Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that are involved in water and electrolyte homeostasis in heart failure. Although both hormones exert almost identical biological actions, the differential regulation of cardiac ANP and BNP mRNA in compensated and overt heart failure is not known. To study the hypothesis that cardiac BNP is more specifically induced in overt heart failure, a large aortocaval shunt of 30 days duration was produced in rats and compared with compensated heart failure. Compensated heart failure was induced either by a small shunt of 30 days duration or by a large shunt of 3 days duration. Both heart failure models were characterized by increased cardiac weight, which was significantly higher in the large-shunt model, and central venous pressure. Left ventricular end-diastolic pressure was elevated only in the overt heart failure group (control: 5.7 +/- 0. 7; small shunt: 8.6 +/- 0.9; large shunt 3 days: 8.5 +/- 1.7; large shunt 30 days: 15.9 +/- 2.6 mmHg; P < 0.01). ANP and BNP plasma concentrations were elevated in both heart failure models. In compensated heart failure, ANP mRNA expression was induced in both ventricles. In contrast, ventricular BNP mRNA expression was not upregulated in any of the compensated heart failure models, whereas it increased in overt heart failure (left ventricle: 359 +/- 104% of control, P < 0.001; right ventricle: 237 +/- 33%, P < 0.01). A similar pattern of mRNA regulation was observed in the atria. These data indicate that, in contrast to ANP, cardiac BNP mRNA expression might be induced specifically in overt heart failure, pointing toward the possible role of BNP as a marker of the transition from compensated to overt heart failure.

The atrial natriuretic peptide (ANP)-C receptor is generally believed to clear ANP; however, the ANP-C receptor may serve to reduce cAMP by inhibiting adenylate cyclase. ANP decreases endothelial permeability in coronary endothelial cell monolayers. We tested the hypothesis that part of this effect might be mediated by the ANP-C receptor. We used an endothelial cell monolayer from rat coronary endothelium and measured albumin flux. We applied either ANP or a ring-deleted ANP (C-ANP), which only stimulates the ANP-C receptor. ANP and C-ANP both decreased permeability from 100 pM to 100 nM by 60 and 30%, respectively. ANP increased endothelial cGMP contents 5.5-fold, whereas C-ANP had no effect. ANP reduced endothelial cAMP contents by 75%, which was only partly blocked by pertussis toxin. C-ANP also reduced cAMP; however, this effect was completely blocked by pertussis toxin. Protein kinase G inhibition blocked the ANP-mediated decrease in permeability by 50%. In contrast, pretreatment with pertussis toxin, in the face of protein kinase G inhibition, blocked the effect completely. C-ANP decreased permeability by half the amount of ANP. This C-ANP effect was completely blocked by pertussis toxin but not by protein kinase G inhibition. Isoproterenol (10 μM) increased permeability by almost 50%, which was completely blocked by ANP but only partially blocked by C-ANP. The C-ANP effect was blocked completely by pertussis toxin. Isoproterenol increased cAMP threefold, which was abolished by ANP. C-ANP reduced the isoproterenol-induced increase in cAMP by 50%. Isoproterenol had no effect on cGMP. We conclude that agonist binding to the ANP-C receptor inhibits cAMP production via a Giprotein-coupled signaling system. This inhibition may contribute to the decreased endothelial permeability evoked by ANP in this system.

Objecti¨e: Recently, deletion of specific genes by so called knock-out techniques has become important for investigating the pathogenesis of various diseases. This form of genetic engineering is widely performed in murine models. There are, however, only a limited number of mouse models available in cardiovascular pathology. The objective of this study, therefore, was to develop a new model of overt congestive heart failure associated with myocardial hypertrophy in the mouse. Methods: Female C57rBL6 mice weighing 19᎐20 g were anesthetized with ether. After abdominal incision, the aorta was temporarily clamped Ž . proximal to the renal arteries. The aorta was then punctured with a needle outer diameter 0.6 mm and the needle was further advanced into the adjacent vena cava. After withdrawal of the needle, the aortic puncture site was sealed with cyanoacrylate glue. The clamp was removed, and the patency of the shunt was visually verified as swelling and mixing of venous and arterial blood in the vena cava. Sham-operated mice served as controls. Results: Perioperative mortality of mice with aortocaval shunt was 42%. Four weeks after shunt induction, mice showed a significant cardiac hypertrophy with a Ž . relative heart weight of 7.5" 0.2 mgr100 g body weight vs. 5.1" 0.7 mgr100 g in control mice, P-0.001 . While no changes in blood pressure and heart rate occurred, left ventricular enddiastolic pressure was significantly increased in mice with shunt, Ž . and left ventricular contractility was impaired from 6331 " 412 to 4170 " 296 mmHgrs P -0.05 . Plasma concentrations of Ž . atrial natriuretic peptide ANP and its second messenger cGMP as humoral markers of heart failure as well as ventricular Ž . expression of ANP-and brain natriuretic peptide BNP -mRNA were significantly increased in mice with shunt compared to control mice. Conclusions: The aortocaval shunt in the mouse constitutes a new model of overt congestive heart failure with impaired hemodynamic parameters and may be a useful tool to investigate the role of particular genes in the development of heart failure. ᮊ

Background: The presence of apoptotic cell death in cardiac myocytes is now well established and the contribution of apoptosis for the development of heart failure has been suggested. However, the mechanism responsible for the induction of apoptosis remains unclear. The present study was designed to investigate the involvement of Fas and caspase 3 in the transition from pressure overload-induced left ventricular hypertrophy (LVH) to left ventricular dysfunction (LVD). Methods: Pressure overload induced LVH (10 days) and LVD (30 days) were induced by thoracic aortic banding. Changes in apoptosis-related genes were studied in rats with thoracic aortic banding. After 10 and 30 days, cardiac Fas mRNA expression was measured by RT-PCR. The mRNA expression of caspase 3 was detected by RNase protection assay. The activity of caspase 3 was measured by fluorometric assay. Protein levels of caspase 3 were measured by Western blot. Results: Rats with aortic banding had increased heart/body weight ratios after 10 and 30 days, compared to controls. Central venous pressure and lung weights were increased, left ventricular contractility was significantly impaired only in rats after 30 days of aortic banding, indicating LVD. Caspase 3 mRNA expression (7.1 F 0.1 vs. 2.8 F 0.4, P < 0.05), caspase 3 activity (1418 F 181 vs. 849 F 154 AU, P < 0.05) as well as caspase 3 protein levels were increased in rats with LVD but not with LVH. Similarly, Fas mRNA was increased in rats with LVD. Conclusions: The activation of Fas and caspase 3 only after 30 days of aortic banding suggests that induction of these pathways may be involved in pressure overload-induced LVD.

Abstract-Growth hormone (GH) application is a new strategy in the treatment of heart failure. However, clinical and experimental investigations have shown contradictory effects of GH on cardiac performance. We tested the hypothesis that GH could improve cardiac and renal function in volume overload-induced heart failure.

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In heart failure, sodium and water retention develop despite elevated plasma levels of atrial natriuretic peptide. Atrial natriuretic peptide is degraded in part by a neutral endopeptidase. Whether neutral endopeptidase inhibition improves sodium and water excretion in heart failure is unknown. We determined the effect of neutral endopeptidase inhibition on plasma levels of atrial natriuretic peptide and the renal response to acute volume expansion in rats with aortocaval shunts and in sham-operated controls. Acute endopeptidase inhibition with SQ 28,603 (30 mg/kg) elevated atrial natriuretic peptide plasma levels in both shunted rats (523 +/- 54 to 1258 +/- 330 pmol/L, P<.05) and controls (184 +/- 28 to 514 +/- 107 pmol/L, P<.05). Urinary cGMP excretion, which reflects renal action, increased in parallel. However, the diuretic and natriuretic responses to acute volume expansion were enhanced only in control rats and not in shunted rats. In contrast to the acute effects, chronic neutral endopeptidase inhibition with SCH 34826 (30 mg/kg twice daily) in shunted rats did not change atrial natriuretic peptide plasma levels or cGMP excretion. Nevertheless, the diuretic and natriuretic responses to acute volume load were increased by chronic endopeptidase inhibition in shunted rats (1789 +/- 154 to 2674 +/- 577 microL/80 min and 99 +/- 31 to 352 +/- 96 micromol/80 min, respectively; P<.05). Chronic endopeptidase inhibition attenuated the cardiac hypertrophic response to aortocaval shunt without changing arterial blood pressure. Our data show that the renal effects of neutral endopeptidase inhibition are not necessarily dependent on changes in atrial natriuretic peptide plasma levels but instead may be mediated by local inhibition of the neutral endopeptidase in the kidney. In addition, chronic endopeptidase inhibition may attenuate heart failure-induced cardiac hypertrophy independent of hemodynamic effects.

Natriuretic peptides are important regulators of vascular resistance and volume and electrolyte homeostasis. The quantification of natriuretic peptide receptor (NPR) mRNA is important for the understanding of the regulation of this humoral system, but is difficult due to low expression of the NPR mRNA. We report here on the evaluation of a polymerase chain reaction (PCR)-aided transcript titration assay for quantification of all three NPR subtypes (NPR-A, NPR-B, and NPR-C) mRNA. A multispecific internal standard RNA with parts of NPR-A, NPR-B, NPR-C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nucleotide sequences was constructed and reverse transcription of standard and sample RNA (400 ng) was performed in parallel for all three NPRs and GAPDH. The specific PCR yielded differently sized products, which were quantified by high performance liquid chromatography (HPLC). The determination of specific mRNA concentrations was not influenced by cDNA input and did not depend on the PCR cycle number. Linearity between sample RNA input and mRNA concentration was demonstrated. Application of the evaluated method showed that the NPR-A mRNA expression was the most abundant of the three natriuretic peptide receptor mRNAs in rat lungs, glomeruli and left ventricles, followed by the NPR-C mRNA and the NPR-B mRNA expression. Thus, the described method allows the reliable quantification of the specific mRNA expression of all three NPRs with small amounts of RNA. The presented method might foster future research on the regulation of this humoral system in cardiovascular and kidney diseases.