Role of angiotensin II AT1 and AT2 subtype receptors in the regulation of atrial natriuretic peptide expression in salt-restricted rats

Previous studies have suggested that angiotensin II modulates ANP secretion and this action appears to be largely independent from its hemodynamic effects. In order to explore the contribution of angiotensin II AT₁ (AT₁r) and AT₂ (AT₂r) receptor subtypes in the regulation of cardiac ANP, we studied the effects of selective antanonists of these receptors on ANP mRNA levels in the cardiac chambers of salt-restricted rats. Thirty-one Sprague-Dawley rats (12 weeks-old) weighing 250–350 g were studied during a low salt regimen and randomly assigned to the following treatment groups: AT₁r-blockade (losartan) (10 mg7kg/day) (n = 18), AT₂r-blockade (PD123319) (50 μg/kg/min) (n = 6), Control (salt-restriction) (n = 7). Treatments were maintained for 7 days; subsequently 12 rats from the AT₁r-blockade group were subdivided in to two groups: AT₁r-blockade (losartan+PD123319) (n = 6) and AT₁r-blockade/vehicle (losartan-vehible) (n = 6), and treated for 7 additional days. Systolic blood pressure was significantly reduced by AT₁r-blockade (p < 0.001), while it was not affected by AT₂r-blockade. Concomitant treatment with both antagonists (AT₁r/AT₂r-blockade) restored blood pressure values to baseline (p < 0.001 vs. AT₁r-blockade, p = n.s. vs Control). Atrial ANP mRNA was reduced by AT₁r-blockade (-42%, p < 0.05) and did not change during AT₂r-blockade alone. On the contrary, concomitant treatment with both antagonists resulted in a further significant inhibition of ANP expression (-65% and -36% vs Control and AT₁r-blockade, respectively, both p < 0.05). ANP expression in ventricles was not affected by any of these treatments. Our results demonstrate that angiotensin II tonically modulates cardiac ANP expression in our experimental model. In particular, angiotensin II receptor subtypes AT₁r and AT₂r regulate atrial ANP mRNA levels through a synergic action and independently from blood pressure changes. Received: 26 May 1999, Returned for revision: 17 June 1999, Revision received: 4 August 1999, Accepted: 26 August 1999     

Ventricular remodeling and transforming growth factor-beta 1 mRNA expression after nontransmural myocardial infarction in rats: effects of angiotensin converting enzyme inhibition and angiotensin II type 1 receptor blockade

With the application of early reperfusion therapy after acute MI, the incidence and importance of nontransmural infarction is increasing. In a rat model with nontransmural infarction, we evaluated 1) the changes of LV dimension, LV interstitial fibrosis and transforming growth factor-β1 (TGF-β1) mRNA expression and 2) the effects of angiotensin converting enzyme (ACE) inhibitor and angiotensin II type 1 (AT1) receptor antagonist treatment. Female Sprague-Dawley rats were subjected to 45 min of coronary occlusion followed by reperfusion, and five days after the operation the animals were randomized to untreated (n = 19), captopril-treated (n = 15) and losartan-treated (n = 14) groups. Twenty-six days after MI, echocardiographic examination revealed a remarkable dilatation of LV. Captopril or losartan treatment reduced the extent of LV cavity dilatation. Collagen volume fractions in noninfarcted septum as well as in peri-infarct area decreased with captopril or losartan treatment, compared to those of the untreated rats. In noninfarcted septum of untreated rats, TGF-β1 mRNA expression increased more than two fold (P < 0.05 vs. pre-MI) 5 and 10 days after MI. Captopril or losartan treatment suppressed the acute induction of TGF-β1 mRNA expressions. These results indicate that ACE inhibitor or AT1 receptor antagonist treatment after nontransmural infarction 1) attenuates LV remodeling as in transmural infarction and 2) decreases interstitial fibrosis at least partly by blocking the acute induction of TGF-β1 mRNA expression. Received: 10 December 1998, Returned for revision: 29 January 1999, Revision received: 15 March 1999, Accepted: 22 March 1999     

Inhibition of carnitine synthesis modulates protein contents of the cardiac sarcoplasmic reticulum Ca2+-ATPase and hexokinase type I in rat hearts with myocardial infarction

It was previously reported than inhibition of carnitine synthesis by 3-(2,2,2-trimethyl-hydrazinium) propionate (MET-88) restores left ventricular (LV) systolic and diastolic function in rats with myocardial infarction (MI). Preservation of the calcium uptake function of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) is one of the possible mechanisms by which MET-88 alleviates hemodynamic dysfunction. To test this hypothesis, the effects of MET-88 on protein content of SERCA2 were evaluated using the same rat model of heart failure. Myocardial protein content of hexokinase, which is one of the key enzymes of glucose utilization, was also measured. Either MET-88 (MET-88 group) or a placebo (MI group) was administered for 20 days to rats with MI induced by coronary artery ligation. The control group underwent sham surgery (no ligation) and received placebo. In LV myocardial homogenates, the myocardial SERCA2 protein content was 32% lower (p<0.05) in the MI group than in the control group. However, in the MET-88 group myocardial SERCA2 content was the same as in the control group. Hexokinase I protein content was 29% lower (p<0.05) in the MI group compared with the control. In contrast, hexokinase II protein content did not differ significantly among the three groups. Consequently, inhibition of carnitine synthesis ameliorates depression of SERCA2 and hexokinase I protein content which may reduce tissue damage caused by MI. Received: 26 July 1999 Returned for 1. revision: 14 September 1999 1. Revision received: 14 December 1999 Returned for 2. revision: 26 January 2000 2. Revision received: 28 February 2000 Accepted: 6 April 2000     

Central AT1 receptors are involved in the enhanced cardiac sympathetic afferent reflex in rats with chronic heart failure

The purpose of this study was to determine if the cardiac sympathetic afferent reflex (CSAR) was augmented in rats with coronary ligation-induced chronic heart failure (CHF), and if central angiotensin II (ANG II) was involved in this enhancement. Under α-chloralose and urethane anesthesia, mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were recorded in sino-aortic denervated and cervical vagotomized rats. An intracerebroventricular cannula was implanted. The CSAR was examined by epicardial application of bradykinin (BK, 0.04 and 0.4 μg in 2.0 μl) or capsaicin (0.04 and 0.4 μg in 2.0 μl) to the anterior and posterior wall of the left ventricle. The CSAR evoked by BK or capsaicin was augmented in rats with CHF. In sham rats, there was no significant difference of the CSAR induced by BK or capsaicin between anterior and posterior epicardial application. However, in rats with CHF, the CSAR induced by BK (0.04 μg) to anterior epicardial application was blunted compared with posterior application. Intracerebroventricular injection of losartan (500 nmol) normalized the enhanced CSAR in rats with CHF, but had no significant effects on the CSAR in sham rats. However, intravenous application of the same dose of losartan only decreased the baseline MAP, but did not alter baseline RSNA or the CSAR in sham or CHF rats. Pre-treatment with epicardial application of lidocaine to the anterior wall abolished the CSAR evoked by application of BK or capsaicin but had no effects on the CSAR evoked by epicardial application of BK or capsaicin to the posterior wall. These results suggest that the CSAR induced by epicardial application of BK and capsaicin is enhanced in the rats with CHF, and the enhanced CSAR is mediated by central AT₁ receptors. Received: 17 December 2001/Returned for revision: 7 January 2002/Revision received: 4 February 2002/Accepted: 26 February 2002     

Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and β-adrenergic stimulation

We previously demonstrated the presence of components for a renin-angiotensin system in fibroblasts cultured from neonatal rat ventricles, the regulation of expression of which has not been studied. Since glucocorticoids and β-adrenergic stimuli have been implicated in cardiac hypertrophy, and function as regulators of the circulating renin-angiotensin system, we examined the effects of dexamethasone and isoproterenol on angiotensinogen mRNA levels and protein secretion in cultured neonatal rat cardiac fibroblasts. Treatment of cardiac fibroblasts for 8 h with 10 μmol/l isoproterenol or 100 nmol/l dexamethasone increased angiotensinogen mRNA levels by 246 ± 7% and 1406 ± 207%, respectively. Over 24 h, dexamethasone and isoproterenol increased angiotensinogen secretion by 148 ± 32% and 123 ± 26%, respectively. Angiotensin II, which has been reported to be a positive regulator of angiotensinogen synthesis and secretion in liver, markedly attenuated the effects of dexamethasone and isoproterenol on angiotensinogen mRNA expression and secretion. In the presence of 1 μmol/l angiotensin II, the stimulation in angiotensinogen secretion observed with dexamethasone and isoproterenol was decreased by 62% and 76%, respectively. The negative feedback of angiotensin II on angiotensinogen expression was primarily mediated through the type one angiotensin II (AT₁) receptor (IC₅₀ = 0.30 ± 0.02 nmol/l). In summary, results from this study demonstrate that angiotensinogen mRNA levels and protein secretion in cardiac fibroblasts are positively regulated by glucocorticoid and β-adrenergic stimulation. In addition, angiotensinogen production by cardiac fibroblasts is under negative feedback control of angiotensin II. Received: 28 October 1999, Returned for revision: 24 November 1999, Revision received: 14 January 2000, Accepted: 26 January 2000     

Electrophysiological characterization of murine myocardial ischemia and infarction

Background Genetically altered mice will provide important insights into a wide variety of processes in cardiovascular physiology underlying myocardial infarction (MI). Comprehensive and accurate analyses of cardiac function in murine models require implementation of the most appropriate techniques and experimental protocols. Objective In this study we present in vivo, whole-animal techniques and experimental protocols for detailed electrophysiological characterization in a mouse model of myocardial ischemia and infarction. Methods FVB mice underwent open-chest surgery for ligation of the left anterior descending coronary artery or sham-operation. By means of echocardiographic imaging, electrocardiography, intracardiac electrophysiology study, and conscious telemetric ECG recording for heart rate variability (HRV) analysis, we evaluated ischemic and post-infarct cardiovascular morphology and function in mice. Results Coronary artery ligation resulted in antero-apical infarction of the left ventricular wall. MI mice showed decreased cardiac function by echocardiography, infarct-typical pattern on ECG, and increased arrhythmia vulnerability during electrophysiological study. Electrophysiological properties were determined comprehensively, but were not altered significantly as a consequence of MI. Autonomic nervous system function, measured by indices of HRV, did not appear altered in mice during ischemia or infarction. Conclusions Cardiac conduction, refractoriness, and heart rate variability appear to remain preserved in a murine model of myocardial ischemia and infarction. Myocardial infarction may increase vulnerability to inducible ventricular tachycardia and atrial fibrillation, similarly to EPS findings in humans. These data may be of value as a reference for comparison with mutant murine models necessitating ischemia or scar to elicit an identifiable phenotype. The limitations of directly extrapolating murine cardiac electrophysiology data to conditions in humans need to be considered. Received: 5 October 2000, Returned for 1. revision: 2 November 2000, 1. Revision received: 24 November 2000, Returned for 2. revision: 28 November 2000, 2. Revision received: 13 December 2000, Accepted: 14 December 2000     

Angiotensin inhibition and coronary autoregulation in a canine model of LV hypertrophy

In humans with hypertension and LV hypertrophy, beneficial effects of angiotensin inhibition may be associated with preserved autoregulatory capacity. We studied the effect of acute angiotensin converting enzyme (ACE) inhibition on coronary autoregulatory pressure-flow relations and transmural distribution of blood flow in sham and LV hypertrophy dogs. Heart/body weight ratio increased (p = 0.001) from 5.5 ± 0.7 in sham to 6.9 ± 0.5 in LV hypertrophy dogs. The lower coronary pressure limit (LPL) on the pressure-flow relation was 47 ± 2 mmHg in sham and 57 ± 6 mmHg (p = 0.001) in LV hypertrophy dogs; after acute ACE-inhibition the LPL was reduced to 40 ± 5 mmHg and 49 ± 6 mmHg (p = 0.001), respectively. Transmural distribution of blood flow was preserved at the LPL in both groups before and after acute ACE-inhibition. Concomitant blockade of prostaglandin and nitric oxide release and bradykinin catabolism had no additional effects on the LPL and distribution of blood flow. After acute ACE-inhibition in LV hypertrophy dogs, distribution of blood flow across the LV wall was preserved and subendocardial vascular reserve was maintained even though the LPL was significantly lower. Preservation of autoregulatory capacity by ACE inhibitors contributes to beneficial outcome in patients with hypertension and LV hypertrophy. Received: 2 October 2001, Returned for 1. revision: 17 October 2001, 1. Revision received: 20 December 2001, Returned for 2. revision: 2 January 2002, 2. Revision received: 14 January 2002, Accepted: 17 January 2002     

The arterial length‐densities under preventive angiotensin‐converting‐enzyme inhibiting treatment in the myocardium of spontaneously hypertensive rats

The length density (L_V) of capillaries is known to be increased in the hearts of spontaneously hypertensive rats (SHR) after high‐dosed but also after low‐dosed subantihypertensive treatment with the ACE‐inhibitor Ramipril administered in utero and post partum. Under the same conditions in the present study only highdose Zabicipril caused an increase of capillary L_V. Under preventive ACE‐inhibition in both high‐dose groups L_V of myocardial arteries was significantly higher. In the low‐dose groups L_V was not significantly increased. The increased arterial L_V in the high‐dose‐group may result from the avoidance of angiotensin II‐induced overabundant growth of myocardial muscle‐mass. Changes in collagen could not be found in any of the experimental groups. (Basic Res Cardiol) Received: 7 April 1997, Returned for 1. revision: 9 June 1997, 1. Revision received: 12 September 1997, Returned for 2. revision: 29 October 1997, 2. Revision received: 31 October 1997, Accepted: 6 November 1997     

Endothelium-dependent vasodilatation in Sprague-Dawley rats with postinfarction hypertrophy: Lack of endothelial dysfunction in vitro

The hypothesis was tested whether postinfarction hypertrophy/congestive heart failure in rats is associated with endothelial dysfunction and increased vascular generation of reduced oxygen species. Myocardial infarction was induced in Sprague-Dawley rats by ligation of the left coronary artery. After 16 weeks, endothelium-dependent (with acetylcholine) and -independent (with sodium nitroprusside) relaxation were studied in isolated aortic rings, and isolated rings from the femoral and mesenteric arteries. The generation of superoxide, hydrogenperoxide, and peroxynitrite was measured in arteries using lucigenin- and luminol-enhanced chemiluminescence techniques. Systolic blood pressure decreased over the 16 week study period as compared to shamoperated control rats; organ weights (lungs, right and left ventricles) significantly increased in coronary artery ligated rats indicating development of congestive heart failure. Surprisingly, concentration response curves with acetylcholine and sodium nitroprusside were almost identical in myocardial infarction rats as compared to control animals, irrespective of which type of vessel was studied (aorta, femoral or mesenteric arteries). In addition, no differences in the production of reduced radical species were found in aortic tissue from heart failure rats as compared to control rats. Received: 3 March 1998, Returned for 1. revision: 20 April 1998, 1. Revision received: 14 May 1998, Returned for 2. revision: 25 June 1998, 2. Revision received: 8 July 1998, Accepted: 9 July 1998     

Ventricular arrhythmias following coronary artery occlusion in rats: is the diabetic heart less or more sensitive to ischaemia?

Rhythm disorders are common complications in diabetic patients, due to their enhanced sensitivity to ischaemia. However, experimental studies are inconsistent, and both higher and lower vulnerability to injury has been reported. Our objectives were to compare susceptibility to ventricular arrhythmias in rats with prolonged duration of diabetes induced hy streptozotocin (45 mg/kg, i. v.), utilising two different models. Following 8 weeks, either anaestetised open-chest rats in vivo or isolated Langendorff-perfused hearts were subjected to 30 min regional zero-flow ischaemia induced by occlusion of LAD coronary artery. In addition, cardiac glycogenolysis and lactate production were measured. In open-chest rats, 90% of the controls exhibited ventricular tachycardia (VT) which represented 55.4% of total arrhythmias, whereby only 19.9% of arrhythmias occurred as VT in 44% of the diabetic rats (P < 0.05 vs controls). Duration of VT and ventricular fibrillation (VF) was reduced from 35.5 ± 11.1 and 224.8 ± 153.9 s in the controls to 4.8 ± 2.5 and 2.2 ± 0.2 s in the diabetics, respectively (P < 0.05). Accordingly, severity of arrhythmias (arrhythmia score, AS) was also lower in the diabetics (2.0 ± 0.38 vs 3.3 ± 0.3 in the controls; P < 0.05). In the isolated hearts, high incidence of VF was decreased in the diabetic hearts, and although VT occurred in almost all of the diabetic hearts, the duration of VT and VF was substantially shorter (61.5 ± 14.5 and 5.5 ± 0.5 s vs 221.5 ± 37 and 398.5 ± 55 s in the controls, respectively; P < 0.05). AS was reduced to 2.9 ± 0.12 from 4.1 ± 0.3 in the controls (P < 0.05). Postischaemic accumulation of lactate was lower in the diabetic than in the non-diabetic myocardium (20.4 ± 1.9 vs 29.5 ± 2.9 μmol/l/g w.wt.; P < 0.05). These results suggest that rat hearts with chronic diabetes, despite some differences in the arrhythmia profiles between the in vivo model and isolated heart preparation, are less sensitive to ischaemic injury and exhibit lower susceptibility to ventricular arrhythmias and reduced accumulation of glycolytic metabolites. Received: 3 April 2000, Returned for 1. revision: 9 May 2000, 1. Revision received: 5 July 2000, Returned for 2. revision: 7 August 2000, 2. Revison received: 11 September 2000, Returned for 3. revision: 27 September 2000, 3. Revision received: 13 October 2000, Accepted: 16 October 2000     

Early rather than delayed administration of lisinopril protects the heart after myocardial infarction in rats

Background:ACE inhibitors have shown beneficial results in several studies after myocardial infarction (MI). However, this studies have shown conflicting results about the ideal starting time of the ACE inhibitors administration after MI and the importance of infarct size. Objectives: This study was designed to assess the long-term effects of lisinopril on mortality, cardiac function, and ventricular fibrosis after MI, in rats. Methods: Lisinopril (20 mg/kg/day) was given on day 1 or 21 days after coronary occlusion in small or large infarctions. Results: The mortality rate was reduced by 39% in early treatment and 30% in delayed treatment in comparison to the untreated rats. Early treatment reduced cardiac dysfunction in small MIs; however, delayed treatment did not. No statistical difference was observed among the groups for large MIs. No statistical difference was observed among the groups with large or small MIs on myocardial hydroxyproline concentration. Conclusions: Both early and delayed treatments with lisinopril increased survival. Treatment exerts no marked effects on fibrosis; early treatment has exerted beneficial influences on cardiac function whereas delayed treatment had no consistent effects. The protective effect of lisinopril is detectable only in small (< 40% of LV) MIs. Received: 6 May 1999, Returned for 1. revision: 28 May 1999, 1. Revision received: 20 July 1999, Returned for 2. revision: 26 August 1999, 2. Revision received: 28 September 1999, Accepted: 29 September 1999     

Beta-adrenergic receptors and intracellular signalling pathway in stunned and non-ischemic regions of pig myocardium

The β-adrenergic pathway may have a role in the pathophysiology of ischemic syndromes characterised by reversible left ventricular dysfunction, such as myocardial stunning and other clinical conditions of unstable angina or coronary spasms, or chronic reversible left ventricular dysfunction, which might be a consequence of repeated events of short-term ischemia (“repetitive stunning”). A partial-to-total occlusion of the left anterior descending coronary artery in pigs was used to induce short periods of ischemia (total ischemic time 12 ± 2 min). Hypokinesis and dyskinesis of the myocardium were considered signs of myocardial dysfunction. We found a maintained function of the β-adrenergic signalling system. Density and affinity of β-adrenergic receptors were not different in stunned and non-ischemic regions, nor were cyclic AMP and cyclic GMP intracellular contents and ratio, nor well as the ratio of stimulatory/inhibitory G protein a subunits. Our findings are in agreement with a maintained β-adrenergic signalling system in the pathophysiology of chronic reversible left ventricular dysfunction. Received: 25 September 2000 / Returned for 1. revision: 9 October 2000 / 1. Revision received: 22 November 2000 / Returned for 2. revision: 7 December 2000 / 2. Revision received: 8 January 2001 / Accepted: 11 January 2001     

A1 adenosine receptor overexpression decreases stunning from anoxia-reoxygenation: role of the mitochondrial K(ATP) channel

Myocardial A₁ adenosine receptor (A₁AR) overexpression protects hearts from ischemia-reperfusion injury; however, the effects during anoxia are unknown. We evaluated responses to anoxia-reoxygenation in wild-type (WT) and transgenic (Trans) hearts with ∼200-fold overexpression of A₁ARs. Langendorff perfused hearts underwent 20 min anoxia followed by 30 min reoxygenation. In WT hearts peak diastolic contracture during anoxia was 45 ± 3 mmHg, diastolic pressure remained elevated at 18 ± 3 mmHg after reoxygenation, and developed pressure recovered to 52 ± 4 % of pre-anoxia. A₁AR overexpression reduced hypoxic contracture to 29 ± 4 mmHg, and improved recovery of diastolic pressure to 8 ± 1 mmHg and developed pressure to 76 ± 3 % of pre-anoxia. Mitochondrial K_ATP blockade with 100 μM 5-hydroxydecanoate (5-HD) increased hypoxic contracture to 73 ± 6 mmHg in WT hearts, reduced post-hypoxic recoveries of both diastolic (40 ± 5 mmHg) and developed pressures (33 ± 3 %). In contrast, 5-HD had no effect on hypoxic contracture (24 ± 8 mmHg), or post-hypoxic diastolic (10 ± 2 mmHg) and developed pressures (74 ± 3 %) in Trans hearts. In summary, (i) A₁AR overexpression improves myocardial tolerance to anoxia-reoxygenation, (ii) intrinsic mitochondrial K_ATP channel activation decreases hypoxic contracture and improves functional recovery in wild-type hearts, and (iii) mitochondrial K_ATP channels do not appear to play a major role in the functional protection from anoxia afforded by A₁AR overexpression. Received: 5 February 2001, Returned for 1. revision: 21 February 2001, 1. Revision received: 20 August 2001, Returned for 2. revision: 3 September 2001, 2. Revision received: 24 October 2001, Accepted: 25 October 2001     

Effects of BIIB513 on ischemia-induced arrhythmias and myocardial infarction in anesthetized rats

Na⁺/H⁺ exchange (NHE) plays an important role in the regulation of the intracellular pH (pH_i) and in cardiac cell injury induced by ischemia and reperfusion. In the present study, we investigated the effects of BIIB513, a selective NHE-1 inhibitor on myocardial ischemia induced arrhythmias and myocardial infarction, provoked by 30 minutes of left main coronary artery occlusion followed by 2 hours of reperfusion in an anesthetized rat model. Intravenous administration of BIIB513 (0.01–3.0 mg/kg) did not induce changes in blood pressure or heart rate. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced ventricular premature beats, ventricular tachycardia, and a complete suppression of ventricular fibrillation down to the dose of 0.1 mg/kg. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced the infarct size with an ED50 value of 0.16 mg/kg. BIIB513 (1.0 mg/kg) given prior to reperfusion also reduced infarct size by 47.3 ± 13.1%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase (CPK). In conclusion, the present study demonstrates the cardioprotective ability of NHE-1 inhibition during myocardial ischemia and reperfusion by reducing serious ventricular arrhythmias and myocardial infarct size in anesthetized rats. Received: 18 November 1999, Returned for 1.revision: 9 December 1999, 1.Revision received: 2 May 2000, Returned for 2.revision: 24 May 2000, 2.Revision received: 5 June 2000, Accepted: 7 June 2000     

Effects of different inotropic interventions on myocardial function in the developing rabbit heart

The development of the mammalian heart is characterized by substantial changes in myocardial performance. We studied the ontogeny of myocardial function with and without various inotropic interventions in the developing isolated, antegrade-perfused rabbit heart (2d, 8d, 14d, 28d, n = 96). Myocardial function was related to the protein expression of the sarcolemmal Na⁺-Ca²⁺ exchanger and to the sarcoplasmic Ca²⁺-ATPase. In neonatal hearts an age-dependent increase in maximal developed pressure velocity (dP/dt_max) by 45 % and peak negative pressure velocity (dP/dt_min) by 75 % within days 2 to 8 were observed. In response to inotropic intervention with isoproterenol, ouabain, calcium and the Na⁺-channel modulator BDF 9148, dP/dt_max and dP/dt_min increased in a concentration dependent manner. Significant differences between neonatal, juvenile and adult hearts could be demonstrated in a repeated measurement ANOVA model on the concentration-response curves for BDF 9148 (dP/dt_max and dP/dt_min), ouabain (dP/dt_min) and calcium (dP/dt_min), but not for isoproterenol. At the maximum isoproterenol concentration of 1 μmol/l, the increase in dP/dt_max and dP/dt_min was significantly higher in adult compared to neonatal hearts (t-test, p < 0.01). The significant decline of the Na⁺-Ca²⁺ exchanger protein expression from neonatal (1822 ± 171) to adult hearts (411 ± 96 S.E.M. [units per 20 μg protein], p < 0.01) was related to an increase in myocardial function (dP/dt_max r = 0.63, p < 0.01, dP/dt_min r = 0.62, p < 0.01). Contractility, relaxation and the observed positive inotropic effects were in general significantly lower in neonatal compared to adult hearts. In the individual heart an increase in contractility and relaxation was related to a decrease in Na⁺-Ca²⁺ exchanger expression. Received: 22 May 2000, Returned for 1. revision: 21 June 2000, 1. Revision received: 27 November 2000, Returned for 2. revision: 19 December 2000, 2. Revision received: 2 January 2001, Returned for 3. revision: 17 January 2001, 3. Revision received: 25 May 2001, Accepted: 11 June 2001     

Alcohol and the risk of myocardial infarction

Epidemiological studies have repeatedly demonstrated a beneficial effect of moderate alcohol consumption on the incidence of coronary heart disease, myocardial infarction and overall mortality. The latter increases with excessive alcohol consumption. Although most epidemiological studies demonstrate a beneficial effect of alcohol consumption independent from the specific kind of alcoholic beverage, there is increasing evidence that wine and in particular red wine might contain pharmacological substances, which prevent atherosclerosis and myocardial infarction independent from the wine ethanol. Pathophysiological mechanisms mediating these beneficial effects include effects of wine phenols and tannins on LDL-cholesterol oxidation status, thrombocyte aggregation, endothelial function and smooth muscle cell proliferation. Identification and characterization of the pharmacologically active substances might provide the stage for the development of new substances to be used in the prevention of coronary artery disease and myocardial infarction. Received: 14 August 2000, Returned for 1. revision: 6 September 2000, 1. Revision received: 23 November 2000, Returned for 2. revision: 5 December 2000, 2. Revision received: 21 December 2000, Accepted: 8 January 2001     

Angiotensin II-augmented migration of VSMCs towards PDGF-BB involves Pyk2 and ERK 1/2 activation

Activation of the local and systemic renin-angiotensin system is directly and indirectly involved in mechanisms of vascular remodeling during chronic hypertension. This study investigated the effect of angiotensin II (AII) on rat vascular smooth muscle cell (VSMC) migration towards platelet-derived growth factor-BB (PDGF-BB) in vitro. Pre-treatment with AII (1 μM) for 48 or 72 h induced a significant increase in PDGF-BB-directed migration by 77 ± 21 % and 58 ± 24 %, respectively (both p < 0.01). This effect was concentration dependent and inhibited by the selective angiotensin receptor type I (AT₁) blocker DUP 753. PDGF-directed migration of VSMCs was significantly inhibited by antibodies against β₃-and β₅-integrins, indicating an important role of these integrins in VSMC migration. However, AII augmented migration was not accompanied by an increased expression of β₃- and β₅-integrin mRNA and protein levels in VSMCs. Inhibition of the mitogen-activated protein kinase ERK 1/2 with PD 98059 (30 μM) completely abolished the effect of AII on PDGF-BB-directed VSMC migration (p < 0.01). The proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are cytoskeleton-associated protein kinases participating in integrin-dependent signaling. Therefore, expression and phosphorylation of these kinases was determined 48 h after AII treatment, revealing a significant increase in Pyk2 and FAK protein levels (up to 2-fold, both p < 0.05) and increased phosphorylation of Pyk2 (2-fold, p < 0.05) and ERK 1/2 (4-fold, p < 0.05) as compared to controls. Furthermore, immunofluorescence and Western blot analysis demonstrated a translocation of Pyk2 from the plasma membrane to the cytosol, as well as a perinuclear enrichment of ERK 1/2 protein 48 h after AII treatment. In conclusion, our data suggest that changes in the levels of Pyk2 and ERK 1/2 phosphorylation, responsible for integrin-dependent signaling, as well as their subcellular translocation are important for the enhanced chemotactic response of VSMCs after AII pre-treatment. Received: 16 July 2001/Returned for 1. revision: 2 August 2001/1. Revision received: 12 December 2001/Returned for 2. revision: 21 January 2002/2. Revision received: 14 February 2002/Accepted: 13 March 2002     

Angiotensin II type 1 receptor expression in human coronary arteries with variable degrees of atherosclerosis

Autopsy specimens of human coronary arteries were collected from 65 men and women ranging in age from 40–76 years of age. We made 209 coronary artery sections, which were graded in terms of severity of atherosclerosis by means of the Stary classification. Sections were stained using an antibody directed against the angiotensin II type 1 (AT1) receptor. We found that in non-atherosclerotic sections, staining was confined to vascular smooth muscle cells in the media. However, with the advent of atherosclerosis, AT1 receptor expression was also present in atherosclerotic plaque and involved other cell types including inflammatory cells and myofibroblasts. We identified a remarkable correlation between AT1 receptor staining and the severity of coronary atherosclerosis as well as intima-media thickness. These human data correspond well to animal models of atherosclerosis indicating an upregulation of AT1 receptor expression in atherosclerotic tissue. Received: 7 June 2001/Returned for 1. revision: 28 June 2001/1. Revision received: 31 July 2001/Returned for 2. revision: 20 August 2001/2. Revision received: 11 March 2002/Accepted: 11 March 2002     

Effect of non-selective endothelin blockade, TAK-044, on the ischemic cellular injury of rat heart

The aim of this study is to evaluate the role of non-selective endothelin blockade (TAK-044) in ischemic myocardial injury. Forty anesthetized rats were separated into four groups: 1) TAK-I group, after preinjection of TAK-044 (3 mg/kg), LAD was ligated for 60 min and reperfused for 60 min; 2) Saline (SAL)-I group, LAD ligation and reperfusion without TAK-044; 3) TAK-C group, sham operated TAK group; 4) SAL-C group, sham-operated SAL group. Myocardium from each group was separated and analyzed by biochemical and ultrastructural procedures. Reperfusion arrhythmia (VT) was observed in 88 % of the SAL-I Group, in contrast to only 36% of the TAK-I group. At the end of reperfusion, hemodynamics indicated no significant differences between these two groups. The Ca⁺⁺-ATPase activity of sarcoplasmic reticulum (SR) was 3.9μmoles Pi/mg protein/h (39 % of SAL-C group) in the SAL-I group, while that in the TAK-I group was significantly higher at 6.1 (54%). The ratio of infarct/risk area was 58% in the SAL-I group and 36% in the TAK-I group. In the ultrastructural observations, irreversibly injured cells of the ischemic portion were reduced significantly from 35% (SAL-I group) to 14% (TAK-I group). Thus, our results indicated that endothelin blockade reduced ischemic cellular injury. The mechanism of this reduction was speculated to be a restistance to ischemic injury in the subcellular levels of the myocardium conferred by a reduction of vascular constriction and improvement of imbalance in the energy supply and demand. Received: 28 July 1998, Returned for 1. revision: 27 August 1998, 1.Revision received: 14 September 1998, Returned for 2. Revision: 1 October 1998, 2. Revision received: 13 October 1998, Returned for 3. Revision: 3 November 1998, 3. Revision received: 1 December 1998, Accepted: 7 December 1998     

Establishment of a long-surviving murine model of myocardial infarction: Qualitative and quantitative conventional microscopic findings during pathological evolution

Summary Ongoing basic molecular analyses are being performed in mice, and a simple long-surviving murine model of myocardial infarction (MI) would be very useful in this regard. Although a few studies have included MI in mice by coronary artery ligation, the induction involves a complex technique and has a relatively high mortality rate. In addition, the identification of the basic pathological sequence is essential to the interpretation of experimental results. We developed a simple technique for the induction of MI in mice and examined qualitative and quantitative conventional microscopic findings during the pathological evolution over a 28-day observation period. Male BALB/c mice weighing approximately 25 – 30 g were anesthetized and then ventilated with a positive pressure ventilator. The heart was exposed by thoracotomy. Left coronary artery occlusion was performed by thermocoagulation using a thermocoagulation knife at the level of the tip of the left atrium. After establishing this surgical method, we used it to induce MI in 71 mice. The operative and postoperative mortality rates of this model were 5.6 % (4/71) and 12.6 % (9/71), respectively. In 3 (5.2%) of the 58 surviving mice, the area of infarct was not sufficient. The infarct area in the remaining 55 mice was 40 ± 9 % of the entire perimeter of the left ventricle. Conventional microscopic examinations with hematoxylin-eosin and Masson-trichrome staining disclosed that all of the characteristic histopathological features of MI occurred 1 – 2 days earlier than those in rats. Our surgical technique provides a sufficient infarct area, with an acceptable mortality rate. The present study clarified the histopathological sequence in this long surviving murine MI model. Received: 2 July 1998, Returned for revision: 19 August 1998, Revision received: 7 October 1998, Accepted: 7 October 1998