In vivo assessment of the electrophysiological integration and arrhythmogenic risk of myocardial cell transplantation strategies.

Cell replacement strategies are promising interventions aiming to improve myocardial performance. Yet, the electrophysiological impact of these approaches has not been elucidated. We assessed the electrophysiological consequences of grafting of two candidate cell types, that is, skeletal myoblasts and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). The fluorescently labeled (DiO) candidate cells were grafted into the rat's left ventricular myocardium. Two weeks later, optical mapping was performed using the Langendorff-perfused rat heart preparation. Images were obtained with appropriate filters to delineate the heart's anatomy, to identify the DiO-labeled cells, and to associate this information with the voltage-mapping data (using the voltage-sensitive dye PGH-I). Histological examination revealed the lack of gap junctions between grafted skeletal myotubes and host cardiomyocytes. In contrast, positive Cx43 immunostaining was observed between donor and host cardiomyocytes in the hESC-CMs-transplanted hearts. Optical mapping demonstrated either normal conduction (four of six) or minimal conduction slowing (two of six) at the hESC-CMs engraftment sites. In contrast, marked slowing of conduction or conduction block was seen (seven of eight) at the myoblast transplantation sites. Ventricular arrhythmias could not be induced in the hESC-CM hearts following programmed electrical stimulation but were inducible in 50% of the myoblast-engrafted hearts. In summary, a unique method for assessment of the electrophysiological impact of myocardial cell therapy is presented. Our results demonstrate the ability of hESC-CMs to functionally integrate with host tissue. In contrast, transplantation of cells that do not form gap junctions (skeletal myoblats) led to localized conduction disturbances and to the generation of a proarrhythmogenic substrate.

Effect of acute and chronic ethanol on atrial fibrillation vulnerability in rats.

BACKGROUND: Even though ethanol consumption has been associated with risk of atrial fibrillation (AF), little is known about how ethanol affects atrial electrophysiology. OBJECTIVE: The purpose of this study was to study the electrophysiological effect of ethanol on rat AF. METHODS: Atrial optical mapping was performed on male Long Evans rat hearts with escalating concentrations of ethanol (0, 1, 2, and 3 mM). In addition, patch-clamp recordings on isolated atrial myocytes were performed. In chronic ethanol study, rats were divided into control and chronic ethanol groups (20% ethanol in drinking water for 6 months). Atrial optical mapping, histology, immunohistochemistry, and reverse transcriptase polymerase chain reaction were performed in chronic rats. RESULTS: Acute ethanol perfusion increased AF vulnerability (0% at 0 mM, 0% at 1 mM, 57.1% at 2 mM, and 100% at 3 mM) in a dose-related response. Ethanol infusion decreased conduction velocities (CVs) in both atria and shortened effective refractory periods (ERP) only in the right atria with increased in dispersion of refractoriness. Action potential duration at 50% and 90% repolarization from right atrial myocytes were shortened, with corresponding increase of sustained potassium current. Chronic ethanol consumption increased AF inducibility (10% control vs 95.2% chronic ethanol). CVs in both atria were significantly decreased. ERP of the right atrium was shortened, and dispersion of ERP was increased. Expression (mRNA) of KCNQ1 and connexin40 were increased, but KCNA5 was decreased in the right atrium of rats exposed to chronic ethanol. CONCLUSION: Acute and chronic exposure to ethanol increases AF vulnerability by slowing CV, shortening right atrial ERP, and increasing dispersion of ERP.

Absence of natriuretic peptide clearance receptor attenuates TGF-ß1-induced selective atrial fibrosis and atrial fibrillation.

Aims: TGF-ß1 plays an important role in atrial fibrosis and atrial fibrillation (AF); previous studies have shown that the atria are more susceptible to TGF-ß1 mediated fibrosis than the ventricles. Natriuretic peptides (NPs) play an important role in cardiac remodelling and fibrosis, but the role of natriuretic peptide clearance (NPR-C) receptor is largely unknown. We investigated the role of NPR-C in modulating TGF-ß1 signalling in the atria. Methods and results: MHC-TGF-ß1 transgenic (TGF-ß1-Tx) mice, which develop isolated atrial fibrosis and AF, were cross-bred with NPR-C knock-out mice (NPR-C-KO). Transverse aortic constriction (TAC) was performed in wild type (Wt) and NPR-C knockout mice to study. Atrial fibrosis and AF inducibility in a pathophysiologic model. Electrophysiology, molecular, and histologic studies were performed in adult mice. siRNA was used to interrogate the interaction between TGF-ß1 and NP signalling pathways in isolated atrial and ventricular fibroblasts/myofibroblasts. NPR-C expression level was 17 ± 5.8-fold higher in the atria compared with the ventricle in Wt mice (P = 0.009). Cross-bred mice demonstrated markedly decreased pSmad2 and collagen expression, atrial fibrosis, and AF compared with TGF-ß1-Tx mice with intact NPR-C. There was a marked reduction in atrial fibrosis gene expression and AF inducibility in the NPR-C-KO-TAC mice compared with Wt-TAC. In isolated fibroblasts, knockdown of NPR-C resulted in a marked reduction of pSmad2 (56 ± 4% and 24 ± 14% reduction in atrial and ventricular fibroblasts, respectively) and collagen (76 ± 15% and 35 ± 23% reduction in atrial and ventricular fibroblasts/myofibroblasts, respectively) in response to TGF-ß1 stimulation. This effect was reversed by simultaneously knocking down NPR-A but not with simultaneous knock down of PKG-1. Conclusion: The differential response to TGF-ß1 stimulated fibrosis between the atria and ventricle are in part mediated by the abundance of NPR-C receptors in the atria.

Left Ventricular Dyssynchrony Predicts the Cardiomyopathy Associated With Premature Ventricular Contractions.

BACKGROUND: The pathophysiology of cardiomyopathy associated with premature ventricular contractions (PVCs) remains unclear. OBJECTIVES: This study prospectively explored cardiomyopathy development in a swine model of paced ectopic beats. METHODS: A total of 35 swine underwent pacemaker implantation. A group exposed to paced bigeminy from the right ventricular apex (RVA) for 14 weeks (RVA PVC) (n = 10) were compared with a group exposed to regular pacing from the RVA at 140 beats/min (RV-140) (n = 5) and a control group (n = 5). To test the role of ectopic beat dyssynchrony, further groups were exposed for 12 weeks to bigeminy from the right ventricular free wall (RVFW PVC) (n = 5), the left ventricular epicardium (LV Epi PVC) (n = 5) or the right atrium (premature atrial complex) (n = 5). RESULTS: After 14 weeks, the mean left ventricular ejection fraction (LVEF) was significantly lower in the RVA PVC group than in the RV-140 or control groups (p < 0.05). LVEF declined significantly in the LV Epi PVC (65.2 ± 2.4% to 39.7 ± 3.0%; p < 0.01) and RVFW PVC (66.1 ± 2.6% to 48.6 ± 2.7%; p < 0.01) groups, with final LVEF significantly lower and ventricular fibrosis significantly higher in the LV Epi PVC group compared with all others (p < 0.05). Protein levels of pRyR2, NCX-1, CaMKII-α, and PLN were up-regulated and levels of SERCA2a were down-regulated in the LV Epi PVC group compared with the control group (p < 0.05). Longer ectopic beat QRS duration and greater LV dyssynchrony were significantly associated with larger declines in LV systolic function. CONCLUSIONS: In a swine model of paced ectopic beats, PVC-induced cardiomyopathy is phenotypically distinct from a tachycardia-induced cardiomyopathy. Cardiomyopathy severity is strongly associated with severity of the hemodynamic derangement associated with the paced ectopic beats, particularly the extent of LV dyssynchrony.

Diffuse fibrosis leads to a decrease in unipolar voltage: Validation in a swine model of premature ventricular contraction-induced cardiomyopathy.

BACKGROUND: Frequent premature ventricular contractions (PVCs) may lead to dilated cardiomyopathy. A leftward shift in the unipolar voltage distribution in patients with cardiomyopathy has also been described and attributed to increased fibrosis. OBJECTIVES: We established a swine model of PVC-induced cardiomyopathy and assessed (1) whether an increase in left ventricular fibrosis occurs and (2) whether increased fibrosis leads to a leftward shift in the unipolar voltage distribution. METHODS: Ten swine underwent implantation of ventricular pacemakers; 6 programmed to deliver a 50% PVC burden and 4 controls without pacing. Voltage maps were acquired at baseline and after 14 weeks of ventricular bigeminy. RESULTS: In the PVC group, left ventricular ejection fraction decreased from 67% ± 7% to 44% ± 15% (P < .05) with no change in controls (71% ± 6% to 73% ± 4%; P = .56). The fifth percentile of the bipolar and unipolar voltage distribution at baseline was 1.63 and 5.36 mV, respectively. In the control group, after 14 weeks of pacing there was no significant change in % bipolar voltage <1.5 mV (pre 1.2% vs post 2.2%; P = .34) or % unipolar voltage <5.5 mV (pre 4.0% vs post 3.5%; P = .20). In the PVC group, there was a significant increase in % unipolar voltage <5.5 mV (5.4% vs 12.6%; P < .01), with a leftward shift in the unipolar voltage distribution. Histologically, % fibrosis was increased in the PVC group (control 1.8% ± 1.3% vs PVC 3.4% ± 2.6%; P < .01). CONCLUSION: PVC-induced cardiomyopathy in swine leads to an increase in interstitial fibrosis and a leftward shift in the unipolar voltage distribution. These findings are consistent with findings in humans with PVC-induced cardiomyopathy.

C-type natriuretic Peptide down-regulates expression of its cognate receptor in rat aortic smooth muscle cells.

The C-type natriuretic (CNP) peptide signals through the type B natriuretic peptide receptor (NPR-B) in vascular smooth muscle cells to activate the particulate guanylyl cyclase activity intrinsic to that receptor and raise cellular cyclic GMP levels. In the present study, we demonstrate that CNP down-regulates the expression of this receptor leading to a reduction in NPR-B activity. Pretreatment of rat aortic smooth muscle cells with CNP reduces NPR-B activity, NPR-B protein levels, NPR2 (NPR-B gene) mRNA levels, and NPR2 promoter activity. The decrease in NPR2 promoter activity is dependent on DNA sequence present between -441 and -134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP. 8-Bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reduced NPR2 mRNA levels and NPR2 promoter activity. Atrial natriuretic peptide, which signals through the type A natriuretic peptide receptor (NPR-A) to increase cyclic GMP levels in these cells, also reduced NPR-B mRNA levels and inhibited NPR-B promoter activity; however, this inhibition was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway. This report provides the first documentation that CNP is capable of autoregulating the expression of its cognate receptor.

Association of a novel single nucleotide polymorphism of the prostacyclin synthase gene with myocardial infarction.

BACKGROUND: Myocardial infarction (MI) is a complex multifactorial and polygenic disorder that is thought to result from an interaction between an individual's genetic makeup and various environmental factors. The purpose of this study was to investigate the association between a novel single nucleotide polymorphism in the prostacyclin synthase gene and MI. METHODS AND RESULTS: By the use of polymerase chain reaction-single-strand conformation polymorphism analysis, we identified a single nucleotide polymorphism, C1117A, in exon 8. This nucleotide change did not cause an amino acid change in codon 373. We performed an association study of the polymorphism in 138 patients and 130 healthy control subjects. Multiple logistic linear regression analysis showed the genotype distributions were significantly different between the control group and the MI group (odds ratio, 2.12; 95% CI, 1.47-3.05, P =.04). The C/C genotype was found more frequently in the MI group than in the control group. CONCLUSIONS: We conclude that the C1117A polymorphism in exon 8 is associated with risk for MI and may be a genetic marker of MI in Japanese persons.

Angiotensin-converting enzyme gene and longevity in the Xin Jiang Uighur autonomous region of China: an association study.

BACKGROUND: Longevity can be regarded as a multifactorial trait that results from an interaction between environmental factors and sets of epistatic alleles that have pleiotropic age-dependent effects. The Hotan district in the Xin Jiang Uighur Autonomous region of China is relatively isolated and is well known for an ethnic group that displays marked longevity. METHODS: We performed a correlation study between the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene and longevity by comparing distributions of the polymorphism between three different ethnic groups in this region. We obtained data from 424 subjects comprising 227 Uighur individuals, 108 Kazakh individuals, and 89 Han individuals. All subjects in the latter two groups ranged in age from 65 to 70 years, whereas the Uighur subjects actually comprised two different age groups: those ranging in age from 59 to 70 years (Uighur older group in Hotan [UOH]) and those ranging in age from 90 to 113 years (Uighur longevity group in Hotan [ULH]). Genomic DNA was extracted from peripheral white blood cells. Polymerase chain reaction was performed to amplify the I/D polymorphic region of the ACE gene. RESULTS: Frequencies of the insertion (I) and deletion (D) alleles were 0.596 (243/408) and 0.404 (165/408) in the Uighur group, 0.606 (130/216) and 0.394 (85/216) in the Kazakh group, and 0.657 (117/178) and 0.343 (61/178) in the Han group. The overall distributions of alleles in these three groups did not differ significantly (chi(2) = 4.6, p =.33). Within the Uighur group, frequency of the D allele was significantly higher in the ULH group (0.448) than in the UOH group (0.355) (p <.04). CONCLUSIONS: This association reflects a genetic influence on differential survival and may point to pleiotropic age-dependent effects on longevity. Our data may help elucidate the relationship between natural longevity and race difference among individuals in the Xin Jiang Uighur Autonomous region of China.

Variable number of tandem repeat of the 5'-flanking region of type-C human natriuretic peptide receptor gene influences blood pressure levels in obesity-associated hypertension.

The gene for natriuretic peptides receptor C (NPRC), which is a candidate susceptibility gene for essential hypertension (EH) or obese hypertension, plays a key role in the regulation of plasma levels and biological effects of natriuretic peptides. The aims of the present study were to find new genetic markers in the 5'-flanking region of the NPRC gene and to assess relationships between variants and phenotypes of EH, including EH in obese patients. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, we discovered a novel six-nucleotide repeat polymorphism located 4 base pairs (bp) upstream of the major transcriptional initiation site. We performed association analysis of this variable number of tandem repeat (VNTR) in 242 EH patients and 212 normotensive controls (NT). Although no significant difference in overall frequency of VNTR was found between NT and EH groups, the blood pressure level of EH patients with the 5/6 genotype was significantly higher in obese subjects. This suggests that the VNTR of the 5'-flanking region of the NPRC gene influences blood pressure levels in obesity-associated hypertension.

Association study between a novel single nucleotide polymorphism of the promoter region of the prostacyclin synthase gene and essential hypertension.

The purpose of this study was to investigate whether an association exists between the promoter region of the prostacyclin synthase gene and essential hypertension (EH). Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a novel single nucleotide polymorphism (SNP), T-192G, in the 5'-flanking region. We performed an association study using the SNP in 200 patients and 200 controls. The allele frequency distribution in the two groups was not significantly different. Thus, this SNP in the PGIS gene is not associated with EH.

A novel missense mutation of exon 3 in the type A human natriuretic peptide receptor gene: possible association with essential hypertension.

The natriuretic peptide (NP) family is involved in regulation of blood pressure and fluid volume. We recently characterized the exon/intron organization of the human type A NP receptor (hNPRA) gene. The aim of this study was to isolate the genetic markers according to the organization of this gene, and to study the association between this gene and essential hypertension. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, we identified a novel missense mutation, M3411, consisting of a methionine (ATG) to isoleucine (ATC) substitution at nucleotide 1023 in exon 3. Computer-aided three-dimensional structural analysis suggested that M341 exists in the loop between two alpha-helices, and that the mutation may influence receptor activities by altering the conformation of the alpha-helices. We performed an association study of the mutation in 210 essential hypertension (EH) patients and 210 normotensive controls. The overall distribution of alleles was not significantly different between the control and EH groups. However, the C/C homozygous genotype was found only in the EH group. The ratio of plasma brain natriuretic peptide (BNP)/mean blood pressure of the C/C genotype was significantly higher than that of the G/G genotype or the G/C genotype. We conclude that the significance of homozygous M3411 mutation in exon 3 is worth investigating for its possible association with EH.

Molecular basis of selective atrial fibrosis due to overexpression of transforming growth factor-ß1.

AIMS: Animal studies show that transforming growth factor-ß1 (TGF-ß1) is an important mediator of atrial fibrosis and atrial fibrillation (AF). This study investigated the role of TGF-ß1 in human AF and the mechanism of atrial-selective fibrosis. METHODS AND RESULTS: Atrial specimens from 17 open heart surgery patients and left atrial and ventricular specimens from 17 explanted hearts were collected to assess the relationship between TGF-ß1, AF, and differential atrial vs. ventricular TGF-ß1 levels. A transgenic mouse model overexpressing active TGF-ß1 was used to study the mechanisms underlying the resultant atrial-selective fibrosis. Higher right atrial total TGF-ß1 levels (2.58 ± 0.16-fold, P < 0.0001) and active TGF-ß1 (3.7 ± 0.7-fold, P = 0.013) were observed in those that developed post-operative AF. Although no ventricular differences were observed, 11 explanted heart failure hearts exhibited higher atrial TGF-ß1 levels than 6 non-failing hearts (2.30 ± 0.87 fold higher, P < 0.001). In the transgenic mouse, TGF-ß1 receptor-1 kinase blockade resulted in decreased atrial expression of fibrosis-related genes. By RNA microarray analyses in that model, 80 genes in the atria and only 2 genes in the ventricle were differentially expressed. Although these mice atria, but not the ventricles, exhibited increased expression of fibrosis-related genes and phosphorylation of Smad2, there were no differences in TGF-ß1 receptor levels or Smads in the atria compared with the ventricles. CONCLUSIONS: TGF-ß1 mediates selective atrial fibrosis in AF that occurs via TGF-ß Receptor 1/2 and the classical Smad pathway. The differential atrial vs. ventricular fibrotic response occurs at the level of TGF-ß1 receptor binding or phosphorylation.

Expression of guanylyl cyclase-B (GC-B/NPR2) receptors in normal human fetal pituitaries and human pituitary adenomas implicates a role for C-type natriuretic peptide.

C-type natriuretic peptide (CNP/Nppc) is expressed at high levels in the anterior pituitary of rats and mice and activates guanylyl cyclase B receptors (GC-B/Npr2) to regulate hormone secretion. Mutations in NPR2/Npr2 can cause achondroplasia, GH deficiency, and female infertility, yet the normal expression profile within the anterior pituitary remains to be established in humans. The current study examined the expression profile and transcriptional regulation of NPR2 and GC-B protein in normal human fetal pituitaries, normal adult pituitaries, and human pituitary adenomas using RT-PCR and immunohistochemistry. Transcriptional regulation of human NPR2 promoter constructs was characterized in anterior pituitary cell lines of gonadotroph, somatolactotroph, and corticotroph origin. NPR2 was detected in all human fetal and adult pituitary samples regardless of age or sex, as well as in all adenoma samples examined regardless of tumor origin. GC-B immunoreactivity was variable in normal pituitary, gonadotrophinomas, and somatotrophinomas. Maximal transcriptional regulation of the NPR2 promoter mapped to a region within -214 bp upstream of the start site in all anterior pituitary cell lines examined. Electrophoretic mobility shift assays revealed that this region contains Sp1/Sp3 response elements. These data are the first to show NPR2 expression in normal human fetal and adult pituitaries and adenomatous pituitary tissue and suggest a role for these receptors in both pituitary development and oncogenesis, introducing a new target to manipulate these processes in pituitary adenomas.

Atrial natriuretic peptide suppresses endothelin gene expression and proliferation in cardiac fibroblasts through a GATA4-dependent mechanism.

AIMS: Atrial natriuretic peptide (ANP) is a hormone that has both antihypertrophic and antifibrotic properties in the heart. We hypothesized that myocyte-derived ANP inhibits endothelin (ET) gene expression in fibroblasts. METHODS AND RESULTS: We have investigated the mechanism(s) involved in the antiproliferative effect of ANP on cardiac fibroblasts in a cell culture model. We found that cardiac myocytes inhibited DNA synthesis in co-cultured cardiac fibroblasts as did treatment with the ET-1 antagonist BQ610. The effect of co-culture was reversed by antibody directed against ANP or the ANP receptor antagonist HS-142-1. ANP inhibited the expression of the ET-1 gene and ET-1 gene promoter activity in cultured fibroblasts. The site of the inhibition was localized to a GATA-binding site positioned between -132 and -135 upstream from the transcription start site. GATA4 expression was demonstrated in cardiac fibroblasts, GATA4 bound the ET-1 promoter both in vitro and in vivo, and siRNA-mediated knockdown of GATA4 inhibited ET-1 expression. ET-1 treatment resulted in increased levels of phospho-serine(105) GATA4 in cardiac fibroblasts and this induction was partially suppressed by co-treatment with ANP. CONCLUSION: Collectively, these findings suggest that locally produced ET-1 serves as an autocrine stimulator of fibroblast proliferation, that ANP produced in neighbouring myocytes serves as a paracrine inhibitor of this proliferation, and that the latter effect operates through a reduction in GATA4 phosphorylation and coincident reduction in GATA4-dependent transcriptional activity.

Transcriptional regulation of type B human natriuretic Peptide receptor gene promoter: dependence on Sp1.

The type B natriuretic peptide receptor (NPR-B) is the cognate receptor for the C-type natriuretic peptide and, as such, is responsible for signaling growth-suppressant activity in vascular smooth muscle cells. Here we report the isolation and characterization of the human (h) NPR-B gene promoter. Using 5' rapid amplification of cDNA ends analysis, we have identified the 5' terminus of the hNPR-B gene transcript approximately 732 base pairs upstream from the presumed translation start site of the protein. We generated a series of 5' deletion mutants linked to a luciferase reporter and introduced these constructs into rat aortic smooth muscle cells or neonatal rat cardiac fibroblasts. Maximal expression was seen with a construct harboring 441 base pairs of 5' flanking sequence. Site-directed mutagenesis of the proximal promoter revealed a series of GC-rich sequences, 5 of which contributed modestly (approximately 25%) to basal hNPR-B promoter activity. Mutation of a sixth GC-rich sequence led to a >90% reduction in promoter activity. This sequence was shown to associate with Sp1 and Sp3 in vitro. The same mutation that resulted in loss of functional activity also resulted in loss of binding activity in vitro. Overexpression of Sp1 or Sp3 in Drosophila Schneider cells resulted in an increase in hNPR-B promoter activity that was completely nullified with the Sp1 binding site mutation described above. These studies provide the first description and characterization of the NPR-B gene promoter and suggest that this promoter's activity is dominated by a single cluster of Sp1-binding elements in the proximal 5' flanking sequence of the gene.

Tumor necrosis factor-alpha inhibits endothelial nitric-oxide synthase gene promoter activity in bovine aortic endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to reduce endothelial nitric-oxide synthase (eNOS) gene expression through post-transcriptional regulation of mRNA stability. The current study documented an independent effect of the cytokine on the eNOS gene promoter. TNF-alpha effected a time- and dose-dependent reduction in activity of a transiently transfected human -1197 eNOS-luciferase reporter. This reduction was inhibited by co-transfection of dominant negative IKKbeta as well as a nonphosphorylatable constitutively suppressive mutant of IkappaB implying involvement of the NFkappaB cascade in the inhibitory effect. The locus of the TNF-alpha-dependent inhibition was traced to two Sp1-binding sites positioned between -109 and -95 and -81 and -67 relative to the transcription start site. Electrophoretic mobility shift analysis and immunoperturbation studies showed evidence for Sp1 and Sp3 binding to each element. TNF-alpha treatment had no effect on the binding pattern to the downstream (-81 to -67) site but did suppress association of Sp1 and Sp3 to the upstream (-109 to -95) site. Collectively, these data indicate that TNF-alpha exerts transcriptional, as well as post-transcriptional, effects on eNOS gene expression and suggest a potential mechanism to account for the endothelial dysfunction that accompanies disorders such as diabetes mellitus and heart failure.

Structure and polymorphisms of the human natriuretic peptide receptor C gene.

Natriuretic peptides (NPs) regulate cardiovascular homeostasis, including natriuresis, diuresis, vasodilation, regulation of endocrine secretion, and inhibition of cellular growth. Atrial natriuretic peptide receptor C (NPRC) has a short cytoplasmic domain that lacks guanylyl cyclase activity. We used information available for the cDNA of human NPRC to amplify products covering all genomic regions of the gene by long polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR. PCR products were sequenced directly after extraction and purification. The human NPRC gene spans > 65 kb and contains eight exons and seven introns. All of the exon-intron junction sequences contain the GT/AG consensus junction sequence. We then used the PCR-single-strand conformation polymorphism (PCR-SSCP) to identify polymorphisms of the human NPRC gene. All eight exons and neighboring introns were analyzed by PCR-SSCP for 96 subjects, and migration variants were observed for intron 1, exon 2, and exon 5. Direct sequencing of these variants revealed the following sequence differences: a C to T transition in intron 1, an A to C transition in exon 2, and a C to T transition in exon 5. PCR-restriction fragment length polymorphism analysis (PCR-RFLP) was used to evaluate all three variations. We have determined the structural organization and identified polymorphic sites in the human NPRC gene. The results of this study will facilitate further genetic analyses of the human NPRC gene function.

Ser9Gly polymorphism in the dopamine D3 receptor gene is not associated with essential hypertension in the Japanese.

BACKGROUND: The Dopamine D3 receptor (DRD3) gene is thought to be involved in essential hypertension (EH) because dopamine inhibits renin secretion via this receptor and because disruption of the DRD3 gene increases blood pressure in mice. EH is a complex, polygenetic disease. Association studies using the candidate gene approach may provide important clues regarding the etiology of hypertension and define a basis for further genetic investigation. Therefore we examined the association between the Ser9Gly polymorphism in the DRD3 gene and EH. MATERIAL/METHODS: One hundred eighty-one patients with EH and 181 age-matched subjects with normal blood pressure were enrolled. Genomic DNA was extracted from peripheral blood leukocytes. Polymerase chain reaction (PCR) was used to amplify the Ser9Gly polymorphic site in the DRD3 gene, and restriction fragment length polymorphism (RFLP) analysis of the PCR product was used to score the A and G alleles. Plasma renin activity and plasma aldosterone concentration were measured in untreated EH subjects. RESULTS: The genotype distribution was in Hardy-Weinberg equilibrium, and was not significantly different between the NT and EH groups. The frequencies of A and G alleles were 0.674 (244/362) and 0.326 (118/362) for the NT group and 0.688 (249/362) and 0.312 (113/362) for the EH group, respectively, and did not differ significantly between the two groups. The genotype did not influence the plasma renin activity and aldosterone concentration in untreated EH patients. CONCLUSIONS: The Ser9Gly polymorphism in the DRD3 gene are not associated with EH. However, our negative result does not exclude the possibility of another variant elsewhere in or near the DRD3 gene in EH.