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Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF)in hearts subjected to hypoxia (12 min) and re-oxygenation (40 min). We studied the effect of putative activators and inhibitors of PKC on the incidence of VF. Hearts exposed to 4β-phorbol, 12, 13-dibutyrate (PDBu), isophorbol or the membrane permeant diacylglycerol analog, t-oleoyl-2-accetyl-acetyl-rac-glycerol (OAG), during the prehypoxic phase had an increased incidence of VF during the hypoxic and reoxygenation periods. The incidence of VF was 90%, 83% and 75% in hearts exposed to PDBu, isophorbol and OAG, respectively (P < 0.05 vs control). Perfusion of hearts with PDBu was associated with a significant increase in the membrane fraction of cardiac PKC activity. In the presence of the inactive phorbol ester 4α-phorbol didecanoate, the incidence of VF was 17% (P > 0.05 vs control). PKC activators were profibrillatory at concentrations that did not affect cardiac function: neither left ventricular developed pressure nor coronary perfusion pressure were affected. The effect of PDBu was antagonized by staurosporine: the incidence of VF was 17% in PDBu + staurosporine treated hearts (P < 0.05 vs control). To further study the porfibrillatory effect of PDBu, hearts were exposed to PDBu in the presence of the ATP-dependent potassium channel antagonist glibenclamide. The latter prevented PDBu-induced VF. The results show that under the conditions employed, PDBu-induce activation of PKC induces redistribution of PKC activity and is associated with the development of VF. Copyright 1993, 1999 Academic Press

We wished to examine selected class I antiarrhythmic agents for their potential to exhibit proarrhythmic or antifibrillatory actions. Quinidine, lidocaine, aprindine, and flecainide were evaluated in an experimental model that made use of rabbit isolated perfused heart. Hearts were stabilized with oxygenated buffer (95 O2/ 5 CO2) with or without pinacidil (1.25 µM) and then were subjected to hypoxia (95 N2/5 CO2) for 12 min, followed by 40-min normoxic perfusion (95 O2/5 CO2). Test drugs were added to the perfusion medium 5 min before hypoxia was induced. Prevention of spontaneous ventricular fibrillation (VF) was determined. To characterize the electrophysiologic effects of the selected antiarrhythmic agents, we determined the changes in threshold current and effective refractory period (ERP) before and after drug treatment. Addition of the potassium channel agonist, pinacidil, to the perfusion medium invariably resulted in VF during the hypoxic interval or shortly after reoxygenation. Pretreatment of the heart with glibenclamide prevented pinacidil-induced VF. Quinidine, aprindine, lidocaine, and flecainide each were studied at a single concentration. The respective drug concentrations were selected to produce comparable changes in ventricular refractory period. Of the class I agents selected for study, only quinidine prolonged the ventricular ERP and provided significant protection against pinacidil-induced VF. In contrast, aprindine and lidocaine decreased ventricular ERP and did not prevent VF induced by the combination of pinacidil and hypoxia. Quinidine, aprindine, and lidocaine did not exhibit proar-rhythmic effects in the presence of hypoxia when pinacidil was not added to the perfusion medium. Flecainide, when added to the perfusion medium without pinacidil elicited proarrhythmic activity leading to VF when the hearts were made hypoxic. Flecainide-induced VF was antagonized by glibenclamide. The data suggest that VF can be provoked by the potassium channel agonist pinacidil or by flecainide under conditions that reduce intracellular ATP concentration. Glibenclamide, a selective antagonist of the KATPchannel prevented the profibrillatory actions of pinacidil and flecainide. Quinidine, but not lidocaine and aprindine, prevented VF induced by pinacidil and hypoxia.

We investigated the effects of the ATP-dependent K+channel antagonist glyburide and the ATP-dependent K+channel agonist pinacidil in a Langendorff-perfused rabbit isolated heart subjected to a period of global hypoxia. A class III antiarrhythmic drug, E-4031, also was studied in this model. These studies aimed to define the mechanism of action of putative profibrillatory actions of pinacidil and the mechanism for the antifibril-latory effect of the class III antiarrhythmic drug, E-4031, in the hypoxic heart. After stabilization and determination of baseline functional parameters under normoxic perfusion conditions (95 O2/5 CO2), hearts were subjected to global hypoxia by switching to a 95 N2/5 CO2saturated perfusion medium for a period of 12 min. After the hypoxic period, normoxia was re-established by switching to the oxygen-carbon dioxide saturated buffer medium for a period of 40 min. The oxygen tension of the perfusion buffer was reduced from approximately 400 mm Hg to below 50 mm Hg during the hypoxic period. All hearts subjected to hypoxia had reduced function: the left ventricular developed pressure and ±dPldtwere reduced significantly. Myocardial tissue ATP concentrations were reduced (>50) in hearts subjected to hypoxia. Under conditions of hypoxic/reoxygenation and in the presence of a low (2.5 mM) potassium concentration ([K+]0), pinacidil (1.25 μM) facilitated the induction of ventricular fibrillation (80 fibrillation in the presence of pinacidil vs. 20 in the absence of pinacidil). Glyburide (10 μM) and E-4031 (1 and 10 μM) significantly reduced the incidence of ventricular fibrillation associated with pinacidil (20 fibrillation in the presence of hypoxia, pinacidil, and glyburide or 10 μME-4031). Opening of the ATP-dependent K +channel by pinacidil under normoxia and low K +also facilitated the induction of ventricular fibrillation (60 ventricular fibrillation). Pinacidil failed to induce ventricular fibrillation under either normoxic or conditions of hypoxic/reoxygenation when the [K +]0was increased to 5.1 mM.The results of this study demonstrate that K +channel activators facilitate the induction of ventricular fibrillation under both normoxic conditions and conditions of hypoxic/reoxygenation when the perfusion buffer K+concentration is reduced.

The presence of amidases cleaving the tripeptide VAL.LEU.ARG.PNA, and liberating from human plasma kininogen substance(s) able to contract uterine smooth muscle and to lower blood pressure (uterus contracting and hypotensive activity), has been demonstrated in vascular and muscle tissues from normally perfused and ischaemic areas of dog hearts. Studies were carried out on blood free preparations of coronary arteries and veins and normally perfused and ischaemic ventricle. All the tissues were found to contain both acid optimum (pH 6) and alkaline optimum (pH> 9) enzymes forming uterus contracting substance (UCS, bioassayed on isolated uterus of rats in oestrus), the highest levels of both activities being found in arterial tissues and the least in ventricle. Enzyme levels in ischaemic or normally perfused ventricle did not differ significantly. Gel filtration (Sephacryl, S-300) of coronary artery extracts gave one peak each of acid optimum enzyme with a molecular weight of 38 300±800 daltons and alkaline optimum enzyme with a molecular weight of 92 100±4000 daltons. Both acid and alkaline enzyme fractions cleaved the tripeptide substrate with pH optima identical to those for UCS formation. The acid optimum activity, both of UCS formation and tripeptide cleavage, was inhibited by pepstatin but not by aprotinin or soybean trypsin inhibitor (SBTI). The alkaline optimum activity was inhibited by aprotinin and SBTI but not pepstatin. Both acid and alkaline optimum enzymes released a hypotensive agent from a plasma protein substrate. The molecular weight and response to inhibitors of the acid optimum enzyme were similar to a cathepsin, and those of the alkaline optimum enzyme were similar to plasma kallikrein.