Kieran E. Brack

Effects of Direct Sympathetic and Vagus Nerve Stimulation on the Physiology of the Whole Heart – A Novel Model of Isolated Langendorff Perfused Rabbit Heart with Intact Dual Autonomic Innervation

A novel isolated Langendorff perfused rabbit heart preparation with intact dual autonomic innervation is described. This preparation allows the study of the effects of direct sympathetic and vagus nerve stimulation on the physiology of the whole heart. These hearts (n= 10) had baseline heart rates of 146 ± 2 beats min−1 which could be increased to 240 ±11 beats min−1 by sympathetic stimulation (15 Hz) and decreased to 74 ± 11 beats min−1 by stimulation of the vagus nerve (right vagus, 7 Hz). This model has the advantage of isolated preparations, with the absence of influence from circulating hormones and haemodynamic reflexes, and also that of in vivo preparations where direct nerve stimulation is possible without the need to use pharmacological agents. Data are presented characterising the preparation with respect to the effects of autonomic nerve stimulation on intrinsic heart rate and atrioventricular conduction at different stimulation frequencies. We show that stimulation of the right and left vagus nerve have differential effects on heart rate and atrioventricular conduction.

Nitric oxide mediates the vagal protective effect on ventricular fibrillation via effects on action potential duration restitution in the rabbit heart

We have previously shown that direct vagus nerve stimulation (VNS) reduces the slope of action potential duration (APD) restitution while simultaneously protecting the heart against induction of ventricular fibrillation (VF) in the absence of any sympathetic activity or tone. In the current study we have examined the role of nitric oxide (NO) in the effect of VNS. Monophasic action potentials were recorded from a left ventricular epicardial site on innervated, isolated rabbit hearts (n= 7). Standard restitution, effective refractory period (ERP) and VF threshold (VFT) were measured at baseline and during VNS in the presence of the NO synthase inhibitor NG‐nitro‐l‐arginine (l‐NA, 200 μm) and during reversing NO blockade with l‐arginine (l‐Arg, 1 mm). Data represent the mean ±s.e.m. The restitution curve was shifted upwards and became less steep with VNS when compared to baseline. l‐NA blocked the effect of VNS whereas l‐Arg restored the effect of VNS. The maximum slope of restitution was reduced from 1.17 ± 0.14 to 0.60 ± 0.09 (50 ± 5%, P < 0.0001) during control, from 0.98 ± 0.14 to 0.93 ± 0.12 (2 ± 10%, P= NS) in the presence of l‐NA and from 1.16 ± 0.17 to 0.50 ± 0.10 (41 ± 9%, P= 0.003) with l‐Arg plus l‐NA. ERP was increased by VNS in control from 119 ± 6 ms to 130 ± 6 ms (10 ± 5%, P= 0.045) and this increase was not affected by l‐NA (120 ± 4 to 133 ± 4 ms, 11 ± 3%, P= 0.0019) or l‐Arg with l‐NA (114 ± 4 to 123 ± 4 ms, 8 ± 2%, P= 0.006). VFT was increased from 3.0 ± 0.3 to 5.8 ± 0.5 mA (98 ± 12%, P= 0.0017) in control, 3.4 ± 0.4 to 3.8 ± 0.5 mA (13 ± 12%, P= 0.6) during perfusion with l‐NA and 2.5 ± 0.4 to 6.0 ± 0.7 mA (175 ± 50%, P= 0.0017) during perfusion with l‐Arg plus l‐NA. Direct VNS increased VFT and flattened the slope of APD restitution curve in this isolated rabbit heart preparation with intact autonomic nerves. These effects were blocked using l‐NA and reversed by replenishing the substrate for NO production with l‐Arg. This is the first study to demonstrate that NO plays an important role in the anti‐fibrillatory effect of VNS on the rabbit ventricle, possibly via effects on APD restitution.

Vagus nerve stimulation protects against ventricular fibrillation independent of muscarinic receptor activation

AIMS The role of the vagus in the ventricle is controversial, although the vagus can protect against ventricular fibrillation (VF) via nitric oxide (NO). This study aims to determine whether the mechanisms involved are dependent on post-ganglionic release and muscarinic receptor activation. For this purpose, NO release and electrophysiological effects of vagus nerve stimulation (VNS) were evaluated in relation to acetylcholine and vasoactive intestinal peptide (VIP). In addition, the role of the coronary endothelium and afferent nerves was tested. METHODS AND RESULTS Using the isolated innervated rabbit heart, we measured ventricular NO release using 4,5-diaminofluorescein (DAF-2) fluorescence and ventricular fibrillation threshold (VFT) during VNS after muscarinic, ganglionic, and VIP inhibition [atropine, hexamethonium, and VIP (6-28), respectively] and after Triton-X endothelial functional dysfunction. The vagal-mediated increases in NO and VFT were not significantly affected (P> 0.05) during (i) atropine perfusion [increase in NO: 196.8 ± 35.2 mV (control) vs. 156.1 ± 20.3 mV (atropine) and VFT 3.1 ± 0.5 mA (control) vs. 2.7 ± 0.4 mA (atropine)], (ii) VIP inhibition-increase in NO: 243.0 ± 42.4 mV (control) vs. 203.9 ± 28.5 mV [VIP(6-28)] and VFT 3.3 ± 0.3 mA (control) vs. 3.9 ± 0.6 mA [VIP(6-28)], or (iii) after endothelial functional dysfunction [increase in NO: 127.7 ± 31.7 mV (control) vs. 172.1 ± 31.5 mV (Triton-X) and VFT 2.6 ± 0.4 mA (control) vs. 2.5 ± 0.5 mA (Triton-X)]. However, the vagal effects were inhibited during ganglionic blockade [increase in NO: 175.1 ± 38.1 mV (control) vs. 0.6 ± 25.3 mV (hexamethonium) and VFT 3.3 ± 0.5 mA (control) vs. -0.3 ± 0.3 mA (hexamethonium)]. CONCLUSIONS We show that the vagal anti-fibrillatory action in the rabbit ventricle occurs via post-ganglionic efferent nerve fibres, independent of muscarinic receptor activation, VIP, and the endothelium. Together with our previous publications, our data support the possibility of a novel ventricular nitrergic parasympathetic innervation and highlight potential for new therapeutic targets to treat ventricular dysrhythmias.

Direct evidence of nitric oxide release from neuronal nitric oxide synthase activation in the left ventricle as a result of cervical vagus nerve stimulation

Information regarding vagal innervation in the cardiac ventricle is limited and the direct effect of vagal stimulation on ventricular myocardial function is controversial. We have recently provided indirect evidence that the anti‐fibrillatory effect of vagus nerve stimulation on the ventricle is mediated by nitric oxide (NO). The aim of this study was to provide direct evidence for the release of nitric oxide in the cardiac ventricle during stimulation of the efferent parasympathetic fibres of the cervical vagus nerve. The isolated innervated rabbit heart was employed with the use of the NO fluorescent indicator 4,5‐diaminofluorescein diacetate (DAF‐2 DA) during stimulation of the cervical vagus nerves and acetylcholine perfusion in the absence and presence of the non‐specific NO synthase inhibitor NG‐nito‐l‐arginine (l‐NNA) and the neuronal NO synthase selective inhibitor 1‐(2‐trifluormethylphenyl)imidazole (TRIM). Using the novel fluorescence method in the beating heart, we have shown that NO‐dependent fluorescence is increased by 0.92 ± 0.26, 1.20 ± 0.30 and 1.91 ± 0.27% (during low, medium and high frequency, respectively) in the ventricle in a stimulation frequency‐dependent manner during vagus nerve stimulation, with comparable increases seen during separate stimulation of the left and right cervical vagus nerves. Background fluorescence is reduced during perfusion with l‐NNA and the increase in fluorescence during high frequency vagal stimulation is inhibited during perfusion with both l‐NNA (1.97 ± 0.35% increase before l‐NNA, 0.00 ± 0.02% during l‐NNA) and TRIM (1.78 ± 0.18% increase before TRIM, −0.11 ± 0.08% during TRIM). Perfusion with 0.1 μm acetylcholine increased NO fluorescence by 0.76 ± 0.09% which was blocked by l‐NNA (change of 0.00 ± 0.03%) but not TRIM (increase of 0.82 ± 0.21%). Activation of cardiac parasympathetic efferent nerve fibres by stimulation of the cervical vagus is associated with NO production and release in the ventricle of the rabbit, via the neuronal isoform of nitric oxide synthase.

Interaction between direct sympathetic and vagus nerve stimulation on heart rate in the isolated rabbit heart

The interaction between the effects of vagus nerve stimulation (VS) and sympathetic stimulation (SS) on intrinsic heart rate was studied in the novel innervated isolated rabbit heart preparation. The effects of background VS, at different frequencies – 2 Hz (low), 5 Hz (medium), 7 Hz (high) – on the chronotropic effects of different frequencies of SS – 2 Hz (low), 5 Hz (medium), 10 Hz (high) – were studied. The experiments were repeated in the reverse direction studying the effects of different levels of background SS on the chronotropic effects of different levels of VS. Background VS reduced the overall positive chronotropic effect of SS at steady state in a frequency dependent manner and the rate of increase in heart rate during low and medium SS (but not high SS) was slowed in the presence of background VS. These results suggest that pre‐ and postjunctional mechanisms may be involved in the sympatho–vagal interaction on heart rate. On the other hand, the chronotropic effect of VS was enhanced in the presence of background SS. Vagal stimulation appears to play a dominant role over sympathetic stimulation in chronotropic effects on the isolated heart. The innervated isolated heart preparation is a valuable model to study the complex mechanisms underlying the interaction between sympathetic and parasympathetic stimulation on cardiac function.

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.

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. IhERG 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 IhERG 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 IhERG 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.

Vagus nerve stimulation inhibits the increase in Ca2+ transient and left ventricular force caused by sympathetic nerve stimulation but has no direct effects alone – epicardial Ca2+ fluorescence studies using fura‐2 AM in the isolated innervated beating rabbit heart

The effects of direct autonomic nerve stimulation on the heart may be quite different to those of perfusion with pharmacological neuromodulating agents. This study was designed to investigate the effect of autonomic nerve stimulation on intracellular calcium fluorescence using fura‐2 AM in the isolated Langendorff‐perfused rabbit heart preparation with intact dual autonomic innervation. The effects of autonomic nerve stimulation on cardiac force and calcium transients were more obvious during intrinsic sinus rhythm. High‐frequency (15 Hz, n= 5) right vagus nerve stimulation (VS) decreased heart rate from 142.7 ± 2.6 to 75.5 ± 10.2 beats min–1 and left ventricular pressure from 36.4 ± 3.2 to 25.9 ± 1.9 mmHg, whilst simultaneously decreasing the diastolic and systolic level of the calcium transient. Direct sympathetic nerve stimulation (7 Hz, n= 8) increased heart rate (from 144.7 ± 10.5 to 213.2 ± 4.9 beats min–1) and left ventricular pressure (from 37.5 ± 3.6 to 43.7 ± 2.8 mmHg), whilst simultaneously increasing the diastolic and systolic level of the calcium transient. During constant ventricular pacing, the high‐frequency right vagus nerve stimulation did not have any direct effect on ventricular force or the calcium transient (n= 8), but was effective in reducing the effect of direct sympathetic nerve stimulation.

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.

The effect of direct autonomic nerve stimulation on left ventricular force in the isolated innervated Langendorff perfused rabbit heart

The relative contribution of the chronotropic effects of stimulating sympathetic and vagus nerves on cardiac inotropic changes in the isolated Langendorff perfused rabbit heart with intact dual autonomic nerves was studied. The force-frequency relationship was investigated, in addition to sympathetic nerve stimulation (SS) at 2 Hz (low), 5 Hz (med) and 10 Hz (high), and left and right vagus nerve stimulation (VS) studied at 2 Hz (low), 5 Hz (med) and 7 Hz (high) with and without right ventricular pacing. It was shown that a biphasic force-frequency relationship is present with a positive relationship at low heart rates and a negative force-frequency relationship at higher heart rates. There was a trend for left- and right-VS to decrease left ventricular pressure with a decrease in heart rate, whilst SS had the opposing effects in a frequency-dependent manner. When heart rate was kept constant, there was no effect from left- or right-VS, while SS increased left ventricular pressure in a frequency-dependent manner. Together these results suggest that SS, left- and right-VS alter left ventricular force by two different mechanisms. Left- and right-VS decrease left ventricular pressure predominantly via chronotropic effects whilst SS increases force predominantly by direct changes in contractility.