The isolated working heart model in infarcted rat hearts.

Congestive heart failure (CHF) is one of the most common causes of death in western countries. The aim of this study was to establish and validate the working heart model in rat hearts with CHF. In the rat model the animals show parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The focus of attention was the evaluation of cardiodynamics (e.g.contractility) in the isolated 'working heart' model. The geometric properties of the left ventricle were measured by planimetry (stereology). Formulae available in the past for determining certain parameters in the working heart model (e.g.external heart work) have to be fitted to the circumstances of the infarcted rat hearts with its different organ properties.CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive rats (SHR/NHsd) by creating a permanent (8 week) occlusion of the left coronary artery, 2 mm distal to the origin from the aorta, by a modified technique (Itter et al. 2004). This resulted in a large infarction of the free left ventricular wall. We were able to establish and adapt a new and predictive working heart model in spontaneously hypertensive rat hearts with myocardial infarction (MI) 8-12 weeks after coronary artery ligation. At this stage the WKY rat did not show any symptoms of CHF. The SHR rat represented characteristic parameters and symptoms that could be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of CHF such as dyspnoea, subcutaneous oedema, palebluish limbs and impaired motion were prominent. On necropsy the SHR showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. In the working heart model the infarcted animals showed reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd than in the Wistar Kyoto rat (WKY/NHsd). The aim for the future is to find a causal therapy of heart failure treatment. At present, only palliative therapy is possible for patients with heart failure. For this reason the working heart model in CHF rat hearts should provide a valuable method for early testing of new therapeutic approaches for patients with CHF.

A model of chronic heart failure in spontaneous hypertensive rats (SHR).

Common models of chronic heart failure (CHF) do not always result in parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The aim of this study was to establish and validate a new model of CHF in the rat. CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive (SHR/NHsd) rats by creating a permanent (8-week) occlusion of the left coronary artery 2 mm distal to the origin from the aorta by a modified technique. This resulted in a large infarction of the free left ventricular wall. The focus of attention was the validation of the geometric properties of the left ventricle and its contractility. The validation of the geometric properties of the left ventricle was done by a non-invasive magnetic resonance imaging (MRI) technique and by planimetry (stereology). Cardiodynamics (e.g. contractility) were evaluated in the isolated 'working heart' model. We were able to establish a new and predictive model of heart failure in the spontaneously hypertensive rat 8 weeks after coronary artery ligation. At this time point, the WKY rat did not show any symptoms of CHF. The model represents characteristic parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of congestive heart failure were prominent, such as dyspnoea, subcutaneous oedema, pale-bluish limbs and impaired motion. Non-invasive sequential measurements by NMR techniques showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. The infarcted animals showed a reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd rat than in the WKY/NHsd rat. Furthermore the infarcted animals showed enhanced levels of hydroxyproline/proline ratios, again much more so in the SHR/NHsd rat than in the WKY/NHsd rat.

Ischemic preconditioning and infarct mass: the effect of hypercholesterolemia and endothelial dysfunction.

In an experimental model of atherosclerosis we investigated whether rabbits fed an atherogenic diet (0.25% cholesterol, 3% coconut oil) develop endothelial dysfunction accompanied with increased infarct mass compared to normal fed rabbits and, whether hypercholesterolemia would interfere with the beneficial outcome of ischemic preconditioning observed in normal rabbits. After four weeks on either a normal or an atherogenic diet, New Zealand White rabbits (n=7 in each group) were subjected to 30 min of myocardial ischemia by occlusion of a branch of the left anterior descending coronary artery (LAD) followed by 2 hours of reperfusion (infarct studies). For ischemic preconditioning experiments, LAD was additionally occluded twice for 5 min followed by 10 min reperfusion before the long-lasting (30 min) ischemia. Infarct mass was evaluated by triphenyl-tetrazolium staining. Besides the assessment of aortic endothelium-dependent function and NO-release, aortic and cardiac vessels were inspected for atherosclerotic lesions. Total cholesterol serum levels in rabbits on an atherogenic diet were significantly higher (15.3+/-2.7 mmol/L) than those on a standard diet (0.65+/-0.08 mmol/L). The aortas and heart vessels were without any histological evidence of atherosclerosis, whereas endothelial dysfunction and significantly reduced calcium-ionophore stimulated endothelial NO-release were found in isolated aortic rings of hypercholesterolemic animals. Rabbits on a standard diet showed an infarct mass (related to the area at risk) of 41+/-33%, which was reduced to 21+/-2% by ischemic preconditioning (49% decrease, p<0.05). In rabbits on an atherogenic diet, infarct mass was significantly increased to 63+/-3% (52% increase versus standard diet). Interestingly, hypercholesterolemia did not affect the beneficial influence of ischemic preconditioning; infarct mass (21+/-3%, p<0.05 vs hypercholesterolemia) was similar to rabbits on a standard diet with ischemic preconditioning. Our results show that experimental hypercholesterolemia increases infarct mass in nonpreconditioned hearts but it does not interfere with the reduction of infarct mass elicited by preconditioning. This may suggest that NO produced by the endothelium is not a prime factor in the cardioprotective mechanism of preconditioning.

Ramiprilat prevents the development of acute coronary endothelial dysfunction in the dog.

We investigated the effect of an infusion of ramiprilat on the development of coronary endothelial dysfunction. In anesthetized dogs, the endothelium-dependent vasodilators acetylcholine (ACh, 5 and 10 microg x min(-1) for 1 min) and serotonin (5-HT, 50 and 100 microg x min(-1) for 1 min) and the endothelium-independent vasodilator nitroglycerin (NTG, 50 and 100 microg x min(-1) for 1 min) were given intracoronarily (i.c.) both prior to and after 60 min of ischemia (I) and 180 min of reperfusion (R) of a coronary artery. During I/R the dogs received i.c. either saline (N = 22) or ramiprilat (40 ng/kg x min(-1), N = 14). At the end of the experiment, a biopsy of the most distal coronary bed was processed for scanning electron microscopy (SEM). Prior to I/R all vasodilators induced a similar dose-related increase in coronary flow in both groups. Following I/R, in controls the responses to ACh and 5-HT were significantly blunted (ACh: -39% and -34%; 5-HT: -48% and -49%); those to NTG were unchanged. Ramiprilat significantly prevented the blunting of the responses to ACh (-5%, and -10%) and 5-HT (-11%, and -19%). SEM of control subepicardial arterioles showed adhesion of leukocytes to the endothelium and crater formation. No craters were seen in the ramiprilat-treated dogs. Thus, an acute infusion of ramiprilat significantly prevents the development of coronary endothelial dysfunction. Additionally, the appearance of crater-like changes on the endothelial surface can be taken as a morphological marker of endothelial dysfunction.

Late treatment with ramipril increases survival in old spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) begin to die from cardiovascular complications at approximately 15 months of age. We tested whether chronic ACE-inhibitor treatment would extend the lifespan of such old animals. We also studied cardiac hypertrophy and function, endothelial function and expression, and activity of NO synthase (eNOS). One hundred 15-month-old SHR were randomized into 3 groups, control (n=10), placebo-treated (n=45), and ramipril-treated with an antihypertensive dose of 1 mg. kg(-1). d(-1) in drinking water (n=45). Ex vivo experiments were performed after 15 months (control) and 21 months, when approximately 80% of the placebo group had died. Late treatment with ramipril significantly extended lifespan of the animals from 21 to 30 months. Fully established cardiac hypertrophy, observed in placebo-treated animals and in controls, was significantly reversed by ramipril treatment. In isolated working hearts, a significantly improved function associated with increased cardiac eNOS expression was seen versus placebo and control hearts. Endothelial dysfunction in isolated aortic rings from control and placebo-treated SHR was significantly improved by ACE inhibition and associated with enhanced NO release. Late treatment of SHR with the ACE inhibitor ramipril extended lifespan from 21 to 30 months, which is comparable to the lifespan of untreated normotensive Wistar-Kyoto rats. This lifespan extension, probably due to blood pressure reduction, correlated with increased eNOS expression and activity followed by a regression of left ventricular hypertrophy and cardiac and vascular dysfunction.

Cardiotonic activity of the water soluble forskolin derivative 8,13-epoxy-6 beta-(piperidinoacetoxy)- 1 alpha, 7 beta, 9 alpha-trihydroxy-labd- 14en-11-one.

8,13-Epoxy-6 beta-(piperidinoacetoxy)-1 alpha,7 beta, 9 alpha-trihydroxy-labd -14en-11-one (HL 706, CAS 114376-11-3) is a water soluble derivative of forskolin with positive inotropic and vasodilating properties. In both in vitro and in vivo preparations, it exhibited significant positive inotropic activity with concomitant increase in heart rate and decrease in mean blood pressure. Though its potency is lower than that of forskolin, its duration of action is more prolonged. In conscious dog experiments, HL 706, administered orally, also showed a dose related increase in LV dP/dtmax. A significant reversal of cardiac failure was attained in anaesthetised dogs subjected to propranolol induced cardiac insufficiency. HL 706, like forskolin increased cAMP by virtue of its adenylate cyclase stimulant activity. Through increase in cAMP it also exhibited non-specific smooth muscle relaxant activity in isolated vascular and ileal preparations. Its therapeutic ratio is quite favourable.

Bradykinin-mediated metabolic effects in isolated perfused rat hearts.

Bradykinin perfusion (BK 1 x 10(-12) to 1 x 10(-8) mol/l) of isolated working rat hearts with postischemic reperfusion arrhythmias induced a reduction of the incidence as well as duration of ventricular fibrillation, improvement of cardiodynamics via increased left ventricular pressure, contractility, and coronary flow without changes in heart rate. These beneficial effects were accompanied by reduced activities of the cytosolic enzymes lactate dehydrogenase and creatine kinase as well as lactate output. In the myocardial tissue lactate content was reduced and the energy rich phosphates increased compared to saline perfused control hearts. Glycogen stores were also preserved. These beneficial effects of BK were concentration-dependently abolished by perfusion of the B2 kinin receptor antagonist HOE 140 and the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA). These results suggest that improved cardiac function during and after myocardial ischemia as well as increased energy rich phophates and glycogen stores are mediated by BK and the subsequent release of NO, shifting myocardial metabolism during ischemia and reperfusion to the glucose pathway which leads to changes indicative for cardioprotection.

Intracardiac generation of angiotensin and its physiologic role.

The emerging recognition of the existence and potential biological significance of local tissue renin-angiotensin systems in a number of organs has fostered interest in a possible intrinsic cardiac renin-angiotensin system. Evidence for such a system was first provided by biochemical measurements of components of the renin-angiotensin system in cardiac tissue. It has recently been demonstrated that the genes coding for renin and angiotensinogen are expressed in all regions of the heart, an essential prerequisite for the postulated intracardiac biosynthesis of these proteins. Moreover, we have shown the presence of a functional and physiologically active pathway for the conversion of angiotensin I to angiotensin II in the beating mammalian heart. This conversion appears to be catalyzed by a specific cardiac converting enzyme that is susceptible to systemically administered converting-enzyme inhibitors. Evidence for the physiologic importance of the cardiac renin-angiotensin system comes from experimental data as well as indirect clinical evidence. The potent coronary vasoconstrictor properties of angiotensin II underscore its possible significance in myocardial ischemia and ischemic heart disease, in particular when viewed in the context of selective local activation. The long-known positive inotropic effects of angiotensin II are based on its direct myotropic properties and on its facilitatory effects on sympathetic neurotransmission and may be of added significance in metabolically compromised states. We have recently demonstrated that locally generated angiotensin may be a dominant etiologic factor in the pathogenesis of reperfusion arrhythmias. In addition, we have found experimental evidence for a deleterious effect of angiotensin II on myocardial metabolism in the setting of regional myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue renin-angiotensin systems: focus on the heart.

Recent findings support the existence of independently functioning, local renin-angiotensin systems in a number of tissues. The clinical importance that inhibitors of the renin-angiotensin system have gained in the treatment of hypertension and cardiac failure, as well as molecular biology data, suggest that a functional, tissue renin-angiotensin system is present in the heart. Using several experimental approaches we present evidence to support the existence and the possible physiopathological relevance of such a cardiac renin-angiotensin system. Tissue angiotensins were documented and quantified in different regions of the heart by high performance liquid chromatography combined with specific radio-immunoassay. Reduction of cardiac angiotensin after administration of converting enzyme inhibitors in nephrectomized animals indicates that these peptides are generated locally. Effects of converting enzyme inhibitors on angiotensin II-dependent facilitation of cardiac sympathetic tone further support this concept and emphasize the potential physiological role of a cardiac renin-angiotensin system. Further, direct evidence for local generation of angiotensin II in the myocardium is provided by assessment of the intracardiac conversion of angiotensin I to angiotensin II and by measurement of the activity of angiotensin converting enzyme in the heart.

Hypotensive action of an intra-nasally applied angiotensin II-antagonist.

Intranasal administration of the specific angiotensin II-antagonist (1-NSuc-5-Val-8-Phg) A II was investigated in anaesthetized rats with different forms of experimentally elevated blood pressure. Renin- or angiotensin II-induced blood pressure increases were markedly reversed by the angiotensin II-antagonist applied intra-nasally. In rats with acute accelerated elevation of blood pressure the analogue induced also a significant decrease. No change could be observed in rats with chronic renal hypertension.

Blood pressure response to central and peripheral injection of angiotensin II and 8-C-phenylglycine analogue of angiotensin II in rats with experimental hypertension.

1. We have compared the effect of central and peripheral administration of angiotensin II and (1-succinamoly-5-valine-8-phenylglycine)angiotensin II on blood pressure of male conscious unrestrained rats with normal blood pressure, and with spontaneous hypertension or chronic renal hypertension. 2. After central and peripheral injection of angiotensin II all rats exhibited a significant dose-related increase in blood pressure. 3. Administration of the analogue was without effect in normotensive rats. Ten-weeks-old rats with spontaneous hypertension showed a significant blood pressure decrease after central injection, but an increase after peripheral injection. This centrally induced decrease could not be observed in spontaneously hypertensive rats 14 weeks old. In these animals the analogue increased the blood pressure. In rats with chronic renal hypertension in contrast to peripheral injection, central administration decreased the pressure significantly. 4. Plasma renin activity was not changed after central injection of the analogue in normotensive rats. 5. These observations suggest the participation of the intrinsic brain isorenin-angiotensin system in central blood pressure regulation in these forms of experimental hypertension.

1,8-disubstituted analogues of (Ile5) and (Val5)angiotensin II: difference in potency and specificity of angiotensin II-antagonistic activity.

Depending on the species, position 5 in angiotensin II is occupied by isoleucine or valine. 1,8-disubstituted analogues of [Ile5]angiotensin II show distinct differences from the corresponding [Val5]angiotensin II analogues in the potency and specificity of their inhibitory action. The syntheses of new analogues are described.

Comparative pharmacology of new specific angiotensin antagonists.

1. The angiotensin II antagonism by newly synthesized 8-C-phenylglycine analogues of [5-isoleucine]angiotensin II in different preparations was investigated in vitro and in vivo. 2. All analogues competitively inhibited the myotropic effect of angiotensin II on the isolated colon ascendens of the guinea-pig and the stomach of the rat. 3. In normotensive dogs, cats, rabbits, guinea-pigs and rats the blood pressure response to infused angiotensin II was inhibited by the antagonists. The angiotensin II-induced fall in renal blood flow in the dog was blocked during infusion of the analogues. Acute renal hypertension in rats was significantly decreased. Of conscious rats variously with normal blood pressures, spontaneous hypertension and chronic renal hypertension, only in the last group could a marked uniform fall in blood pressure be demonstrated. The central pressor effect of angiotensin II was also inhibited in conscious rats. 4. 8-C-Phenylglycine analogues of [5-isoleucine]-angiotensin II exhibit a specific antagonistic activity to endogenous and exogenous angiotensin II.