Autonomic neuro-cardiac profile of electrical, structural and neuronal remodeling in myocardial infarction-induced heart failure.

Aims: Heart failure is a clinical syndrome typified by abnormal autonomic tone, impaired ventricular function, and increased arrhythmic vulnerability. This study aims to examine electrophysiological, structural and neuronal remodeling following myocardial infarction in a rabbit heart failure model to establish its neuro-cardiac profile. Methods and results: Weight-matched adult male New Zealand White rabbits (3.2 ± 0.1 kg, n = 25) were randomized to have coronary ligation surgeries (HF group, n = 13) or sham procedures (SHM group, n = 12). Transthoracic echocardiography was performed six weeks post-operatively. On week 8, dual-innervated Langendorff-perfused heart preparations were set up for terminal experiments. Seventeen hearts (HF group, n = 10) underwent ex-vivo cardiac MRI. Twenty-two hearts (HF group, n = 7) were examined histologically. Electrical remodeling and abnormal autonomic profile were evident in HF rabbits with exaggerated sympathetic and attenuated vagal effect on ventricular fibrillation threshold, ventricular refractoriness and restitution curves, in addition to increased spatial restitution dispersion. Histologically, there was significant neuronal enlargement at the heart hila and conus arteriosus in HF. Structural remodeling was characterized by quantifiable myocardial scarring, enlarged left ventricles, altered ventricular geometry and impaired contractility. Conclusion: In an infarct-induced rabbit heart failure model, extensive structural, neuronal and electrophysiological remodeling in conjunction with abnormal autonomic profile provide substrates for ventricular arrhythmias.

The Effects of Vagus Nerve Stimulation on Ventricular Electrophysiology and Nitric Oxide Release in the Rabbit Heart.

Background: Abnormal autonomic activity including impaired parasympathetic control is a known hallmark of heart failure (HF). Vagus nerve stimulation (VNS) has been shown to reduce the susceptibility of the heart to ventricular fibrillation, however the precise underlying mechanisms are not well understood and the detailed stimulation parameters needed to improve patient outcomes clinically are currently inconclusive. Objective: To investigate NO release and cardiac electrophysiological effects of electrical stimulation of the vagus nerve at varying parameters using the isolated innervated rabbit heart preparation. Methods: The right cervical vagus nerve was electrically stimulated in the innervated isolated rabbit heart preparation (n = 30). Heart rate (HR), effective refractory period (ERP), ventricular fibrillation threshold (VFT) and electrical restitution were measured as well as NO release from the left ventricle. Results: High voltage with low frequency VNS resulted in the most significant reduction in HR (by -20.6 ± 3.3%, -25.7 ± 3.0% and -30.5 ± 3.0% at 0.1, 1 and 2 ms pulse widths, with minimal increase in NO release. Low voltage and high frequency VNS significantly altered NO release in the left ventricle, whilst significantly flattening the slope of restitution and significantly increasing VFT. HR changes however using low voltage, high frequency VNS were minimal at 20Hz (to 138.5 ± 7.7 bpm (-7.3 ± 2.0%) at 1 ms pulse width and 141.1 ± 6.6 bpm (-4.4 ± 1.1%) at 2 ms pulse width). Conclusion: The protective effects of the VNS are independent of HR reductions demonstrating the likelihood of such effects being as a result of the modulation of more than one molecular pathway. Altering the parameters of VNS impacts neural fibre recruitment in the ventricle; influencing changes in ventricular electrophysiology, the protective effect of VNS against VF and the release of NO from the left ventricle.

Effects of sympatho-vagal interaction on ventricular electrophysiology and their modulation during beta-blockade.

AIMS: The effects of sympatho-vagal interaction on heart rate (HR) changes are characterized by vagal dominance resulting in accentuated antagonism. Complex autonomic modulation of ventricular electrophysiology may exert prognostic arrhythmic impact. We examined the effects of concurrent sympathetic (SNS) and vagus (VNS) nerve stimulation on ventricular fibrillation threshold (VFT) and standard restitution (RT) in an isolated rabbit heart preparation with intact dual autonomic innervation, with and without beta-blockade. METHODS AND RESULTS: Monophasic action potentials were recorded from left ventricular epicardial surface of dual-innervated isolated heart preparations from New Zealand white rabbits (n = 18). HR, VFT and RT were measured during different stimulation protocols (Protocol 1: VNS-SNS; Protocol 2: SNS-VNS) involving low- and high-frequency stimulations. A sub-study of Protocol 2 was performed in the presence of metoprolol tartrate. In both protocols, HR changes were characterized by vagal-dominant bradycardic component, affirming accentuated antagonism. During concurrent high-frequency VNS (HV), SNS prevails in lowering VFT in a frequency-sensitive manner during low (LS) or high (HS)-frequency stimulations (HV-LS: -2.8 ± 0.8 mA; HV-HS: -4.0 ± 0.9 mA, p < .05 vs. HV), with accompanying steepening of relative RT slope gradients (HV-LS: 223.54 ± 37.41%; HV-HS: 295.20 ± 60.86%, p < .05 vs. HV). In protocol 2, low (LV) and high (HV) vagal stimulations during concurrent HS raised VFT (HS-LV: 1.0 ± 0.4 mA; HS-HV: 3.0 ± 0.6 mA, p < .05 vs HS) with associated flattening of RT slopes (HS-LV: 32.40 ± 4.97%;HS-HV: 38.07 ± 6.37%; p < .05 vs HS). Metoprolol abolished accentuated antagonism in HR changes, reduced VFT and flattened RT globally during SNS-VNS. CONCLUSIONS: Accentuated antagonism is absent in ventricular electrophysiological changes during sympatho-vagal interaction with sympathetic effect prevailing, suggesting a different mechanism at the ventricular level from heart rate effects. Metoprolol nullified accentuated antagonism with additional anti-fibrillatory effect beyond adrenergic blockade during sympatho-vagal stimulations.

Different paths, same destination: divergent action potential responses produce conserved cardiac fight-or-flight response in mouse and rabbit hearts.

KEY POINTS: Cardiac electrophysiology and Ca2+ handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses. ABSTRACT: Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P < 0.05) followed by shortening (46.5 ± 9.1 ms at t = 60 s, P = NS vs. t = 0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by ß-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight-or-flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly.

Data highlighting the effects of spinal segmental stimulation of preganglionic sympathetic neurons on the electrophysiology of the rabbit heart.

This article presents data highlighting the functional selectivity of cardiac preganglionic sympathetic neurons in the rabbit heart. Specifically, the data draw attention to the role of each spinal segmental outflow on cardiac electrophysiology and the influence of each segment on cardiac excitability through investigating markers of arrhythmia such as electrical restitution. This data holds importance for exploring whether the preganglionic sympathetic neurons have functionally distinct pathways to the heart and whether some spinal segmental outflows have a greater potential for arrhythmia generation than others. Discussion of the data can be found in Chauhan et al. (2018) [1].

Electrophysiological effects of nicotinic and electrical stimulation of intrinsic cardiac ganglia in the absence of extrinsic autonomic nerves in the rabbit heart.

BACKGROUND: The intrinsic cardiac nervous system is a rich network of cardiac nerves that converge to form distinct ganglia and extend across the heart and is capable of influencing cardiac function. OBJECTIVE: The goals of this study were to provide a complete picture of the neurotransmitter/neuromodulator profile of the rabbit intrinsic cardiac nervous system and to determine the influence of spatially divergent ganglia on cardiac electrophysiology. METHODS: Nicotinic or electrical stimulation was applied at discrete sites of the intrinsic cardiac nerve plexus in the Langendorff-perfused rabbit heart. Functional effects on sinus rate and atrioventricular conduction were measured. Immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase, and/or neuronal nitric oxide synthase (nNOS) was performed using whole mount preparations. RESULTS: Stimulation within all ganglia produced either bradycardia, tachycardia, or a biphasic brady-tachycardia. Electrical stimulation of the right atrial and right neuronal cluster regions produced the largest chronotropic responses. Significant prolongation of atrioventricular conduction was predominant at the pulmonary vein-caudal vein region. Neurons immunoreactive (IR) only for ChAT, tyrosine hydroxylase, or nNOS were consistently located within the limits of the hilum and at the roots of the right cranial and right pulmonary veins. ChAT-IR neurons were most abundant (1946 ± 668 neurons). Neurons IR only for nNOS were distributed within ganglia. CONCLUSION: Stimulation of intrinsic ganglia, shown to be of phenotypic complexity but predominantly of cholinergic nature, indicates that clusters of neurons are capable of independent selective effects on cardiac electrophysiology, therefore providing a potential therapeutic target for the prevention and treatment of cardiac disease.

Functional selectivity of cardiac preganglionic sympathetic neurones in the rabbit heart.

BACKGROUND: Studies have shown regional and functional selectivity of cardiac postganglionic neurones indicating there might exist a similar heterogeneity in spinal segmental preganglionic neurones, which requires further investigation. METHODS: Right and left sympathetic chains were electrically stimulated from T6 to T1 in the innervated isolated rabbit heart preparation (n = 18). Sinus rate, left ventricular pressure, retrograde ventriculo-atrial conduction, monophasic action potential duration, effective refractory period, ventricular fibrillation threshold and electrical restitution were measured. RESULTS: Right sympathetic stimulation had a greater influence on heart rate (T1-T2: right; 59.9 ±â€¯6.0%, left; 41.1 ±â€¯5.6% P < 0.001) and left stimulation had greater effects on left ventricular pressure (T1-T2: right; 20.7 ±â€¯3.2%, left; 40.3 ±â€¯5.4%, P < 0.01) and ventriculo-atrial conduction (T1-T2: right; -6.8 ±â€¯1.1%, left; -15.5 ±â€¯0.2%) at all levels, with greater effects at rostral levels (T1-T3). Left sympathetic stimulation caused shorter monophasic action potentials at the base (T4-T5: right; 119.3 ±â€¯2.7 ms, left; 114.7 ±â€¯2.5 ms. P < 0.05) and apex (T4-T5: right; 118.8 ±â€¯1.2 ms, left; 114.6 ±â€¯2.6 ms. P < 0.05), greater shortening of effective refractory period (T4-T5: right; -3.6 ±â€¯1.3%, left; -7.7 ±â€¯1.8%. P < 0.05), a steeper maximum slope of restitution (T4-T5 base: right; 1.3 ±â€¯0.2, left; 1.8 ±â€¯0.2. P < 0.01. T4-T5 apex: right; 1.0 ±â€¯0.2, left; 1.6 ±â€¯0.3. P < 0.05) and a greater decrease in ventricular fibrillation threshold (T4-T5: right; -22.3 ±â€¯6.8%, left;-39.0 ±â€¯1.7%), with dominant effects at caudal levels (T4-T6). CONCLUSIONS: The preganglionic sympathetic efferent axons show functionally distinct pathways to the heart. The caudal segments (T4-T6) of the left sympathetic chain had a greater potential for arrhythmia generation and hence could pose a target for more focused clinical treatments for impairments in cardiac function.

The heart's 'little brain' controlling cardiac function in the rabbit.

NEW FINDINGS: What is the topic of this review? The topic of the review is the intrinsic cardiac nervous system in the rabbit. What advances does it highlight? The anatomy of rabbit intrinsic ganglia is similar to that of other species, including humans. Immunohistochemistry confirms the presence of cholinergic and adrenergic neurones, with a striking arrangement of neuronal nitric oxide synthase-positive cell bodies. Activation of atrial ganglia produces effects on local and remote regions. Heart disease is a primary cause of mortality in the developed world, and it is well recognized that neural mechanisms play an important role in many cardiac pathologies. The role of extrinsic autonomic nerves has traditionally attracted the most attention. However, there is a rich intrinsic innervation of the heart, including numerous cardiac ganglia (ganglionic plexuses), that has the potential to affect cardiac function independently as well as to influence the actions of the extrinsic nerves. To investigate this, an isolated, perfused, innervated rabbit Langendorff heart preparation was considered the best option. Although ganglionic plexuses have been well described for several species, there was no full description of the anatomy and histochemistry of rabbit hearts. To this end, rabbit intrinsic ganglia were located using acetylcholinesterase histology (n = 33 hearts). This revealed six generalized ganglionic regions, defined as a left neuronal complex above the left pulmonary vein, a right neuronal complex around the base of right cranial vein, three scattered in the dorsal right atrium and a region containing numerous ventricular ganglia located on the conus arteriosus. Using immunohistochemistry, neurons were found to contain choline acetyltransferase or tyrosine hydroxylase and/or neuronal nitric oxide synthase in differing amounts (choline acetyltransferase > tyrosine hydroxylase > neuronal nitric oxide synthase). The function of rabbit intrinsic ganglia was investigated using a bolus application of nicotine or electrical stimulation at each of the above sites whilst measuring heart rate and atrioventricular conduction. Nicotine applied to different ganglionic plexuses caused a bradycardia, a tachycardia or a mixture of the two, with the right atrial plexus producing the largest chronotropic responses. Electrical stimulation at these sites induced only a bradycardia. Atrioventricular conduction was modestly changed by nicotine, the main response being a prolongation. Electrical stimulation produced significant prolongation of atrioventricular conduction, particularly when the right neuronal complex was stimulated. These studies show that the intrinsic plexuses of the heart are important and could be crucial for understanding impairments of cardiac function. Additionally, they provide a strong basis from which to progress using the isolated, innervated rabbit heart preparation.

hERG potassium channel blockade by the HCN channel inhibitor bradycardic agent ivabradine.

BACKGROUND: Ivabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodal HCN-channels. This study investigated the propensity of ivabradine to interact with KCNH2-encoded human Ether-à-go-go-Related Gene (hERG) potassium channels, which strongly influence ventricular repolarization and susceptibility to torsades de pointes arrhythmia. METHODS AND RESULTS: Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Ih ERG was inhibited with an IC50 of 2.07 µmol/L for the hERG 1a isoform and 3.31 µmol/L for coexpressed hERG 1a/1b. The voltage and time-dependent characteristics of Ih ERG block were consistent with preferential gated-state-dependent channel block. Inhibition was partially attenuated by the N588K inactivation-mutant and the S624A pore-helix mutant and was strongly reduced by the Y652A and F656A S6 helix mutants. In docking simulations to a MthK-based homology model of hERG, the 2 aromatic rings of the drug could form multiple π-π interactions with the aromatic side chains of both Y652 and F656. In monophasic action potential (MAP) recordings from guinea-pig Langendorff-perfused hearts, ivabradine delayed ventricular repolarization and produced a steepening of the MAPD90 restitution curve. CONCLUSIONS: Ivabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate between hERG and HCN channels: it inhibits Ih ERG with similar potency to that reported for native If and HCN channels, with S6 binding determinants resembling those observed for HCN4. These findings may have important implications both clinically and for future bradycardic drug design.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Innervation of sinoatrial nodal cardiomyocytes in mouse. A combined approach using immunofluorescent and electron microscopy.

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart preparations demonstrates a particularly dense and complex network of nerve fibres and cardiomyocytes which are positive to the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4-positive cardiomyocytes) in the sinoatrial node region and adjacent areas around the root of right cranial vein. The present study was designed to investigate the morphologic and histochemical pattern of nerve fibres and HCN4-positive cardiomyocytes using fluorescent techniques and/or electron microscopy. Adrenergic and cholinergic nerve fibres together with HCN4-positive cardiomyocytes were identified using primary antibodies for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and the HCN4 channel respectively. Amid HCN4-positive cardiomyocytes, fluorescence and electron microscopy data demonstrated a dense distribution of nerve fibres immunoreactive for ChAT and TH. In addition, novel electron microscopy data revealed that the mouse sinoatrial node contained exclusively unmyelinated nerve fibres, in which the majority of axons possess varicosities with clear mediatory vesicles that can be classified as cholinergic. Synapses occurred without any clear terminal connection with the effector cell, i.e. these synapes were of "en passant" type. In general, the morphologic pattern of innervation of mouse HCN4-positive cardiomyocytes identified using electron microscopy corresponds well to the dense network of nerve fibres demonstrated by fluorescent immunohistochemistry in mouse sinoatrial node and adjacent areas. The complex and extraordinarily dense innervation of HCN4-positive cardiomyocytes in mouse sinoatrial node underpins the importance of neural regulation for the cardiac conduction system. Based on the present observations, it is concluded that the occurrence of numerous nerve fibres nearby atrial cardiomyocytes serves as a novel reliable extracellular criterion for discrimination of SA nodal cardiomyocytes using electron microscopy.

Morphological pattern of intrinsic nerve plexus distributed on the rabbit heart and interatrial septum.

Although the rabbit is routinely used as the animal model of choice to investigate cardiac electrophysiology, the neuroanatomy of the rabbit heart is not well documented. The aim of this study was to examine the topography of the intrinsic nerve plexus located on the rabbit heart surface and interatrial septum stained histochemically for acetylcholinesterase using pressure-distended whole hearts and whole-mount preparations from 33 Californian rabbits. Mediastinal cardiac nerves entered the venous part of the heart along the root of the right cranial vein (superior caval vein) and at the bifurcation of the pulmonary trunk. The accessing nerves of the venous part of the heart passed into the nerve plexus of heart hilum at the heart base. Nerves approaching the heart extended epicardially and innervated the atria, interatrial septum and ventricles by five nerve subplexuses, i.e. left and middle dorsal, dorsal right atrial, ventral right and left atrial subplexuses. Numerous nerves accessed the arterial part of the arterial part of the heart hilum between the aorta and pulmonary trunk, and distributed onto ventricles by the left and right coronary subplexuses. Clusters of intrinsic cardiac neurons were concentrated at the heart base at the roots of pulmonary veins with some positioned on the infundibulum. The mean number of intrinsic neurons in the rabbit heart is not significantly affected by aging: 2200 ± 262 (range 1517-2788; aged) vs. 2118 ± 108 (range 1513-2822; juvenile). In conclusion, despite anatomic differences in the distribution of intrinsic cardiac neurons and the presence of well-developed nerve plexus within the heart hilum, the topography of all seven subplexuses of the intrinsic nerve plexus in rabbit heart corresponds rather well to other mammalian species, including humans.

Cardiac contractility modulation increases action potential duration dispersion and decreases ventricular fibrillation threshold via ß1-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 ß1-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 ß1-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 (r(2)=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.

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

Blebbistatin (BS) is a recently discovered inhibitor of the myosin II isoform and has been adopted as the mechanical uncoupler of choice for optical mapping, because previous studies suggest that BS has no significant cardiac electrophysiological effects in a number of species. The aim of this study was to determine whether BS affects cardiac electrophysiology in isolated New Zealand White rabbit hearts. Langendorff-perfused hearts (n=39) in constant-flow mode had left ventricular monophasic action potential duration (MAPD) measured at apical and basal regions during constant pacing (300 ms cycle length). Standard action potential duration restitution was obtained using the single extrastimulus method with measurement of the maximal restitution slope. Ventricular fibrillation threshold was measured as the minimal current inducing sustained ventricular fibrillation with burst pacing (30 stimuli, at 30 ms intervals). Optical action potentials were recorded using the voltage-sensitive dye di-4-ANEPPS. Measurements were taken at baseline and after 60 min perfusion with BS (5 µm). Blebbistatin significantly prolonged left ventricular apical (mean±SEM; from 129.9±2.9 to 170.7±4.1 ms, P<0.001, n=8) and basal MAPD (from 135.0±2.3 to 163.3±5.6 ms, P<0.001) and effective refractory period (from 141.3±4.8 to 175.6±3.7 ms, P<0.001) whilst increasing the maximal slope of restitution (apex, from 0.79±0.09 to 1.57±0.16, P<0.001; and base, from 0.71±0.06 to 1.44±0.24, P<0.001) and ventricular fibrillation threshold (from 5.3±1.1 to 17.0±2.9 mA, P<0.001). In other hearts, blebbistatin significantly prolonged optically recorded action potentials (from 136.5±6.3 to 173.0±7.9 ms, P<0.05, n=4). In control experiments, the increase of MAPD with blebbistatin was present whether the hearts were perfused in constant-pressure mode (n=5) or in unloaded conditions (n=5). These data show that blebbistatin significantly affects cardiac electrophysiology. Its use in optical mapping studies should be treated with caution.

Mechanisms underlying the autonomic modulation of ventricular fibrillation initiation--tentative prophylactic properties of vagus nerve stimulation on malignant arrhythmias in heart failure.

Classical physiology teaches that vagal post-ganglionic nerves modulate the heart via acetylcholine acting at muscarinic receptors, whilst it is accepted that vagus nerve stimulation (VNS) slows heart rate, atrioventricular conduction and decreases atrial contraction; there is continued controversy as to whether the vagus has any significant direct effect on ventricular performance. Despite this, there is a significant body of evidence from experimental and clinical studies, demonstrating that the vagus nerve has an anti-arrhythmic action, protecting against induced and spontaneously occurring ventricular arrhythmias. Over 100 years ago Einbrodt first demonstrated that direct cervical VNS significantly increased the threshold for experimentally induced ventricular fibrillation. A large body of evidence has subsequently been collected supporting the existence of an anti-arrhythmic effect of the vagus on the ventricle. The development of prognostic indicators of heart rate variability and baroreceptor reflex sensitivity--measures of parasympathetic tone and reflex activation respectively--and the more recent interest in chronic VNS therapy are a direct consequence of the earlier experimental studies. Despite this, mechanisms underlying the anti-arrhythmic actions of the vagus nerve have not been fully characterised and are not well understood. This review summarises historical and recently published data to highlight the importance of this powerful endogenous protective phenomenon.

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.