Tonino Stati

Na+/H+ exchange inhibition attenuates left ventricular remodeling and preserves systolic function in pressure‐overloaded hearts

Cardiac hypertrophy is a homeostatic response to elevated afterload. Na+/H+ exchanger (NHE) inhibition reduces the hypertrophic response in animal models of left ventricular hypertrophy (LVH) and myocardial infarction. We examined the effect of chronic treatment with cariporide, a selective inhibitor of Na+/H+ exchanger isoform 1 (NHE‐1), on left ventricular (LV) systolic and diastolic function under pressure overload conditions. Male CD‐1 mice were randomized to receive either a control diet or an identical diet supplemented with 6000 p.p.m. of cariporide. Cardiac pressure overload was induced by thoracic aortic banding. LV dimension and systolic and diastolic function were assessed in sham and banded mice by echocardiography and cardiac catheterization 2 and 5 weeks after surgery. Histological analysis was also performed. After 2 weeks of pressure overload, the vehicle‐treated banded mice (Veh‐Bd) had enhanced normalized LV weight (about +50%) and normal chamber size and function, whereas cariporide‐treated banded mice (Car‐Bd) showed a preserved contractility and systolic function despite a marked attenuation of LVH. Diastolic function did not differ significantly among groups. After 5 weeks, the Veh‐Bd developed LV chamber enlargement and systolic dysfunction as evidenced by a 16% increase in LV end‐diastolic diameter, a 36% decrease in myocardial contractility, and a 26% reduction in percent fractional shortening. In contrast, Car‐Bd showed an attenuated increase in LV mass, normal chamber size, and a maintained systolic function. A distinct histological feature was that in banded mice, cariporide attenuated the development of cardiomyocyte hypertrophy but not the attendant myocardial fibrosis. In conclusion, the results of the present study indicate that (i) the hypertrophic response to pressure overload is dependent on NHE‐1 activity, and (ii) at the 5‐week stage, banding‐induced deterioration of LV performance is prevented by NHE‐1 inhibition.

Pressure overload causes cardiac hypertrophy in β1-adrenergic and β2-adrenergic receptor double knockout mice

Objective Cardiac hypertrophy arises as an adaptive response to increased afterload. Studies in knockout mice have shown that catecholamines, but not α1-adrenergic receptors, are necessary for such an adaptation to occur. However, whether β-adrenergic receptors are critical for the development of cardiac hypertrophy in response to pressure overload is not known at this time. Methods and results Pressure overload was induced by transverse aortic banding in β1-adrenergic and β2-adrenergic receptor double knockout (DβKO) mice, in which the predominant cardiac β-adrenergic receptor subtypes are lacking. Chronic pressure overload for 4 weeks induced cardiac hypertrophy in both DβKO and wild-type mice. There were no significant differences between banded mice in left ventricular weight to body weight ratio, in the left ventricular wall thickness, in the cardiomyocyte size or in the expression levels of the load-sensitive cardiac genes such as ANF and β-MHC. Additionally, the left ventricular systolic pressure, an index of afterload, and cardiac contractility, evaluated as dp/dtmax, the maximal slope of systolic pressure increment, and Ees, end-systolic elastance, were increased at a similar level in both wild-type and DβKO banded mice, and were significantly greater than in sham controls. Conclusion Despite chronic activation of the cardiac β-adrenergic system being sufficient to induce a pathological hypertrophy, we show that β1-adrenergic and β2-adrenergic receptors are not an obligatory component of the signaling pathway that links the increased afterload to the development of cardiac hypertrophy.

Propranolol causes a paradoxical enhancement of cardiomyocyte foetal gene response to hypertrophic stimuli.

Pathological cardiac hypertrophy is associated with the expression of a gene profile reminiscent of foetal development. The non selective β‐adrenoceptor antagonist propranolol is able to blunt cardiomyocyte hypertrophic response in pressure‐overloaded hearts. It remains to be determined whether propranolol also attenuates the expression of hypertrophy‐associated foetal genes.

cAMP-mediated β-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes

The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.

Propranolol promotes Egr1 gene expression in cardiomyocytes via β-adrenoceptors

Recent research has revealed that propranolol, a beta-adrenoceptor antagonist, causes extracellular signal-regulated kinase (ERK) cascade activation, nuclear translocation of phospho-ERK and increased transcriptional activity in cultured cell lines. Given the importance of beta-adrenoceptor antagonists in the treatment of heart failure, we evaluated the capability of propranolol of promoting the ERK-dependent gene expression at the cardiomyocyte level. To this end, the gene expression of the early growth response factor 1 (Egr1), a well-recognized indicator of nuclear extracellular signal-regulated kinase 1/2 (ERK1/2) activation, was assessed by quantitative real-time RT-PCR in vivo as well as in vitro experiments. Propranolol, administered at the dose of 10 mg/kg/day in C57BL/6 mice, caused a approximately 19-fold increase of Egr1 mRNA expression in left ventricular myocardium along with a approximately 2.1-fold increase of Egr1 protein expression. Isoproterenol, a nonselective beta-adrenoceptor agonist, also increased Egr1 mRNA and protein expression but to a lesser degree. Remarkably, isoproterenol administration was associated with the development of cardiac hypertrophy, whereas propranolol-treated mice showed a completely normal cardiac morphology. The effect of propranolol on Egr1 mRNA expression was abrogated in mice lacking beta(1)- and beta(2)-adrenoceptors indicating that propranolol increases Egr1 mRNA expression in a beta-adrenoceptor-dependent manner. The role of beta-adrenoceptors was further confirmed by showing that propranolol was able to increase Egr1 mRNA and protein levels in cultured neonatal cardiomyocytes. Collectively, these results indicate that propranolol promotes Egr1 gene expression in cardiomyocytes via beta-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes.

Nitric oxide (NO) modulation of PAF‐induced cardiopulmonary action: interaction between NO synthase and cyclo‐oxygenase‐2 pathways

To further investigate into the mechanisms of PAF‐induced cardiopulmonary actions, we examined the effects of the nitric oxide synthase (NOS) inhibitor L‐Nω‐nitro‐L‐arginine (L‐NNA), of the specific cyclooxygenase‐2 (COX‐2) inhibitor NS 398, and of the combined presence of both COX and NOS inhibitors on the PAF responses in the heart lung preparation of guinea‐pig (HLP). In HLPs perfused with homologous blood, dose‐response curves for the haemodynamic and bronchial effects of PAF (1 – 32 ng) were carried out in the absence or presence of L‐NNA (200 μM). L‐NNA caused an increase in the resting pulmonary arterial pressure (PAP) without affecting the other basal values, and strongly potentiated the bronchoconstriction and pulmonary hypertension elicited by PAF. An enhancement of the PAF‐induced actions on right atrial pressure (RAP) and cardiac output (CO) was also observed. All the effects of L‐NNA were antagonized by L‐arginine (2 mM). The presence of L‐NNA in the perfusing blood of HLPs failed to affect the pulmonary hypertensive and bronchoconstrictor responses induced by the thromboxane A2 mimetic U46619 (0.05 – 1.6 μg), 5‐hydroxytryptamine (0.1 – 1.6 μg), and histamine (0.1 – 1.6 μg), thus suggesting that these PAF secondary mediators are not responsible for the hyper‐responsiveness to PAF induced by L‐NNA. Blocking COX‐2 pathway with NS 398 (15 – 30 μM) did not alter the cardiopulmonary resting variables. However, a reduction of the PAF‐mediated pulmonary hypertension, but not of bronchoconstriction, was observed. When L‐NNA was added to the perfusing medium of HLPs pre‐treated with NS 398 or with indomethacin (15 μM), the basal PAP values were enhanced. However, in the combined presence of COX and NOS inhibitors, only a slight increase in the hypertensive responses to the highest doses of PAF was observed, whereas the PAF mediated actions at bronchial and cardiac level were unaffected. This study indicates that (i) the cardiopulmonary actions induced by PAF are specifically modulated by endogenous NO through the NOS pathway, and (ii) COX‐2 isoform is involved in the pulmonary hypertensive, but not bronchoconstrictor, effects of PAF. Furthermore, an interaction between PAF stimulated COX, particularly COX‐2, and NOS pathways appears to take a functional role at both bronchial and cardiovascular level.

β-Blockers promote angiogenesis in the mouse aortic ring assay.

Abstract: Recent results indicate that the reduction of &bgr;-adrenergic signaling impairs angiogenesis under ischemic conditions. Because angiogenesis may occur in the absence of ischemia, it remains to be determined whether and how &bgr;-adrenergic signaling regulates angiogenesis, which develops under normoxic conditions. The effect of &bgr;-adrenergic ligands on angiogenesis was investigated using 3-dimensional cultures of mouse aortic rings embedded in collagen type I, in which luminized microvessels develop in response to vascular endothelial growth factor (VEGF). Under normoxic conditions, both isoproterenol, a &bgr;-adrenergic receptor (&bgr;-AR) agonist, and forskolin, an adenylate cyclase activator, were unable to influence aortic microvessel sprouting. On the contrary, treatment with propranolol, a &bgr;-AR antagonist, caused an approximately 70% increase in VEGF-mediated microvessel sprouting. This effect was abolished in rings from both double &bgr;-AR and &bgr;1-AR knockout mice, but not in rings from &bgr;2-AR knockout mice. Significant increases in microvessel sprouting were also observed when mouse aortic rings from C57BL/6 mice were treated with the &bgr;1-AR–selective antagonists metoprolol and bisoprolol or with the &bgr;2-AR–selective antagonist ICI 118,551. Conversely, carvedilol, a nonselective &bgr;-AR antagonist, was unable to affect aortic sprouting. These findings suggest that some &bgr;-blockers display proangiogenic activity through a mechanism that is independent of their ability to antagonize catecholamine action. The present results also identify a new function for &bgr;-AR signaling as a facilitator for VEGF-mediated angiogenesis and have implications for understanding the mechanisms that regulate angiogenic responses under normoxic conditions.

Propranolol enhances cell cycle‐related gene expression in pressure overloaded hearts

BACKGROUND AND PURPOSE Cell cycle regulators are regarded as essential for cardiomyocyte hypertrophic growth. Given that the β‐adrenoceptor antagonist propranolol blunts cardiomyocyte hypertrophic growth, we determined whether propranolol alters the expression of cell cycle‐related genes in mouse hearts subjected to pressure overload.

Biphasic effects of propranolol on tumour growth in B16F10 melanoma-bearing mice

Propranolol is a vasoactive drug that shows antiangiogenic and antitumour activities in melanoma. However, it is unknown whether these activities are dose‐dependent and whether there is a relationship between systemic vascular effects of propranolol and anti‐melanoma activity.

Iron excretion in iron dextran-overloaded mice

BACKGROUND Iron homeostasis in humans is tightly regulated by mechanisms aimed to conserve iron for reutilisation, with a negligible role played by excretory mechanisms. In a previous study we found that mice have an astonishing ability to tolerate very high doses of parenterally administered iron dextran. Whether this ability is linked to the existence of an excretory pathway remains to be ascertained. MATERIALS AND METHODS Iron overload was generated by intraperitoneal injections of iron dextran (1 g/kg) administered once a week for 8 weeks in two different mouse strains (C57bl/6 and B6D2F1). Urinary and faecal iron excretion was assessed by inductively coupling plasma-mass spectrometry, whereas cardiac and liver architecture was evaluated by echocardiography and histological methods. For both strains, 24-hour faeces and urine samples were collected and iron concentration was determined on days 0, 1 and 2 after iron administration. RESULTS In iron-overloaded C57bl/6 mice, the faecal iron concentration increased by 218% and 157% on days 1 and 2, respectively (p<0.01). The iron excreted represented a loss of 14% of total iron administered. Similar but smaller changes was also found in B6D2F1 mice. Conversely, we found no significant changes in the concentration of iron in the urine in either of the strains of mice. In both strains, histological examination showed accumulation of iron in the liver and heart which tended to decrease over time. CONCLUSIONS This study indicates that mice have a mechanism for removal of excess body iron and provides insights into the possible mechanisms of excretion.