Left Ventricular Atrial Natriuretic Peptide Research Articles

Effect of cardiac resynchronization therapy on left ventricular fibrosis-related mRNA expression profile in patients with end-stage heart failure

Abstract Background When indicated, cardiac resynchronization therapy (biventricular pacing, CRT) decreases mortality in patients with heart failure (HF) and reduced ejection fraction, especially in those with non-ischemic cardiomyopathy. This is reflected by relatively rapid improvement of left ventricular (LV) end-diastolic diameter (LVEDD) and LV ejection fraction (LVEF) indicating reverse remodelling. These LV structural and functional improvements are accompanied by characteristic changes in LV gene expression profile. However, whether beneficial gene expression alterations related to biventricular pacing are sustained independently of structural and functional reverse remodelling is unclear. Purpose We aimed to compare LV fibrosis-related mRNA expression profile in end-stage HF patients with idiopathic dilated cardiomyopathy (DCM) who were not on CRT versus to those on CRT. Methods Left ventricular myocardial samples were harvested from end-stage HF patients undergoing heart transplantation (HTX). Inclusion criteria were negative family history of DCM, negative coronarography (i.e. non-ischemic), no relevant comorbidity (e.g. diabetes, hypertension) and no history of myocarditis. Accordingly, the following patient groups were included: 1.) DCM (n=12, 17% female, mean age [±standard deviation] 46.8±11.8 years) without CRT and 2.) CRT-DCM (n=12, 42% female, mean age 47.8±12.3 years) which comprised DCM patients on active CRT for mean 3.2±2.4 years until HTX. LV RNA was extracted and subjected to a commercially available mRNA expression panel interrogating 760 genes related to the development and regulation of fibrosis. Normalization to 10 housekeeping genes and batch corrections were conducted as per protocol. LV mRNA expression of atrial-natriuretic peptide (ANP) was quantified using qRT-PCR. Results Markers of reverse remodelling including LVEDD (73.4±8.3 mm vs 75.4±9.9 mm), LVEF (21.9±3.7% vs 18.5±6.8%) and LV ANP mRNA expression (arbitrary units: 1.05±1.80 vs 1.04±0.88) were comparable between DCM and CRT-DCM patients (all P>0.05), respectively. High-throughput mRNA expression screening revealed significant (all P<0.001) downregulation of 3 genes proven to be implicated in adverse LV remodelling: alpha catalytic subunit of protein phosphatase 2 (PPP2CA), interleukin 20 receptor subunit beta (IL20RB) and lipoprotein lipase (LPL). According to pathway analysis using directed significance scores, CRT was associated with collective upregulation of genes modifying complement activation (SERPING1, C1S, CFH) and collective downregulation of genes promoting cell proliferation (PPP2CA, ANAPC7, HSP90AA1, CSNK2B). Conclusions Independently of structural and functional reverse remodelling, CRT might be associated with slightly favourable LV expression profile of genes related to the regulation and development of fibrosis. This suggests that biventricular pacing might be beneficial on the molecular level beyond improvement of LV structure and function. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Fund of Hungary, Higher Education Institutional Excellence Programme of the Ministry of Human Capacities of Hungary

Metoprolol Treatment Lowers Thrombospondin‐4 Expression in Rats with Myocardial Infarction and Left Ventricular Hypertrophy

Thrombospondins are matrix proteins linked to extracellular matrix remodelling but their precise role in the heart is not known. In this study, we characterised left ventricular thrombospondin-1 and -4 expression in rats treated with a beta-blocker metoprolol during the remodelling process in response to pressure overload and acute myocardial infarction. Left ventricular thrombospondin-1 and thrombospondin-4 mRNA levels increased 8.4-fold (p < 0.001) and 7.3-fold (p < 0.001) post-infarction, respectively. Metoprolol infusion by osmotic minipumps (1.5 mg/kg/hr) for 2 weeks after myocardial infarction decreased thrombospondin-1 and thrombospondin-4 mRNA levels (55% and 50%, respectively), improved left ventricular function, and attenuated left ventricular remodelling with reduction of left ventricular atrial natriuretic peptide and brain natriuretic peptide gene expression. Thrombospondin-1 and -4 mRNA levels correlated positively with echocardiographic parameters of left ventricular remodelling as well as with atrial natriuretic peptide and brain natriuretic peptide gene expression. Moreover, there was a negative correlation between left ventricular ejection fraction and thrombospondin-1 mRNA levels. In 12-month-old spontaneously hypertensive rats with left ventricular hypertrophy, metoprolol decreased left ventricular thrombospondin-4 levels and attenuated remodelling while thrombospondin-1, atrial natriuretic peptide and brain natriuretic peptide mRNA levels as well as left ventricular function remained unchanged. In metoprolol-treated spontaneously hypertensive rats, thrombospondin-4 gene expression correlated with parameters of left ventricular remodelling, while no correlations between thrombospondins and natriuretic peptides were observed. These results indicate that thrombospondin-1 expression is linked exclusively to left ventricular remodelling process post-infarction while thrombospondin-4 associates with myocardial remodelling both after myocardial infarction and in hypertensive heart disease suggesting that thrombospondins may have unique roles in extracellular matrix remodelling process.

Treatment with atorvastatin partially protects the rat heart from harmful catecholamine effects

Atorvastatin blunts the response of cardiomyocytes to catecholamines by reducing isoprenylation of G gamma subunits. We examined whether atorvastatin exerts similar effects in vivo and protects the rat heart from harmful effects of catecholamines. Rats were treated with atorvastatin (1 or 10 mg/kg x day) or H(2)O for 14 days per gavage. All three animal groups were subjected to restraint stress on day 10 and to infusions of isoprenaline (ISO; 1 mg/kg x day) or NaCl via minipumps for the last 4 days. Heart rate was measured by telemetry, left ventricular atrial natriuretic peptide (ANP) transcript levels by RT-PCR, and left atrial contractile function in organ baths. Heart rate was similar in all six study groups. In animals pre-treated with water, infusion of ISO induced an increase in heart-to-body weight ratio (HW/BW) by approximately 20%, an increase in ANP mRNA by approximately 350%, and a reduction in the inotropic effect of isoprenaline in left atrium by approximately 50%. In animals pre-treated with high-dose atorvastatin, the effects of ISO on HW/BW, ANP, and left atrial force were approximately 40, 50, and 40% smaller, respectively. Low dose atorvastatin had similar, albeit smaller effects. Atorvastatin treatment of NaCl-infused rats had only marginal effects. In cardiac homogenates from atorvastatin-treated rats (both NaCl- and ISO-infused), G gamma and G alpha(s) were partially translocated from the membrane to the cytosol. In the rat heart, treatment with atorvastatin results in translocation of cardiac membrane G gamma and G alpha(s) to the cytosol. This mechanism might contribute to protecting the heart from harm induced by chronic isoprenaline infusion without affecting heart rate.

Atrial natriuretic peptide behaviour and myocyte hypertrophic profile in combined pressure and volume-induced cardiac hypertrophy

To investigate cardiomyocyte hypertrophy and hormonal profile in cardiac hypertrophy resulting from sequentially applied overloads. We studied Sprague-Dawley rats with renovascular hypertension (RV), where pressure overload predominates, or deoxycorticosterone acetate (DOCA)-salt (DS), where volume overload predominates, at 2 and 4 weeks of treatment, and the combination of both models in inverse sequence: RV 2 weeks/DS 2 weeks (RV2/DS2) and DS 2 weeks/RV 2 weeks (DS2/RV2), and their sham controls (Sh). Blood pressure and cardiomyocyte diameter increased to a similar extent in RV and DS at 2 and 4 weeks and in combined models. Cardiomyocyte length increased remarkably in the DS4 group. Circulating atrial natriuretic peptide (ANP) was elevated in all hypertensive groups after 2 and 4 weeks. The RV2/DS2 group showed similar plasma ANP levels to RV4, but DS2/RV2 exhibited a three-fold increase in ANP levels (P<0.001 versus Sh4, DS2 and DS4). Atrial ANP mRNA remained unchanged in all groups. DS treatment alone or in combination with RV increased left ventricular ANP mRNA, meanwhile only RV treatment increased left ventricular B-type natriuretic peptide (BNP) mRNA. Ventricular ANP expression levels, but not circulating ANP, correlated with both cardiomyocyte diameter (r=0.859, P<0.01) and length (r=0.848, P<0.01). Renal expression of natriuretic peptide receptor C (NPR-C) was unchanged in RV4 but decreased to a similar extent in the DS4 group and both combined treatments. Morphometric patterns seem to be more related to the paracrine function of the heart than to the secretion of ANP and the endocrine function. Plasma ANP in the DS2/RV2 group could indicate a different evolution of the remodelling process. ANP expression seems to be a more sensitive marker for volume than for pressure overload.

Involvement of endogenous ouabain-like compound in the cardiac hypertrophic process in vivo

The Na +/K +-ATPase inhibitor ouabain has been shown to trigger hypertrophic growth of cultured cardiomyocytes; however, the significance of endogenous ouabain-like compound (OLC) in the hypertrophic process in vivo is unknown. Here we characterized the involvement of OLC in left ventricular (LV) hypertrophy induced by norepinephrine (NE) and angiotensin II (Ang II) infusions in rats. Administration of NE (300 μg/kg/h) via subcutanously implanted osmotic minipumps for 72 h resulted in a significant increase in left ventricular weight to body weight (LVW/BW) ratio ( P < 0.001) and a substantial up-regulation of atrial natriuretic peptide (ANP) gene expression (13.2-fold, P < 0.001). NE infusion induced a transient increase in plasma OLC levels at 12 h ( P < 0.05), which returned to control levels by 72 h. Adrenalectomy markedly reduced both basal and NE-induced increase in plasma OLC levels. LVW/BW ratio was not modulated by adrenalectomy; however, ANP gene expression was blunted by 44% ( P < 0.01) and 47% ( P < 0.05) at 12 and 72 h, respectively. In agreement, adrenalectomy reduced up-regulation of ANP without affecting LV mass in rats infused with Ang II (33 μg/kg/h). Administration of exogenous ouabain (1 nM to 100 μM) for 24 h had no effect on ANP gene expression in cultured neonatal rat ventricular myocytes. However, the up-regulation of ANP mRNA levels induced by the α-adrenergic agonist phenylephrine (1 μM) was markedly enhanced by ouabain (100 μM) (5.6-fold vs. 9.6-fold, P < 0.01). These data show that OLC as an adrenal-derived factor may be required for the induction LV ANP gene expression during the hypertrophic process.

Rat chromosome 19 transfer from SHR ameliorates hypertension, salt-sensitivity, cardiovascular and renal organ damage in salt-sensitive Dahl rats

Unlike Dahl salt-sensitive (SS) rats, some strains of spontaneously hypertensive (SHR) rats develop only minor organ damage even when exposed to high-salt diet. In previous linkage studies, we identified quantitative trait loci on rat chromosome 19 (RNO19) linked to the SHR allele suggesting a protective effect against salt-induced hypertensive organ damage in SS. To test the relevance of this finding, we generated and characterized a consomic strain SS-19SHR in which RNO19 from SHR was introgressed into the susceptible background of SS. We compared the effects of low-salt (0.2% NaCl) and high-salt (4% NaCl) diet exposure for 8 weeks on the development of hypertension and target organ damage in male consomic and SS animals (n=14-20, each). Systolic blood pressure, relative left ventricular weight and urinary protein excretion were significantly lower in SS-19SHR compared to SS under both low-salt and high-salt diet (P < 0.05, respectively). Left ventricular atrial natriuretic peptide mRNA expression showed a more pronounced 4.5-fold increase in SS compared to SS-19 (two-fold) after high-salt (P < 0.05). In comparison to low diet, high-salt exposure induced a significant increase in vascular aortic hypertrophy index, left ventricular interstitial fibrosis (+210%) and perivascular fibrosis (+195%) in SS but not in consomic SS-19SHR (P < 0.05, respectively). These results demonstrate a strong protective effect of RNO19 from SHR on the development of hypertension, salt-sensitivity, cardiovascular and renal organ damage in SS. In particular, we demonstrate a genetic effect protecting against the development of cardiac fibrosis in salt-sensitive hypertension.

Additive effects of combined blockade of AT1receptor and HMG-CoA reductase on left ventricular remodeling in infarcted rats

Both angiotensin receptor antagonists and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to attenuate cardiomyocyte hypertrophy after myocardial infarction. Whether combination treatment may be superior to either drug alone on cardiomyocyte hypertrophy remains unclear. After ligation of the left anterior descending artery, rats were randomized to both, one, or neither of the angiotensin receptor antagonists olmesartan (0.01, 0.1, 1, and 2 mg.kg-1.day-1) and HMG-CoA reductase inhibitor pravastatin (5 mg.kg-1.day-1) for 4 wk. Each drug, when given alone, decreased cardiomyocyte sizes isolated by enzymatic dissociation at the border zone when compared with vehicles. However, compared with either drug alone, combined olmesartan and pravastatin prevent cardiomyocyte hypertrophy to a larger extent, which was further confirmed by downregulation of the left ventricular atrial natriuretic peptide mRNA. The myocardial endothelin-1 levels at the border zone were 6.5-fold higher (P<0.0001) in the vehicle group compared with the sham group, which can be inhibited after pravastatin administration. Combination treatment significantly attenuated cardiomyocyte hypertrophy in a dose-dependent manner, although tissue endothelin-1 levels remained stable in combination groups of different olmesartan doses. Measurements of the arrhythmic score mirrored those of cardiomyocyte hypertrophy. Dual therapy with pravastatin and olmesartan, which produced an additive reduction in cardiomyocyte hypertrophy and cardiac fibrosis after myocardial infarction through different mechanisms, decreases the propensity of the heart to arrhythmogenesis. Pravastatin administration provided favorable ventricular remodeling, probably through decreased tissue endothelin-1 level. In contrast, olmesartan-related attenuated cardiomyocyte hypertrophy is independent of endothelin-1 pathway.

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.

Aliskiren, a Human Renin Inhibitor, Ameliorates Cardiac and Renal Damage in Double-Transgenic Rats

We tested the hypothesis that the renin inhibitor aliskiren ameliorates organ damage in rats transgenic for human renin and angiotensinogen genes (double transgenic rat [dTGR]). Six-week-old dTGR were matched by albuminuria (2 mg per day) and divided into 5 groups. Untreated dTGR were compared with aliskiren (3 and 0.3 mg/kg per day)-treated and valsartan (Val; 10 and 1 mg/kg per day)-treated rats. Treatment was from week 6 through week 9. At week 6, all groups had elevated systolic blood pressure (BP). Untreated dTGR showed increased BP (202+/-4 mm Hg), serum creatinine, and albuminuria (34+/-5.7 mg per day) at week 7. At week 9, both doses of aliskiren lowered BP (115+/-6 and 139+/-5 mm Hg) and albuminuria (0.4+/-0.1 and 1.6+/-0.6 mg per day) and normalized serum creatinine. Although high-dose Val lowered BP (148+/-4 mm Hg) and albuminuria (2.1+/-0.7 mg per day), low-dose Val reduced BP (182+/-3 mm Hg) and albuminuria (24+/-3.8 mg per day) to a lesser extent. Mortality was 100% in untreated dTGR and 26% in Val (1 mg/kg per day) treated rats, whereas in all other groups, survival was 100%. dTGR treated with low-dose Val had cardiac hypertrophy (4.4+/-0.1 mg/g), increased left ventricular (LV) wall thickness, and diastolic dysfunction. LV atrial natriuretic peptide and beta-myosin heavy chain mRNA, albuminuria, fibrosis, and cell infiltration were also increased. In contrast, both aliskiren doses and the high-dose Val lowered BP to a similar extent and more effectively than low-dose Val. We conclude that in dTGR, equieffective antihypertensive doses of Val or aliskiren attenuated end-organ damage. Thus, renin inhibition compares favorably to angiotensin receptor blockade in reversing organ damage in dTGR.

Differential expression of cardiac ANP and BNP in a rabbit model of progressive left ventricular dysfunction.

Activation of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) is considered a hallmark of myocardial remodeling. To determine magnitude and relative proportion of activation during the progression to heart failure, we assessed ANP and BNP gene expression in atrial and left ventricular (LV) tissue in a newly developed model of progressive rapid ventricular pacing-induced heart failure in rabbits. Six animals underwent progressive pacing with incremental rates (330 beats per min (bpm) to 380 bpm over 30 days), resulting in congestive heart failure (CHF). Five animals underwent pacing at 330 bpm for 10 days only (early LV dysfunction, ELVD) and five additional animals served as control group (CTRL). ELVD was characterized by decreased mean arterial pressure (P=0.05 vs. CTRL) as well as significantly impaired LV function (LV fractional shortening (FS) P<0.01 vs. CTRL) and dilatation (P<0.01 vs. CTRL). CHF was characterized by further decreased mean arterial pressure (P<0.01 vs. ELVD), further impaired LV function (FS P<0.03 vs. ELVD) and dilatation (P<0.01 vs. CTRL). In control animals, significant ANP expression was observed only in atrial tissue (P<0.02 vs. BNP) while BNP expression was ubiquitous but marginal (LV P<0.05 vs. ANP). In ELVD, activation of ANP (atria and LV P<0.05 vs. CTRL) and BNP (atria P<0.05 vs. CTRL, LV n.s.) was observed. In CHF, LV-BNP increased further markedly (P<0.01 vs. CTRL, P<0.05 vs. ELVD) while atrial ANP and BNP expression as well as LV ANP expression remained unchanged (all P=n.s. vs. ELVD). The current studies demonstrate differential activation of atrial and LV ANP and BNP under normal conditions and during the progression to heart failure and provide a molecular basis for the superiority of BNP as marker of LV dysfunction and CHF.

Relationship between left ventricular wall stress and ANP gene expression during the evolution of rapid ventricular pacing-induced heart failure in the dog.

We have recently described a modified model of progressive rapid ventricular pacing-induced heart failure which evolves over a period of 38 days. To further characterize left ventricular remodeling during the progression of heart failure, we assessed left ventricular geometry, wall stress, and atrial natriuretic peptide (ANP) gene expression and protein content during control conditions, asymptomatic left ventricular dysfunction, and overt congestive heart failure (CHF). Although asymptomatic left ventricular dysfunction was characterized by a significant increase in systolic and diastolic left ventricular dimension (+30% and +6%, respectively, P<0.05 each) and a marked increase in left ventricular systolic wall stress (+68%, P<0.01), left ventricular ANP gene expression was unchanged as compared to control. In contrast, strong left ventricular ANP gene expression (+449%, P<0.05) was observed during overt CHF in the absence of further significant increases in left ventricular systolic wall stress. The onset of strong left ventricular ANP gene expression was associated with increased ANP content (+88%, P<0.05) and left ventricular mass index (+13%, P<0.05). In contrast, left atrial ANP gene expression and left ventricular diastolic wall stress increased progressively during asymptomatic left ventricular dysfunction (+39%, P=n.s. and +131%, P<0.01) and overt CHF (+76% and +336% vs. control, P<0.01 each). Progressive rapid ventricular pacing is associated with the induction of left ventricular ANP gene expression and protein synthesis exclusively during overt CHF. The current studies provide new insight into the temporal pattern of ANP-activation and the disparity between left ventricular systolic wall stress and ANP-activation in a large animal model of progressive CHF.

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.

Effect of Ovariectomy and Estrogen Replacement on Cardiovascular Disease in Heart Failure-Prone SHHF/Mcc- fa cp Rats

The importance of endogenous and exogenous estrogen levels to the development of cardiovascular disease in women in controversial. The purpose of our study was to examine the effect of estrogen on the development of hypertension, cardiac hypertrophy, ventricular function, and gene expression for atrial natriuretic peptide (ANP) and components of the renin angiotensin system in spontaneously hypertensive heart failure rats (SHHF/Mcc- facp). Development of hypertension was prevented in 3-month-old ovariectomized rats receiving subcutaneous 17 β -estradiol implants (EST) compared to ovariectomized (OVX) and controls (CON). EST had the least left ventricular hypertrophy, CON were intermediate, and OVX had the most (P<0.05), correlating well with systolic blood pressure. OVX had significantly lower percentage V1myosin isoform compared to EST and CON, indicating reversion to a more immature phenotype associated with hypertrophy. Similarly, OVX had decreased percentage left ventricular shortening fraction by echocardiography compared to EST and CON. These changes were not accompanied by alterations in plasma ANP, or in expression of mRNA for left ventricular ANP, renal renin, or hepatic angiotensinogen. Serum angiotensin converting enzyme activity was lower in EST compared to CON or OVX. When 17β -estradiol was given to 17-month-old rats that had naturally ceased estrous cycling, there was no effect on hypertension, progression of cardiac functional decline, or survival. In conclusion, estradiol treatment given prior to the development of hypertension in SHHF prevented left ventricular hypertrophy and hypertension. Development of congestive heart failure was not delayed if 17 β -estradiol was begun in the post-menopausal period. Effectiveness of estrogen therapy may depend on age or whether hypertension is already established at the time treatment is begun.

Regulation of the rat atrial natriuretic peptide gene after acute imposition of left ventricular pressure overload.

The upregulation of left ventricular (LV) atrial natriuretic peptide (ANP) mRNA is a highly conserved marker of cardiac hypertrophy. The aim of this study was to further examine the pathway leading to ANP induction during pressure overload of the heart. Systolic wall stress was imposed acutely on isovolumetrically beating rat hearts in a Langendorff apparatus (sigma-=300 x 10[3] dyne/cm2). Northern and Western blots revealed that elevated wall stress induced LV c-fos and c-jun mRNAs (3.5- and 3-fold, P<.05 after 60 minutes), c-Fos and c-Jun proteins (3.9- and 4.3-fold, P<.05 after 120 minutes), as well as ANP mRNA (2.2-fold, P<.05 after 120 minutes). ANP upregulation was prevented by inhibition of protein synthesis (cycloheximide). Electrophoresis mobility shift assays were performed to link c-Fos and c-Jun (ie, components of the heterodimeric transcription factor AP-1) and ANP induction. A putative AP-1 binding site within the rat ANP promoter (nucleotides -512 to -473) bound specifically to nuclear proteins of wall stress-stimulated hearts. Antibodies directed against c-Fos protein resulted in a shift of this DNA/protein complex, suggesting physical interaction between AP-1 and the ANP promoter. Myocardial transfection of promoter constructs revealed that after acute imposition of wall stress, this AP-1 site enhanced a reporter gene (8- to 10-fold compared with a minimal promoter, P<.05). Interestingly, nuclear extracts of stimulated hearts as well as pure AP-1 protein bound to a putative CRE site (nucleotides -613 to -584) as well. Like the AP-1 site, this cAMP-responsible element (CRE) site was found to enhance the transfected ANP promoter/reporter gene significantly (17.5-fold, P<.05). Mutation of either AP-1 or CRE sites did not decrease reporter gene activity, whereas mutation of both resulted in loss of inducibility. These experiments suggest that LV ANP regulation after acute wall stress includes the activation of AP-1 and/or CRE cis acting elements. However, the transient nature of c-fos and c-jun upregulation also suggests that AP-1 is not the only mediator of ANP induction in LV hypertrophy.

Angiotensin II receptor gene expression in hypertrophied left ventricles of rat hearts

To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventricles of rats subjected to ventricular pressure overloading induced by aortic banding for 6 weeks and then 6 weeks medical treatment. Aortic banding was related to an increase in relative weight of the left ventricle from 1.73 +/- 0.06 (sham-operated) to 2.81 +/- 0.25 g/kg, an increase in beta-myosin: alpha-myosin messenger RNA (mRNA) ratio from 0.30 +/- 0.02 to 1.94 +/- 0.55 and an 18-fold increase in left ventricular atrial natriuretic peptide mRNA levels. In contrast, left ventricular pressure overload hypertrophy was not related to a significant change in the abundance of AT1a and AT1b mRNA, which were expressed in a relative ratio of 5:1. Similarly, the abundance of AT2 mRNA was not significantly changed in hypertrophied ventricles. In rats receiving the angiotensin II AT, receptor antagonist losartan (40 mg/kg) for 6 weeks after banding, relative heart weights were 2.39 +/- 0.14 g/kg, the beta-myosin: alpha-myosin ratio was 1.04 +/- 0.20 and atrial natriuretic peptide mRNA levels displayed a blunted increase (11-fold over sham-treated controls), documenting a significant amelioration of left ventricular hypertrophy by blockade of the AT1 receptor. Losartan treatment in parallel did not affect AT1a, AT1b and AT2 receptor mRNA levels, which were not different from those in vehicle-treated or sham-treated controls. These findings confirm that left ventricular hypertrophy in the rat is associated with increased ventricular expression of beta-myosin and of atrial natriuretic peptide and with reduced expression of alpha-myosin. Despite these significant changes in cardiac gene expression no alteration was observed in AT1a, AT1b and AT2 receptor mRNA levels.

Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction.

We have demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone predominantly synthesized in and secreted from the ventricle. We have also reported that, compared with atrial natriuretic peptide (ANP), the plasma concentration of BNP is increased to a greater degree in patients with congestive heart failure and more rapidly in patients with acute myocardial infarction (AMI). To investigate ventricular gene expression of BNP in AMI, we analyzed plasma and ventricular BNP concentrations along with ventricular BNP mRNA in rats with AMI produced by coronary artery ligation. The BNP concentration in the left ventricle increased about 2-fold as early as 12 hours postinfarction and 5-fold 1 day postinfarction compared with sham-operated rats, whereas left ventricular ANP concentration remained unchanged within 1 day. The tissue concentration of BNP increased in the noninfarcted region as well as in the infarcted region. The surviving myocytes in and around the necrotic tissues in the infarcted region were intensely stained with the anti-BNP antiserum, indicating augmented production in the remaining myocytes in the infarcts. The BNP concentration in the right ventricle also increased about 10-fold 12 hours postinfarction, whereas the ANP concentration remained unchanged within 12 hours. Northern blot analysis revealed that BNP mRNA expression was augmented 3-fold in the left ventricle as early as 4 hours postinfarction. In contrast, ANP mRNA expression was unchanged. Reflecting the rapid induction of ventricular BNP production, the plasma BNP concentration rose to about 100 pg/mL 12 hours postinfarction (sham-operated rats, < 70 pg/mL). These results demonstrate the rapid induction of ventricular BNP gene expression in rats with AMI compared with ANP and suggest that BNP gene expression in the ventricle is regulated distinctively from ANP gene expression against acute ventricular overload. They also suggest that the BNP gene can be one of the acutely responsive cardiac genes for the ventricular overload and suggest a possible pathophysiological role of BNP distinct from ANP in AMI.

Blockade of the renin-angiotensin system in cardiac pressure-overload hypertrophy in rats.

Left ventricular hypertrophy in response to pressure overload may be modified by neurohumoral activation. To investigate the contribution of the renin-angiotensin system, we studied rats after banding of the ascending aorta that developed severe left ventricular hypertrophy associated with normal plasma renin but elevated cardiac angiotensin-converting enzyme (ACE) levels. Rats were treated with vehicle, ACE inhibitor (ramipril), angiotensin II type 1 receptor antagonist (losartan), or vasodilator (hydralazine) during weeks 7 through 12 after aortic banding. A significant regression of left ventricular mass index as determined by serial echocardiography was observed in ramipril- and losartan-treated groups during weeks 9 through 12 after banding, whereas hypertrophy further increased in vehicle- and hydralazine-treated groups. Twelve weeks after banding, relative left ventricular weights and myocyte widths were markedly increased in vehicle- and hydralazine-treated groups, whereas ramipril and losartan significantly reduced these parameters. In addition, molecular adaptations in left ventricular hypertrophy, such as upregulation of left ventricular atrial natriuretic peptide and downregulation of sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels, were blunted by ramipril or losartan treatment. Hypertrophic regression was associated with reduced mortality in rats treated with ramipril (11%) and losartan (13%) versus hydralazine (20%) and vehicle (31%). Thus, the renin-angiotensin system may be involved in the maintenance of chronic left ventricular hypertrophy. Blockade of the system may result in regression of the hypertrophic phenotype and improve survival in rats despite persistent pressure overload.

Changes of atrial natriuretic peptide and its messenger RNA with development and regression of cardiac hypertrophy in renovascular hypertensive rats.

We assessed the changes in atrial natriuretic peptide (ANP) and its messenger RNA (mRNA) levels in atria and ventricles in relation to hemodynamic factors during antihypertensive treatments in two-kidney, one-clip renovascular hypertensive rats (RHRs). Hypertension of 10-week duration caused a twofold increase in the left ventricular weight/body weight ratio, a significant increase in left ventricular end-diastolic pressure, and an eightfold increase in left ventricular ANP mRNA levels in RHRs, as compared with the levels in control rats. Uninephrectomy or 4 weeks of treatment with the converting enzyme inhibitor enalapril reduced the blood pressure to the control level, with the complete reversal of left ventricular hypertrophy, left ventricular end-diastolic pressure, and ANP mRNA levels. Four weeks of treatment with the arterial vasodilator hydralazine significantly, but not completely, reduced the high blood pressure, but it did not influence left ventricular hypertrophy, end-diastolic pressure, and ANP mRNA levels. The increased ANP synthesis observed in the right ventricles of RHRs also reverted to the control level by uninephrectomy or enalapril treatment, but not by hydralazine, with a time course similar to that of left ventricular ANP. In addition, uninephrectomy caused the left and right ventricular ANP and ANP mRNA levels of RHRs to fall to the levels of control rats as early as 1 week, despite persistent left ventricular hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

Atrial Natriuretic Peptide in Plasma, Atria, Ventricles, and Hypothalamus of Long-Evans and Vasopressin- Deficient Brattleboro Rats*

To evaluate the role of vasopressin in the regulation of atrial natriuretic peptide (ANP) secretion, the plasma, atrial, ventricular, and hypothalamic levels of ANP were measured in Long-Evans (LE) and vasopressin-deficient Brattleboro (DI) rats. Total atrial immunoreactive ANP (IR-ANP) as well as right auricular IR-ANP concentration were higher in the DI than in the LE rats, whereas no significant difference was noted in left auricular IR-ANP concentration. In the left ventricle of DI rats, the IR-ANP concentration was 82% greater than that in the LE rats, while no substantial difference was found in the right ventricular IR-ANP concentration between the strains. Normal LE rats had low levels of left ventricular ANP mRNA and barely detectable ANP mRNA in the right ventricle, DI rats showed a 3-fold greater ANP mRNA concentration in the left ventricle than age-matched LE controls, and ANP mRNA levels were also increased in the left auricle of DI rats. The hypothalamic IR-ANP content, but not the concentration, was significantly increased in the DI compared to the LE rats. Despite increased cardiac IR-ANP and ANP mRNA levels, plasma IR-ANP concentrations were similar in the conscious DI rats (97 +/- 9 pg/ml; n = 13) and the LE rats (95 +/- 8 pg/ml; n = 15). Volume expansion (1.1 ml/100 g BW of 0.9% saline, iv) increased right atrial pressure and caused a significant rise in plasma IR-ANP in both strains (P less than 0.01). Elevations of plasma IR-ANP concentrations caused by volume loading were comparable in LE and DI rats in either the absence or presence of exogenous vasopressin (5 ng/kg.min, iv), which, when infused alone, did not significantly influence the plasma IR-ANP concentration. However, the relation between the change in IR-ANP concentration and the change in right atrial pressure shifted to the left, and thus, for a given increase in right atrial pressure, a greater amount of IR-ANP was released in the vasopressin-treated rats than in the control animals. These results demonstrate that although acute volume expansion does not necessarily require endogenous vasopressin for the ANP secretory response, vasopressin increased the ability of volume expansion to induce ANP release, thus modulating the direct mechanical stimulus-induced ANP secretion. The increased left ventricular levels of immunoreactive ANP and augmentation of ANP mRNA levels in Brattleboro rats despite normal left ventricular weight to body weight ratio show that increased ANP gene expression may occur in the ventricles independently of hypertrophy.

Atrial natriuretic peptide in a rat model of cardiac failure. Atrial and ventricular mRNA, atrial content, plasma levels, and effect of volume loading.

This study examined the relation between synthesis, atrial storage, and plasma levels of atrial natriuretic peptide (ANP), and it examined plasma ANP levels and hemodynamic output in response to volume expansion in a rat model of myocardial infarction and failure. Arterial ANP concentrations did not correlate linearly with infarct size, but they did show an abrupt increase when infarct size exceeded 30% of the left ventricle, similar to the abrupt increase of left ventricular end-diastolic pressure with infarct size greater than 30%. Consequently, a close relation was found between plasma ANP levels and left ventricular end-diastolic pressure (n = 23, r = 0.89, p less than 0.001). Atrial ANP content per gram of tissue but not ANP content per pair of atria was reduced in rats with large infarcts (greater than 40%, p less than 0.05 vs. control animals). ANP mRNA level per pair of atria (related to total atrial RNA), determined by liquid hybridization (controlled by northern blot analysis), was increased by 38% in infarcted rats (p less than 0.05 vs. controls), but the ratio of atrial ANP mRNA relative to atrial beta-actin mRNA levels was not increased. Right and left ventricular ANP mRNA level increased by 90% and 380%, respectively, far exceeding the concomitant increase in beta-actin mRNA (+26% in the left ventricle). Plasma ANP increased with volume loading in controls and rats with moderate infarcts but not in rats with large infarcts despite a similar increase in right atrial pressure (compared with control animals); thus, the relation of delta ANP/delta right atrial pressure exerted by volume loading decreased in rats with large infarcts. Similarly, the response of cardiac output and renal blood flow (determined by radioactive microspheres) to volume loading was attenuated in rats with large infarcts. Thus, in this model of chronic cardiac failure, the activation of the ANP system is closely coupled with the increase in intracardiac pressures without correlating linearly to the extent of myocardial loss. Second, in severe cardiac failure, additional stimulation such as volume loading may elicit only an attenuated ANP secretion response, for example, due to saturation of the ANP receptor sensing system or to a limited transformation rate of pro-ANP. Third, the increase in atrial ANP synthesis and the increase in atrial ANP gene expression seems limited; however, substantial specific ANP gene expression occurs in the ventricles, which, in turn, may contribute to increased plasma ANP levels in chronic heart failure.