James Winter

The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart

•  What is the central question of this study? Does the mechanical uncoupler blebbistatin affect cardiac electrophysiology? •  What is the main finding and what is its importance? Blebbistatin significantly affects cardiac ventricular electrophysiology and induction of ventricular fibrillation. This is a new finding that has serious implications for optical mapping studies where blebbistatin is used to inhibit cardiac contraction.

Differential cardiac responses to unilateral sympathetic nerve stimulation in the isolated innervated rabbit heart

The heart receives both a left and right sympathetic innervation. Currently there is no description of an in vitro whole heart preparation for comparing the influence of each sympathetic supply on cardiac function. The aim was to establish the viability of using an in vitro model to investigate the effects of left and right sympathetic chain stimulation (LSS/RSS). For this purpose the upper sympathetic chain on each side was isolated and bipolar stimulating electrodes were attached between T2-T3 and electrically insulated from surrounding tissue in a Langendorff innervated rabbit heart preparation (n=8). Heart rate (HR) was investigated during sinus rhythm, whilst dromotropic, inotropic and ventricular electrophysiological effects were measured during constant pacing (250 bpm). All responses exhibited linear increases with increases in stimulation frequency (2-10 Hz). The change in HR was larger during RSS than LSS (P<0.01), increasing by 78±9 bpm and 49±8 bpm respectively (10 Hz, baseline; 145±7 bpm). Left ventricular pressure was increased from a baseline of 50±4 mmHg, by 22±5 mmHg (LSS, 10 Hz) and 4±1 mmHg (RSS, 10 Hz) respectively (P<0.001). LSS, but not RSS, caused a shortening of basal and apical monophasic action potential duration (MAPD90). We demonstrate that RSS exerts a greater effect at the sinoatrial node and LSS at the left ventricle. The study confirms previous experiments on dogs and cats, provides quantitative data on the comparative influence of right and left sympathetic nerves and demonstrates the feasibility of isolating and stimulating the ipsilateral cardiac sympathetic supply in an in vitro innervated rabbit heart preparation.

Cardiac contractility modulation in the treatment of heart failure: initial results and unanswered questions

Despite advances in our understanding of the pathology of heart failure modern conventional pharmacological therapies have proved insufficient. The application of electrical impulses during the refractory period of the cardiac contractile cycle, so‐called cardiac contractility modulation (CCM), presents a new therapeutic approach with promising results demonstrated in clinical trials to date. The mechanism by which CCM exerts its effects remains inconclusive with conflicting reports. This article provides a concise review of the experimental and clinical CCM studies conducted to date with particular focus on areas of controversy and unexplored avenues of interest with this novel electrical therapy.

The acute inotropic effects of cardiac contractility modulation (CCM) are associated with action potential duration shortening and mediated by β1-adrenoceptor signalling

Despite promising results in clinical trials conducted to date, little is known about how cardiac contractile modulation (CCM) mediated inotropic enhancement occurs and how CCM affects the electrophysiological characteristics of the heart. The aims of the present study were to 1) investigate how the stimulation parameters of the CCM signal and the location of stimulus delivery influence the contractile response, 2) characterise the effect of CCM on ventricular electrophysiology, and 3) investigate the potential physiological mechanisms underlying these acute inotropic and electrophysiological effects. Experiments were conducted in isolated rabbit hearts with simultaneous measurement of ventricular contractility and monophasic action potential duration (MAPD). Biphasic square wave pulses were applied to the left ventricle, timed to coincide with the absolute refractory period. CCM mediated responses were assessed over a range of signal amplitudes (2–30 mA), durations (2–15 ms) and delays from the activation of the locally recorded monophasic action potential (0–30 ms). Responses were assessed during perfusion with the β1-adrenoceptor antagonist metoprolol (1.8 μM) and HMR 1556 (500 nM), an inhibitor of the slow delayed rectifying potassium current. Norepinephrine content was collected and assessed by ELISA from samples of coronary effluent collected during CCM. CCM induced a significant increase in left ventricular pressure (LVP) in a manner dependent upon the amplitude and duration of the CCM signal but independent of the delay of the stimulus within the action potential plateau and was associated with an increase in norepinephrine in coronary effluent (Mean: 46 ± 9 pg/ml). CCM promoted a shortening of MAPD-90% close to the site of stimulation (− 19 ± 3%) but had no effect on those recorded at distant sites (0 ± 1%). The increase in LVP (4.7 ± 1.8 vs. 0.7 ± 0.9%, P < 0.01) and shortening of local MAPD-90% (− 15 ± 3 vs. 1 ± 1%, P < 0.01) was abolished with metoprolol. Perfusion with HMR 1556 caused a significant inhibition of local MAPD shortening (− 27 ± 2 vs. − 21 ± 3 ms, P < 0.05). CCM is associated with a shortening of ventricular MAPD in a manner dependent upon β-adrenoceptor stimulation resulting from catecholamine release, a finding which may be of clinical significance in regard to the development of malignant ventricular arrhythmias. This article is part of a Special Issue entitled Possible Editorial.

Cardiac contractility modulation increases action potential duration dispersion and decreases ventricular fibrillation threshold via β1-adrenoceptor activation in the crystalloid perfused normal rabbit heart.

Background/objectives Cardiac contractility modulation (CCM) is a new treatment being developed for heart failure (HF) involving application of electrical current during the absolute refractory period. We have previously shown that CCM increases ventricular force through β1-adrenoceptor activation in the whole heart, a potential pro-arrhythmic mechanism. This study aimed to investigate the effect of CCM on ventricular fibrillation susceptibility. Methods Experiments were conducted in isolated New Zealand white rabbit hearts (2.0–2.5 kg, n = 25). The effects of CCM (± 20 mA, 10 ms phase duration) on the left ventricular basal and apical monophasic action potential duration (MAPD) were assessed during constant pacing (200 bpm). Ventricular fibrillation threshold (VFT) was defined as the minimum current required to induce sustained VF with rapid pacing (30 × 30 ms). Protocols were repeated during perfusion of the β1-adrenoceptor antagonist metoprolol (1.8 μM). In separate hearts, the dynamic and spatial electrophysiological effects of CCM were assessed using optical mapping with di-4-ANEPPS. Results CCM significantly shortened MAPD close to the stimulation site (Basal: 102 ± 5 [CCM] vs. 131 ± 6 [Control] ms, P < 0.001). VFT was reduced during CCM (2.6 ± 0.6 [CCM] vs. 6.1 ± 0.8 [Control] mA, P < 0.01) and was correlated (r2 = 0.40, P < 0.01) with increased MAPD dispersion (26 ± 4 [CCM] vs. 5 ± 1 [Control] ms, P < 0.01) (n = 8). Optical mapping revealed greater spread of CCM induced MAPD shortening during basal vs. apical stimulation. CCM effects were abolished by metoprolol and exogenous acetylcholine. No evidence for direct electrotonic modulation of APD was found, with APD adaptation occurring secondary to adrenergic stimulation. Conclusions CCM decreases VFT in a manner associated with increased MAPD dispersion in the crystalloid perfused normal rabbit heart.

Restitution slope is principally determined by steady-state action potential duration

Aims The steepness of the action potential duration (APD) restitution curve and local tissue refractoriness are both thought to play important roles in arrhythmogenesis. Despite this, there has been little recognition of the apparent association between steady-state APD and the slope of the restitution curve. The objective of this study was to test the hypothesis that restitution slope is determined by APD and to examine the relationship between restitution slope, refractoriness and susceptibility to VF. Methods and results Experiments were conducted in isolated hearts and ventricular myocytes from adult guinea pigs and rabbits. Restitution curves were measured under control conditions and following intervention to prolong (clofilium, veratridine, bretylium, low [Ca]e, chronic transverse aortic constriction) or shorten (catecholamines, rapid pacing) ventricular APD. Despite markedly differing mechanisms of action, all interventions that prolonged the action potential led to a steepening of the restitution curve (and vice versa). Normalizing the restitution curve as a % of steady-state APD abolished the difference in restitution curves with all interventions. Effects on restitution were preserved when APD was modulated by current injection in myocytes pre-treated with the calcium chelator BAPTA-AM – to abolish the intracellular calcium transient. The non-linear relation between APD and the rate of repolarization of the action potential is shown to underpin the common influence of APD on the slope of the restitution curve. Susceptibility to VF was found to parallel changes in APD/refractoriness, rather than restitution slope. Conclusion(s) Steady-state APD is the principal determinant of the slope of the ventricular electrical restitution curve. In the absence of post-repolarization refractoriness, factors that prolong the action potential would be expected to steepen the restitution curve. However, concomitant changes in tissue refractoriness act to reduce susceptibility to sustained VF. Dependence on steady-state APD may contribute to the failure of restitution slope to predict sudden cardiac death.

Geometrical considerations in cardiac electrophysiology and arrhythmogenesis

The rate of repolarization (RRepol) and so the duration of the cardiac action potential are determined by the balance of inward and outward currents across the cardiac membrane (net ionic current). Plotting action potential duration (APD) as a function of the RRepol reveals an inverse non-linear relationship, arising from the geometric association between these two factors. From the RRepol-APD relationship, it can be observed that a longer action potential will exhibit a greater propensity to shorten, or prolong, for a given change in the RRepol (i.e. net ionic current), when compared with one that is initially shorter. This observation has recently been used to explain why so many interventions that prolong the action potential exert a greater effect at slow rates (reverse rate-dependence). In this article, we will discuss the broader implications of this simple principle and examine how common experimental observations on the electrical behaviour of the myocardium may be explained in terms of the RRepol-APD relationship. An argument is made, with supporting published evidence, that the non-linear relationship between the RRepol and APD is a fundamental, and largely overlooked, property of the myocardium. The RRepol-APD relationship appears to explain why interventions and disease with seemingly disparate mechanisms of action have similar electrophysiological consequences. Furthermore, the RRepol-APD relationship predicts that prolongation of the action potential, by slowing repolarization, will promote conditions of dynamic electrical instability, exacerbating several electrophysiological phenomena associated with arrhythmogenesis, namely, the rate dependence of dispersion of repolarization, APD restitution, and electrical alternans.

Vagal modulation of dispersion of repolarisation in the rabbit heart

Bradycardia is a risk factor for arrhythmia in several disorders, including acquired long QT syndrome, whereby slowing of heart rate facilitates ectopic activity and torsade de pointes. Slowing of rate is associated with an increase in the spatiotemporal dispersion of ventricular repolarisation (DOR) in electrically paced hearts. However, there have been conflicting reports on the effect of the vagus nerve, which mediates the physiological slowing of heart rate, on DOR. The aim of this study was to investigate the effect of vagus nerve stimulation (VNS) on the heterogeneity of ventricular repolarisation, as assessed using the T-wave peak-to-end interval (TpTe) and monophasic action potentials (MAPs), in normal hearts and in hearts with acquired long QT syndrome. Experiments were conducted in an isolated innervated rabbit heart preparation. The effect of VNS on cardiac electrograms, MAPs and ventricular function was investigated in control and following perfusion of E4031 (50nmol/L); an inhibitor of the rapid delayed rectifying potassium current. VNS was associated with a stimulation frequency-dependent bradycardia (-74±6 [10Hz] vs. -25±4bpm [2Hz], P<0.05). VNS prolonged the TpTe interval (29±1 vs. 20±2ms, P<0.05) and increased T-wave amplitude (1.7±0.3 vs. 0.7±0.2mV, P<0.05) in association with increased apicobasal DOR. The effects of VNS were exacerbated by E4031, with a greater prolongation of TpTe (ΔTpTe 42±6 vs. 8±1ms, P<0.05) and max-min apicobasal time of repolarisation (TRepol; 45±11 vs. 5±2ms, P<0.05). ΔTpTe was strongly correlated with the Δmax-minTRepol (r(2)=0.87, P<0.05) and TpTe was prolonged to a greater degree in hearts exhibiting spontaneous ventricular tachyarrhythmia. Rate dependent differences in regional action potential prolongation were replicated using computational models. These data demonstrate that VNS increases ventricular DOR and that the effects of the vagus nerve on ventricular electrophysiology are exacerbated in pharmacologically acquired long QT syndrome.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS. Objective: To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated. Methods and Results: Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (IKs), sufficient to abolish IKs-mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart. Conclusions: SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased IKs.

Reversal of cardiac vagal effects of physostigmine by adjunctive muscarinic blockade

Pre-treatment with reversible acetylcholinesterase (AChE) inhibitors is an effective strategy for reducing lethality following organophosphate nerve agent exposure. AChE inhibition may have unwanted cardiac side effects, which could be negated by adjunctive anti-cholinergic therapy. The aims of the present study were to examine the concentration-dependent effects of physostigmine on cardiac responses to vagus nerve stimulation (VNS), to test whether adjunctive treatment with hyoscine can reverse these effects and to assess the functional interaction and electrophysiological consequences of a combined pre-treatment. Studies were performed in an isolated innervated rabbit heart preparation. The reduction in heart rate with VNS was augmented by physostigmine (1-1000nmol/L), in a concentration-dependent manner - with an EC50 of 19nmol/L. Hyoscine was shown to be effective at blocking the cardiac responses to VNS with an IC50 of 11nmol/L. With concomitant perfusion of physostigmine, the concentration-response curve for hyoscine was shifted downward and to the right, increasing the concentration of hyoscine required to normalise (to control values) the effects of physostigmine on heart rate. At the lowest concentration of hyoscine examined (1nmol/L) a modest potentiation of heart rate response to VNS (+15±3%) was observed. We found no evidence of cardiac dysfunction or severe electrophysiological abnormalities with either physostigmine or hyoscine alone, or as a combined drug-therapy. The main finding of this study is that hyoscine, at concentrations greater than 10-8M, is effective at reversing the functional effects of physostigmine on the heart. However, low-concentrations of hyoscine may augment cardiac parasympathetic control.