A new low molecular weight, MnII-containing scavenger of superoxide anion protects cardiac muscle cells from hypoxia/reoxygenation injury.

Reperfusion injury after oxygen starvation is a key pathogenic step in ischemic diseases. It mainly consists in oxidative stress, related to mitochondrial derangement and enhanced generation of reactive oxygen species (ROS), mainly superoxide anion (O2(•2)), and peroxynitrite by cells exposed to hypoxia. This in vitro study evaluates whether Mn(II)(4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate).2H2O, or Mn(II)(Me2DO2A), a new low molecular weight, Mn(II)-containing O2(•) scavenger, has a direct protective action on H9c2 rat cardiac muscle cells subjected to hypoxia and reoxygenation. Mn(II)(Me2DO2A) (1 and 10 µmol/l) was added to the culture medium at reoxygenation and maintained for 2 h. In parallel experiments, the inactive congener Zn(II)(Me2DO2A), in which Zn(II) replaced the functional Mn(II) center in the same organic scaffold, was used as negative control. Mn(II)(Me2DO2A) (10 µmol/l) significantly increased cardiac muscle cell viability (trypan blue assay), improved mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test, membrane potential Δψ), reduced apoptosis (mitochondrial permeability transition pore opening, caspase-3, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay), decreased intracellular ROS levels (2',7'-dichlorodihydrofluorescein diacetate and MitoSOX assays), and decreased protein nitroxidation (nitrotyrosine [NT] expression) and DNA oxidation (8-hydroxy-deoxyguanosine levels). Of note, Zn(II)(Me2DO2A) had no protective effect. The mechanism of Mn(II)(Me2DO2A) relies on concentration-dependent removal of harmful O2(•) generated at reoxygenation from dysfunctional mitochondria in hypoxia-induced cells, as indicated by the MitoSOX assay. This study suggests that Mn(II)(Me2DO2A) is a promising antioxidant drug capable of reducing O2(•)-mediated cell oxidative stress which occurs at reoxygenation after hypoxia. In perspective, Mn(II)(Me2DO2A) might be used to reduce ischemia-reperfusion organ damage in acute vascular diseases, as well as to extend the viability of explanted organs before transplantation.

New onset of electrocardiographic abnormalities heralding hypertrophic cardiomyopathy in an adult athlete.

A 41-year-old male asymptomatic athlete with unremarkable personal and family history of heart disease underwent annual preparticipation screening. No abnormalities were noted on prior testing. On this occasion, a 12-lead electrocardiogram showed diffused and marked repolarization abnormalities. He was therefore referred for echocardiography, which showed moderate asymmetric hypertrophy localized at the mid-apical portions of the left ventricular anterolateral wall. Cardiac magnetic resonance confirmed the diagnosis of hypertrophic cardiomyopathy. Re-evaluation of the electrocardiogram performed the previous year revealed a completely normal tracing.

Activation of cannabinoid receptors prevents antigen-induced asthma-like reaction in guinea pigs.

In this study we evaluated the effects of the CB1/CB2 cannabinoid receptor agonist CP55, 940 (CP) on antigen-induced asthma-like reaction in sensitized guinea pigs and we tested the ability of the specific CB2 receptor antagonist SR144528 (SR) and CB1 receptor antagonist AM251 (AM) to interfere with the effects of CP. Ovalbumin-sensitized guinea pigs placed in a respiratory chamber were challenged with the antigen given by aerosol. CP (0.4 mg/kg b.wt.) was given i.p. 3 hrs before ovalbumin challenge. Sixty minutes before CP administration, some animals were treated i.p. with either AM, or SR, or both (0.1 mg/kg b.wt.). Respiratory parameters were recorded and quantified. Lung tissue specimens were then taken for histopathological and morphometric analyses and for eosinophilic major basic protein immunohistochemistry. Moreover, myeloperoxidase activity, 8-hydroxy-2-deoxyguanosine, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) levels, and CB1 and CB2 receptor protein expression by Western blotting were evaluated in lung tissue extracts. In the bronchoalveolar lavage fluid, the levels of prostaglandin D2 and tumour necrosis factor-alpha TNF-alpha were measured. Ovalbumin challenge caused marked abnormalities in the respiratory, morphological and biochemical parameters assayed. Treatment with CP significantly reduced these abnormalities. Pre-treatment with SR, AM or both reverted the protective effects of CP, indicating that both CB1 and CB2 receptors are involved in lung protection. The noted treatments did not change the expression of cannabinoid receptor proteins, as shown by Western blotting. These findings suggest that targeting cannabinoid receptors could be a novel preventative therapeutic strategy in asthmatic patients.

Reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism.

Altered nitric oxide (NO) production/release is involved in gastrointestinal motor disorders occurring in dystrophic (mdx) mice. Since the hormone relaxin (RLX) can upregulate NO biosynthesis, its effects on spontaneous motility and NO synthase (NOS) expression in the ileum of dystrophic (mdx) mice were investigated. Mechanical responses of ileal preparations were recorded in vitro via force-displacement transducers. Evaluation of the expression of NOS isoforms was performed by immunohistochemistry and Western blot. Normal and mdx mice were distributed into three groups: untreated, RLX pretreated, and vehicle pretreated. Ileal preparations from the untreated animals showed spontaneous muscular contractions whose amplitude was significantly higher in mdx than in normal mice. Addition of RLX, alone or together with l-arginine, to the bath medium depressed the amplitude of the contractions in the mdx mice, thus reestablishing a motility pattern typical of the normal mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) or the guanylate cyclase inhibitor ODQ reversed the effects of RLX. In RLX-pretreated mdx mice, the amplitude of spontaneous motility was reduced, thus resembling that of the normal mice, and NOS II expression in the muscle coat was increased in respect to the vehicle-pretreated mdx animals. These results indicate that RLX can reverse the altered ileal motility of mdx mice to a normal pattern, likely by upregulating NOS II expression and NO biosynthesis in the ileal smooth muscle.

The vasorelaxant hormone relaxin induces changes in liver sinusoid microcirculation: a morphologic study in the rat.

This study shows that specialized contractile endothelial cells exist in rat liver sinusoids which may be involved in the local control of hemodynamics and which are sensitive to vasoactive agents, including the vasorelaxant hormone relaxin. Male rats were treated with 10 microg relaxin for 4 days; phosphate-buffered saline (PBS)-treated rats were the controls. For comparison, rats treated with relaxin together with the NO-synthase inhibitor N(omega)-nitro-l -arginine methyl ester (L-NAME), and rats treated with the vasodilator taurodeoxycholic acid or the vasoconstrictor ethanol were investigated. Liver fragments were studied morphologically and morphometrically. In the control rats, peculiar contractile cells were present in the endothelial lining. These cells had abundant myofilaments and formed cytoplasmic blebs projecting into and often occluding the lumen. In the ethanol-treated rats, sinusoids were constricted and filled with cytoplasmic blebs. In the relaxin-treated rats, sinusoids were markedly dilated and the cytoplasmic blebs nearly disappeared. Similar findings were observed in the taurodeoxycholic acid-treated rats. The effects of relaxin were blunted by L-NAME, suggesting that the relaxin action involves an NO-mediated mechanism.

Relaxin causes changes of the liver. In vivo studies in rats.

During pregnancy, the liver undergoes metabolic adjustments directed to fulfil the needs of the mother and the growing fetus. This study was designed to verify whether relaxin, a hormone related to pregnancy, may induce histochemical and ultrastructural modifications of hepatocytes which can be related to metabolic changes. Estrogen-primed female rats were treated with relaxin (10 microg in repository vehicle) for 18 h. Additional male rats were treated with relaxin (10 microg/day in PBS) for 4 days. Appropriate vehicle-treated rats were used as controls. After fasting, the rats were killed and liver fragments were processed for light and electron microscopy and for computer-assisted morphometry of PAS-positive glycogen deposits and acid phosphatase-reactive organelles. In both sexes, the relaxin-treated rats underwent a significant decrease in the amount of glycogen in the hepatocytes as compared with the controls. These changes were accompanied by an increase in smooth endoplasmic reticulum, endocytosis vesicles and lysosomes. These findings show that relaxin promotes glycogen depletion and induces morphological changes of hepatocytes which are consistent with functional activation. It is suggested that relaxin might play an important role in hepatic metabolic adjustments occurring during pregnancy.

Relaxin promotes differentiation of human breast cancer cells MCF-7 transplanted into nude mice.

Previous studies showed that the hormone relaxin acts on human breast cancer MCF-7 cells in vitro by modulating cell proliferation and promoting cell differentiation toward a duct epithelial phenotype. The present study was designed to investigate whether relaxin retains these properties when acting in vivo on MCF-7 cell tumors developed in athymic nude mice. Mice bearing MCF-7 cell tumors transplanted under the mammary fat pad and estrogenized to sustain tumor growth were treated systemically with relaxin (10 microg/day) for 19 days. Vehicle-treated mice were used as controls. Thirty days later, the mice were sacrificed and tumor fragments were analyzed by light and electron microscopy and immunocytochemistry. Measurements of tumor volume were recorded weekly for the overall experimental period. The results obtained indicate that relaxin treatment promotes differentiation of tumor cells towards both myoepithelial-like and epithelial-like cells, as judged by the ultrastructural features of the cells and by the increased expression of smooth muscle actin and cadherins. Measurements of tumor size and of the number of cycling cells show that relaxin, at the doses and times of exposure used in this study, does not significantly influence tumor growth and cell proliferation.

Relaxin up-regulates the nitric oxide biosynthetic pathway in the mouse uterus: involvement in the inhibition of myometrial contractility.

The uterus is a site of nitric oxide (NO) production and expresses NO synthases (NOS), which are up-regulated during pregnancy. NO induces uterine quiescence, which is deemed necessary for the maintenance of pregnancy. Relaxin is known to promote uterine quiescence. Relaxin has also been shown to stimulate NO production in several targets. In this study we investigated the effects of relaxin on the NO biosynthetic pathway of the mouse uterus. Estrogenized mice were treated with relaxin (2 microg) for 18 h, and the uterine horns were used for determination of immunoreactive endothelial-type NOS and inducible NOS. Moreover, uterine strips from estrogenized mice were placed in an organ bath, and the effect of relaxin on K+-induced contracture was evaluated in the presence or absence of the NOS inhibitor nitro-L-arginine. Relaxin increases the expression of endothelial-type NOS in surface epithelium, glands, endometrial stromal cells, and myometrium, leaving inducible NOS expression unaffected. Moreover, relaxin inhibits myometrial contractility, and this effect is blunted by nitro-L-arginine, thus indicating that the L-arginine-NO pathway is involved in the relaxant action of relaxin on the myometrium. Because relaxin is elevated during pregnancy, it is suggested that relaxin has a physiological role in the up-regulation of uterine NO biosynthesis during pregnancy.

Aortic root dilatation in young men with normally functioning bicuspid aortic valves.

OBJECTIVE: To evaluate the dimensions of the aortic root in a selected population of young males with isolated normally functioning bicuspid aortic valve. DESIGN AND SETTING: Echocardiographic and Doppler evaluation of conscripts with bicuspid aortic valve at the time of military pre-enrolment screening in two military hospitals. SUBJECTS AND METHODS: 66 consecutive young men with a normally functioning bicuspid aortic valve were studied to assess aortic size at four aortic levels: annulus, sinuses of Valsalva, supra-aortic ridge, and proximal ascending aorta; 70 consecutive normal young subjects, matched for age and body surface area, were used as controls. RESULTS: In men with a bicuspid aortic valve, the diameter of the aortic root was significantly larger than in controls at the sinuses (3.16 (0.37) v 2.87 (0.31) cm, p < 0.001), at the supra-aortic ridge (2.64 (0.46) v 2.47 (0.28) cm, p = 0.01), and at the level of the proximal ascending aorta (3.12 (0.48) v 2.69 (0.28) cm, p < 0.001). The prevalence of aortic root dilatation was 7.5% at the annulus (5/66), 19.6% at the sinuses (13/66), 15% at the supra-aortic ridge (10/66), and 43.9% at the ascending aorta (29/66); 32 subjects (48%) had aortic root dimensions comparable with controls, while 34 (52%) had definitely abnormal aortic root dimensions. CONCLUSIONS: Aortic root enlargement in people with a bicuspid aortic valve occurs independently of haemodynamic abnormalities, age, and body size. However, there appear to be different subgroups of young adults with bicuspid aortic valves, one of which is characterised by aortic dilatation, possibly caused by a congenital abnormality of the aortic wall.

Postextrasystolic potentiation and dobutamine echocardiography in predicting recovery of myocardial function after coronary bypass revascularization.

BACKGROUND: Identification of viable but hibernating myocardium remains a relevant issue in the current era of myocardial revascularization. Echocardiography can be helpful in detecting reversible contractile dysfunction and optimizing the selection of patients for coronary bypass surgery. METHODS AND RESULTS: Eighty-four consecutive candidates for bypass surgery with chronic multivessel coronary artery disease were screened, and 60 were included in this prospective study. Preoperative evaluation of a reversible contractile dysfunction in asynergic myocardial regions was performed by dobutamine infusion at 5 (low dose) and 10 (intermediate dose) microg x kg(-1) x min(-1) with each stage lasting at least 5 minutes; postextrasystolic potentiation (PESP), with a coupling interval ranging from 500 to 300 ms with a progressive 10-ms decrease; or a combination of both dobutamine infusion and PESP. Sensitivity (92% versus 86%) and predictive accuracy (89% versus 84%) were higher with PESP than dobutamine (P=.009 and P=.001, respectively), but the combination did not improve sensitivity or accuracy. Dobutamine induced ischemic dysfunction in 15% of patients at the intermediate dose; however, the low dose resulted in loss of sensitivity. CONCLUSIONS: PESP echocardiography is a useful and cost-effective method to identify viable myocardium in patients with multivessel coronary disease undergoing revascularization and is more sensitive and accurate than dobutamine infusion.

Evidence of myocardial dysfunction in Bartter's syndrome.

Bartter's syndrome (BS) is characterized by arterial normohypotension despite biochemical and hormonal abnormalities generally associated with hypertension. An abnormal intracellular calcium homeostasis due to a reduced capacity to increase intracellular calcium has been demonstrated by us in BS and proposed as the main pathophysiological factor of the vascular hyporeactivity in BS. The present study was designed to assess whether this altered intracellular calcium homeostasis could also impair contractile recruitment at the myocyte level. Left-ventricular function of patients with BS and normal subjects (C) were studied by quantitative 2-D echocardiography at rest and by postextrasystolic potentiation (PESP), an inotropic stimulus able to recruit the maximal contractile reserve. A group of patients with hypokalemia other than BS (PB) was also included in the study to evaluate the effect of hypokalemia on myocardial contractile recruitment. Baseline left-ventricular end-diastolic volume (EDV) and ejection fraction (EF) did not differ in the 3 groups: EDV: 62 +/- 6 vs. 64 +/- 9 and 60 +/- 12 ml/m2; EF: 64 +/- 9 vs. 67 +/- 8 and 64 +/- 8%. PESP determines an increase of EF in C and PB: 82 +/- 5%, p < 0.01 and 76 +/- 6%, p < 0.01, while in BS it is unchanged: 69 +/- 9% and is reduced in comparison with the increment of myocardial function shown by C and PB (p < 0.01). This study is the first demonstration in BS of a depressed inotropic recruitment causing an exercise-induced left-ventricular dysfunction likely due to an abnormal intracellular calcium homeostasis in the myocytes.

[The prevalence of the Wolff-Parkinson-White syndrome in a population of 116,542 young males]. / Prevalenza della sindrome di Wolff-Parkinson-White in una popolazione di 116.452 giovani maschi.

Since 1988 to 1992 we analyzed 116,452 consecutive 12-lead electrocardiograms belonging to the entire cohort of 18-year old young boys resident in Padova, Treviso, Rovigo, Venezia, Belluno area (2,834,000 inhabitants). We identified 173 cases of overt WPW pattern (short PR interval, delta wave, anomalous configuration of QRS complex) with a calculated incidence of 1.48/1000. Accessory pathway location was: left free wall (70 patients), right free wall (39 patients), postero-septal (37 patients), antero-septal (15 patients) and undetermined (12 patients). Sixty patients (34.6%) complained of different symptoms as palpitations, near syncope and dizziness. Fifty-three patients (30.6%) went in a regular sport activity. Twenty-four hour Holter monitoring (41 patients) and exercise stress test (43 patients) did not show sustained tachyarrhythmias; intermittent preexcitation was recorded in 23 and 32 patients, respectively. Two-dimensional echocardiogram (68 out of 173) was normal in 44 patients, while 24 showed minor cardiac abnormalities with two major disease; mitral valve prolapse was diagnosed in 8 patients. On the basis of transesophageal (24 patients) or intracardiac (5 patients) electrophysiologic study, 11 patients were considered at high risk for sudden death. Eight of them suffered from spontaneous symptoms.

Myocardial dysfunction and abnormal left ventricular exercise response in autonomic diabetic patients.

In diabetic patients, the pathophysiologic mechanisms of exercise-induced left ventricular (LV) dysfunction remain controversial. In this study, the role of myocardial contractility recruitment in determining an abnormal LV response to isometric or dynamic exercise has been investigated in 14 diabetic patients with autonomic dysfunction. Ischemic heart disease was excluded by the absence of LV wall motion abnormalities induced by isotonic and isometric exercise and by coronary angiography. Left ventricular and myocardial function were studied at rest, and during isometric and isotonic exercise, by two-dimensional echocardiography; moreover, recruitment of an inotropic reserve was assessed by postextrasystolic potentiation at rest and at peak handgrip. An abnormal response of LV ejection fraction to isometric (9/14) or to dynamic (8/14) exercise was frequent in study patients. In these patients, baseline myocardial contractility was normal, and the significant increase in ejection fraction by postextrasystolic potentiation indicated a normal contractile reserve (65 +/- 7% vs. 74 +/- 6%, p = 0.001). Nevertheless, the downward displacement of LV ejection fraction-systolic wall stress relationships during exercise suggests an inadequate increase in myocardial contractility. However, the abnormal ejection fraction at peak handgrip was completely reversed by postextrasystolic potentiation (67 +/- 6% vs. 58.1 +/- 10%, p = 0.008), a potent inotropic stimulation independent of the integrity of adrenergic cardiac receptors. A defective inotropic recruitment, despite the presence of a normal LV contractile reserve, plays an important role in deexercise LV dysfunction in diabetic patients with autonomic neuropathy.

Nifedipine in asymptomatic patients with severe aortic regurgitation and normal left ventricular function.

BACKGROUND: Vasodilator therapy with nifedipine reduces left ventricular volume and mass and increases the ejection fraction in asymptomatic patients with severe aortic regurgitation. METHODS: To assess whether vasodilator therapy reduces or delays the need for valve replacement, we randomly assigned 143 asymptomatic patients with isolated, severe aortic regurgitation and normal left ventricular systolic function to receive either nifedipine (20 mg twice daily, 69 patients) or digoxin (0.25 mg daily, 74 patients). RESULTS: By actuarial analysis, we determined that after six years a mean (+/- SD) of 34 +/- 6 percent of the patients in the digoxin group had undergone valve replacement, as compared with only 15 +/- 3 percent of those in the nifedipine group (P < 0.001). In the digoxin group, valve replacement (in a total of 20 patients) was performed because of left ventricular dysfunction (ejection fraction < 50 percent) in 75 percent, left ventricular dysfunction plus symptoms in 10 percent, and symptoms alone in 15 percent. In the nifedipine group, all six patients who underwent valve replacement did so because of the development of left ventricular dysfunction. In addition, all the patients in both groups who underwent aortic-valve replacement had an increase of 15 percent or more in the left ventricular end-diastolic volume index. After aortic-valve replacement, 12 of the 16 patients (75 percent) in the digoxin group and all six patients in the nifedipine group who had had an abnormal left ventricular ejection fraction before surgery had a normal ejection fraction. CONCLUSIONS: Long-term vasodilator therapy with nifedipine reduces or delays the need for aortic-valve replacement in asymptomatic patients with severe aortic regurgitation and normal left ventricular systolic function.

Reversible contractile dysfunction of viable myocardium. Implications for decision making process in post-infarction patients.

Myocardial dysfunction and silent myocardial ischemia have been identified as important prognostic factors following acute myocardial infarction, also in low risk patients. In postinfarction patients, impaired left ventricular function is the result of fixed scar and reversible contractile dysfunction of viable stunning or hibernating myocardium. Post-extrasystolic potentiation (PESP) during 2-dimensional echocardiographic monitoring may be used to detect the presence of viable myocardium in asynergic myocardial segments. Incidence of reversible contractile dysfunction is a very common phenomenon at predischarge examination after myocardial infarction in asymptomatic patients, and it is independent on the persistence of silent ischemia. A progressive loss of myocardial viability occurs over the first year following the acute phase despite the maintenance of an asymptomatic clinical status. This phenomenon is associated with significant dilatation of the left ventricle. Moreover, silent ischemia is strongly related with this progressive loss of myocardial viability and left ventricular dilatation. Thus, it becomes evident that the most important role of medical and interventional approaches consists of limiting the acute necrosis by reperfusion and in preventing the loss of viable chronically hypoperfused myocardium that appears to be a major factor of left ventricular remodeling and changes over time of prognostication in individual patients. Finally, the presence of viable myocardium by PESP in the arterial zone at risk is highly predictive of 4-year mortality, particularly in patients with low ejection fraction (< 40%), and identifies patients who are suitable candidates for revascularization after myocardial infarction.

Left ventricular function and prognosis after myocardial infarction: rationale for therapeutic strategies.

Prognosis after acute myocardial infarction is strongly associated with left ventricular dysfunction. However, asynergy does not necessarily imply loss of viability and myocardial necrosis. In fact, two different patterns of contractile dysfunction, possibly coexisting, have been shown after acute myocardial infarction: Stunning and hibernation represent distinct patterns of contractile dysfunction that share the character of reversibility. It is noteworthy, then, to identify the presence of these two conditions at the bedside and to develop medical treatment to effect recovery of myocardial dysfunction. This strategy has the potential to ameliorate the outcome of patients after acute myocardial infarction by improving left ventricular function. Beta-blocker therapy significantly reduces mortality and the incidence of reinfarction after an acute myocardial infarction: These benefits result from the prevention of sudden death, the reduction of the extent of myocardial injury during the acute phase, and a further antiischemic action. Nevertheless, beta-blocker therapy increases left ventricular dilatation. Recent experimental and clinical data show that ACE inhibitors confer positive therapeutic effects after myocardial infarction by reducing the extent of left ventricular dilation, by reducing mortality, and by improving the clinical outcome. Not all patients, however, can be subjected to this therapeutical approach because of the possible detrimental effects produced by hypotension and by block of neurohormonal activation, sometimes truly compensatory in the early phase. Therefore, it would be interesting to suggest a combination therapy of a beta-blocker with a vasodilator agent (ACE inhibitor or calcium-channel blocker.