Impact of AT2 receptor deficiency on postnatal cardiovascular development.

BACKGROUND: The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of α-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca(2+) transient in response to isoprenaline when stimulated concomitantly with angiotensin II. CONCLUSION: The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.

Dual mechanisms of HNO generation by a nitroxyl prodrug of the diazeniumdiolate (NONOate) class.

Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)═NO(-)Na(+) (1, IPA/NO) with BrCH(2)OAc produced a stable derivative of structure iPrHN-N(O)═NO-CH(2)OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH(3)-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and ß-adrenergically desensitized ventricular myocytes.

Contractile dysfunction and diminished response to ß-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 µg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca2+ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by+40-200% in a concentration-dependent manner (EC50 ∼55 µM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute ß-adrenergic stimulation with ISO (1 µM). NCA (60 µM) increased peak sarcomere shortening and Ca2+ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca2+ sensitivity and sarcoplasmic reticulum (SR) Ca2+ cycling. Importantly, NCA did not affect diastolic Ca2+ or SR Ca2+ content, as assessed by rapid caffeine application. NCA (45 µM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and ß-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.