B-type Natriuretic Peptide mRNA Levels Research Articles

Suppression of B-type natriuretic peptide gene expression in cardiomyocytes under anoxic conditions

Several cell biology studies have focused on the effects of hypoxic environments on cardiomyocytes. However, the effect of anoxic conditions on cardiomyocytes remains largely unexplored. In the present study, we investigated the direct effects of anoxia on B-type natriuretic peptide (BNP) gene expression in cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) were exposed to anoxia using an airtight chamber saturated with 95 % N2/5 % CO2. BNP mRNA levels in NRCM were substantially reduced after more than 8 h of anoxia exposure, whereas after reoxygenation, BNP gene expression levels recovered in a time-dependent manner and significantly increased after 24 h of reoxygenation. BNP mRNA levels suppressed under anoxic conditions were significantly increased by aldosterone-induced activation of sodium-proton exchanger 1 (NHE1), which was canceled by an NHE1 inhibitor, suggesting that anoxia reduces BNP gene expression, at least in part, in an NHE1-dependent manner. In summary, we found that BNP gene expression in cardiomyocytes decreases under anoxic conditions, in contrast to previous research findings that BNP expression increases under hypoxic conditions. These findings reveal a new insight that, within a single heart tissue in various cardiovascular diseases, such as myocardial infarction, the biological responses of cardiomyocytes are fundamentally different in regions of anoxia and hypoxia.

Reversal effects of low-dose imatinib compared with sunitinib on monocrotaline-induced pulmonary and right ventricular remodeling in rats

High-dose imatinib reverses cardiopulmonary remodeling but adverse effects limit its clinical use. Efficacy of the multi-kinase inhibitor sunitinib remains questionable. We compared anti-remodeling effects of imatinib with sunitinib on monocrotaline-induced right ventricular (RV) hypertrophy and pulmonary arterial remodeling in rats, focusing on a lower dose. Fourteen days after monocrotaline injection, oral gavage of imatinib (5, 15, or 50mg/kg), sunitinib (0.3, 1, 3, or 10mg/kg), or water for 14days was started. RV hypertrophy and b-type natriuretic peptide mRNA levels were significantly and dose-dependently reduced, much greater in imatinib- than sunitinib-treated groups. Imatinib normalized muscularization of 20–50μm intra-acinar pulmonary arteries more significantly than sunitinib. At transcript levels, sunitinib significantly upregulated pulmonary nestin, and downregulated platelet-derived growth factor receptor beta (PDGFR-β), fibroblast growth factor receptor 1, vascular endothelial growth factor receptor-2 and vascular endothelial growth factor (VEGF)-A, but not Raf-1 proto-oncogene serine/threonine kinase mRNAs. Sunitinib also suppressed VEGF-A, but not phosphorylated extra-cellular-signal-related kinase (ERK)-1/2 protein expression. The sole PDGFR-β antagonism of imatinib resulted in significant Raf-1 mRNA and phosphorylated ERK-1/2 protein downregulation, suggesting that the equivocal reversal effect of sunitinib may be due to its VEGF signaling inhibition in the lung. Imatinib's greater dose-dependent reversal on cardiopulmonary remodeling may make a low dose suitable for PAH treatment.

Hydrogen (H2) Inhibits Isoproterenol-Induced Cardiac Hypertrophy via Antioxidative Pathways.

Background and Purpose: Hydrogen (H2) has been shown to have a strong antioxidant effect on preventing oxidative stress-related diseases. The goal of the present study is to determine the pharmacodynamics of H2 in a model of isoproterenol (ISO)-induced cardiac hypertrophy.Methods: Mice (C57BL/6J; 8–10 weeks of age) were randomly assigned to four groups: Control group (n = 10), ISO group (n = 12), ISO plus H2 group (n = 12), and H2 group (n = 12). Mice received H2 (1 ml/100g/day, intraperitoneal injection) for 7 days before ISO (0.5 mg/100g/day, subcutaneous injection) infusion, and then received ISO with or without H2 for another 7 days. Then, cardiac function was evaluated by echocardiography. Cardiac hypertrophy was reflected by heart weight/body weight, gross morphology of hearts, and heart sections stained with hematoxylin and eosin, and relative atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) mRNA levels. Cardiac reactive oxygen species (ROS), 3-nitrotyrosine and p67 (phox) levels were analyzed by dihydroethidium staining, immunohistochemistry and Western blotting, respectively. For in vitro study, H9c2 cardiomyocytes were pretreated with H2-rich medium for 30 min, and then treated with ISO (10 μM) for the indicated time. The medium and ISO were re-changed every 24 h. Cardiomyocyte surface areas, relative ANP and BNP mRNA levels, the expression of 3-nitrotyrosine, and the dissipation of mitochondrial membrane potential (MMP) were examined. Moreover, the expression of extracellular signal-regulated kinase1/2 (ERK1/2), p-ERK1/2, p38, p-p38, c-Jun NH2-terminal kinase (JNK), and p-JNK were measured by Western blotting both in vivo and in vitro.Results: Intraperitoneal injection of H2 prevented cardiac hypertrophy and improved cardiac function in ISO-infused mice. H2-rich medium blocked ISO-mediated cardiomyocytes hypertrophy in vitro. H2 blocked the excessive expression of NADPH oxidase and the accumulation of ROS, attenuated the decrease of MMP, and inhibited ROS-sensitive ERK1/2, p38, and JNK signaling pathways.Conclusion: H2 inhibits ISO-induced cardiac/cardiomyocytes hypertrophy both in vivo and in vitro, and improves the impaired left ventricular function. H2 exerts its protective effects partially through blocking ROS-sensitive ERK1/2, p38, and JNK signaling pathways.

Regulation of B-type natriuretic peptide synthesis by insulin in obesity in male mice.

Human studies suggest that insulin resistance and obesity are associated with a decrease in B-type natriuretic peptide (BNP) plasma concentrations. The objective of the study was to gain insights into the mechanisms involved in the association between insulin resistance and decreased BNP plasma concentrations. Mice fed a high-fat, high-fructose (HFHF) diet for 4 weeks developed mild obesity and systemic insulin resistance. Elevated plasma concentrations of insulin, glucose and triglycerides were noted. The HFHF diet was also associated with myocardial insulin resistance, characterized by an impaired response of the phosphoinositide 3-kinase-AKT (PI3K-AKT) pathway to insulin in the left ventricle. Myocardial BNP expression and protein were decreased in HFHF-fed mice compared with control animals. Exposure of cardiomyocytes to 100 nm insulin activated PI3K-AKT signalling (15 min) and induced a 1.9 ± 0.3-fold increase in BNP gene expression (6 h). Prolonged exposure of cardiomyocytes to a high insulin concentration (100 nm) for 48 h induced insulin resistance, characterized by an impaired response of the PI3K-AKT signalling pathway and a decreased response of the BNP gene expression to insulin. The decreased response in BNP gene expression was reproduced by treating cardiomyocytes for 7 h with a PI3-kinase inhibitor (wortmannin). In conclusion, HFHF diet in vivo, prolonged exposure to an elevated concentration of insulin or inhibition of the PI3K-AKT pathway in vitro all decrease BNP mRNA levels; this decrease may in turn contribute to the decreased BNP peptide concentrations in plasma observed in insulin-resistant individuals.

B-type natriuretic peptide expression and cardioprotection is regulated by Akt dependent signaling at early reperfusion

Exogenously administered B-type natriuretic peptide (BNP) has been shown to offer cardioprotection through activation of particulate guanylyl cyclase (pGC), protein kinase G (PKG) and KATP channel opening. The current study explores if cardioprotection afforded by short intermittent BNP administration involves PI3K/Akt/p70s6k dependent signaling, and whether this signaling pathway may participate in regulation of BNP mRNA expression at early reperfusion. Isolated Langendorff perfused rat hearts were subjected to 30min of regional ischemia and 120min of reperfusion (IR). Applying intermittent 3×30s infusion of BNP peptide in a postconditioning like manner (BNPPost) reduced infarct size by >50% compared to controls (BNPPost 17±2% vs. control 42±4%, p<0.001). Co-treatment with inhibitors of the PI3K/Akt/p70s6k pathway (wortmannin, SH-6 and rapamycin) completely abolished the infarct-limiting effect of BNP postconditioning (BNPPost+Wi 36±5%, BNPPost+SH-6 41±4%, BNPPost+Rap 37±6% vs. BNPPost 17±2%, p<0.001). Inhibition of natriuretic peptide receptors (NPR) by isatin also abrogated BNPPost cardioprotection (BNPPost+isatin 46±2% vs. BNPPost 17±2%, p<0.001). BNPPost also significantly phosphorylated Akt and p70s6k at early reperfusion, and Akt phosphorylation was inhibited by SH-6 and isatin. Myocardial BNP mRNA levels in the area at risk (AA) were significantly elevated at early reperfusion as compared to the non-ischemic area (ANA) (Ctr(AA) 2.7±0.5 vs. Ctr(ANA) 1.2±0.2, p<0.05) and the ischemic control tissue (Ctr(AA) 2.7±0.5 vs. ischemia 1.0±0.1, p<0.05). Additional experiments also revealed a significant higher BNP mRNA level in ischemic postconditioned (IPost) hearts as compared to ischemic controls (IPost 6.7±1.3 vs. ischemia 1.0±0.2, p<0.05), but showed no difference from controls run in parallel (Ctr 5.4±0.8). Akt inhibition by SH-6 completely abrogated this elevation (IPost 6.7±1.3 vs. IPost+SH-6 1.8±0.7, p<0.05) (Ctr 5.4±0.8 vs. SH-6 1.5±0.9, p<0.05). In conclusion, Akt dependent signaling is involved in mediating the cardioprotection afforded by intermittent BNP infusion at early reperfusion, and may also participate in regulation of reperfusion induced BNP expression.

Cardiac Natriuretic Peptide Gene Expression and Plasma Concentrations During the First 72 Hours of Life in Piglets

Plasma measurement of cardiac natriuretic peptides constitutes promising markers of congenital heart disease. However, concentrations change rapidly and dramatically during the first days after delivery even in healthy neonates, which complicates clinical interpretation. It is unknown whether these changes in plasma concentrations are explained by corresponding changes in the cardiac gene expression. We quantified the chamber-specific mRNA levels of ANP (A-type natriuretic peptide) and BNP (B-type natriuretic peptide) and plasma pro-ANP and BNP-32 concentrations in healthy piglets during the first 72 hours of life (from 2 litters, n = 44). Chamber-specific ANP and BNP mRNA levels reflected hemodynamic neonate changes at birth but did not correlate with circulating natriuretic peptide concentrations. However, plasma pro-ANP and creatinine concentrations were closely correlated (P < .0001; r = 0.73). Plasma pro-ANP levels were highest on the day of delivery (5580 pmol/L [4320-6786] decreasing to 2484 pmol/L [1602-2898] after 72 hours, P < .0001). During the 72 hours, gel chromatography suggested that the translational products in circulation and in atrial tissue were immature, ie, unprocessed pro-ANP. In contrast to pro-ANP, BNP-32 plasma concentrations were low at delivery and peaked after 48 hours (12 [10.5-20.6] vs. 88.8 [71.7-101.4] pmol/L, P < .0001). To conclude, ANP and BNP gene expression differs considerably between cardiac chambers in the first 72 hours of life in healthy piglets, resembling the transition from fetal to neonate circulation. However, the cardiac gene expression does not explain plasma concentrations.

Abstract P310: Connective Tissue Growth Factor Antibody Protects from Adverse Left Ventricular Remodeling Following Myocardial Infarction

Ischemic heart disease (IHD) is the most common cause of death in most Western countries, but the therapies for IHD are lacking. We aimed to determine the potential of connective tissue growth factor (CTGF) antibody in protecting myocardium from long-term deleterious effects of myocardial infarction. Healthy 8-week old mice were subjected to myocardial infarction (MI) and at 1 week post-MI the animals were randomized for treatment with vehicle, metoprolol or CTGF monoclonal antibody. After six weeks of treatment, CTGF antibody treated animals showed significantly better left ventricular (LV) systolic function than the vehicle-treated animals. LV diastolic function or LV dimensions were not affected by treatments. Analysis of cardiac sections revealed no difference in infarct scar size between the experimental groups. However, RT-PCR analysis revealed a decrease in the expression levels of fibrosis-related genes in non-ischemic area in the hearts of CTGF antibody treated animals. In addition, cardiomyocyte size was significantly reduced in LV sections of CTGF antibody treated animals compared to vehicle-treated animals. This was accompanied with a significant decrease in B-type natriuretic peptide mRNA levels in the LV, whereas expression levels of atrial natriuretic peptide or beta-myosin heavy chain were similar in MI groups. There was also a slight decrease in mRNA levels of interleukin-6 and CTGF in the hearts of CTGF antibody treated animals. Treatments had no effect on mRNA levels of transforming growth factor-β1, tumor necrosis factor-α and vascular endothelial growth factor A following MI. In conclusion, CTGF antibody treatment attenuates LV hypertrophy and improves LV systolic function following MI. Blocking CTGF function by a specific antibody may be useful for prevention of adverse myocardial remodeling and heart failure.

Key roles of endothelin-1 and p38 MAPK in the regulation of atrial stretch response

Mechanisms regulating stretch response in the left ventricle are investigated in detail but not well understood in atrial myocardium. Hypertrophic growth of atrial myocardium contributes to the pathogenesis of atrial fibrillation. In this study, we sought to elucidate mechanisms of stretch-induced activation of key signaling pathways and hypertrophy-associated genes in rat atria. Stretching of isolated atria induced a rapid increase in phosphorylation of p38 MAPK and ERK and induced a p38 MAPK-dependent increase in DNA binding activity of transcription factors Elk-1 and GATA-4. Inhibition of the ERK pathway had no effect on the cardiac transcription factors studied. Stretch-induced increase in atrial contractile function was substantially enhanced by inhibition of p38 MAPK. p38 MAPK also regulated stretch-induced increase in c-fos, β-myosin heavy chain, B-type natriuretic peptide mRNA levels, and atrial natriuretic peptide secretion in isolated atria. Various autocrine/paracrine factors are known to mediate the stretch response in the left ventricle. Stretching of isolated atria resulted in a robust increase in endothelin-1 (ET-1) mRNA levels, while apelin and adrenomedullin signaling cascades were downregulated. Administration of mixed ET(A/B) receptor antagonist bosentan attenuated the stretch-induced activation of GATA-4 in isolated atria, whereas ANG II receptor type-1 antagonist CV-11974 had no effect. Moreover, analysis of RNA from intact atrial and ventricular myocardium revealed significantly higher mRNA levels of ET(A) receptor and ET converting enzyme-1 in atrial compared with ventricular myocardium. In conclusion, our findings identify the local ET-1 system and p38 MAPK as key regulators of load-induced hypertrophic response in isolated rat atria.

Perinatal profile of ventricular overload markers in congenital diaphragmatic hernia

Background In congenital diaphragmatic hernia (CDH), pulmonary hypertension increases right ventricle (RV) afterload, which could impair heart function and contribute to poor outcome for most affected infants. Nevertheless, the real significance of vascular pulmonary alterations in perinatal hemodynamics is largely unknown. It is defined that ventricular pressure overload induces increased myocardium gene expression of B-type natriuretic peptide (BNP) and components of the renin-angiotensinogen and endothelin (ET)–1 systems. Our aim was to evaluate perinatal myocardium expression of these genes associated with ventricular pressure overload in a nitrofen-induced CDH rat model. Methods In the nitrofen-induced CDH rat model, fetuses from dated pregnant Sprague-Dawley rats at 15.5, 17.5, 19.5 and 21.5 days postcoitum as well as newborn pups were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from the RV and left ventricle (LV) were processed for quantification of messenger RNA (mRNA) of BNP, angiotensinogen, and ET-1. Results The perinatal expression of BNP, angiotensinogen, and ET-1 mRNA in the RV and LV of the control group revealed daily changes. During gestation, the expression of BNP and angiotensinogen mRNA underwent significant oscillation compared with control in both nitrofen-exposed fetuses, although we cannot identify significant differences between the nitrofen and CDH groups. After birth, we found a significant increasing expression of all studied genes only in the RV of CDH pups. Conclusions Perinatal myocardial quantification of BNP, angiotensinogen, and ET-1 mRNA levels suggests that both nitrofen-exposed and control pups revealed prenatal variations of expression of the studied genes. Moreover, CDH is associated with significant molecular alterations only in the RV after birth.

Exercise training prevents development of cardiac contractile dysfunction in hypertensive TG(mREN2)27 rats

Angiotensin II (Ang II) contributes to cardiac remodeling and left ventricular (LV) dysfunction. In contrast, exercise may have beneficial effects on LV structure and function. We investigated the effects of low-intensity exercise training (ET) on in-vivo cardiac function in hypertensive TG(mREN-2)27 rats (Ren2), which develop LV hypertrophy and dysfunction. Ren2 rats and Sprague-Dawley (SD) controls (4 to 5 weeks) began treadmill exercise every day for 5 to 6 weeks. Cardiac function was evaluated by echocardiography. Cardiac output and stroke volume were increased by ET in both the 8-week-old SD and Ren2. Slope of mitral deceleration time, a noninvasive measure of diastolic function, was lower in the Ren2 rats, but not changed by ET. LV collagen deposition, as assessed by hydroxyproline assay, was not affected by rat strain or ET at 10 to 11 weeks of age. LV B-type natriuretic peptide mRNA levels were higher in the Ren2 rats (100%), but not affected by ET. Both α (∼14.5-fold) and β (∼2.5-fold) myosin heavy chain mRNA were higher in the LV of Ren2 rats ( P < .05), but were not changed by ET. Low-intensity exercise training in Ren2 rats, a model of Ang-II-mediated hypertension, maintains cardiac index and stroke volume in the presence of impaired diastolic function at 8 weeks of age.

Cardiac BNP gene activation by angiotensin II in vivo

The transcription factors involved in the activation of cardiac gene expression by angiotensin II (Ang II) in vivo are not well understood. Here we studied the contribution of transcriptional elements to the activation of the cardiac B-type natriuretic peptide (BNP) gene promoter by Ang II in conscious rats and in angiotensin II type 1 receptor (AT1R) transgenic mice. Rat BNP luciferase reporter gene constructs were injected into the left ventricular wall. The mean luciferase activity was 1.8-fold higher ( P < 0.05) in the ventricles of animals subjected to 2-week Ang II infusion as compared with vehicle infusion. Our results indicate that GATA binding sites at −90 and −81 in the rat BNP promoter are essential for the in vivo response to Ang II. The GATA factor binding to these sites is GATA-4. BNP mRNA levels and GATA-4 binding activity are also increased in the hypertrophied hearts of aged AT1R transgenic mice.

Distinct modulation of angiotensin II-induced early left ventricular hypertrophic gene programming by dietary fat type

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

Posttranscriptional control of BNP gene expression in angiotensin II-induced hypertension.

B-type natriuretic peptide (BNP) plasma concentrations are raised in patients with heart failure. In several experimental models of cardiac overload, however, BNP mRNA and plasma BNP peptide levels are normal, despite the persistent increase in blood pressure and ventricular hypertrophy. In this study, the role of transcriptional mechanisms in the regulation of BNP gene expression were studied in angiotensin (Ang) II-induced hypertension by injecting DNA constructs containing the BNP promoter (-2200 to 75 bp of the transcriptional start site) linked to luciferase reporter into rat myocardium. Ang II was administered to conscious rats via intravenous infusion for 2 hours or by subcutaneous minipumps for 6 hours, 12 hours, 3 days, 1 week, and 2 weeks. Ang II increased blood pressure and cardiac mass and induced changes in diastolic function. The left ventricular BNP mRNA levels increased 2.2-fold (P<0.001) at 2 hours and peaked at 12 hours (5.2-fold, P<0.001). Thereafter, BNP mRNA levels decreased (1.8-fold induction at 3 days, P<0.05) and returned to control levels at 1 week, despite persistent hypertension and myocardial hypertrophy. Left ventricular BNP peptide concentrations followed the changes in BNP mRNA levels. The BNP promoter was activated 2.7-fold (P<0.05) at 2 hours and remained upregulated up to 2 weeks (2.8-fold, P<0.05) during Ang II infusion, except at 12 hours. These results indicate that posttranscriptional control plays a major role in the regulation of ventricular BNP gene expression in Ang II-induced hypertension.

GATA4 mediates activation of the B-type natriuretic peptide gene expression in response to hemodynamic stress.

To identify the mechanisms that couple hemodynamic stress to alterations in cardiac gene expression, DNA constructs containing the rat B-type natriuretic peptide (BNP) promoter were injected into the myocardium of rats, which underwent bilateral nephrectomy or were sham-operated. Ventricular BNP mRNA levels were induced about 4-fold; and the BNP reporter construct containing the proximal 2200 bp, 5-fold, in response to 1-d nephrectomy. Deletion of sequences between bp -2200 and -114 did not affect basal or inducible activity of the BNP promoter. An activator protein-1-like site and two tandem GATA elements are located within this 114-bp sequence. Both deletion and mutation of the AP-1-like motif decreased basal activity but did not abolish the response to nephrectomy. In contrast, mutation or deletion of -90 bp GATA-sites abrogated the response to hemodynamic stress. The importance of these GATA elements to BNP promoter activation was further confirmed by the corresponding 38-bp oligonucleotide conferring hemodynamic stress responsiveness to a minimal BNP promoter. In gel mobility shift assays, nephrectomy increased left ventricular BNP GATA4 binding activity significantly. In conclusion, GATA elements are necessary and sufficient to confer transcriptional activation of BNP gene in response to hemodynamic stress.

Overexpression of sarcolemmal calcium pump attenuates induction of cardiac gene expression in response to ET-1.

The function of the plasma membrane calmodulin-dependent calcium ATPase (PMCA) in myocardium is unknown. PMCA is localized in caveolae, 50- to 100-nm membrane invaginations, which also contain receptors for endothelin-1 (ET-1) and various other ligands. PMCA has been suggested to play a role in regulation of caveolar signal transduction. We studied the effects of the hypertrophic agonist ET-1 and increased coronary perfusion pressure on cardiac synthesis of B-type natriuretic peptide (BNP) in transgenic rats overexpressing the human PMCA 4CI in isolated perfused heart preparation. ET-1 infusion for 2 h increased BNP mRNA levels twofold in left ventricles (LV) of nontransgenic rats, whereas no increase was noted in PMCA rat hearts. Similar responses were seen in adrenomedullin and c-fos mRNA levels, and in immunoreactive BNP secretion. Increased mechanical load produced by elevated perfusion pressure induced similar 1.5- to 1.6-fold increases in LV BNP mRNA in both nontransgenic and PMCA rat hearts. These results show that cardiac overexpression of PMCA attenuates ET-1-stimulated early induction of cardiac gene expression, suggesting that PMCA may modulate myocardial growth responses.

Differential regulation of cardiac adrenomedullin and natriuretic peptide gene expression by AT1 receptor antagonism and ACE inhibition in normotensive and hypertensive rats.

To study the effects of long-term treatment with the type 1 angiotensin (AT1) receptor antagonist losartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril, on cardiac adrenomedullin (ADM), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression. Spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were given losartan (15 mg/kg per day) or enalapril (4 mg/kg per day) orally for 10 weeks. The effects of drugs on systolic blood pressure, cardiac hypertrophy, ANP, BNP and ADM mRNA and immunoreactive-ANP (IR)-ANP, IR-BNP and IR-ADM levels in the left ventricle and atria were compared. Losartan and enalapril treatments completely inhibited the increase of systolic blood pressure occurring with ageing in SHR. The ratio of heart to body weight was reduced in both losartan- and enalapril-treated SHR and WKY rats. Treatment with losartan or enalapril reduced left ventricular ANP mRNA and IR-ANP in both strains, and ventricular BNP mRNA levels in SHR rats. Inhibition of ACE, AT1 receptor antagonism, changes in blood pressure or cardiac mass had no effect on left ventricular ADM gene expression in SHR and WKY rats. In addition, atrial IR-ANP and IR-ADM levels increased in SHR whereas IR-BNP levels decreased in WKY and SHR rats in response to drug treatments. Our results show that ventricular ADM synthesis is an insensitive marker of changes in haemodynamic load or cardiac hypertrophy. Furthermore, the expression of ADM, ANP and BNP genes is differently regulated both in the left ventricle and atria in response to AT1 receptor antagonism and ACE inhibition.

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132 ± 6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA ( P < 0.001) and IR-BNP ( P < 0.05) levels in 2-month old rats. There was also a 1.5-fold ( P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% ( P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% ( P < 0.05) and 39 % ( P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% ( P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.

Endothelin-1 is involved in stretch-induced early activation of B-type natriuretic peptide gene expression in atrial but not in ventricular myocytes: acute effects of mixed ET(A)/ET(B) and AT1 receptor antagonists in vivo and in vitro.

The precise role of paracrine and autocrine factors in mechanical load-induced activation of cardiac gene expression is unknown. Here we report the effects of endothelin-1 (ET-1) and angiotensin II (Ang II) receptor antagonism on acute pressure overload-induced activation of cardiac B-type natriuretic peptide (BNP) gene expression in spontaneously hypertensive rats (SHRs) in vivo and on mechanical stretch-induced increase in atrial BNP gene expression in vitro. Acute pressure overload produced in conscious SHRs by infusion of arginine8-vasopressin (0.05 microg x kg(-1) x min(-1)) for 2 hours resulted in an increase in BNP mRNA levels in the left ventricle as well as in the atrium. Bolus injections of bosentan (mixed ET(A)/ET(B) receptor antagonist, 10 mg/kg I.V.) but not losartan (AT1 receptor antagonist, 10 mg/kg I.V.) blocked the increase of the BNP mRNA levels produced by pressure overload in the left atria, whereas the elevation of BNP mRNA levels was similar (a 1.9-fold increase) in the left ventricles of vehicle-, losartan-, and bosentan-infused SHRs. In an isolated perfused rat heart preparation, infusion of bosentan (1 micromol/L) for 2 hours inhibited the mechanical stretch-induced increase in BNP mRNA levels in the right atria, whereas an AT1 receptor antagonist, CV-11974 (10 nmol/L), had no effect. The findings of the present study demonstrate that Ang II and ET-1 are not obligatorily required for stretch to trigger the increased BNP gene expression in ventricular myocytes in vivo. In contrast, mechanical load on the atrial myocytes did initiate an ET-1-dependent expression of BNP gene showing that endogenous ET-1 production differentially regulates BNP gene expression in atrial and ventricular myocytes.

Involvement of transcriptional and posttranscriptional mechanisms in cardiac overload-induced increase of B-type natriuretic peptide gene expression.

The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (P < .05) and 2.2-fold (P < .01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8 +/- 2.2 to 20.1 +/- 1.2 ng/mg, P < .05). Actinomycin D (10 micrograms/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (P < .05) and 2 hours (P < .001), whereas a protein synthesis inhibitor, cycloheximide (90 micrograms/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch-induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 mumol/L) and tyrphostin A25 (3 mumol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 mumol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These resuls demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.

Induction of cardiac natriuretic peptide gene expression in rats trained in hypobaric hypoxic conditions.

Adaptation of cardiac muscle to prolonged hypobaric hypoxia (770-740 mbar, 2,250-2,550 m), endurance training, and their combination was studied in rats by investigating the gene expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in atria and ventricles. Rats were assigned into the following groups according to the barometric conditions and physical activity; normobaric sedentary (NS), normobaric training, hypobaric sedentary (HS), and hypobaric training (HT). Experimental periods were 10, 21, and 56 days; the groups at 91 days served as recovery groups from exposure to and training in normobaric and hypobaric conditions for 56 days. The right ventricular hypertrophy in HT rats at 10 days and 56 days was associated with elevated BNP mRNA levels (2.1- and 1.7-fold, P < 0.05, respectively), whereas hypobaric exposure without training was not sufficient to significantly increase ventricular BNP gene expression, although it lead to hypertrophy of the right ventricle. Right and left atrial BNP mRNA levels were also increased (up to 3.9-fold, P < 0.01) in 10-day HS and 10-day HT groups. ANP mRNA levels in right ventricle and left ventricular epicardium were over twofold higher (P < 0.05-0.01) in 10-day HS and 10-day HT groups in comparison to 10-day NS group. Plasma immunoreactive ANP concentration was increased (P < 0.05) in both hypobaric groups up to 21 days. The results show that exposure to hypobaric hypoxia itself and endurance training in hypobaric, hypoxic conditions lead to a marked early increase in ventricular and atrial ANP and BNP mRNA levels. The adaptational response to hypoxia was more pronounced when the oxygen availability was lowered additionally by endurance training carried out in hypobaric hypoxic conditions.