Blockade of A1 adenosine receptors prevents the ischaemia-induced sensitisation of adenylyl cyclase: evidence for a protein kinase C-mediated pathway

Objective: Acute myocardial ischaemia leads to a transient sensitisation of adenylyl cyclase which may contribute to the occurrence of malignant arrhythmias and the propagation of myocardial necrosis. It is prevented by blockade of protein kinase C (PKC) which is activated in early ischaemia as shown by its translocation from the cytosol to the plasma membranes. Translocation of PKC may also occur in ischaemic preconditioning, a process thought to be induced by activation of adenosine A₁ receptors. In this study it was investigated whether A₁ adenosine receptors may be involved in the sensitisation of adenylyl cyclase and the activation of PKC induced by ischaemia. Methods:Isolated rat hearts were perfused with the specific A₁ adenosine antagonist 8-caclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM) or adenosine (1 μM) prior to ischaemia induced by stop of perfusion for 5 and 10 min. Adenylyl cyclase activity was determined in plasma membranes stimulated by forskolin or stimulated via β-receptors by isoproterenol. Total PKC activity was measured in purified plasma membranes and in the cytosolic fraction using histone III-S as a substrate. Results:Myocardial ischaemia induced a β-receptor-independent sensitisation of adenylyl cyclase (forskolin-stimulated activity 515 ± 55 vs. 384 ± 30 pmol/min/mg protein) which was completely blocked by pre-perfusion with DPCPX (385 ± 23 vs. 386 ± 24 pmol/min/mg protein). DPCPX alone did not alter the responsiveness of adenylyl cyclase to stimulation. The stimulated adenylyl cyclase activity was increased by 20% after pre-perfusion with adenosine, mimicking the ischaemia-induced sensitisation. The effect of adenosine was not augmented by additional ischaemia. PKC activity was translocated from the cytosol to the plasma membranes by acute ischaemia, indicating an activation of the enzyme. This effect was completely abolished by DPCPX. Conclusion: These data demonstrate that in the rat heart the sensitisation of adenylyl cyclase in acute myocardial ischaemia is dependent on activation of A₁ adenosine receptors. It is suggested that the sensitisation of adenylyl cyclase by adenosine or ischaemia might be mediated by an activation of PKC. Received: 23 October 1998, Returned for revision: 19 November 1998, Revision received: 8 February 1999, Accepted: 22 February 1999     

Potential role of endothelin-1 and endothelin antagonists in cardiovascular diseases

The endothelins comprise a family of three isopeptides ET-1, ET-2 and ET-3, whereby ET-1 appears to be the most relevant in humans. They act in a paracrine manner on ET_A and ET_B receptors. ET-1 plays an important role in the cardiovascular system. In addition, it modulates vasomotion and growth processes, and it participates in thrombogenesis and neutrophil adhesion. This review summarizes some of the current literature pertaining to the physiological and pathophysiological significance of ET-1, focusing the assets and drawbacks of elevated ET-1 levels. In this regard, modulation of the endothelin system by either receptor blockade or by inhibition of endothelin converting enzyme is expected to provide novel therapeutic drug strategies. Received: 13 September 1999, Returned for revision: 2 November 1999, Revision received: 2 March 2000, Accepted: 23 March 2000     

Contribution of I<Subscript>K,ADO</Subscript> to the negative dromotropic effect of adenosine

Objective Despite the pathophysiological and therapeutic significance of the negative dromotropic effect of adenosine, its underlying ionic mechanism, and specifically the role of the adenosine-activated K⁺ current (I_K,ADO) is not experimentally defined. Therefore, we studied the contribution of I_K,ADO to the negative dromotropic effect of adenosine. Methods Effects of adenosine on single atrioventricular nodal and left atrial myocytes from rabbits were studied using the whole cell configuration of the patch clamp technique. Complementary experiments were done in rabbit and guinea pig isolated hearts instrumented to measure the atrium-to-His bundle interval. Results In contrast to its effect in atrial myocytes, Ba²⁺ selectively and completely blocked I_K,ADO at membrane potentials from −70 to 0 mV in atrioventricular nodal myocytes and abolished the adenosine-induced leftward shift of the reversal membrane potential. Ba²⁺ alone did not significantly prolong the A–H interval, but markedly attenuated the A–H interval prolongation caused by adenosine. In guinea pig heart, EC₅₀ values (pD₂± SEM) for adenosine-induced atrium-to-His bundle interval prolongation were 3.3 μmol/L (5.48 ± 0.04) and 13.2 μmol/L (4.88 ± 0.05, P < 0.001) in the absence and presence of Ba²⁺, respectively. Despite species-dependent differences in sensitivities to adenosine (guinea pig > rabbit), the relative contribution of adenosine-activated K⁺ current to the atrium-to-His bundle interval prolongation was nearly identical. In guinea pig hearts it ranged from 37.8 % (P = 0.013) to 72.5 % (P < 0.001) at 2 to 6 μmol/L adenosine, respectively. Conclusion I_K,ADO contributes significantly to the negative dromotropic effect of adenosine, but predominantly at relatively high concentrations of the nucleoside. Received: 16 November 2001/Returned for 1. revision: 12 December 2001/1. Revision received: 20 December 2001/Returned for 2. revision: 2 January 2002/2. Revision received: 14 January 2002/Accepted: 21 January 2002     

Delayed cardioprotection in a human cardiomyocyte-derived cell line: the role of adenosine, p38MAP kinase and mitochondrial KATP

Evidence of delayed preconditioning (PC) in man is limited. Adenosine is proposed as a trigger via action on the A₁ receptor in many species and the mitochondrial K_ATP channel is a likely end effector. We examined the ability of a brief, simulated ischemic episode on day one to provide delayed cardioprotection against lethal, simulated ischemia on day two in a human cardiac cell line with reference to the role of adenosine, the p38MAP kinase signalling pathway and mitochondrial K_ATP channel. Results: PC and adenosine administered on day 1 protected against cell death on day 2 as measured by LDH release and propidium iodide (PI) exclusion: (%LDH release: PC: 12.1 ± 1.1%, ADO: 11.9 ± 2.0% vs control: 36.4 ± 1.1%, %PI positive: PC: 14.6% ± 1.4%, ADO: 17.9 ± 2.0% vs control: 34.4 ± 2.0% respectively). This protection is abolished by treatment with SB203580 prior to the protective stimulus on day 1: [PC + SB (%LDH release 28.6 ± 2.8%, %PI positive 34.7 ± 2.2%) and ADO + SB (%LDH release 25.3 ± 2.9%; %PI positive 33.7 ± 7.3%)]. Similarly 5-hydroxydecanoate abolished protection, when given immediately prior to lethal simulated ischemia on day 2: [PC + 5-HD; (%LDH release 31.9 ± 4.8%; %PI positive 29.5 ± 2.0%) and ADO + 5-HD (%LDH release 36.9 ± 4.0%; %PI positive 34.8 ± 2%)]. Conclusion: In this model delayed PC can be mimicked by adenosine and involves the p38MAP kinase pathway and the mitochondrial K_ATP channel. Received: 2 November 1999, Returned for revision: 24 November 1999, Revision received: 23 December 1999, Accepted: 20 January 2000     

A new rat model of small vessel stenting

Objectives: Restenosis is the major complication of coronary angioplasty and stenting. In addition, the small vessel diameter represents a major limitation to the wide use of the technology. The aim of this study was to assess the feasibility and the vascular response of stent deployment in rat small vessels. Methods: In 40 Wistar rats (500–550 g) a Nir stent crimped on a 1.5 mm Comet angioplasty balloon catheter was deployed at high pressure in the common carotid artery. Neointimal area, neointima/media ratio and the arterial dimension were assessed immediately and at 7, 14, 21, and 28 days after stenting. Results: After stent deployment, the neointimal area and the neointima/media ratio increased progressively and peaked at 14 days (p < 0.05 vs 0 and 7 days). Alpha-actin-positive cells were found circumferentially organized on the lumen surface. At 21 and 28 days after stenting, the neointima and the neointima/media ratio were not statistically different compared with the results obtained fourteen days after stent deployment. No significant differences in the area of external elastic lamina were observed during the study period. In contrast, the internal lumen area was reduced significantly at 14, 21, and 28 days after the stent deployment. Subacute thrombosis rate after stent implantation was 26.5%. Conclusions: The results of this study demonstrated that the balloon expandable stents can be safely placed into rat arteries and the reduction of the internal arterial lumen observed after stent deployment was only due to the neointima formation whereas remodeling did not occur. Received: 5 August 1999, Returned for 1. revision: 6 October 1999, 1. Revision received: 23 November 1999, Returned for 2. revision: 7 December 1999, 2. Revision received: 22 December 1999, Accepted: 6 January 2000     

A role of PKC in the improvement of energy metabolism in preconditioned heart

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function. Received: 29 January 1999, Returned for 1. revision: 26 February 1999, 1. Revision received: 27 April 1999, Returned for 2. revision: 18 May 1999, 2. Revision received: 12 July 1999, Returned for 3. revision: 26 July 1999, 3. Revision received: 25 October 1999, Accepted: 3 November 1999     

Characterisation of the function of adult guinea-pig ventricular myocytes following co-culture with neonatal rat myocytes

Adult guinea-pig myocytes were co-cultured with a layer of spontaneously contracting neonatal rat myocytes based on a method described by Weisensee D. (In Vitro Cell Dev Biol 31: 190–195, 1995). Contractile studies were performed on freshly isolated, 24 and 48 h co-cultured adult guinea-pig myocytes to investigate whether alterations in contractile function had occurred. No difference was found between freshly isolated and 24 h co-cultured adult guinea-pig myocytes in terms of sensitivity to calcium, isoprenaline, frequency response and beat duration. After 48 h, the frequency response was depressed (P<0.02) and the beat was prolonged (P<0.05) when compared to that of freshly isolated myocytes. In the presence of the SR Ca²⁺ ATPase inhibitor, thapsigargin, the beat was significantly prolonged (P=0.003) in 24 h co-cultured myocytes but not in freshly isolated myocytes. These findings show that adult guinea-pig myocytes can be maintained in co-culture with neonatal rat myocytes with little change in contractile function for 24 h but after this time contractile function begins to deteriorate. Received: 3 March 1998, Returned for 1. revision: 25 March 1998, 1. Revision received: 26 May 1998, Returned for 2. revision: 18 June 1998, 2. Revision received: 23 July 1998, Accepted: 23 July 1998     

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo

Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes –E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field. Received: 1 October 2001, Returned for 1. revision: 18 October 2001, 1. Revision received: 19 November 2001, Returned for 2. revision: 6 December 2001, 2. Revision received: 13 February 2002, Accepted: 6 March 2002     

The upregulation of endothelin and its receptors in porcine coronary arteries in a double balloon injury model of restenosis

Endothelin, a potent vasoconstrictor, mitogen, and stimulant of collagen synthesis, is reported to be increased after vascular injury. We tested the hypothesis that tissue endothelin levels and its receptor expression are increased following double balloon injury in a porcine coronary artery model of restenosis. Male miniature swine maintained on a hyperlipidemic diet underwent oversized balloon injury to both the proximal right coronary artery and left circumflex coronary artery. Two weeks following the initial injury, the arteries were repeat injured at the same site and subsequently harvested four weeks later. Proximal balloon injured (BI) and distal non-balloon-injured (NBI) segments from the same artery were collected. Tissue endothelin-1 (ET-1) levels were measured by ELISA. Endothelin receptors were assayed by radioligand binding using ¹²⁵I-ET-1 and also immunolabeling. Tissue endothelin levels were 4–5 fold greater in BI arteries as compared to NBI. There was a significant increase in tissue ET-1 levels and endothelin receptor binding following double balloon injury relative to NBI control arteries. Western blots showed an increase expression of ET_A receptor protein in injured vessels compared to non-injured arteries. Immunohistochemistry using an ET_A receptor specific antibody confirmed increased receptor density following balloon injury. Thus, in an in vivo double balloon injury model for coronary artery restenosis, the response to vascular injury is increased tissue ET-1 content and upregulation of ET_A receptor density associated with increased receptor protein. Received: 1 March 1999, Returned for revision: 12 April 1999, Revision received: 11 May 1999, Accepted: 28 May 1999     

Chronic effects of ACE-inhibition (quinapril) and angiotensin-II type-1 receptor blockade (losartan) on atrial natriuretic peptide in brain nuclei of rats with experimental myocardial infarction

Alterations of the central nervous system may be important for imbalance of cardiovascular and fluid regulation in heart failure. The central renin-angiotensin and atrial natriuretic peptide (ANP) systems act as mutual antagonists. The effects of angiotensin converting enzyme (ACE) inhibition (quinapril, 6 mg/kg/day) and angiotensin II type 1 (AT₁) receptor blockade (losartan, 10 mg/kg/day) on ANP levels in 18 selected, microdissected brain nuclei were determined in sham-operated rats and rats with left ventricular dysfunction 8 weeks after myocardial infarction (MI). Plasma ANP tended to increase in MI rats and was further increased by quinapril. ANP was decreased in 12 brain areas of MI rats. ANP concentration was also significantly decreased by quinapril in six brain nuclei including subfornical organ and organum vasculosum laminae terminalis (areas lacking blood-brain barrier), and by losartan in 16 brain nuclei outside and within the blood-brain barrier in sham operated rats. However, both quinapril and losartan prevented a further reduction of central ANP as a result of myocardial infarction. These data suggest that there are effects on central ANP that result from chronic left ventricular dysfunction as well as an ACE-inhibitor and AT₁-antagonist. Mechanisms and consequences of central ANP depression remain unclear. They could, however, support systemic vasoconstriction and sodium and fluid retention. Received: 8 October 1999, Returned for 1. revision: 28 December 1999, 1. Revision received: 2 March 2000, Returned for 2. revision: 17 April 2000, 2. Revision received: 20 October 2000, Accepted: 9 November 2000     

Effect of atrial dilatation on electrophysiologic properties and inducibility of atrial fibrillation

Introduction: Atrial dilatation may play an important role in the occurrence of atrial fibrillation (AF) in clinical situations. However, the electrophysiologic characteristics of dilated atria are still unclear. Methods and results: In 18 isolated Langendorff-perfused canine hearts (14.6 ± 2.2 kg), we measured atrial effective refractory periods (ERPs) at four different sites, conduction velocity and percentage of slow conduction on the right atrium (using a high-density electrode plaque), and assessed the inducibility of AF at the baseline (0 cm H₂O) and high (15 cm H₂O) atrial pressure. The atrial ERPs did not change significantly, but the dispersion of ERP increased significantly (40 ± 18 vs 25 ± 9 vs ms, p = 0.01) during high atrial pressure. The percentage of slow conduction (< 25 cm/s) over the mapping area, and the inducibility of AF increased during high atrial pressure (23.7 ± 10.2 % vs 32.1 ± 12.5 %, p = 0.02). The AF inducibility significantly correlated with the ERP dispersion (R = 0.75, p < 0.001) and maximal percentage of slow conduction (R = 0.88, p < 0.001). Furthermore, ERPs were significantly shorter in the induced AF group than those without induced AF (68 ± 17 vs 84 ± 16 ms, P < 0.05). Conclusions: The increased inhomogenity in atrial electrophysiological properties during atrial dilatation contributed to the inducibility of AF. Received: 26 November 2001?Returned for 1. revision: 2 January 2002?1. Revision received: 11 February 2002?Returned for 2. revision: 25 March 2002?2. Revision received: 6 May 2002?Returned for 3. revision: 10 June 2002?3. Revision received: 21 August 2002?Accepted: 11 September 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     

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     

5-HD abolishes ischemic preconditioning independently of monophasic action potential duration in the heart

Objective: Blocking of the K_ATP channel with either glibenclamide or 5-hydroxydecanoate (5-HD) has been shown to abolish the infarct reducing effect of ischemic preconditioning (IPC) in hearts from several species, but the results in rat and rabbit have been equivocal. In this study we investigated if 5-HD could abolish IPC in rat and rabbit and further if IPC or IPV + 5-HD were affecting action potential duration in the rabbit heart. Methods: The rat hearts were isolated and retrogradely perfused on a Langendorff perfusion apparatus with Krebs-Henseleit buffer. The rabbit experiments were performed in an in situ model. Rat and rabbit hearts were subjected to 30 min regional ischemia by ligating a coronary artery followed by 120 min (rat) or 150 min (rabbit) of reperfusion. The preconditioning protocol was one or three cycles of 5 min ischemia plus 5 min reperfusion in the rat and one cycle of 5 min ischemia plus 10 min reperfusion in the rabbit. In the rat 5-HD was added to the reservoir before ischemic preconditioning in different concentrations, and in the rabbit 5-HD was given as a bolus 5 mg/kg intraventricularly 2 min before the preconditioning ischemia. In the rabbit epicardial monophasic action potential duration at 50% repolarization (MAPD₅₀) was measured at 1, 2 and 5 min in each of the ischemic periods using a contact pressure electrode. Infarcts were measured with tetrazolium staining and risk zone volumes with fluorescent microspheres. Results: All data are presented as infarct size in % of risk zone volume (mean ± SEM). In the rat 200 μM of 5-HD abolished the protective effect of one cycle of IPC (28.6 ± 4.7 versus 8.4 ± 0.8) and 500M of 5-HD abolished three cycles of IPC (50.7 ± 7.8 versus 8.4 ± 2.0). Control was 40.9 ± 2.8. In the rabbit 5-HD abolished IPC (41.2 ± 7.2 versus 8.1 ± 3.2). Control was 53.5 ± 12.4. MAPD₅₀ were significantly more shortened compared to control at 1 and 2 min into the 30 min ischemia for the IPC and IPC+5-HD. Conclusions: We conclude that 5-HD abolishes ischemic preconditioning when given before the preconditioning ischemia in both rat and rabbit but does not abolish into ischemia induced shortening of the action potential duration in the rabbit; thus, a role for the mitochondrial K_ATP channel and not the sarcolemmal K_ATP channel in the protective mechanism behind IPC is probable. Received: 15 July 1999, Returned for 1. revision: 17 August 1999, 1. Revision received: 13 September 1999, Returned for 2. revision: 12 October 1999, 2. Revision received: 3 November 1999, Accepted: 17 November 1999     

Cesium chloride induced ventricular arrhythmias in dogs: three-dimensional activation patterns and their relation to the cesium dose applied

Introduction: Cesium chloride has widely been used in experimental models to produce various ventricular arrhythmias. The study was designed to evaluate whether type and mechanism of these arrhythmias are dose-dependent. Methods: In 7 dogs with acute AV-block, 60 pins containing 4 bipolar electrodes each were inserted into both ventricles to provide 240 endo-, epi- and midmyocardial recording sites. A computerized mapping system was used to determine three-dimensional activation patterns of ventricular arrhythmias induced by three injections of 1 mmol/kg cesium chloride at 20 minute intervals. Results:Out of all arrhythmias induced, 25 ventricular extrasystoles, 31 monomorphic and 47 polymorphic ventricular tachycardias were mapped. Nonsustained ventricular tachycardias were readily inducible by a single bolus of cesium chloride, whereas sustained episodes required repetitive injections (1.45 ± 0.61 vs. 2.61 ± 0.57 doses, p < 0.05). Polymorphic tachycardias were observed more commonly than monomorphic tachycardias (87 vs. 31). Initiation and maintenance of cesium induced arrhythmias were exclusively based on focal mechanisms originating from the subendocardium, irrespective of morphology and dosage. All monomorphic arrhythmias were caused by repetitive firing of single immobile foci located in either the right or the left ventricle. Bi- and multifocal mechanisms, however, were found to underlie the polymorphic episodes. Conclusions: Although there is a dose-dependence as to the sustenance of mono- or polymorphic tachycardias, this does not reflect on the three-dimensional activation pattern of cesium induced arrhythmias, which are due to mono- or multifocal activation originating from the subendocardium. Received: 6 August 1999, Returned for 1. revision: 8 October 1999, 1. Revision received: 27 October 1999, Returned for 2. Revision: 24 November 1999, 2. Revision received: 9 December 1999, Accepted: 9 December 1999     

Cyclic GMP attenuates cyclic AMP-stimulate inotropy and oxygen consumption in control and hypertrophic hearts

We tested the hypothesis that increasing myocardial cyclic GMP would attenuate cyclic AMP induced positive inotropy and O₂ consumption, in part, through changes in cyclic AMP and that renal hypertension-induced cardiac hypertrophy (HYP) would alter this relationship. Anesthetized, open chest rabbits (N = 48) were divided into four groups of control (CON) and HYP animals which received vehicle (VEH), isoproterenol 10⁻⁶M (ISO), 3-morpholinosyndnonimine 10⁻⁴M, (SIN-1), or a combination of ISO+SIN-1. Coronary blood flow (micro-spheres) and O₂ extraction (microspectrophotometry) were used to determine O₂ consumption in both subepicardium (EPI) and subendocardium (ENDO). Left ventricular change in wall thickness (%) was increased significantly by ISO in both CON (16 ± 4 to 31 ± 6) and HYP (17 ± 2 to 24 ±3). Percent change in wall thickness was similar in the CON, SIN-1, and ISO+SIN-1 groups. Myocardial O₂ consumption (ml O₂/min/100 g) was increased by ISO in CON (10.3 ± 1.0 to 13.6 ± 2.0 EPI; 10.9 ± 1.0 17.1 ±1.7 ENDO) and HYP (8.2 ± 1.4 to 12.3 ± 2.2 EPI; 6.6 ± 1.4 to 14.8 ± 1.8 ENDO). Oxygen consumption was unaffected by SIN-1 in CON and HYP animals. ISO+SIN-1 caused attenuated ISO-induced increases in O₂ consumption in CON in EPI and ENDO, and in EPI in HYP. Cyclic GMP (pmol/g) was unchanged by ISO in CON and HYP, and increased by SIN-1 in CON (8.1 ± 1.3 to 19.2 ± 2.3 EPI) and HYP (9.1 ± 1.5 to 12.8 ± 2.0 EPI). Cyclic GMP remained elevated with ISO+SIN-1 in both groups. Cyclic AMP (pmol/g) was increased significantly by ISO in CON (496 ± 43 to 725 ± 106 EPI; 534 ± 44 to 756 ± 148 ENDO) and insignificantly in HYP (435 ± 50 to 566 ± 35 EPI; 497 ± 51 to 583 ± 47 ENDO). Cyclic AMP levels were unaffected by SIN-1 in either group. Isoproterenol induced increases in cyclic AMP were blunted by ISO+SIN-1 in CON (496 ± 43 to 537 ± 59 EPI) and not affected in HYP. The current study demonstrated attenuation of cyclic AMP mediated increased inotropy and O₂ consumption by increasing cyclic GMP, which appeared, in part, related to cyclic GMP-induced reduction in cyclic AMP. This effect of cyclic GMP on cyclic AMP was not observed in myocardial hypertrophy. Received: 4 January 1999, Returned for 1. revision: 29 January 1999, 1. Revision received: 30 March 1999, Returned for 2. Revision: 3 May 1999, 2. Revision received: 3 May 1999, Returned for 3. Revision: 12 May 1999, 3. Revision received: 23 June 1999, Returned for final revision: 7 July 1999, Accepted: 22 July 1999     

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     

Endogenous adenosine suppresses norepinephrine-induced ventricular arrhythmias in rat heart

Adenosine is an antiarrhythmic substance particularly effective in catecholamine-dependent tachycardias. Although endogenous adenosine substantially accumulates in catecholamine-stimulated hearts, little is known about the antiarrhythmic potency of endogenous adenosine in this condition. Therefore, we sought to demonstrate a potential antifibrillatory effect of endogenous adenosine either by blockade of adenosine receptors with 8-phenyltheophylline (8-PT) or by suppression of endogenous adenosine release with nitrobenzyl-6-thioinosine (NBTI). The study was performed in spontaneously beating Langendorff-perfused rat hearts. Adenosine release into the effluent was determined by HPLC methods. Catecholamine stimulation was induced by perfusing the hearts with norepinephrine (1 μmol/l) for 30 min, which caused ventricular tachycardia (VT) in 31% and ventricular fibrillation (VF) in 25% of control hearts (n=35). When 8-PT (10 μmol/l) was added to the perfusion buffer prior to norepinephrine, the incidence of VT and VF increased to 79 and 68%, respectively. The addition of 8-PT did not affect the catecholamine-dependent formation of adenosine. Perfusion of the hearts with NBTI (10 μmol/l) prior to norepinephrine reduced adenosine release and increased the occurrence of both VT (65%) and VF (40%). In summary, the results indicate that adenosine is an endogenous antiarrhythmic substance, which accumulates in catecholamine-stimulated myocardium to a level, which effectively suppresses the occurrence of ventricular arrhythmias. Received: 26 November 1997, Returned for 1. revision: 18 December 1997, 1. Revision received: 15 January 1998, Returned for 2. revision: 29 January 1998, 2. Revision received: 18 February 1998, Accepted: 18 February 1998     

Phase angle convergence of multiple monophasic action potential reordings precedes spontaneous termination of ventricular fibrillation

Aims To elucidate the mechanism of spontaneous termination of ventricular fibrillation (VF) and to define an indicator of its occurrence, the phase angle, a novel measure to assess synchrony of activation, was evaluated. Methods and results In 7 isolated rabbit hearts, 7 monophasic action potentials were recorded simultaneously. Ventricular fibrillation was induced by T wave shocks. Cycle lengths (CL) and phase angles between all 7 recordings were analyzed until spontaneous termination or shock-induced defibrillation. Average phase angle was calculated as activation time difference to a reference channel and expressed as a fraction of the reference channel's CL with 1 equaling a complete CL. Initial CLs and phase angles were similar in sustained and terminating episodes (CL: 141±16 ms vs 142±24 ms, phase angle: 0.244±0.11 vs 0.263±0.1, p=NS). During spontaneous termination, CL increased slightly by 7%. Average phase angle converged gradually over the last three activations before termination of ventricular fibrillation by 22–48% (p<0.0005), eventually resulting in phase angles similar to paced rhythms directly prior to spontaneous termination of ventricular fibrillation. Conclusions Gradual synchronization of activation is part of the electrophysiological mechanism resulting in spontaneous ventricular fibrillation termination and can be detected three activations before termination. Phase angle convergence may be useful to detect spontaneous termination of ventricular fibrillation. Received: 24 October 1997, Returned for 1. revision: 24 November 1997, 1. Revision received: 21 January 1998, Returned for 2. revision: 4 March 1998, 2. Revision received: 29 April 1998, Returned for 3. revision: 25 May 1998, 3. Revision received: 15 June 1998, Accepted: 17 June 1998     

Reduction of oxygen delivery during post-ischemic reperfusion protects the isolated guinea pig heart

Objective: To further characterise the influence of oxygen delivery during early reperfusion (first 5 min) in the isolated guinea pig heart, three modes of coronary reperfusion were chosen, differing with respect to reperfusion flow and arterial PO₂. Methods: Isolated working guinea pig hearts underwent ischemia and reperfusion (15 min each). Reperfusion was at constant pressure (Group 1, 60 mmHg, n = 7) or at constant flow (Group 2, 5 ml/min, n = 7) with a PO₂ of 600 mmHg. Group 3 (n = 8) was reperfused at 5 ml/min with a PO₂ of 300 mmHg for 5 min and a PO₂ of 600 mmHg thereafter. Lactate release and oxygen consumption were determined during reperfusion. Glutathione release served to assess myocardial oxidative stress. Results: After ischemia, hearts in Group 1 (mean coronary flow 14.4±1.1 ml/min during the first 5 min of reperfusion) performed external heart work at 31 ± 2 % of the pre-ischemic level. Performance in Group 2 recovered to 50 ± 3 % and in Group 3 to 68 ± 3 %. Myocardial oxygen consumption during early reperfusion (2nd min) was lowest in Group 3 (1.9 μmol/min) and highest in Group 1 (8.3 μmol/min). No difference in lactate release was observed. Release of glutathione during the first 5 min of reperfusion was 43.8 ± 7.9 nmol in Group 1, but only 3.6 ± 0.7 in Group 2 (p < 0.05). Conclusions: In isolated guinea pig hearts, controlled oxygen delivery during post-ischemic reperfusion by both, reduction of coronary flow and PO₂, improves recovery of pump function. The effect is accompanied by less oxidative stress, as indicated by lowered rates of glutathione release. Received: 1 December 1998, Returned for 1. revision: 4 January 1999, 1. Revision received: 28 January 1999, Returned for 2. revision: 8 February 1999, 2. Revision received: 18 February 1999, Accepted: 2 March 1999