The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias.

The 'Lambeth Conventions' is a guidance document, written in 1987 (Walker et al., 1988), intended to be of practical value in the investigation of experimental arrhythmias induced by ischaemia, infarction, and reperfusion. This is an update, expanded to include guidance on the study of supraventricular arrhythmias, drug-induced arrhythmias, heritable arrhythmias, and advances in our knowledge in core areas since 1987. We have updated the guidance on the design and execution of experiments and the definition, classification, quantification, and analysis of all types of arrhythmias. Investigators are encouraged to adopt the conventions and test their validity in the hope that this will improve uniformity and interlaboratory comparisons, aid clinical research, facilitate antiarrhythmic drug discovery and safety assessment, and improve antiarrhythmic drug deployment for different cardiac conditions. We note that there is a gap between some definitions proposed here and their conventional clinical counterparts, and encourage the research necessary to bridge that translational gap. A web link offers the chance to vote and comment on the new conventions (https://bscr.wufoo.com/forms/z7x0x5/).

Susceptibility to ischemia-induced ventricular fibrillation in isolated female rat hearts varies moderately with estrous cycle stage.

INTRODUCTION: The vast majority of studies employing the isolated perfused rat heart model to study ischemic arrhythmias have used male rats only. The objective of this study was to determine the susceptibility to ischemia-induced ventricular fibrillation (VF) in isolated female rat hearts in each stage of the estrous cycle that corresponds with a different endogenous reproductive hormonal environment. METHODS: Hearts were isolated from female rats under pentobarbital anesthesia and perfused with modified Krebs solution containing 3mM K(+). Experiments were grouped according to estrous cycle stage that was determined by prior vaginal lavage (n=10-13 per group). A group of male rat hearts was used as the control. Regional ischemia was induced by coronary ligation and maintained for 30min. The incidence of VF was determined from the ECG. RESULTS: The incidence of VF in male hearts was 100%, while the incidence of VF in female hearts was also high but varied moderately with stage of the estrous cycle (diestrus 70%, metestrus 100%, proestrus 90%, estrus 69%; P>0.05). Compared to male hearts, the onset of VF was similar in all groups except for hearts excised from rats in proestrus, in which it was delayed. There was no difference between groups in an arrhythmia score, ischemic zone size, or baseline electrocardiographic or hemodynamic variables. DISCUSSION: In conclusion, the susceptibility of isolated female rat hearts to ischemic VF is comparable to that of male rat hearts, meaning that isolated female rat hearts can be used as controls in studies to assess antiarrhythmic drug efficacy. Since female rats can be used for isolated heart studies of ischemic VF, the need to cull female rats is reduced. However, the variation in VF susceptibility in female rat hearts that is associated with the different stages of the estrous cycle may affect statistical power that could potentially lead to Type II statistical errors. This problem can be prevented with careful randomization.

Reduced antiarrhythmic efficacy of verapamil in isolated rat hearts in the presence of elevated extracellular calcium.

An isolated heart method that has been proposed to aid in ascertaining the involvement of L-type calcium channel blockade in the mechanism of action of novel antiarrhythmic drugs involves increasing the calcium concentration in the perfusion buffer. The purpose of this study was to determine the validity of this method using an established L-type calcium channel blocker, verapamil. Isolated rat hearts were perfused with normal calcium (1.4 mM) Krebs solution containing drug vehicle only, a normal calcium solution containing verapamil (300 nM), or a high calcium (2.8 mM) solution containing verapamil. The occurrence of ventricular fibrillation during a subsequent period of regional myocardial ischemia was monitored. The incidence of ventricular fibrillation was significantly reduced from 80% in controls to 20% by perfusion with verapamil in normal calcium Krebs solution (P < 0.05). Perfusion with the high calcium solution increased the incidence of ventricular fibrillation in the presence of verapamil to 40% (P > 0.05 versus controls). We conclude that the antiarrhythmic effect of verapamil in isolated hearts can be attenuated by increasing the calcium content of the perfusion solution, but a twofold increase in the calcium concentration failed to fully restore susceptibility to ventricular fibrillation to that observed in verapamil-free controls.

Mitochondrial uncoupling agents trigger ventricular fibrillation in isolated rat hearts.

Sudden cardiac death resulting from ventricular fibrillation (VF) remains a major cause of mortality. The purpose of this study was to investigate the roles of loss of oxidative phosphorylation and activation of the mitochondrial ATP-sensitive K+ channel and permeability transition pore in VF development during myocardial ischemia by using mitochondrial uncoupling agents (carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol) and channel blockers (5-hydroxydecanoate and cyclosporine A) at concentrations that have been demonstrated to block the intended targets selectively. Isolated rat hearts (n = 8 per group) were perfused with 0.3 µM carbonyl cyanide m-chlorophenylhydrazone, 100 µM 2,4-dinitrophenol, 0.2 µM cyclosporine A, 100 µM 5-hydroxydecanoate, or vehicle solution and regional ischemia induced after 10 minutes. Carbonyl cyanide m-chlorophenylhydrazone and 2,4 dinitrophenol caused profound QT shortening and triggered VF in 100% of hearts before ischemia. During ischemia, neither cyclosporine A (88%) nor 5-hydroxydecanoate (100%) reduced VF incidence compared with control (100% VF). In separate hearts, carbonyl cyanide m-chlorophenylhydrazone decreased tissue ATP content, and glibenclamide or glimepiride delayed the QT shortening and onset of VF triggered by carbonyl cyanide m-chlorophenylhydrazone. In conclusion, mitochondrial uncoupling agents trigger VF, likely as a result of ATP depletion with subsequent activation of sarcolemmal ATP-sensitive K+ currents. The mechanism of VF in ischemia does not involve activation of the mitochondrial ATP-sensitive K+ channel or permeability transition pore.

Druggable targets for sudden cardiac death prevention: lessons from the past and strategies for the future.

Sudden cardiac death (SCD) is most commonly caused by ventricular fibrillation (VF). The single largest cohort of victims is the population with little or no prior overt heart disease. Effective prevention will require long-term prophylaxis by drugs in large numbers of people identified by risk factors. This means that safe as well as effective drugs are required. Drugs with overt effects on cardiac electrophysiology have failed in the clinic owing to poor effectiveness and/or adverse effects. This article examines possible new drug targets. We have focused on acute myocardial ischaemia as it is the most strikingly proarrhythmic pathology, and the most common cause of coronary artery disease-related VF and SCD according to inferences from epidemiology, drug trials and decades of animal research. To set the scene we have briefly explored drugs that have failed in the clinic in order to identify possible targets that have been overlooked or underexploited. We conclude that the best strategy is identification of pathology-specific targets that render drugs active only where and when their action is required.

Complex adrenergic and inflammatory mechanisms contribute to phase 2 ventricular arrhythmias in anaesthetized rats.

BACKGROUND AND PURPOSE: The mechanisms responsible for phase 2 (infarct-related) ventricular arrhythmias remain unclear. We have investigated the role of alpha(1) and beta(1) adrenoceptor activation and the interaction of this with infarct neutrophil accumulation, in anaesthetized rats. EXPERIMENTAL APPROACH: Neutrophil-replete Sprague-Dawley rats (n = 8-9 per group) were anaesthetized and randomized to receive vehicle, prazosin (0.5 mg.kg(-1) i.v.), atenolol (4 mg.kg(-1) i.v.) or their combination prior to left main coronary artery occlusion. A further group was depleted of neutrophils and received both atenolol and prazosin. Coronary ligation in all groups was maintained for 240 min. KEY RESULTS: Atenolol and prazosin treatment lowered heart rates and blood pressures respectively, but neither agent given alone affected the incidence of phase 2 ventricular tachycardia or fibrillation. However, co-administration of atenolol with prazosin reduced phase 2 ventricular premature beats (log(10)-transformed totals were 1.25 +/- 0.26 vs. 2.43 +/- 0.18 in controls; P < 0.05). Neutrophil depletion attenuated this antiarrhythmic effect (log(10)-transformed total ventricular premature beats were 1.66 +/- 0.35; P > 0.05 vs. controls). CONCLUSIONS AND IMPLICATIONS: Phase 2 arrhythmias appear to depend in part on a complex interaction between catecholamines and neutrophils. A model of this interaction is proposed.

Ca2+/calmodulin-dependent protein kinase IIdelta and protein kinase D overexpression reinforce the histone deacetylase 5 redistribution in heart failure.

Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKIIdelta and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5-green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (F(nuc)/F(cyto) 3.3+/-0.3 vs 7.2+/-0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKIIdelta(C) expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP F(nuc)/F(cyto) toward control, and simultaneous inhibition restored F(nuc)/F(cyto) to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKIIdelta(B), or CaMKIIdelta(C) reduced baseline HDAC5 F(nuc)/F(cyto) in control myocytes (3.4+/-0.5, 3.8+/-0.5, and 5.2+/-0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Neutrophil ablation with anti-serum does not protect against phase 2 ventricular arrhythmias in anaesthetised rats with myocardial infarction.

OBJECTIVES: Arrhythmias, including ventricular fibrillation (VF), occur in two phases after coronary obstruction, the first during the reversible stage of acute myocardial ischaemia (phase 1) and the second during evolution of the infarct (phase 2). We tested the hypothesis that phase 2 arrhythmias are mediated by actions of neutrophils accumulating within the infarct. METHODS: Male rats (n=10 per group) were randomized to receive 2 ml/kg i.p. of either rabbit anti-rat neutrophil anti-serum or normal rabbit serum. After 17 h, single stage left coronary artery ligation was performed under pentobarbitone anaesthesia, and ischaemia was maintained for 240 min. RESULTS: Anti-serum pretreatment caused almost total neutropenia, reducing neutrophils in circulating blood from 2096+/-274x10(3) to 8+/-8x10(3) per ml (p<0.05). It also blocked neutrophil accumulation in the infarct, reducing cardiac myeloperoxidase activity from 74.7+/-27.4 to 9+/-3 mU per mg protein (p<0.05). Despite this, there was no significant difference between control and anti-serum-treated rats in the incidence of phase 2 VF (30% in each group) tachycardia (VT; 60% vs 80%) or number of ventricular premature beats (VPBs). CONCLUSION: Neutrophil accumulation within the evolving myocardial infarct does not mediate phase 2 VF.

Actions of flecainide on susceptibility to phase-2 ventricular arrhythmias during infarct evolution in rat isolated perfused hearts.

The mechanism of flecainide-induced unexpected death remains uncertain. Phase-2 ventricular arrhythmias occur during infarct evolution. We examined whether flecainide (0.74 and 1.48 microM, representing the peak unbound plasma and total blood concentrations, respectively, at 'therapeutic' dosage) has proarrhythmic activity on phase-2 arrhythmia susceptibility during infarct evolution. To achieve this, we used the Langendorff-perfused rat heart preparation (n=8 per group) in which baseline phase-2 arrhythmia susceptibility is low. Left main coronary occlusion evoked phase-1 (acute ischaemia-induced) ventricular arrhythmias including fibrillation (VF) in all hearts. By 90 min, hearts were relatively arrhythmia-free. Randomized and blinded switch of perfusion to flecainide at 90 min caused no increase over baseline in the incidence of VF, tachycardia (VT) or premature beats (VPB) during the following 150 min of ischaemia, or during reperfusion (begun 240 min after the onset of ischaemia). In separate hearts, catecholamines (313 nM norepinephrine and 75 nM epinephrine) were co-perfused with flecainide from 90 min of ischaemia. Catecholamine perfusion increased heart rate, coronary flow and QT interval, and shortened PR interval (all P<0.05), actions that were not altered by flecainide. Catecholamine perfusion caused a weak nonsignificant increase in phase-2 VPB, VT and VF incidence, but there was no proarrhythmic interaction with flecainide. In conclusion, the present findings suggest that the increased risk of death associated with clinical use of flecainide is not due to facilitation of phase-2 ventricular arrhythmias.

Phase 2 ventricular arrhythmias in acute myocardial infarction: a neglected target for therapeutic antiarrhythmic drug development and for safety pharmacology evaluation.

Ventricular fibrillation (VF), a cause of sudden cardiac death (SCD) in the setting of acute myocardial infarction (MI), remains a major therapeutic challenge. In humans, VF may occur within minutes or hours after the onset of chest pain, so its precise timing in relation to the onset of ischaemia is variable. Moreover, because VF usually occurs unobserved, out of hospital, and is usually lethal in the absence of intervention, its precise timing of onset is actually unknown in most patients. In animal models, the timing of susceptibility to VF is much better characterised. It occurs in two distinct phases. Early VF (defined as phase 1 VF, with possible subphases 1a and 1b in some animal species) occurs during the first 30 min of ischaemia when most myocardial injury is still reversible. Late VF, defined as phase 2 VF, occurs when myocardial necrosis is becoming established (after more than 90 min of ischaemia). Although much is known about the mechanisms and pharmacology of phase 1 VF, little is known about phase 2 VF. By reviewing a range of different types of data we have outlined the likely mechanisms and clinical relevance of phase 2 VF, and have evaluated possible future directions to help evolve a strategy for its suppression by drugs. The possibility that a proarrhythmic effect on phase 2 VF contributes to the adverse cardiac effects of certain cardiac and noncardiac drugs is also discussed in relation to the emerging field of safety pharmacology. It is concluded that suppression of phase 2 as well as phase 1 VF will almost certainly be necessary if drugs of the future are to achieve what drugs of the past and present have failed to achieve: full protection against SCD. Likewise, safety will require avoidance of exacerbation of phase 2 as well as phase 1 VF.

Cardioprotective efficacy of zoniporide, a potent and selective inhibitor of Na+/H+ exchanger isoform 1, in an experimental model of cardiopulmonary bypass.

1. We determined (1) the inhibitory potency of zoniporide against the native Na(+)/H(+) exchanger isoform 1 (NHE1) that is expressed in adult rat ventricular myocytes and platelets, and (2) the cardioprotective efficacy of zoniporide in isolated, blood-perfused adult rat hearts subjected to cardioplegic arrest, hypothermic ischaemia (150 min at 25 degrees C) and normothermic reperfusion (60 min at 37 degrees C). 2. In isolated myocytes, in which NHE1 activity was determined directly by measurement of H(+) efflux rate following intracellular acidification, zoniporide produced a dose-dependent inhibition of such activity (IC(50) 73 nm at 25 degrees C). A comparable NHE1-inhibitory potency was retained at 37 degrees C. 3. In platelets, in which the rate of cell swelling was used as a surrogate index of NHE1 activity, this was again inhibited by zoniporide (IC(50) 67 nm at 25 degrees C). 4. In the isolated heart model, administration of zoniporide (loading bolus of 1 mg kg(-1) i.v. plus continuous infusion at 1.98 mg kg(-1) h(-1) i.v.) to the support animal achieved a free plasma drug concentration of >/=1 microm. At this dose, zoniporide afforded significant cardioprotective benefit relative to vehicle treatment, with improved preservation of left ventricular end-diastolic and developed pressures and coronary perfusion pressure during reperfusion. Myocardial myeloperoxidase activity was also attenuated by zoniporide treatment, indicating reduced neutrophil accumulation. 5. These data show that zoniporide (1) is a potent inhibitor of native NHE1 activity in ventricular myocytes and platelets, and (2) affords significant cardioprotective benefit during ischaemia and reperfusion in an experimental model that mimics several distinctive features of human cardioplegic arrest with cardiopulmonary bypass.

The isolated blood-perfused rat heart: an inappropriate model for the study of ischaemia- and infarction-related ventricular fibrillation.

1. Well-characterized in vivo and in vitro models exist for the study of ischaemia- and infarction-related ventricular fibrillation (VF). In rats in vivo, VF appears to occur in distinct acute ischaemia- (early) and infarction-related (late) phases. Interestingly, isolated buffer-perfused rat hearts do not develop late VF. This raises the possibility that unidentified components of the blood may be responsible for late VF. We thus sought to characterize an isolated blood-perfused rat heart in order to investigate the possible influence of blood components on arrhythmias arising from ischaemia and infarction. 2. Hearts, excised from male Wistar rats, were perfused in the Langendorff mode with blood from support rats (male Wistar, 350-430 g) via an extracorporeal circuit. Perfused hearts underwent left coronary artery occlusion for 240 min or a sham procedure (n=10 group(-1)). 3. Only 10% of ischaemic hearts developed late VF (90-240 min). Tissue myeloperoxidase activity (an index of neutrophil accumulation) increased during ischaemia from 0.017+/-0.004 (six fresh hearts) to 0.056+/-0.005 units mg protein(-1) (P<0.05) at 240 min, but values were similar in sham hearts (0.083+/-0.013). Likewise, the decline (-1 vs 240 min of ischaemia shown) in circulating total white blood cells from 6.8+/-0.5 to 1.9+/-0.2 x 10(3) micro l(-1) and in platelets from 441+/-32 to 274+/-16 x 10(3) micro l(-1) (both P<0.05) was similar in time-matched sham hearts (data not shown). 4 Surprisingly, only 10% of ischaemic hearts developed early VF (0-90 min), although the incidence of early ventricular tachycardia was 100% in these hearts (P<0.05 vs sham hearts). Blood K+ values were normal (hyperkalaemia suppresses VF). 5 Although late VF was absent in blood-perfused hearts, it would be premature to conclude from this that late VF is not mediated by blood components. This is because the similar neutrophil accumulation in ischaemic and sham hearts, the decline in numbers of circulating blood components, and the unexpected paucity of early VF all question the validity of the model.

Independent contribution of catecholamines to arrhythmogenesis during evolving infarction in the isolated rat heart.

Ventricular fibrillation (VF) in conscious rats with coronary artery ligation occurs in two phases, before (phase 1) and after (phase 2) 90 min of ischaemia respectively. The mechanisms of phase 2 VF are not established. Interestingly, phase 2 VF is absent in isolated (denervated) buffer-perfused rat hearts. We investigated whether catecholamine supplementation (to mimic sympathetic drive) was sufficient to restore phase 2 VF in such hearts. Isolated rat hearts (n=10 per group) underwent coronary ligation for 240 min. At 90 min, during a period of relative electrical stability, the perfusion solution was switched from standard (Krebs) to identical solution or Krebs containing catecholamines (313 nM noradrenaline and 75 nM adrenaline) with or without 10 microM trimazosin (an alpha(1)-adrenoceptor antagonist) or 10 microM atenolol (a beta(1)-adrenoceptor antagonist). Although in all groups the incidence of phase 1 VF was high (80 - 100%), the temporal distribution of VF was monophasic, i.e. only one heart in one group developed phase 2 VF (P=NS). Other ventricular arrhythmias (e.g., tachycardia; VT) exhibited a similar temporal distribution. Nevertheless, haemodynamic changes confirmed sympathomimetic effects of catecholamines, e.g., heart rate was increased from 278+/-7 beats min(-1) in controls to 335+/-8 beats min(-1) (P<0.05) by catecholamines, an effect that could be blocked by atenolol (285+/-7 beats min(-1)) but not by trimazosin (342+/-12 beats min(-1)). Coronary flow was correspondingly increased from 7.7+/-0.7 ml min(-1) g(-1) to 16.5+/-1.3 ml min(-1) g(-1) (P<0.05); this effect could be blocked by atenolol (8.1+/-0.6 ml min(-1) g(-1)) and was enhanced by trimazosin (20.7+/-2.4 ml min(-1) g(-1)). In conclusion, despite evidence of adequate alpha- and beta-adrenoceptor activation, catecholamine supplementation to isolated buffer-perfused rat hearts was insufficient to restore phase 2 VF. It therefore appears unlikely that catecholamines alone mediate phase 2 VF.