No abstract

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.

No abstract

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell-type-specific functions. In the human heart, we observed an endogenous carboxyl-terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z-line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl-terminal ahnak fragment (ahnak-C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak-C2 to actin filaments induced filament bundling into paracrystalline-like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak-C2 attenuated the decline in isometric force development upon repeated contraction-relaxation cycles. Our results suggest that the carboxyl-terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Stored cardiac pro-atrial natriuretic peptide (pro-ANP) is converted to ANP and released upon stretch from the atria into the circulation. Corin is a serin protease with pro-ANP-converting properties and may be the rate-limiting enzyme in ANP release. This study was aimed to clone and sequence corin in the rat and to analyze corin mRNA expression in heart failure when ANP release upon stretch is blunted. Full-length cDNA of rat corin was obtained from atrial RNA by RT-PCR and sequenced. Tissue distribution as well as regulation of corin mRNA expression in the atria were determined by RT-PCR and RNase protection assay. Heart failure was induced by an infrarenal aortocaval shunt. Stretch was applied to the left atrium in a working heart modus, and ANP was measured in the perfusates. The sequence of rat corin cDNA was found to be 93.6% homologous to mouse corin cDNA. Corin mRNA was expressed almost exclusively in the heart with highest concentrations in both atria. The aortocaval shunt led to cardiac hypertrophy and heart failure. Stretch-induced ANP release was blunted in shunt animals (control 1,195 +/- 197 fmol.min(-1).g(-1); shunt: 639 +/- 99 fmol.min(-1).g(-1), P < 0.05). Corin mRNA expression was decreased in both atria in shunt animals [right atrium: control 0.638 +/- 0.004 arbitrary units (AU), shunt 0.566 +/- 0.014 AU, P < 0.001; left atrium: control 0.564 +/- 0.009 AU, shunt 0.464 +/- 0.009 AU, P < 0.001]. Downregulation of atrial corin mRNA expression may be a novel mechanism for the blunted ANP release in heart failure.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

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-Natriuretic peptides mediate their physiologic effects through activation of membrane-bound, guanylyl cyclase-coupled receptors (NPRs). Receptor dimerization is an important feature of signal transduction. This study was aimed at characterizing structurally important residues of the extracellular ligand-binding domain of NPR-B for receptor dimerization and cGMP generation. Deletion mutagenesis was used to replace cysteine residues at positions 53 (C53S), 417 (C417S), and 426 (C426S) by serine. Receptor expression, dimerization, whole-cell cGMP response, and guanylyl cyclase activity of membrane fractions were determined in stably transfected COS-7 cells. C53S, C417S, and C426S mutants were expressed and found to form disulfide-bridged covalent dimers. In contrast to NPR-B and C53S, C417S and C426S mutants displayed constitutive activity in whole cells (C417S, 146Ϯ12%, PϽ0.01; C426S, 153Ϯ7% of ligand-independent NPR-B cGMP generation, PϽ0.01). The cGMP response of C417S and C426S mutants in whole cells was dose dependent and Ϸ4 times lower than that in NPR-B, whereas it was blunted in C53S-transfected cells (1 mol/L CNP, NPR-B 2868Ϯ436%; C53S, 206Ϯ16% of control, PϽ0.001 vs NPR-B, C417S, and C426S). Guanylyl cyclase assay in transfected cells confirmed the constitutive activity of C417S and C426S mutants. These data suggest that receptor dimerization by covalent disulfide bridges alters ligand-independent as well as ligand-dependent receptor activity. Localization of the crosslink in relation to the cell membrane is important for configuration of the extracellular domain and the consecutive signal transduction.