Natriuretic Peptide Precursor A Research Articles

Electrophysiologic and molecular mechanisms of a frameshift NPPA mutation linked with familial atrial fibrillation

A frameshift (fs) mutation in the natriuretic peptide precursor A (NPPA) gene, encoding a mutant atrial natriuretic peptide (Mut-ANP), has been linked with familial atrial fibrillation (AF) but the underlying mechanisms by which the mutation causes AF remain unclear. We engineered 2 transgenic (TG) mouse lines expressing the wild-type (WT)-NPPA gene (H-WT-NPPA) and the human fs-Mut-NPPA gene (H-fsMut-NPPA) to test the hypothesis that mice overexpressing the human NPPA mutation are more susceptible to AF and elucidate the underlying electrophysiologic and molecular mechanisms. Transthoracic echocardiography and surface electrocardiography (ECG) were performed in H-fsMut-NPPA, H-WT-NPPA, and Non-TG mice. Invasive electrophysiology, immunohistochemistry, Western blotting and patch clamping of membrane potentials were performed. To examine the role of the Mut-ANP in ion channel remodeling, we measured plasma cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity in the 3 groups of mice. In H-fsMut-NPPA mice mean arterial pressure (MAP) was reduced when compared to H-WT-NPPA and Non-TG mice. Furthermore, injection of synthetic fs-Mut-ANP lowered the MAP in H-WT-NPPA and Non-TG mice while synthetic WT-ANP had no effect on MAP in the 3 groups of mice. ECG characterization revealed significantly prolonged QRS duration in H-fsMut-NPPA mice when compared to the other two groups. Trans-Esophageal (TE) atrial pacing of H-fsMut-NPPA mice showed increased AF burden and AF episodes when compared with H-WT-NPPA or Non-TG mice. The cardiac Na+ (NaV1.5) and Ca2+ (CaV1.2/CaV1.3) channel expression and currents (INa, ICaL) and action potential durations (APD90/APD50/APD20) were significantly reduced in H-fsMut-NPPA mice while the rectifier K+ channel current (IKs) was markedly increased when compared to the other 2 groups of mice. In addition, plasma cGMP levels were only increased in H-fsMut-NPPA mice with a corresponding reduction in plasma cAMP levels and PKA activity. In summary, we showed that mice overexpressing an AF-linked NPPA mutation are more prone to develop AF and this risk is mediated in part by remodeling of the cardiac Na+, Ca2+ and K+ channels creating an electrophysiologic substrate for reentrant AF.

Prediction of Molecular Mechanisms for LianXia NingXin Formula: A Network Pharmacology Study.

Objectives: Network pharmacological methods were used to investigate the underlying molecular mechanisms of LianXia NingXin (LXNX) formula, a Chinese prescription, to treat coronary heart disease (CHD) and disease phenotypes (CHD related diseases and symptoms).Methods: The different seed gene lists associated with the herbs of LXNX formula, the CHD co-morbid diseases and symptoms which were relieved by the LXNX formula (co-morbid diseases and symptoms) were curated manually from biomedical databases and published biomedical literatures. Module enrichment analysis was used to identify CHD-related disease modules in the protein–protein interaction (PPI) network which were also associated to the targets of LXNX formula (LXNX formula’s CHD modules). The molecular characteristics of LXNX formula’s CHD modules were investigated via functional enrichment analysis in terms of gene ontology and pathways. We performed shortest path analysis to explore the interactions between the drug targets of LXNX formula and CHD related disease phenotypes (e.g., co-morbid diseases and symptoms).Results: We identified two significant CHD related disease modules (i.e., M146 and M203), which were targeted by the herbs of LXNX formula. Pathway and GO term functional analysis results indicated that G-protein coupled receptor signaling pathways (GPCR) of M146 and cellular protein metabolic process of M203 are important functional pathways for the respective module. This is further confirmed by the shortest path analysis between the drug targets of LXNX formula and the aforementioned disease modules. In addition, corticotropin releasing hormone (CRH) and natriuretic peptide precursor A (NPPA) are the only two LXNX formula target proteins with the low shortest path length (on average shorter than 3) to their respective CHD module and co-morbid disease and symptom gene groups.Conclusion: G-protein coupled receptor signaling pathway and cellular protein metabolic process are the key LXNX formula’s pathways to treat CHD disease phenotypes, in which CRH and NPPA are the two key drug targets of LXNX formula. Further evidences from Chinese herb pharmacological databases indicate that Pinellia ternata (Banxia) has relatively strong adjustive functions on the two key targets.

Left ventricular pressure-volume measurements and myocardial gene expression profile in type 2 diabetic Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat, a non-obese model of type 2 diabetes mellitus (T2DM), was generated by the selective inbreeding of glucose-intolerant Wistar rats. This is a convenient model for studying diabetes-induced cardiomyopathy independently from the effects of the metabolic syndrome. We investigated the myocardial functional and structural changes and underlying molecular pathomechanisms of short-term and mild T2DM. The presence of DM was confirmed by an impaired oral glucose tolerance in the GK rats compared with the age-matched nondiabetic Wistar rats. Data from cardiac catheterization showed that in GK rats, although the systolic indexes were not altered, the diastolic stiffness was increased compared with nondiabetics (end-diastolic-pressure-volume-relationship: 0.12 ± 0.04 vs. 0.05 ± 0.01 mmHg/μl, P < 0.05). Additionally, DM was associated with left-ventricular hypertrophy and histological evidence of increased myocardial fibrosis. The plasma pro-B-type natriuretic peptide, the cardiac troponin-T, glucose, and the urinary glucose concentrations were significantly higher in GK rats. Among the 125 genes surveyed using PCR arrays, DM significantly altered the expression of five genes [upregulation of natriuretic peptide precursor-A and connective tissue growth factor, downregulation of c-reactive protein, interleukin-1β, and tumor necrosis factor (TNF)-α mRNA-level]. Of the altered genes, which were evaluated by Western blot, only TNF-α protein expression was significantly decreased. The ECG recordings revealed no significant differences. In conclusion, while systolic dysfunction, myocardial inflammation, and abnormal electrical conduction remain absent, short-term and mild T2DM induce the alteration of cardiac TNF-α at both the mRNA and protein levels. Further assessments are required to reveal if TNF-α plays a role in the early stage of diabetic cardiomyopathy development.

Association of genetic variation in the natriuretic peptide system and left ventricular mass and blood pressure in newborns.

The natriuretic peptides play a key role in the modulation of left ventricular mass (LVM) and blood pressure (BP). We hypothesised that NPPA (natriuretic peptide precursor A gene), NPPB (natriuretic peptide precursor B gene), and NPPC (natriuretic peptide precursor C gene) are candidate genes possibly involved in the development or modulation of LVM at early life. To assess the relationship between NPPA, NPPB, and NPPC gene polymorphisms with LVM and BP in newborns. A total of 206 healthy newborns were studied by two-dimensional M-mode echocardiography. The polymorphisms NPPA rs5065, NPPB rs198389, and NPPC rs5268 were characterised. Newborns carrying the C allele of the NPPB polymorphism had significantly lower LVM/body surface area (BSA) and LVM/body weight (BW) values when compared with newborns' homozygotes for the T allele (41.76 g/m2 vs. 48.31 g/m2, p adjusted = 0.044 and 2.78 g/kg vs. 3.26 g/kg, p adjusted = 0.031, respectively). An association was observed between NPPA genotype and systolic BP, diastolic BP, and mean arterial pressure ≥ 90th percentile (p = 0.029, p = 0.0048, p = 0.004, respectively). Also an association was observed for systolic BP ≥ 90th percentile for NPPB (p = 0.016). The present study shows that the NPPB gene polymorphism is associated with modulation of LVM in newborns. The NPPA and NPPB gene polymorphisms are associated with BP.

The expression of NPPA splice variants during mouse cardiac development.

Natriuretic peptide precursor-A (NPPA) is an early and specific marker for functional myocardium of the embryonic heart. NPPA gene encodes for a precursor of atrial natriuretic peptide (ANP). So far, three alternatively spliced variants have been reported for NPPA in human. In mouse, no alternatively spliced transcript of NPPA has been reported. In the current study, we investigated the expression of NPPA gene during cardiac differentiation of mouse adipose-tissue-derived stem cells (ADSCs) and embryonic stem (ES) cells. As revealed by reverse-transcription polymerase chain reaction analysis, 2-week-differentiated cells expressed some cardiac-specific makers, including ANP. Three additional intron-retained splice variants of NPPA were also detected during cardiac differentiation of the ADSCs and ES cells. In addition, we detected three intron-retained splice variants of NPPA in 8.5-day mouse embryonic heart. In the mature cardiomyocytes of 1-week-old mice, only the correctly spliced isoform of NPPA gene was expressed. Freshly isolated stromal vascular fraction also expressed one intron-retained isoform of NPPA gene. In conclusion, our findings have provided evidence for the expression of intron-retained splices of NPPA mRNA during the early stages of mouse cardiogenesis as well as in the mouse adipose tissue.

Atrial natriuretic peptide gene - a potential biomarker for long QT syndrome.

This study highlights the possible implication of NPPA (natriuretic peptide precursor A) gene in the etiology of Long QT syndrome (LQTS) by population-based as well as familial study. Three SNPs of NPPA - C-664G, C1363A and T1766C were examined by molecular analyses in LQTS, controls and first degree relatives (FDRs). This study revealed a possible association of 1364 C>A SNP 'C' allele with LQTS (p = 0.0013). All three SNPs were in tight linkage disequilibrium. The familial study highlights the association of NPPA SNP with cLQTS and implicating it as a potential biomarker in South Indian population.

Electroanatomic mapping and late gadolinium enhancement MRI in a genetic model of arrhythmogenic atrial cardiomyopathy.

Although atrial arrhythmias may have genetic causes, very few data are available on evaluation of the arrhythmic substrate in genetic atrial diseases in humans. In this study, we evaluate the nature and evolution of the atrial arrhythmic substrate in a genetic atrial cardiomyopathy. Repeated electroanatomic mapping and tomographic evaluations were used to investigate the evolving arrhythmic substrate in 5 patients with isolated arrhythmogenic atrial cardiomyopathy, caused by Natriuretic Peptide Precursor A (NPPA) gene mutation. Atrial fibrosis was assessed using late gadolinium enhancement magnetic resonance imaging (LGE-MRI). The substrate of atrial tachycardia (AT) and atrial fibrillation (AF) was biatrial dilatation with patchy areas of low voltage and atrial wall scarring (in the right atrium: 68.5% ± 6.0% and 22.2% ± 10.2%, respectively). The evolution of the arrhythmic patterns to sinus node disease with atrial standstill (AS) was associated with giant atria with extensive low voltage and atrial scarring areas (in the right atrium: 99.5% ± 0.7% and 57.5% ± 33.2%, respectively). LGE-MRI-proven biatrial fibrosis (Utah stage IV) was associated with AS. Atrial conduction was slow and heterogeneous, with lines of conduction blocks. The progressive extension and spatial distribution of the scarring/fibrosis were strictly associated with the different types of arrhythmias. The evolution of the amount and distribution of atrial scarring/fibrosis constitutes the structural substrate for the different types of atrial arrhythmias in a pure genetic model of arrhythmogenic atrial cardiomyopathy.

Atrial Natriuretic Peptide and Type 2 Diabetes Development – Biomarker and Genotype Association Study

BackgroundWe have recently shown that low plasma levels of mid-regional atrial natriuretic peptide (MR-ANP) predict development of diabetes and glucose progression over time, independently of known risk factors for diabetes development. However, since MR-ANP levels might be influenced by unknown factors causing diabetes, we cannot rule out that such relationship might be confounded. Previous studies have shown an association of a single nucleotide polymorphism rs5068 on the natriuretic peptide precursor A (NPPA) locus gene with higher levels of circulating ANP. Since gene variants are inherited randomly and not subject to confounding, we aimed to investigate whether the variant rs5068 within the NPPA locus is associated with incident type 2 diabetes.MethodsWe genotyped the variant rs5068 within the NPPA locus in 27,307 individuals without known diabetes from the Malmö Diet Cancer Study. Incident diabetes was retrieved through national and regional registers (median follow-up time of 14 years, 2,823 incident diabetes cases).ResultsIn Cox regression analysis adjusted for age, sex and BMI, we found that the carriers of at least one copy of the G allele of rs5068 had lower likelihood of incident diabetes within 14 years (HR = 0.88, 95% CI 0.78–0.99, p = 0.037).ConclusionOur results indicate a role of the ANP system in the etiology of type 2 diabetes and might help provide insight in the metabolic actions of natriuretic peptides and the pathophysiology of type 2 diabetes.

Atrial natriuretic peptide and type 2 diabetes development, evidence of causal association from the prospective Malmo diet and cancer study

Purpose: We have recently shown that low plasma levels of mid-regional atrial natriuretic peptide (MR-ANP) predict development of future diabetes and glucose progression over time, independently of known risk factors for diabetes development. However since MR-ANP levels might be influenced by other unknown factors causing diabetes, we cannot rule out that such relationship might be confounded. Previous studies have shown an association of a single nucleotide polymorphism rs5068 on the natriuretic peptide precursor A (NPPA) locus gene with higher levels of circulating MR-ANP. Since gene variants are inherited randomly and not subject to confounding, we aimed to investigate whether the variant rs5068 within the NPPA locus is associated with incident type 2 diabetes in order to prove a causal link between the ANP metabolism and type 2 diabetes. Methods: We genotyped the variant rs5068 within the NPPA locus in 27 308 individuals without known diabetes (mean age 58 years; 60% women). Incident diabetes was retrieved through five national registers (median follow-up time of 14 years, 2823 incident diabetes cases). Results: In Cox regression analysis adjusted for age, sex and BMI, we found that the minor allele of rs5068 was inversely associated with incident diabetes (HR=0.88, 95% CI 0.78-0.99). View this table: Association of rs5068 and incid Conclusion: Our results offer an indication for a potential causal role of the ANP system in the etiology of type 2 diabetes.

Abstract 19074: NPPA Overexpression in Mice Increases Susceptibility to Atrial Fibrillation

Introduction: Mutations in natriuretic peptide precursor A (NPPA) gene, which encodes atrial natriuretic peptide (ANP), have been linked with familial atrial fibrillation (AF). Although the precise mechanism by which NPPA mutations cause AF remains unclear, a shortening of the atrial action potential was demonstrated in an isolated rat heart model. Here, we overexpressed NPPA in a mouse model testing the hypothesis that these mice would be more susceptible to AF. Methods: A triple FLAG-tag (FLAG) was fused in-frame with the 3’ end of the either the wild-type (WT) human NPPA (WT-NPPA-FLAG) cDNA or the mutant NPPA peptide containing the COOH-terminal 12 amino acid extension (mut-NPPA-FLAG) isolated from individuals with familial AF. The FLAG-tag does not diminish NPPA biological activity. The expression of the mutated protein was confirmed by measuring mRNA by polymerase-chain reaction (PCR). We used transesophageal atrial pacing (15 second bursts from 50 to 15 ms) to assess inducibility and duration of AF. Results: Serial weekly transesophageal pacing starting at 16 weeks of age was performed in WT-NPPA-FLAG and mut-NPPA-FLAG mice. Mut-NPPA-FLAG mice had inducible AF at 16 weeks of age in 61±0.1% compared to WT-NPPA-FLAG (n=6 mice, P&lt;0.05). In addition, induced AF was longer lasting in the mut-NPPA-FLAG as compared to WT (AF burden: 19.2±2.9 [mut] sec vs. 12±4.1 s [WT], P&lt;0.05). Conclusions: We show that humanized NPPA overexpression mice are more susceptible to AF with longer AF episodes compared with WT using esophageal pacing.

17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes

17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

Gene Network Profiling Before and After Transplantation in Alcoholic Cirrhosis Liver Transplant Recipients

The main objective of this study was to define a gene network profile network in liver transplant recipients with alcoholic cirrhosis before and after liver transplantation. Genes were selected from data obtained in a previous study of liver transplant recipients with alcoholic cirrhosis. Selected up-regulated genes were further validated by quantitative real-time polymerase chain reaction in different groups of liver transplant recipients with alcoholic cirrhosis (n=5). Selected genes up-regulated before transplantation were: TNFRSF9 (tumor necrosis factor [TNF] receptor superfamily, member 9); IL2RB (interleukin-2 receptor beta); BCL2L2 (BCL2-like 2); NOX5 (NADPH) oxidase, EF-hand calcium binding domain 5); PEX5 (peroxisomal biogenesis factor 5); PPARG (peroxisome proliferator-activated receptor gamma); NIBP (IKK2 binding protein); NKIRAS2 (NFKappaBeta inhibitor interacting Ras-like 2); IL4 (interleukin-4); IL-4R (interleukin 4 receptor); ADH1A (alcohol dehydrogenase 1A, class 1); ALDH1L1 (aldehyde dehydrogenase 1 family, member L1); MPO (myeloperoxidase); NPPA (natriuretic peptide precursor A); BCL2A1 (BCL2-related protein A1); GADD45A (growth arrest and DNA-damage-inducible alpha); TEGT (Bax inhibitor 1); PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide); IFNGR2 (interferon gamma receptor 2); JAK2 (Janus Kinase 2); FAS (Fas, TNF receptor superfamily, member 6); TANK (TRAF family member-associated NFKB activator); TTRAP (TRAF and TNF receptor-associated protein); and ANXA5 (annexin A5).

Gradients of natriuretic peptide precursor A (NPPA) in oviduct and of natriuretic peptide receptor 1 (NPR1) in spermatozoon are involved in mouse sperm chemotaxis and fertilization

Selective spermatozoa movement from storage of the oviduct to fertilization site is suggested to be a result of chemotaxis. In the present study, Natriuretic peptide precursor A (NPPA) induced sperm chemotaxis in capillaries and enhanced intracellular Ca(2+) level, both of which could be blocked by the Natriuretic Peptide Receptor 1 (NPR1) inhibitor anantin and the cGMP-dependent protein kinase (PKG) inhibitors, KT5823 and Rp-8-Br-PET-cGMPS. NPPA also increased spermatozoa kinetic parameters of VAP, VSL, LIN, STR, and BCF. Only 2.0% of positive staining for NPR1 was detected in fresh spermatozoa. The positive rate was increased in capacitated spermatozoa (20.5%), and further increased in spermatozoa of NPPA treatment (70.2%). Nppa mRNA level in the ampullae was significantly higher compared with that in isthmus and uterotubal junction, and NPPA protein had an ascending gradient (AG) from the uterotubal junction to ampullae in gonadotropin-treated mice. NPPA induced sperm chemotaxis in diestrus oviducts without a NPPA gradient, and sperm chemotaxis occurred in the oviducts of gonadotropin-treated mice. These effects were inhibited by anantin. Meanwhile, sperm chemotaxis also occurred in unilateral ovariectomized oviducts of gonadotropin-treated mice, in which the possible effect of follicular fluid and oocyte-cumulus mass were eliminated when ovulation occurs. Furthermore, anantin significantly decreased the rate of fertilization in a dose-dependent manner (0.1 µM, 57.1%; 1 µM, 33.8%) compared with control (78.5%). These results suggest that a NPPA gradient originating in the oviduct induces sperm chemotaxis by binding to its receptor NPR1 and then activating PKG pathway, and plays a physiological role in fertilization.

Transactivation in Drosophila of human enhancers by human transcription factors involved in congenital heart diseases

Background: The human transcription factors (TFs) GATA4, NKX2.5 and TBX5 form part of the core network necessary to build a human heart and are involved in Congenital Heart Diseases (CHDs). The human natriuretic peptide precursor A (NPPA) and α-myosin heavy chain 6 (MYH6) genes are downstream effectors involved in cardiogenesis that have been demonstrated to be in vitro targets of such TFs. Results: To study the interactions between these human TFs and their target enhancers in vivo, we overexpressed them in the whole Drosophila cardiac tube using the UAS/GAL4 system. We observed that all three TFs up-regulate their natural target enhancers in Drosophila and cause developmental defects when overexpressed in eyes and wings. Conclusions: A strong potential of the present model might be the development of combinatorial and mutational assays to study the interactions between human TFs and their natural target promoters, which are not easily undertaken in tissue culture cells because of the variability in transfection efficiency, especially when multiple constructs are used. Thus, this novel system could be used to determine in vivo the genetic nature of the human mutant forms of these TFs, setting up a powerful tool to unravel the molecular genetic mechanisms that lead to CHDs. Developmental Dynamics 241:190–199, 2012. © 2011 Wiley Periodicals, Inc.Key findingsThe mammalian transcription factors GATA4, Nkx2.5 and TBX5 are able to transactivate the Nppa and Myh6 human enhancers in Drosophila embryos.The endogenous Drosophila homologs of GATA4, Nkx2.5 and TBX5 are not transactivated by GATA4, Nkx2.5 or TBX5, ruling out a possible endogenous regulatory circuit.Overexpression in the mesoderm of GATA4, Nkx2.5 or TBX5 is lethal and overexpression in eyes and wings causes reduced organs.

The role of natriuretic peptide precursor A gene polymorphism in the development of coronary heart disease in Chinese Han population

Natriuretic peptide precursor A (NPPA) is synthesized, stored, and released by atrial myocytes. Previous studies have shown that NPPA plays a significant role in the regulation of coronary circulation and in atherosclerosis. Rs5065 NPPA gene polymorphism leads to the translation of NPPA with two additional arginines and has been suggested to be associated with salt-sensitive hypertension. The purpose of the present study was to investigate the relationship between the rs5065 NPPA gene polymorphism and the risk of coronary heart disease (CHD) in Chinese Han population. We genotyped the single nucleotide polymorphism (SNP) rs5065 NPPA in the human NPPA gene in 1861 sex- and age-matched subjects, comprising of 904 CHD cases and 957 controls of Chinese Han population. Genotyping of SNP was performed with Taqman SNP allelic discrimination assays by means of an ABI 7900HT. Our study showed that the frequencies of rs5065 NPPA C allele in the case and the control groups were 0.012 and 0.005, respectively. There was significant difference in C allele frequency distribution between the two groups (OR = 2.607, 95% CI: 1.197–5.678, P = 0.012). In the case group, there was significant difference between smokers and nonsmokers with subjects carrying C allele (P = 0.037), and no significant difference in gender, age, fasting total cholesterol (TC), triglycerides (TG), fasting plasma glucose (FPG), body mass index (BMI), and blood pressure (BP) between the cases and the controls (P>0.05). Our results suggest that the C allele of rs5065 NPPA gene polymorphism may be associated with the risk of CHD.

Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation

Mutations in multiple genes have been implicated in familial atrial fibrillation (AF), but the underlying mechanisms, and thus implications for therapy, remain ill-defined. Among 231 participants in the Vanderbilt AF Registry, we found a mutation in KCNQ1 (encoding the α-subunit of slow delayed rectifier potassium current [ I Ks]) and separately a mutation in natriuretic peptide precursor A ( NPPA) gene (encoding atrial natriuretic peptide, ANP), both segregating with early onset lone AF in different kindreds. The functional effects of these mutations yielded strikingly similar I Ks “gain-of-function.” In Chinese Hamster Ovary (CHO) cells, coexpression of mutant KCNQ1 with its ancillary subunit KCNE1 generated ∼ 3-fold larger currents that activated much faster than wild-type (WT)- I Ks. Application of the WT NPPA peptide fragment produced similar changes in WT- I Ks, and these were exaggerated with the mutant NPPA S64R peptide fragment. Anantin, a competitive ANP receptor antagonist, completely inhibited the changes in I Ks gating observed with NPPA S64R. Computational simulations identified accelerated transitions into open states as the mechanism for variant I Ks gating. Incorporating these I Ks changes into computed human atrial action potentials (AP) resulted in 37% shortening (120 vs. 192 ms at 300 ms cycle length), reflecting loss of the phase II dome which is dependent on L-type calcium channel current. We found striking functional similarities due to mutations in KCNQ1 and NPPA genes which led to I Ks “gain-of-function”, atrial AP shortening, and consequently altered calcium current as a common mechanism between diverse familial AF syndromes.

Association of 77 polymorphisms in 52 candidate genes with blood pressure progression and incident hypertension: the Women's Genome Health Study

Genetic risk factors for essential hypertension are largely unknown. The aim of the present study was to assess the association of 77 previously characterized gene variants in 52 candidate genes from various biological pathways with blood pressure (BP) progression and incident hypertension. We analyzed data from 18 738 white women who participated in a prospective cohort study and were free of hypertension at baseline. BP progression at 48 months and incident hypertension during the entire follow-up according to the different genotypes were assessed by logistic regression and Cox proportional-hazards models, respectively. At 48 months of follow-up, 7889 of 16 635 women (47.4%) had BP progression. Only three of 70 polymorphisms with a minor allele frequency of at least 2% had a significant association with BP progression. The odds ratio [95% confidence interval (CI)] for 5,10-methylenetetrahydrofolate reductase (MTHFR) rs1801133 (minor allele T), natriuretic peptide precursor A (NPPA) rs5063 (minor allele A) and NPPA rs5065 (minor allele C) were 1.05 (1.00-1.10), 0.84 (0.76-0.94) and 0.93 (0.88-1.00), respectively. After adjustment for multiple testing using the false discovery rate, only the NPPA rs5063 association remained significant. During a median follow-up of 9.8 years, 5540 of 18 738 women developed incident hypertension. Only five of 70 polymorphisms were significantly associated with incident hypertension. The hazard ratio (95% CI) for interleukin 6 (IL-6) rs1800795 (minor allele C), MTHFR rs1801133, NPPA rs5063, nitric oxide synthase 3 rs1799983 (minor allele T) and transforming growth factor, beta 1 rs1800469 (minor allele T) were 0.96 (0.92-1.00), 1.06 (1.02-1.10), 0.88 (0.80-0.96), 1.05 (1.01-1.09) and 1.05 (1.01-1.10), respectively. After adjustment for multiple testing, none of these associations remained significant. NPPA gene polymorphisms may have a role in BP progression and incident hypertension. Our data also provide moderate confirmatory evidence of association between MTHFR rs1801133 and the development of hypertension.

A review of the role of atrial natriuretic peptide gene polymorphisms in hypertension and its sequelae

The natriuretic peptide precursor A (NPPA) gene, found on chromosome 1p36, encodes the precursor from which atrial natriuretic polypeptide (ANP) is derived. Due to the action of ANP, it is thought that the NPPA gene is involved in the control of blood pressure. Animal studies have shown that genetically reduced ANP concentration leads to salt-sensitive hypertension, whereas genetically increased ANP concentration leads to hypotension. These studies have encouraged researchers to search the human NPPA gene for polymorphisms that contribute to hypertension and its sequelae such as stroke and cardiovascular disease. This report provides a comprehensive review of studies exploring NPPA polymorphisms in relation to hypertension and hypertension-related outcomes.

Abstract 1477: Identification and Functional Analysis of Protein Interaction Partners of the Estrogen Receptor Alpha in the Human Heart

Background and aims: Estrogens (E2) are important regulators of physiological and pathological processes in various target tissues, including the human heart. Most of the biological actions of E2 are mediated by nuclear steroid receptors, the estrogen receptor alpha (ERα) and beta (ERα), which function as hormone-inducible transcription factors. ERs act in concert with other regulatory elements to mediate estrogenic effects. So far, only few cofactors of ERs have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and functionally analyzed the interaction partners of ERα. Methods: The yeast two hybrid screen (YTH) was performed in the presence of E2 with ERα as bait in a human heart cDNA library. Co-immunoprecipitation analysis (Co-IP) with human atrial proteins was carried out to confirm the interaction in a mammalian system. Immunofluorescence (IF) methods were employed on human atrial tissue samples as well as in cotransfected AC16 cells to analyze the co-localisation of ERα and its binding partners. Promoter reporter assays were performed to show the regulation of the identified interaction partner by ERα. Results: Yeast two hybrid screening of a human heart library revealed that ERα interacts with the atrial natriuretic peptide precursor A (NPPA). Retransformation experiments showed that NPPA can interact with ERα full-length and the ERα-EF domain in an E2-dependent manner. The LxxLL-motif of NPPA was found to interact with the ERα-EF domain in yeast. The interaction of NPPA and ERα was also confirmed using proteins derived from human atrium by Co-IP. IF analysis showed that E2 treatment of AC16 cells led to an increased translocation of NPPA and ERα in the nucleus of AC16 cells and also their co-localization there. Furthermore, expression analysis with NPPA luciferase promoter constructs showed a significant increase in the promoter activity in the presence of E2/ERα. Conclusion: We showed the interaction of ERα and NPPA in an E2-dependent manner. Our findings suggest that the E2-activated ERα and NPPA complex binds to specific regions on the NPPA promoter leading to transcriptional regulation of the NPPA gene.

Evaluation of CXCL9 and CXCL10 as circulating biomarkers of human cardiac allograft rejection

BackgroundCardiac allograft rejection remains a significant clinical problem in the early phase after heart transplantation and requires frequent surveillance with endomyocardial biopsy. However, this is an invasive procedure, which is unpleasant for the patient and carries a certain risk. Therefore, a sensitive non-invasive biomarker of acute rejection would be desirable.MethodsEndomyocardial tissue samples and serum were obtained in connection with clinical biopsies from twenty consecutive heart transplant patients followed for six months. A rejection episode was observed in 14 patients (11 men and 3 women) and biopsies obtained before, during and after the episode were identified. Endomyocardial RNA, from three patients, matching these three points in time were analysed with DNA microarray. Genes showing up-regulation during rejection followed by normalization after the rejection episode were evaluated further with real-time RT-PCR. Finally, ELISA was performed to investigate whether change in gene-regulation during graft rejection was reflected in altered concentrations of the encoded protein in serum.ResultsThree potential cardiac allograft rejection biomarker genes, chemokine (C-X-C motif) ligand 9 (CXCL9), chemokine (C-X-C motif) ligand 10 (CXCL10) and Natriuretic peptide precursor A (NPPA), from the DNA microarray analysis were selected for further evaluation. CXCL9 was significantly upregulated during rejection (p < 0.05) and CXCL10 displayed a similar pattern without reaching statistical significance. Serum levels of CXCL9 and CXCL10 were measured by ELISA in samples from 10 patients before, during and after cardiac rejection. There were no changes in CXCL9 and CXCL10 serum concentrations during cardiac rejection. Both chemokines displayed large individual variations in the selected samples, but the serum levels between the two chemokines correlated (p < 0.001).ConclusionWe conclude, that despite a distinct up-regulation of CXCL9 mRNA in human hearts during cardiac allograft rejection, this was not reflected in the serum levels of the encoded protein. Thus, in contrast to previous suggestions, serum CXCL9 does not appear to be a promising serum biomarker for cardiac allograft rejection.