak, M.A. Asanov, M.Yu. Sinitsky

The Impact of Natriuretic Peptides on Heart Development, Homeostasis, and Disease.

During mammalian heart development, the clustered genes encoding peptide hormones, Natriuretic Peptide A (NPPA; ANP) and B (NPPB; BNP), are transcriptionally co-regulated and co-expressed predominately in the atrial and ventricular trabecular cardiomyocytes. After birth, expression of NPPA and a natural antisense transcript NPPA-AS1 becomes restricted to the atrial cardiomyocytes. Both NPPA and NPPB are induced by cardiac stress and serve as markers for cardiovascular dysfunction or injury. NPPB gene products are extensively used as diagnostic and prognostic biomarkers for various cardiovascular disorders. Membrane-localized guanylyl cyclase receptors on many cell types throughout the body mediate the signaling of the natriuretic peptide ligands through the generation of intracellular cGMP, which interacts with and modulates the activity of cGMP-activated kinase and other enzymes and ion channels. The natriuretic peptide system plays a fundamental role in cardio-renal homeostasis, and its potent diuretic and vasodilatory effects provide compensatory mechanisms in cardiac pathophysiological conditions and heart failure. In addition, both peptides, but also CNP, have important intracardiac actions during heart development and homeostasis independent of the systemic functions. Exploration of the intracardiac functions may provide new leads for the therapeutic utility of natriuretic peptide-mediated signaling in heart diseases and rhythm disorders. Here, we review recent insights into the regulation of expression and intracardiac functions of NPPA and NPPB during heart development, homeostasis, and disease.

Spatial transcriptomic analysis identifies a border zone with localized expression of brain natriuretic peptide in the inferobasal region of the left ventricle in patients with mitral valve prolapse

Abstract Background/Introduction Mitral valve prolapse (MVP) is a common cause of mitral regurgitation (MR), adverse left ventricular (LV) remodeling and arrhythmias. MVP induces mechanical stretch to the inferobasal (IB) myocardium and papillary muscles. The underlying regional and molecular pathology of MVP-induced LV remodeling remains incompletely understood. Purpose The aim was to characterize the molecular effects of mechanical stretch by comparing the regional gene expression of the IB region of LV with the interventricular septum in patients with MVP. Methods Myocardial biopsies were obtained from the IB LV (i.e. between papillary muscles) and the interventricular septum during mitral valve repair surgery for MVP-induced primary MR. The degree of cardiac fibrosis was detected and quantified by histology. We used spatial transcriptomics (ST) that - in contrast to traditional analyses of tissue homogenates - allows regional expressional analysis. Results Automated clustering of ST results correlated with histological staining of fibrosis in the IB and septal regions (R=0.97, P<0.0001), demonstrating good automated identification of fibrotic regions. The extent of fibrosis was similar between the inferobasal region and the septal control in the respective patients with MVP as determined by histological analysis and ST. The localized fibrotic areas of the inferobasal region showed decreased expression of cardiac myocytegenes such as RYR2, TNNI3, TNNC1 or TPM1, and increased expression of fibrous and EndMT markers such as COL3A1, BGN, SM22 or FN1. In the IB region, we identified a transition zone between healthy myocardium and fibrous tissue. This transition zone showed increased expression of the NPPB gene encoding brain natriuretic peptide (BNP), whereas NPPB was not increased in the septal region of the same patients. The upregulation of NPPB (6.4-fold, P=0.004) in the inferobasal region compared to the septal control was confirmed by qPCR in biopsies from patients with MVP. The identified transition zones were characterized by increased expression of genes such as IGFBP2 and LTBP2, which have been described to be associated with adverse LV remodeling in heart failure. Interestingly, the serum NT-proBNP levels (mean 276.8 ± 121.6 pg/ml) of the patients correlated with NPPB levels from the inferobasal region (R=0.76, P=0.01, N=9), whereas no correlation was found with NPPB levels from the septal control regions (R=0.17, P=0.65) of the same patients. Conclusions ST analysis in MVP identifies a transition zone of the inferobasal region of LV which is exposed to high mechanical stretch. This transition zone - but not the inferobasal fibrotic area nor the LV septum - is characterized by a specific increase in NBBP expression that correlates with increased NT-ProBNP serum concentrations. These special sequencing results indicate that mechanical stretch induces highly localized myocardial pathologies with systemic consequences.

Abstract 15078: Atrial and Brain Natriuretic Peptides Expression and Release in Pulmonary Hypertension

Introduction: Precapillary pulmonary hypertension (PH) is characterized by progressive overload for the right ventricle and right heart failure (RHF). N-terminal Brain Natriuretic Peptides (NTproBNP) and Atrial Natriuretic Peptides (ANP) are secreted from cardiomyocytes upon stretch. NTproBNP is used as biomarker for RHF. Aim: To study the association between ANP and NTproBNP and pressure overload in blood, right heart tissue and induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from PH-patients. Methods: We analyzed NTproBNP in 344 PH-patients, 9 controls, and in 9 PH-patients before and 6-months after pulmonary endarterectomy (PEA). We measured NPPA and NPPB gene expression on end-stage human right ventricular (RV) and right atrial (RA) samples from 8 PH-patients and 5 controls. In addition, we performed immunofluoresence staining for ANP and BNP on RA tissue (N=4) and RV tissue (N=10) in PH and controls (N=5). iPSC-CMs from 1 male and 1 female PH-patients were stretched for 24h at 1Hz and 10% on the Flexcell FX-6000 system, and NTproBNP release was measured on static and stretched iPSC-CMs. NTproBNP levels were measured through ECLIA. Results: NTproBNP serum levels were elevated in PH-patients versus controls (918 vs 25 pg/mL, Fig1A). Pressure unloading in CTEPH patients did not result in a decrease in NTproBNP (Fig1B). In RV tissue of PH-patients NPPA expression was upregulated, but NPPB was not (Fig1C). ANP and BNP content was higher in RA and RV tissue of PH-patients (Fig1D-E). Finally, ~5x higher NTproBNP was released from male vs female iPSC-CMs under static conditions and after mechanical stretch (Fig1F). Conclusions: ANP and BNP showed important differences at gene and protein levels in relation to pressure overload. To confirm current results, further analyses on atrial and ventricular iPSC-CMs will be performed, in addition to mid-range proANP measurements in patients’ blood and iPSC-CMs conditioned medium.

Abstract P2100: Highly Localized Myocardial Expression Of Brain Natriuretic Peptide In Patients With Mitral Valve Prolapse

Mitral valve prolapse (MVP) is a common cause of mitral regurgitation (MR), adverse left ventricular (LV) remodeling and arrhythmias. MVP induces mechanical stretch to the inferobasal (INF) myocardium and papillary muscles. The underlying regional and molecular pathology of MVP-induced LV remodeling remains incompletely understood. The aim was to characterize the molecular effects of mechanical stretch by comparing the regional gene expression of the INF region of LV in patients with MVP. Biopsies were obtained from the INF LV and the interventricular septum during mitral valve repair surgery for MVP-induced primary MR. Spatial transcriptome sequencing results correlated with histological staining of fibrosis in the INF and septal regions (R=0.97, P<0.0001). The extent of fibrosis was similar between the INF region and the septal control in the respective patients with MVP. The localized fibrotic areas of the INF region showed decreased expression of cardiac myocyte genes and increased expression of fibrous and EndMT markers. In the INF region, we identified a transition zone between healthy myocardium and fibrous tissue. This transition zone showed increased expression of the NPPB gene encoding brain natriuretic peptide (BNP), whereas NPPB was not increased in the septal region of the same patients, and was confirmed by qPCR (NPPB +6.4-fold, N=11, P=0.004). The INF border zone is characterized by increase expression of NPPB, IGFBP2 and MFGE8, which have been described to be associated with adverse LV remodeling in heart failure. The serum NT-proBNP levels (mean 276.8 ± 121.6 pg/ml) of the patients correlated with NPPB levels from the INF region (R=0.76, P=0.01, N=9), whereas no correlation was found with NPPB levels from the septal control regions (R=0.17, P=0.65) of the same patients. In conclusion, spatial transcriptome analysis in MVP patients identifies a transition zone of the INF region of LV which is exposed to high mechanical stretch. This localized transition zone has a specific gene expression profile, e.g. with increased NBBP expression that correlates with increased NT-ProBNP serum concentrations. Our results indicate that mechanical stretch induces highly localized myocardial pathologies with systemic consequences.

The underlying mechanism of transcription factor IRF1, PRDM1, and ZNF263 involved in the regulation of NPPB rs3753581 on pulse pressure hypertension

To investigate the correlation between NPPB gene variants and pulse pressure hypertension and the underlying regulatory mechanisms and try to confirm that NPPB may be a potential molecular target of gene therapy for pulse pressure hypertension. A total of 898 participants were recruited from the First Affiliated Hospital of Fujian Medical University and the plasmids with differential expression of NPPB were constructed. Genotype distribution of NPPB(rs3753581, rs198388, and rs198389)was analyzed and the expression of N-terminal pro-B-type natriuretic peptide(NT-proBNP) and renin-angiotensin -aldosterone system(RAAS) related indicators were identified in the groups studied. According to a genotype analysis, there was a significant difference in the genotype distribution of NPPB rs3753581 among the groups (P = 0.034). In logistic regression analysis, NPPB rs3753581 TT was associated with a 1.8-fold greater risk of pulse pressure hypertension than NPPB rs3753581 GG (odds ratio = 1.801; 95% confidence interval: 1.070–3.032; P = 0.027). The expression of NT-proBNP and RAAS related indicators in clinical and laboratory samples showed striking differences. The activity of firefly and Renilla luciferase in pGL-3-NPPB-luc (-1299G) was higher than pGL-3-NPPBmut-luc(-1299 T)(P < 0.05). The binding of NPPB gene promoter rs3753581 (-1299G) with transcription factors IRF1, PRDM1, and ZNF263 was predicted and validated by the bioinformatics software TESS and chromatin immunoprecipitation(P < 0.05). NPPB rs3753581 was correlated with genetic susceptibility to pulse pressure hypertension and the transcription factors IRF1, PRDM1, and ZNF263 may be involved in the regulation of NPPB rs3753581 promoter (-1299G) on the expression of NT-proBNP/RAAS.

Association of NPPB rs198389 and NPPA rs5068 single-nucleotide polymorphisms with natriuretic peptide levels and heart failure progression risks in patients with atrial fibrillation

Natriuretic peptides (NUPs) are the strongest predictors of poor prognosis in patients with heart failure (HF). Single-nucleotide polymorphisms (SNPs) rs198389 of the NPPB gene and rs5068 of the NPPA gene are associated with altered levels of NUP. The role of candidate gene polymorphisms in the activity of the NUP system and the association of NPPA/ NPPB SNPs with the risk of cardiovascular disease (CVD) in individuals with HF and atrial fibrillation (AF) is not well understood.The study aims to evaluate the allele and genotype frequencies of NPPA rs5068 and NPPB rs198389 SNPs in a selective sample of the Belarusian population, to determine the relationship of these SNPs with NUP concentrations, and to assess the prognostic significance of these SNPs on the risk of HF hospitalization in patients with HF and permanent AF.The study involved 187 patients. The main group included 152 patients with HF with left ventricular ejection fraction (LVEF) &lt; 50 %. Group 1 included 48 patients with HF and AF; group 2 – 51 patients with HF and sinus rhythm (SR) and 35 patients in the control group. The levels of atrial and brain natriuretic peptides (ANP and BNP) and the N-terminal fragment of the brain natriuretic peptide (NT-proBNP) were determined. A genetic testing of polymorphic loci of the rs5068 NPPA gene and the rs198389 NPPB gene was performed. The primary endpoint of the study was hospitalization due to HF progression.The average observation period was 12.1 [from 9 to 14] months. The distribution of the genotype and allele frequencies of rs198389 NPPB and rs5068 NPPA in HF patients with LVEF &lt; 50 % is comparable to that in individuals without CVD. In patients with HF and persistent AF, the minor allele C rs198389 NPPB is associated with higher BNP levels compared to patients with HF and SR (542 [333.7; 909.4] pg/ml versus 247.3 [244; 365.2] pg/ml; p &lt; 0.05), but it has no relationship with the NT-proBNP level. In patients with HF and permanent AF, the ANP levels are not associated with rs5068 NPPA.The frequency of the T allele rs198389 NPPB in hospitalized patients was significantly lower compared to patients who were not hospitalized (22 patients (44 %) versus 83 patients (62 %); p = 0.04). The presence of the C allele rs198389 NPPB was associated with a higher risk of HF progression in patients with HF and AF, the odds ratio (OR) = 2.071 [95 % CI from 1.072 to 4.001], p &lt; 0.05.

Abstract P2027: Short-term Ambient Particulate Exposure And Social Defeat Cause Reductions In Heart And Pulmonary Function

Deployed service members and first responders are exposed to environments containing high levels of air pollution and psychological distress. Thus, we sought to investigate the interaction of these factors and whether the combination of PM and stress exposures exacerbate cardiac and lung dysfunction in a mouse model. Male C57BL/6 mice were exposed to filtered air (FA) or PM (≤2.5 μm) at concentrations of 137.7 ± 70.76 μg/m 3 (daily mean ± standard deviation) for five days/week for three weeks. A subset of each group was stressed via exposure to a retired male aggressor for two hours/day, 3-5 nights/week (FA-stress and PM-stress groups). Echocardiography revealed no changes in left ventricle (LV) systolic or diastolic indices, but a significant reduction in right ventricle (RV) stroke volume and cardiac output in the PM, FA-stress and PM-stress groups compared to the FA group (P&lt;0.05 via two-way ANOVA). Pressure-volume loops displayed significantly reduced end-systolic elastance for the PM and PM-stress groups, but not for the FA-stress group when compared to FA controls, indicating reduced contractility (Ees, P&lt;0.05 via two-way ANOVA). Data on respiratory mechanics was acquired using the flexiVent FX2 (SCIREQ Inc.) system. PM exposure caused an increase in lung resistance (Rrs), central airway resistance (Rn), and tissue damping (G) compared to FA (P&lt;0.05 via two-way ANOVA at individual doses of methacholine). Also, the FA-stress and PM-stress groups experienced increased lung elastance (Ers), Rrs, Rn, and G compared to FA mice (P&lt;0.05 via two-way ANOVA at individual doses). Tail-vein blood pressure measurements showed an increased diastolic pressure induced by PM exposure, but not by stress (P&lt;0.05 via two-way ANOVA). RT-qPCR data revealed a significant decrease in RV NPPB gene expression and an increased RV Col1A1 expression for PM-exposed mice compared to FA (via two-way ANOVA). Additionally, RT-qPCR demonstrated that PM decreased the expression of many inflammatory markers (TNF-α, CCL2, IL6, and IL-1β) in the LV and RV relative to FA (P&lt;0.05 via two-way ANOVA). Differences in NPPB and Col1A1 expression suggest increased fibrosis in the RV. Calculating capillary to myocyte ratio from a wheat germ agglutinin stain, there was an increase in capillary density of PM-exposed mice compared to FA (P&lt;0.05 via t-test). Taken together, short-term exposure to PM with/without psychological distress impairs respiratory mechanics and RV function. Molecular analyses indicated changes in inflammation in both the LV and RV between the FA and PM groups, with additional changes in RV fibrosis. These results suggest that even short-term exposure to PM, including in occupational settings such as military burn pits, can cause notable cardiopulmonary dysfunction. This work was supported by the Department of Veterans Affairs Airborne Hazards and Burn Pits Center of Excellence

The CR9 element is a novel mechanical load-responsive enhancer that regulates natriuretic peptide genes expression.

Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.

Unexpectedly Low Natriuretic Peptide Levels in Patients With Heart Failure

ObjectivesThe purpose of this study was to determine the frequency of unexpectedly low natriuretic peptide (NP) levels in a clinical population. BackgroundHigher NP concentrations are typically observed as a compensatory response to elevated cardiac wall stress. Under these conditions, low NP levels may be indicative of a “NP deficiency.” MethodsWe identified 3 clinical scenarios in which high B-type natriuretic peptide (BNP) levels would be expected: 1) hospitalization for heart failure (HF); 2) abnormal cardiac structure or function; or 3) abnormal hemodynamics. In Vanderbilt’s electronic health record, 47,970 adult patients had BNP measurements. A total of 13,613 patients had at least 1 of the 3 conditions (hospitalized HF, n = 9,153; abnormal cardiac structure/function, n = 7,041; abnormal hemodynamics, n = 363). We quantified the frequency of low BNP levels. We performed whole exome sequencing of the NPPB gene in a subset of 9 patients. ResultsVery low BNP levels (<50 pg/ml) were observed in 4.9%, 14.0%, and 16.3% of patients with hospitalized HF, abnormal cardiac structure/function, or abnormal hemodynamics, respectively. A small proportion (0.1% to 1.1%) in each group had BNP levels below detection limits. Higher body mass index was the strongest predictor of unexpectedly low BNP. Exome sequencing did not reveal coding variation predicted to alter detection of BNP by clinical assays. ConclusionsA subset of patients with confirmed HF or cardiac dysfunction have unexpectedly low BNP levels. Obesity is the strongest correlate of unexpectedly low BNP levels. Our findings support the possible existence of NP deficiency, which may render some individuals more susceptible to volume or pressure overload.

P4450A cardio-metabolic phenotype in carriers of NPPA (rs5086) and NPPB (rs198389) gene variants in the general population: the dominant role of ANP

P4450A cardio-metabolic phenotype in carriers of NPPA (rs5086) and NPPB (rs198389) gene variants in the general population: the dominant role of ANP

Sex Differences of the Natriuretic Peptide Polymorphism Associated With Angiographic Coronary Atherosclerosis.

BackgroundPolymorphisms within natriuretic peptide (NP) genes have been associated with clinical outcomes for cardiovascular disease (CVD), but no previous study has compared the effect of these polymorphisms between men and women. This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in key genes of the NP system and coronary angiographic outcomes, with the focus on the sexual dimorphism in the effects of these SNPs.MethodsPatients undergoing clinically indicated coronary angiography (n = 513, 328 men and 185 women) were consented and genotyped for NPPA rs5065, NPPB rs198389 and NPR2 rs10758325. Patients were stratified into having normal coronaries, non-obstructive coronary artery disease (CAD) or obstructive CAD, based on the highest stenosis in any epicardial artery. Average luminal narrowing across all four arteries was derived to represent the overall atherosclerotic burden.ResultsThe frequency of NPPB rs198389 AA genotype was significantly higher in women with obstructive CAD (P = 0.014). The same association was not observed in males. With respect to atherosclerotic burden, an association was found between the AA genotype and average luminal narrowing in women (P = 0.005), but not in men.ConclusionsThe current study identified an association between an SNP of the NPPB gene and coronary atherosclerotic burden through angiographic evidence in women but not in men. These results suggest that B-type natriuretic peptide (BNP) may have more important involvement in the development of CAD in women compared to men, and as such, genotyping of the NPPB gene may serve as a potential biomarker to identify women with high risk for CAD.

Genetic variability in the system of natriuretic B peptide and principal toxicological parameters in workers exposed to lead.

The study was aimed at evaluating the influence of selected polymorphisms of natriuretic peptide B precursor (NPPB) and natriuretic peptide receptor C (NPR3) genes on blood lead concentration (Pb-B) and blood zinc protoporphyrin concentration (ZnPP) in persons occupationally exposed to lead. Investigations were conducted on 360 persons (mean age: 44.49±9.62years), workers exposed to lead compounds. The analysis examined four polymorphisms of BNP gene, i.e.,: rs198388, rs198389, rs632793, and rs6676300; as well as one polymorphism of receptor C for natriuretic peptides, i.e., rs1421811. Heterozygosity in locus rs632793 of NPPB gene may result in higher concentrations of Pb-B, while allele A in locus rs632793 of NPPB gene seems to determine higher concentrations of ZnPP in persons occupationally exposed to lead. Workers exposed to lead and carrying allele C in locus rs198388 of NPPB gene, particularly in the heterozygotic setup, seem to be predisposed to present higher concentrations of ZnPP. Carriership of A allele in locus rs198389 of NPPB gene probably determines higher concentrations of ZnPP in study group. In summary, among persons occupationally exposed to lead, certain relationships were demonstrated between rs632793, rs198388 and rs198389 polymorphisms of NPPB gene and principal toxicological parameters characterizing exposure to lead.

O9. The functional role of natriuretic peptides in preeclampsia

Background Natriuretic peptide family is believed to have evolved for the common homeostatic purpose of volume, osmosis and pressure regulation of the circulatory system. The activities of natriuretic peptides and endothelins are associated with each other. In the last three decades many members of the natriuretic peptide family was identified. BNP is the member of this family and with the sympathetic nervous system and other hormones they play key roles, like an endogeneous system in the regulation of the body fluid homeostasis and blood pressure. In the last years the potential use of the elevated BNP levels for diagnosis of pre-eclampsia was examined. There is a variable tandem repeat polymorphism in the 5 ′ -flanking region of the natriuretic peptide precursor B gene (NPPB). A previous study showed association of the (TTTC) small tandem repeat (STR) variants of this gene and essential hypertension in females. Methods In our studies blood samples were collected from healthy pregnant normotensive women ( n =235) and patients having severe pre-eclampsia ( n =220). DNA was isolated and fluorescent PCR and DNA fragment analysis was performed for the detection of (TTTC) repeats. The concentration of the BNP was measured by fluorescence immunoassay -Triage BNP test- method. Results The overall distribution of alleles and genotypes was significantly different between the control and pre-eclamptic groups. We detected 12 different (TTTC) repeats on the NPPB gene in the studied population. The pre-eclamptics were a homogeneous population, with only 10 types of alleles and 20 types of genotypes, contrary to control group with 12 types of alleles and 32 types of genotypes. The 11 homozygote patients have a higher frequency among the severe-pre-eclamptics. I found higher levels of BNP in those who had the genotype 11 homozygotes in both groups, significantly higher in pre-eclamptics. The concentration of the BNP is higher pre-eclamptic pregnancies, it shows association with the (TTTC) genotypes. The concentration of BNP was higher in early onset pre-eclamptic patients than in late onset pre-eclamptics. Intrauterine growth restriction shows no connection with BNP levels in severe pre-eclampsia. There was a significant inverse correlation between plasma BNP levels of preeclamptic patients and sodium and total protein concentrations and a significant positive correlation was observed between plasma levels of BNP and 24h proteinuria. The cut-off value Conclusions We introduced a F-PCR and DNA fragment analysis method for the fast and reliable detection of the STR in the 5 ′ -flanking region of the natriuretic peptide precursor B gene. Further investigations may help to understand the details of molecular biology, biochemistry and clinical relevance of natriuretic peptides.

GW25-e2317 Association study between NPPB gene polymorphisms and essential hypertension in Hainan Li-risk population

GW25-e2317 Association study between NPPB gene polymorphisms and essential hypertension in Hainan Li-risk population

Abstract 162: Single Nucleotide Polymorphism of the B-Type Natriuretic Peptide Helps Predict the Presence of Significant Coronary Artery Disease

Early detection and diagnosis of coronary artery disease (CAD) is crucial in reducing the morbidity and mortality. The clinical standard for detecting CAD is by angiography which is associated with rare but important clinical risks. Recent studies have shown that up to 40% of patients referred for angiography do not have significant disease. This discrepancy between referral and diagnosis may be improved by the utilization of genetic screening. A single nucleotide polymorphisms (SNP) of the B-type natriuretic peptide gene (NPPB), rs198389, was previously found to be associated with several cardiovascular diseases. Our objective was to determine whether detection of genetic variation could contribute to better selection of patients referred for angiography. We hypothesized that an SNP analysis of the NPPB gene may help differentiate patients with significant disease from those with non-significant CAD. Ninety-three patients referred for coronary angiography at the Kingston General Hospital Cardiac Catheterization Lab were consented for genetic screening. Blood samples were collected during angiography procedure. Genomic DNA was isolated from leukocytes, and screened for SNPs using a real-time PCR-based TaqMan SNP Assay. We found that more males were referred for coronary angiography than females: 69% versus 31%. Two out of ten males (20%) were found to have no or minimal CAD. In contrast, 48% of females were found to have non-significant CAD. Older age (≥ 69 years) was deemed to be a significant predictor of CAD in the total recruited population (odds ratio of 3.4), but no age difference was found between healthy and diseased females. Additionally, a mutation of the B-type natriuretic peptide gene (NPPB) was found to be a significant predictor of CAD in the younger population (&lt; 69 years), with an odds ratio of 5.9. We found a significant difference between patterns of CAD development in males and females, suggesting that different diagnostic criteria should be used depending upon gender. Moreover, younger individuals with two copies of the major allele for the NPPB SNP were more likely to develop CAD, making this SNP a potential factor in the prediction of CAD in younger population.

Abstract 19016: ALK2 and ALK3 BMP Type I Receptors Are Required for the Development of Cardiac Hypertrophy

Bone Morphogenetic Proteins (BMP) signaling is required for the development of cardiac hypertrophy. BMP signaling mediates its action via BMP type I and type II receptors. We sought to determine which type I receptors are required for cardiac hypertrophy. We studied BMP signaling in neonatal rat cardiomyocytes (NRCs) stimulated with phenylephrine (PE) or isoproterenol (ISO). In comparison with untreated NRCs, PE (10 μM) and ISO (10 μM) induced hypertrophy, as reflected by increases in cell size (457±50 and 401±35 vs 254±23 μm2, p&lt;0.01) and expression of the gene encoding B-type natriuretic peptide (NPPB; 4.0±1.0 and 3.2±0.8-fold, p&lt;0.01). PE and ISO induced expression of genes specifying BMP2 (6.7±0.5 and 5.6±0.6-fold), BMP7 (4.1±0.4 and 3.6±0.9-fold), and the BMP target gene, Id-1, (3.7±0.1 and 4.2±1.0-fold, p&lt;0.001). PE and ISO also induced phosphorylation of BMP-responsive Smads 1 and 5 and increased activity of a BMP response element-luciferase reporter (14.5±0.8 and 11.9±1.1 vs 5.4±0.4, p&lt;0.01). Incubation of NRCs with the BMP inhibitors, LDN193189 (100 nM) or noggin (100 ng/ml), markedly suppressed the ability of PE and ISO to increase cell size, NPPB gene expression, and Smad1/5 phosphorylation. qRT-PCR analysis revealed that ALK2 and ALK3 were highly expressed in NRC, whereas ALK1 and ALK6 were expressed at very low levels. siRNA-mediated silencing of ALK2 or ALK3, but not ALK1 or ALK6, blocked PE-induced hypertrophy in NRCs. Using cardiomyocytes from ALK1flox/flox and ALK2flox/flox neonatal mice infected with Ad-Cre (to delete the floxed allele) or Ad-RFP (as a control) revealed that PE-induced hypertrophy requires Alk2 but not Alk1. Based on these observations, we conclude that BMP signaling is upregulated in NRCs in response to hypertrophic stimuli. ALK2 and ALK3 appear to be the BMP type I receptors which are required for the development of hypertrophy in neonatal cardiac myocytes.

PP005. Natriuretic peptide precursor B gene (TTTC)N microsatellite polymorphism and elevated BNP levels in early onset pre-eclampsia

There is a variable tandem repeat (TTTC) polymorphism in the 5(')-flanking region of the natriuretic peptide precursor B gene (NPPB), which shows association with severe pre-eclampsia. The 11-repeat carrier frequency is significantly higher in severe pre-eclamptic patients. The 11/11 genotype carriers in the pre-eclamptics has significantly higher BNP levels. Our aim was to identify this polymorphism in samples of early onset pre-eclamptic (EOP) patients, late onset pre-eclamptic patients and healthy controls. We also compared the natriuretic peptide B (BNP) concentrations. Blood samples were collected from healthy pregnant normotensive women (n=235) and women with pre-eclampsia (n=220). DNA was isolated and fluorescent PCR and DNA fragment analysis was performed for the detection of (TTTC) repeats. The plasma BNP concentration was measured by fluorescence immunoassay method using Triage BNP (Alere, San Diego). We detected 12 different repeats on the NPPB gene. The overall distribution of alleles and genotypes was significantly different between the control and pre-eclamptic groups. The 11/11 genotype carriers showed a significantly higher frequency in early onset pre-eclamptic patients than in the healthy pregnant controls (p=0.026). Calculated odds ratio (OR) was 1.694 (95% CI: 1.06-2.70). In contrast the 11/11 genotype carrier frequency was not significantly higher in the late onset pre-eclamptic group than in the control group (p=0.5308), adjusted OR 1.22 (95% CI: 0.7138-2.086). In the early onset pre-eclamptics the 11/11 genotype showed a significantly higher frequency than in the late onset group (p=0.0039) OR: 6.00. The concentration of the BNP was 9.75pg/ml in the healthy controls and 32.40pg/ml in the pre-eclamptic group (p<0.0001). The 11/11 genotype carriers had significantly higher BNP levels in the pre-eclamptic group. The early onset pre-eclamptic patients had a significantly higher BNP concentration (40.55pg/ml) than in the late onset pre-eclamptic patients (24.1pg/ml) (p=0.013). The NPPB gene (TTTC) microsatellite polymorphism in the 5(')-flanking region showed a significant difference in the distribution of alleles and genotypes between early onset and late onset pre-eclamptic patients in an ethnically homogeneous population. The concentration of the BNP was higher in early onset pre-eclamptic women, and it showed association with the genotypes.

In vivo natriuretic peptide reporter assay identifies chemical modifiers of hypertrophic cardiomyopathy signalling

Despite increased understanding of the fundamental biology regulating cardiomyocyte hypertrophy and heart failure, it has been challenging to find novel chemical or genetic modifiers of these pathways. Traditional cell-based methods do not model the complexity of an intact cardiovascular system and mammalian models are not readily adaptable to chemical or genetic screens. Our objective was to create an in vivo model suitable for chemical and genetic screens for hypertrophy and heart failure modifiers. Using the developing zebrafish, we established that the cardiac natriuretic peptide genes (nppa and nppb), known markers of cardiomyocyte hypertrophy and heart failure, were induced in the embryonic heart by pathological cardiac stimuli. This pathological induction was distinct from the developmental regulation of these genes. We created a luciferase-based transgenic reporter line that accurately modelled the pathological induction patterns of the zebrafish nppb gene. Utilizing this reporter line, we were able to show remarkable conservation of pharmacological responses between the larval zebrafish heart and adult mammalian models. By performing a focused screen of chemical agents, we were able to show a distinct response of a genetic model of hypertrophic cardiomyopathy to the histone deacetylase inhibitor, Trichostatin A, and the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126. We believe this in vivo reporter line will offer a unique approach to the identification of novel chemical or genetic regulators of myocardial hypertrophy and heart failure.

Natriuretic peptide precursor B gene (TTTC)n microsatellite polymorphism in pre-eclampsia

BackgroundThere is a variable tandem repeat polymorphism in the 5′-flanking region of the natriuretic peptide precursor B gene (NPPB). A previous study showed association of the (TTTC) small tandem repeat (STR) variants of this gene and essential hypertension. Our aim was to identify this polymorphism in samples of pre-eclamptic patients and healthy controls. We also compared the natriuretic peptide B (BNP) concentrations. MethodsBlood samples were collected from healthy pregnant normotensive women (n=235) and women with pre-eclampsia (n=220). DNA was isolated and fluorescent PCR and DNA fragment analysis was performed for the detection of (TTTC) repeats. The plasma BNP concentration was measured by fluorescence immunoassay method. ResultsWe detected 12 different (TTTC) repeats on the NPPB gene in the studied population. The overall distribution of alleles and genotypes was significantly different between the control and pre-eclamptic groups. The number of 10-repeat genotype carriers showed significantly lower frequency in pre-eclamptics than in the healthy pregnant controls (p=0.032). After adjustment for confounding factors pre-pregnancy BMI, maternal age, primiparity and smoking, the calculated odds ratio (OR) was 0.19 (95% CI: 0.04–0.87). Similarly, the 12-repeat genotype carriers showed significantly lower frequency in pre-eclamptics than in the healthy pregnants (p=0.037; adjusted OR: 0.53 (95% CI: 0.29–0.96)). In contrast the 11-repeat genotype carrier frequency was significantly higher in the pre-eclamptic than in the healthy pregnant group (p<0.001; adjusted OR 2.91 (95% CI: 1.75–4.84)).The concentration of the BNP was 9.75pg/ml in the healthy controls and 32.40pg/ml in the pre-eclamptic group (p<0.0001). The 11/11 genotype carriers had significantly higher BNP levels in both groups. ConclusionsThe NPPB gene (TTTC) microsatellite polymorphism in the 5′-flanking region showed significant difference in the distribution of alleles and genotypes between healthy pregnant controls and pre-eclamptic patients in an ethnically homogeneous population. The concentration of the BNP was higher in pre-eclamptic women, and it showed association with the (TTTC) genotypes. We introduced an F-PCR and DNA fragment analysis method for the fast and reliable detection of this STR.