Rp-cAMPS has ho effect on adenosine A1 receptors in guinea-pig cardiomyocytes

Rp-cAMPS, a protein kinase A inhibitor, is used in the investigation of the cAMP-dependent systems. A report by Musgrave et al. (11) has suggested that Rp-cAMPS may also act on adenosine receptors. To determine whether this occurs in guinea-pig ventricular myocytes, Rp-cAMPS was applied in the presence and absence of DCPCX, an adenosine A₁ receptor antagonist. The isoprenaline-induced response was significantly decreased by Rp-cAMPS and the effect was not altered by the presence of DCPCX. Therefore Rp-cAMPS has no effect on cell contraction via adenosine A₁ receptors and can reliably be used to investigate cyclic AMP-dependent systems in isolated cardiac myocytes. Received: 23 April 1999, Returned for 1. revision: 26 May 1999, 1. Revision received: 23 August 1999, Returned for 2. revision: 28 September 1999, 2. Revision received: 15 November 1999, Accepted: 17 November 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     

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     

Early intervention with a potent endothelin-A/endothelin-B receptor antagonist aggravates left ventricular remodeling after myocardial infarction in rats

Intervention with selective endothelin (ET)A receptor antagonists within 24 h after myocardial infarction (MI) in rats has been reported to aggravate left ventricular (LV) remodeling. In contrast, beneficial effects are reported when initiation of treatment is delayed 7 days or more after MI. However, bosentan, a mixed ET_A/ET_B receptor antagonist with low affinity for the ET receptors, has been shown to exert beneficial effects independent of the time point of initiation of treatment after MI. The aim of the present study was to investigate to what extent early intervention with a mixed ET_A/ET_B receptor antagonist with higher affinity at the ET receptors (SB 209670) would also exert beneficial effects on postinfarction LV remodeling. After ligation of the left coronary artery, rats were randomized to treatment with SB 209670 (6.25 mg·kg⁻¹ SC b.i.d., n = 10) or vehicle (n = 12) for 26 days, starting 48 h after MI. Treatment with SB 209670 adversely affected the postinfarction remodeling process causing further dilatation of the LV (LV end-diastolic diameter: 10.4 ± 0.5 vs 9.1 ± 0.2 mm; LV end-systolic diameter: 8.5 ± 0.4 vs 7.2 ± 0.2 mm, P < 0.05). However, SB 209670 did not significantly affect infarct size, compensatory cardiac hypertrophy, nor the myocardial mRNA levels of procollagen type I and III, and prolyl 4-hydroxylase and lysyl oxidase, 2 important enzymes affecting collagen secretion, stability and functionality. In addition, SB 209670 had no significant effects on LV collagen cross-linking or extent of fibrosis. Thus, our data demonstrate that early intervention with a potent, mixed ET_A/ET_B receptor antagonist after MI may promote dilatation of the LV without significant alterations of infarct size and extracellular matrix composition. Our data support the notion that the timing of initiation of ET receptor antagonism after MI is critical and that potent ET receptor antagonists may be harmful during the first few days after MI. Received: 1 September 2001, Returned for revision: 13 September 2001, Revision received: 6 December 2001, Accepted: 21 December 2001     

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     

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     

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     

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     

Cardiac isoform of α-2 macroglobin, a novel serum protein, may induce cardiac hypertrophy in rats

Earlier studies from this laboratory have identified a novel high molecular weight (182 kDa) serum protein suggested to be involved in the development of cardiac hypertrophy. In the present case the role of this novel serum protein in the development of pressure-induced cardiac hypertrophy and the molecular events associated with it in experimental rats has been investigated. Multiple injections of this purified protein intravenously (through tail vein) into the normal animals lead to the development of cardiac hypertrophy and this is accompanied by an induction of muscle specific genes such as that of MLC2 and β-MHC characteristics of pressure overloaded heart. Further, the hypertrophy-specific serum protein has been found to be identical to rat α-2 macroglobulin (α-2M) in molecular weight (182 kDa) and in its appearance in blood serum. α-2M is an acute phase serum protein that increases markedly after inflammatory stimuli in hepatocytes in liver and gets secreted into the blood. The studies at present suggest that the 182kDa serum protein that appeared during the early stage of development of cardiac hypertrophy in aorta constricted rats is a glycoprotein localized in the heart that showed immunological cross reactivity with α-2M and is expressed in the heart as evinced by Northern blot analysis. Further this protein showed certain differences from rat α-2M under denaturing conditions in isoelectric focusing and partial peptide mapping. Partial peptide sequencing of the internal peptides of tryptic digest of 182kDa showed 100% identity of the sequences with α-2M sequences. Rat α-2M does not, however, have any influence on the development of cardiac hypertrophy and its antibody does not cross react with the 182 kDa protein. These data suggest that the 182 kDa protein that may play an indispensable role in the development of cardiac hypertrophy in experimental rats in cardiac specific, and may be an isoform of liver α-2M belonging to macroglobulin family. Received: 10 December 1998, Returned for 1. revision: 15 January 1999, 1. Revision received: 2 September 1999, Returned for 2. revision: 29 September 1999, 2. Revision received: 15 February 2000, Returned for 3. revision: 20 April 2000, 3. Revision received: 30 June 2000, Accepted: 5 July 2000     

Differential G protein receptor kinase 2 expression in compensated hypertrophy and heart failure after myocardial infarction in the rat

The onset of heart failure is associated with characteristic changes in myocardial expression of G protein receptor kinase 2 (GRK2). Although, GRK2 significantly contributes to the regulation of myocardial function in the failing heart, the GRK2 expression during cardiac hypertrophy without heart failure remains to be explored. We here report a differential expression of GRK2 in cardiac hypertrophy with or without heart failure in response to a myocardial infarction in the rat. Postmyocardial infarction animals were divided into two groups depending on the absence or presence of pulmonary edema, which is a manifestation of heart failure. Remarkably, cardiac GRK2 expression and activity were inhibited in animals with cardiac hypertrophy without heart failure, whereas animals with heart failure had elevated GRK2. Thus, three weeks after the infarction cardiac GRK2 expression in animals with hypertrophy alone was decreased to 0.34 of control, whereas in the group of animals with heart failure GRK2 expression was 1.89-fold higher than in sham-operated animals. GRK2 activity was affected in a similar way, three and nine weeks after the infarction cardiac GRK2 activity was reduced to 0.58 and 0.62 in animals with hypertrophy without heart failure when compared to sham operated animals. By contrast, GRK2 activity was increased by 1.32- and 1.21-fold three and nine weeks postinfarction in animals with heart failure when compared to sham animals. These data suggest that GRK2 expression is differentially regulated in hypertrophic, non-failing and hypertrophic, failing hearts. Received: 26 August 2002, Returned for 1. revision: 9 September 2002, 1. Revision received: 25 September 2002, Returned for 2. revision: 24 October 2002, 2. Revision received: 3 November 2002, Accepted: 9 November 2002 Correspondence to: S. P. Sheikh     

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     

Mechanisms involved in coronary artery dilatation during respiratory acidosis in the isolated perfused rat heart

A rat Langendorff heart preparation, perfused at constant pressure, was used to evaluate the role of K_ATP channels in respiratory acidosis-induced coronary hyperemia. Prior administration of glibenclamide, an inhibitor of K_ATP channels, reduced basal flow rates and eliminated the hyperemia associated with hypercapnia. These results implicate K_ATP channels as a functional link in the respiratory acidosis-induced increase in coronary flow. Received: 28 June 1999, Returned for revision: 11 August 1999, Revision received: 28 August 1999, Accepted: 29 September 1999     

The expression of heat shock protein 60 in myocardium of patients with chronic atrial fibrillation

Objective Cardiomyocytes respond to stress with the expression of different heat shock proteins (HSP). HSP60 is induced by various stress factors. The aim of this study was to investigate the expression of HSP60 in human atrial fibrillation (AF). Method Right atrial samples from 14 patients undergoing elective cardiac surgery were excised and immediately frozen in liquid nitrogen. Eight patients had chronic AF and six patients were in sinus rhythm. The HSP60 protein level was determined by SDS-PAGE, Western blot and quantified by optical densitometry according to the immunoreactive bands of actin. Results In myocardial samples from patients with chronic AF, we found a more than 2.5-fold increase in HSP60 expression compared to atrial myocardium of patients in sinus rhythm. Conclusion This result indicates an up regulation of HSP60 in response to chronic atrial fibrillation Received: 31 October 2001, Returned for 1. revision: 20 Novemver 2001, 1. Revision received: 12 December 2001, Returned for 2. revision: 3 January 2002, 2. Revision received: 25 January 2002, Accepted: 6 February 2002     

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     

Delayed attenuation of myocardial ischemia with repeated exercise in subjects with stable angina: a possible model for the second window of protection?

Aims: A delayed myocardial protection extends between 24 and 96 h after ischemic preconditioning in animals. To test this phenomenon in humans, subjects with stable angina were subjected to exercise test-induced myocardial ischemia and the effect of this “preconditioning” ischemic insult on the exercise-induced myocardial ischemia with the re-exercise after 24–96 hours was studied. Methods and results: Forty-eight males with a history of infarction and positive exercise test were recruited to the study. After baseline symptom-limited exercise test, the subjects were randomized to four experimental groups (n=12/group). The groups were allowed to recover for 24 h, 48 h, 72 h or 96 h before performing the second exercise test. Variables analyzed were heart rate-systolic blood pressure product at 1 mm ST segment depression, time to 1 mm ST segment depression, maximum ST segment depression, exercise duration, and the total ischemic time. There were no intergroup differences in baseline values for these variables. All variables were significantly improved at 24 h, the improvement peaked usually at 48 h (maximum increase in the variables by 31–46%), and the variables returned to baseline by 96 h after the first test. Conclusions: The exercise-induced ischemia caused transient attenuation of myocardial ischemia with re-exercise. Although the time-window and the time-course of this effect shows striking resemblance to those of the delayed preconditioning in animals, its mechanism remains speculative. The most probable mechanisms that may be involved include increased myocardial perfusion and/or some adaptive changes in the myocardium, the delayed preconditioning being one possibility. Received: 2 February 2000 Returned for 1. revision: 23 February 2000 1. Revision received: 3 April 2000 Returned for 2. revision: 3 May 2000 2. Revision received: 20 May 2000 Accepted: 26 May 2000     

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     

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     

Effects of preconditioning on myocardial interstitial levels of ATP and its catabolites during regional ischemia and reperfusion in the rat

The interstitial accumulation of adenine nucleotide breakdown products (ANBP) in the myocardium during ischemia has been shown to provide a useful index of the ischemic injury, whereas reperfusion ANBP washout rate has been regarded as an index of reperfusion damage. The purpose of this study was, using cardiac microdialysis, to examine in the rat model of regional ischemia/reperfusion the relationship between the duration of ischemia and these indices and to assess the profile of interstitial ATP concentrations and the beneficial effects of ischemic preconditioning (IP). The rats underwent 10, 20, 30 or 40 min of coronary artery occlusion and 50 min of reperfusion. Regional ischemia, with its duration, provoked a progressive increase in dialysate ANBP in the ischemic zone. The rate of purine washout during reperfusion exponentially declined with an increase in duration of the ischemic period. IP, induced by three 5-min episodes of ischemia, each separated by 5 min of reperfusion, significantly reduced the accumulation of ANBP during the 30-min period of sustained ischemia and resulted in a marked acceleration of reperfusion ANBP washout, indicating the improvement of postischemic microcirculation. These effects were suggested to be, at least in part, responsible for the infarct size limitation observed. Using the relationship between the duration of ischemia and ANBP washout rate, it could be demonstrated that IP produced similar facilitation of purine washout as shortening of the ischemic period in nonpreconditioned rats from 30 to approximately 7 min. Regional 20-min ischemia induced an early peak increase in interstitial fluid ATP which correlated with the maximal incidence of ventricular arrhythmias, whereas IP abolished both ATP release and arrhythmias during the sustained ischemia. These findings suggest that ATP may be an important mediator of ischemia-induced ventricular arrhythmias. Received: 20 April 1999, Returned for 1. revision: 11 May 1999, 1. Revision received: 23 June 1999, Returned for 2. revision:15 July 1999, 2. Revision received: 27 September 1999, Accepted: 29 September 1999     

A new screening method to diagnose coronary artery disease using multichannel magnetocardiogram and simple exercise

Background: Magnetocardiography (MCG) is a non-contact mapping technique to record cardiac action currents. The Master's two-step electrocardiogram (ECG) test is a simple exercise method for screening coronary artery disease (CAD), but it is inadequate concerning the sensitivity. Our aim was to develop a new screening method using multichannel MCG instead of ECG. Methods: Thirty subjects (aged 54 ± 16 years, 27 males), 17 of whom had CAD confirmed by coronary angiography, underwent the Master's exercise ECG test. After the exercise, MCG signals were acquired every minute during recovery with a 64-channel MCG system (MC-6400, Hitachi Ltd). We integrated tangential components of the MCG signals within QRS (during 20, 40, 80, and 120 ms centering on R-wave peak) immediately after exercise (Iex) and 5 minutes after exercise (Irec). The exercise-induced change of currents [(Iex-Irec)/Irec] was determined and normalized for each channel, and the maximal change among 64 channels, maximal QRS integral change, was used as a diagnostic index for myocardial ischemia. Results: The maximal QRS integral change during 40 ms was significantly higher in the CAD group than in the control group (0.81 ± 0.51 vs. 0.36 ± 0.19, p < 0.01). A sensitivity and specificity for predicting CAD by the change > 0.44 were 82 % and 85 %, respectively, yielding a diagnostic accuracy of 83 %. The conventional Master's ECG test identified the CAD patients with a diagnostic accuracy of 63 % (sensitivity 47 %, specificity 85 %). Conclusion: The Master's two-step exercise test with a 64-channel MCG system showed the high diagnostic accuracy, despite of non-contact recording and simple exercise. The magnetic field in the depolarization process has the potential to detect the subtle myocardial ischemia induced by exercise. Received: 20 June 2002, Returned for 1. revision: 11 July 2002, 1. Revision received: 3 September 2002, Returned for 2. revision: 25 September 2002, 2. Revision received: 10 October 2002, Accepted: 14 October 2002 Correspondence to: S. Nakatani, MD, PhD     

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