The pharmacological treatment of congestive heart failure, a condition associated with a high incidence of ventricular dysrhythmias and sudden cardiac death, is a challenging task for clinicians. Elevated concentrations of adenosine 3′,5′-cyclic monophosphate (cAMP) have been implicated in the genesis of ischaemia-induced dysrhythmias and the lowering of the fibrillation threshold in the dog, pig and baboon heart , in the isolated rat heart , and in human beings . Direct evidence for the dysrhythmogenicity of cAMP was provided by the decrease in the ventricular fibrillation threshold noted during perfusion of the isolated rat heart by dibutyryl cAMP, the permanent form of cAMP . An infusion of cAMP analogues or agents increasing myocardial cAMP at the visible border of the ischaemic zone in pig hearts can provoke ventricular tachycardia (VT) or ventricular fibrillation . Furthermore, elevation of ventricular guanosine 3′,5′-cyclic monophosphate (cGMP) as well as cAMP increases ischaemia-induced ventricular dysrhythmias in the anaesthetized rat [6,7]. In contrast, it has been suggested that cGMP can be an endogenous intracellular cardioprotector and its actions may account for the low susceptibility to ventricular fibrillation normally encountered in hearts reperfused after sustained ischaemia . On the other hand, it is known that congestive heart failure is associated with a high incidence of dysrhythmias that complicate the management of heart failure and contribute to mortality and morbidity . Numerous treatments have been proposed and tested for the long-term management of chronic heart failure to provide adequate symptomatic relief and protection from sudden death in all patients. Phosphodiesterase (PDE) inhibitors, which raise cAMP content by blocking its hydrolysis, improve the haemodynamic status of heart failure via inotropic or vasodilatory effects, or both, and these drugs have a considerable efficacy in the treatment of congestive heart failure, particularly for inotropic support after cardiac surgery [10,11]. However, chronic administration of these drugs affects survival adversely . In addition, a high dose of milrinone, a selective PDE III inhibitor, increases dysrhythmias during acute myocardial ischaemia in rat . Therefore, the purpose of the present study was to evaluate the effects of different PDE inhibitors on ventricular dysrhythmias and cyclic nucleotide content by use of a model of ischaemia-induced dysrhythmias in the pentobarbital anaesthetized rat. The drugs chosen were pimobendan, a selective PDE III inhibitor, rolipram, a selective PDE IV inhibitor, and zaprinast, a selective PDE V inhibitor.
Surgical and recording procedures
The animals were cared for in accordance with our Local Ethics Committee and the National Institute of Health Guidelines. The method used to produce coronary artery occlusion in anaesthetized rats has been fully described previously . Briefly, male Sprague-Dawley rats (250-350 g) were anaesthetized with sodium pentobarbital (60 mg kg−1 intraperitoneally). One-hundred-and-forty rats were used in the study. The trachea was cannulated and the animals artificially ventilated with a miniature Ideal® respiration pump (Harvard, Rodent ventilator: 47 breaths min−1, tidal volume 4 mL). The femoral vein was cannulated for drug administration and the carotid artery cannulated for direct measurement of arterial pressure. Mean arterial pressure was calculated as diastolic pressure + one-third pulse pressure. A left thoracotomy was performed and the fourth rib removed. After the pericardium was opened, the heart was exteriorized and a fine silk ligature placed around the left anterior descending coronary artery (the ligature was not tightened at this stage). The heart was then returned to the chest cavity and left to stabilize for 15 min before starting the experimental protocol.
Animals with a mean arterial pressure <70 mmHg after placement of the ligature were excluded from the experiments as were those that demonstrated dysrhythmias before the ligature being tied. Following coronary ligation, animals that died within the first 5 min due to a progressive but complete loss of arterial pressure without demonstrating dysrhythmias were also excluded.
After the stabilization period, a drug or physiological saline was administered intravenously (i.v.) as an initial bolus dose followed immediately by a continuous infusion. The three drugs used in this study were given at two different doses: rolipram (30 μg kg−1 + 3 μg kg−1 min−1, or 100 μg kg−1 + 10 μg kg−1 min−1); pimobendan (300 μg kg−1 + 30 μg kg−1 min−1, or 1 mg kg−1 + 0.1 mg kg−1 min−1); and zaprinast (300 μg kg−1 + 30 μg kg−1 min−1, or 1 mg kg−1 + 0.1 mg kg−1 min−1). Control animals were treated only with the appropriated solvents: 25% v/v polyethylene glycol 300 in water, or 10% v/v dimethylsulphoxide in water. The coronary artery was occluded 15 min after commencing drug administration and myocardial ischaemia was maintained for 30 min. Over a 30 min ischaemic period, heart rate and mean arterial pressure were recorded.
To study ventricular cyclic nucleotide content (n = 70), the same protocol described above was followed, with the exception that 30 min following coronary artery ligation the heart was removed. Hearts were rapidly washed in ice-cold saline, the free wall of the left ventricle (ischaemic tissue) and the right ventricle (comprising the right ventricle and the intraventricular septum, non-ischaemic tissue) were frozen and stored at −80°C until the day of assay. The time taken to excise the heart, dissect and freeze was approximately 15 s.
Determination of cyclic nucleotide content
cAMP and cGMP were determined by means of cAMP-3H or cGMP-3H by radioimmunoassay following the indications of the manufacturer (Amersham International, Amersham, UK). Tissues were extracted in 0.3 mol perchloric acid (in a ratio of 1:10 w/v) with a Polytron® homogenizer. Extracts were centrifuged at 12 000 rpm for 15 min using a Biofuge A® centrifuge (Heraus, Berlin, Germany). Supernatant was treated with potassium hydroxide solution until pH 7.5 was reached. The samples were centrifuged once and the supernatant was used for cAMP and cGMP analysis. The sensitivity of the assay for cAMP and cGMP was 2 and 10 pmol mL−1, respectively. Protein was determined according to a Lowry modified procedure .
Determination of dysrrhythmias
The number of premature ventricular beats (PVB) and the incidence and duration of VT - a succession of seven or more PVB - and ventricular fibrillation (chaotic ventricular contraction associated with a complete collapse of arterial pressure) were counted 20 min before ligature without drug (control), 15 min before ligature with drug or solvent (−15 min) and during 30 min after the ligature.
Rolipram was dissolved in polyethylene glycol. Pimobendan (Boehringer Ingelheim, Spain) and zaprinast (Rhone-Poulenc Rorer, London, UK) were dissolved in dimethylsulphoxide. The drugs were administrated as an i.v. bolus followed by a continuous infusion. Control animals were treated with their respective solvents.
All data are the mean ± SD. Data were analysed by one-way analysis of variance followed by the Newman-Keuls post hoc test. The incidence of dysrhythmias was analysed by non-parametric t-test and the observed mortality in drug-treated groups was compared with the corresponding control group by Fisher's exact test.
Effect upon ischaemia-induced dysrhythmias
There were no significant changes in the PVB count and duration of ventricular fibrillation with any of the drugs used compared with controls. Both doses of rolipram did not induce significant changes in the incidence of dysrhythmias or in mortality compared with the control group. In contrast, the incidence of ventricular fibrillation was increased in rats that received higher doses of pimobendan (88%, P < 0.05) or zaprinast (89%, P < 0.05) resulting in an increased in mortality (Fig. 1).
Table 1 shows the total PVB count, the duration of VT and ventricular fibrillation in survivors from the 30 min ischaemic period, solvents and the treated groups. Both doses of rolipram and pimobendan (1 mg kg−1 + 0.1 mg kg−1 min−1) significantly (P < 0.05 and <0.01) decreased the duration of VT. However, low-dose pimobendan and both low- and high-dose zaprinast did not significantly affect this variable.
Coronary artery occlusion did not induce haemodynamic changes in the rats treated with solvents only. Rolipram and the higher doses of pimobendan did not change the heart rate or arterial pressure. However, the low dose of pimobendan caused significant decreases of heart rate and arterial pressure 10 min after coronary ligature and remained low for 30 min after ligature. Similarly, the high dose of zaprinast decreased heart rate 15 min before coronary artery occlusion and after 20 min after the ligature (Tables 2 and 3).
Effect of cyclic nucleotide concentrations
In this series of experiments, significant differences (P < 0.01) in cAMP content were observed between the ischaemic left ventricle and non-ischaemic right ventricle in the groups treated with solvent only (Fig. 2). None of the drugs used had significant effects on the cAMP content in the left or right ventricle (Fig. 2).
On the other hand, significant differences in cGMP content were observed between the ischaemic left ventricle and the non-ischaemic right ventricle in the solvent groups. The higher doses of pimobendan or zaprinast (i.e. 1 mg kg−1 + 0.1 mg kg−1 min−1) increased the cGMP content in the left ventricle (0.031 ± 0.002 pmol mg−1, P < 0.01; 0.027 ± 0.001 pmol mg−1, P < 0.05), respectively, and in the right ventricle (0.076 ± 0.06 pmol mg−1, P < 0.001; 0.047 ± 0.009 pmol mg−1, P < 0.05) compared with the solvent group (Fig. 2).
In anaesthetized rats undergoing coronary artery ligation, the PVB count observed at the 30 min postischaemic period was lower than that found by other investigators, although the overall mortality and incidence of dysrhythmias was similar to that previously reported [5,14,16-19]. The cyclic nucleotide concentrations obtained in the present study were similar to those described for the anaesthetized rat [5,13].
The results of the present study indicate the that rolipram inhibitor of Type IV PDE significantly reduced the duration of dysrrhythmias without any changes in the cAMP or cGMP content in the ischaemic left ventricle. In contrast, the high doses of pimobendan or zaprinast exacerbated dysrhythmias during acute myocardial ischaemia. Furthermore, the mortality due to sustained ventricular fibrillation was significantly increased in the groups that received the higher dose of pimobendan or zaprinast. In addition, the high doses of these drugs enhanced the cyclic nucleotide concentrations in this model of ischaemia-induced dysrhythmia. There is evidence to suggest that drug-induced elevations in cyclic nucleotides caused by either selective PDE III inhibition, β-adrenoceptor activation or direct activation of adenylate cyclase are associated with an increased incidence of dysrhythmias [1,3,4,20]. In addition, clinical studies have suggested that PDE III inhibitors such as amrinone and milrinone can have dysrhythmogenic effects during myocardial ischaemia or reperfusion [13,21,23]. Moreover, the higher dose of pimobendan and zaprinast that exacerbated the dysrhythmias increases the cGMP content in the left ventricle, suggesting that cGMP could be implicated in ischaemia-induced dysrhythmias. There is supporting experimental evidence that infusion of cGMP enhances ischaemia-induced dysrhythmias, which raises the possibility of a dysrhythmogenic effect of cGMP in this model . In contrast with the present data, a previous study  showed that in the anaesthetized rat, ischaemia-induced dysrhythmias did not modify the cAMP or cGMP content in ischaemic ventricular tissue. In addition, milrinone increased the cAMP content in the left ventricle without changes in the incidence of dysrhythmias, suggesting that cyclic nucleotides are not involved in this model of ischaemia. These differences could be due to the different protocol used. In the previous study, the samples for the subsequent cyclic nucleotide analysis were taken at 15 min or earlier as appropriate. In our study, the samples were taken at 30 min after ligature. In addition, the results obtained with rolipram and zaprinast were in contrast with another study  in the anaesthetized rabbit that described an exacerbation of dysrhythmias following myocardial ischaemia and reperfusion with rolipram - whereas zaprinast had no significant effects. These differences may represent an intrinsic difference in excitation-contraction coupling between rat and rabbit myocardium and, therefore, a consequent difference in the effects of PDE inhibitors between species.
With respect to cyclic nucleotide concentrations in dysrhythmogenesis, previous studies in the cat , pig  and baboon  suggest that cAMP concentrations rose in ischaemic tissue and that this rise preceded the onset of fibrillation. However, it has been observed in isolated rat hearts that increases in cAMP occur 30-45 min after ischaemia and that the rise in cAMP was substrate-dependent and inhibited by glucose . It has recently reported that the mechanism by which cAMP is elevated has a profound influence on the fibrillation threshold in isolated rat hearts. Large increases in cAMP caused by forskolin did not cause such a large decrease in the fibrillation threshold as that produced following the smaller increases in cAMP caused by isoprotenolol. Compartmentalization of cAMP may be an explanation for these effects . Unfortunately, research in this area has focused upon the relationship between myocardial cAMP and dysrhythmias, and there is little information about the effect of cGMP and ischaemia-induced dysrhythmias. In this way, it has been shown that cGMP plays an important role in the maintenance and/or genesis of atrial fibrillation . In contrast, it has been suggested that the reduction of the cGMP synthesis increases the susceptibility to reperfusion-induced ventricular fibrillation after sustained ischaemia, suggesting that this nucleotide can be an endogenous intracellular cardioprotector .
In conclusion, our results suggest that the administration of the Class III PDE inhibitor pimobendan and the Class V PDE zaprinast are associated with raised myocardial cGMP content in the anaesthetized rat ischaemic left ventricle animal model, whereas the Class IV PDE inhibitor rolipram produces no corresponding rise in cGMP. Both pimobendan and zaprinast administration were associated with an increased mortality from dysrhythmias, an effect not shown with rolipram. We provide evidence that a rise in intracellular cGMP may be important in the aetiology of ischaemia-induced dysrhythmias. However, it is possible that other mechanisms in addition to cyclic nucleotides have been implicated in ischaemiainduced dysrhythmias [26,27].
This study was supported by the Fondo de Investigaciones de la Seguridad Social 98/0584, Spain. Rolipram was a gift from Messrs Almirall, C/Cardener, 68-74, E-08024 Barcelona, Spain.
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