Kieran Brack - Academia.edu (original) (raw)

Papers by Kieran Brack

Biophysical Journal, 2014

Nitric oxide (NO) signalling pathways are reported to regulate cardiac repolarisation. This was i... more Nitric oxide (NO) signalling pathways are reported to regulate cardiac repolarisation. This was investigated using BAY 60-2770, which activates soluble guanylyl cyclase (sGC) in a NO and haem independent manner to generate cGMP. Action potentials (APs) and currents were recorded from isolated guinea pig ventricular myocytes at 37 o C using the perforated patch clamp technique. APs were stimulated at 2 Hz. The slow and rapid delayed rectifier potassium currents, I Ks and I Kr were measured as tail currents elicited by a voltage step from þ40 mV to À50 mV. The following experiments were carried out in the presence of 100 mM IBMX (3-Isobutyl-1-methylxanthine) a non-selective phosphodiesterase (PDE) inhibitor. 1 mM BAY 60-2770 lengthened APD 90 by 15.7 ms (p<0.05, n=7), suggesting that sGC activation and an increase in cGMP inhibits repolarising currents. Further experiments showed that I Ks was inhibited by BAY 60-2770. IBMX alone enhanced I Ks by 77 5 9 % (p<0.001, n=8) and subsequent addition of BAY 60-2770 reduced the IBMX dependent enhancement of I Ks by 68 5 2 % (p<0.001, n=8). This suggests that PDE inhibition permits I Ks to be inhibited by cGMP dependent NO signalling pathways. The sGC mediated inhibition of I Ks by BAY 60-2770 is unlikely to be due to phosphorylation by protein kinase G (PKG) because the inhibition remained in the presence of 100 nM KT-5823, a PKG inhibitor and because PKG activation with 100 mM 8-bromo-cGMP had no effect on I Ks . I Kr was not regulated by PDE inhibition, sGC activation or PKG activation. Overall, these findings suggest that cGMP dependent NO signalling pathways regulate I Ks , but not I Kr , via a PKG independent mechanism.

Heart rhythm : the official journal of the Heart Rhythm Society, 2014

Journal of Molecular and Cellular Cardiology, 2014

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart prep... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;en passant&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; 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.

Annals of Anatomy - Anatomischer Anzeiger, 2014

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtai... more 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.

The Journal of Physiology, 2007

We have previously shown that direct vagus nerve stimulation (VNS) reduces the slope of action po... more 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 N G -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.

The Journal of Physiology, 2009

Information regarding vagal innervation in the cardiac ventricle is limited and the direct effect... more 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 N G -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.

Journal of Anatomy, 2014

Although the rabbit is routinely used as the animal model of choice to investigate cardiac electr... more 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 AE 262 (range 1517-2788; aged) vs. 2118 AE 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.

International Journal of Cardiology, 2014

Background/objectives: Cardiac contractility modulation (CCM) is a new treatment being developed ... more 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 b 0.001). VFT was reduced during CCM (2.6 ± 0.6 [CCM] vs. 6.1 ± 0.8 [Control] mA, P b 0.01) and was correlated (r 2 = 0.40, P b 0.01) with increased MAPD dispersion (26 ± 4 [CCM] vs. 5 ± 1 [Control] ms, P b 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.

Heart Rhythm, 2012

The sympathetic nervous system is thought to play a role in the genesis of ventricular tachyarrhy... more The sympathetic nervous system is thought to play a role in the genesis of ventricular tachyarrhythmias (VT). Left and added right cardiac sympathectomy have been shown to reduce the burden of arrhythmias in the setting of a VT storm. However, the contribution of the right stellate ganglion (RSG) and the left stellate ganglion (LSG) to the innervation of the anterior left ventricular (LV) wall is not well understood. To evaluate the innervation of the anterior LV wall by the LSG and the RSG. The heart and stellate ganglia were exposed via sternotomy in pigs with normal hearts (n = 8). A 20-electrode catheter was placed on the anterior LV wall to record activation recovery interval (ARI), a surrogate measure of action potential duration. A microdialysis catheter was inserted in a similar location to sample interstitial norepinephrine (NE) content. ARI and NE measurements were recorded at baseline and during LSG and RSG stimulation. LSG stimulation shortened ARI by 17.1% ± 10.5% (mean ± standard error), while RSG stimulation shortened ARI by 42.1% ± 15.7%, P = .04 (LSG vs RSG). LSG stimulation increased interstitial NE levels by 200% ± 65%, while RSG stimulation increased the NE content by 260% ± 40% (P = .012). LSG stimulation increased dispersion in ARI from 376.0 ± 83.7 ms(2) to 1242.5 ± 566 ms(2) (P = .03) and caused ventricular fibrillation in 2 pigs. During RSG stimulation, dispersion increased from 419 ± 65.8 to 474.8 ± 81 ms(2) (P = .4). Both the LSG and the RSG provide significant innervation to the anterior LV wall as demonstrated by both ARI shortening and NE concentrations. LSG stimulation significantly increases ARI dispersion. This study provides mechanistic insight into the beneficial effects of left sympathectomy and the additional role of right sympathectomy in reducing arrhythmias in patients with anterior myocardial scars and VT storm.

Heart Failure Reviews, 2013

Classical physiology teaches that vagal postganglionic nerves modulate the heart via acetylcholin... more Classical physiology teaches that vagal postganglionic 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.

Experimental Physiology, 2013

Blebbistatin significantly affects cardiac ventricular electrophysiology and induction of ventric... more 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.

Experimental Physiology, 2004

The interaction between the effects of vagus nerve stimulation (VS) and sympathetic stimulation (... more 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.

Cardiovascular Research, 2011

The role of the vagus in the ventricle is controversial, although the vagus can protect against v... more 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 postganglionic 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.

Biophysical Journal, 2013

sex difference of QT intervals and risks of arrhythmia. We here seek the underlying mechanisms on... more sex difference of QT intervals and risks of arrhythmia. We here seek the underlying mechanisms on the regulation of I Ca,L in patch-clamped guinea pig ventricular myocytes. An external application of P 4 (100 nM) suppressed I Ca,L about 30 % (100 nM P 4 ; 5854 %, n=13, without P 4 (time control); 8452 %, n=5). In the presence of a specific PDE2 inhibitor, EHNA (30 mM), P 4 suppressed I Ca,L to 8455 % (n=7), which is the same extent with the time control. Pharmacological studies revealed that hydrolysis of cAMP by a cGMPstimulated phosphodiesterase (PDE2) involves in the P 4 -induced I Ca,L suppression. However the underlying mechanisms of the I Ca,L suppression remain to be solved. Thus, we employed sucrose density gradient fractionation experiments to examine localization of the molecules in this system, which is dependent of lipid-raft composition. We found that substantial fraction of Ca V1.2 and PDE2 clustered in caveolae fractions, but PDE3 clustered in extra-caveolae fractions. Furthermore, a proximity ligation assay (Olink) revealed that Ca V1.2 interacted both with PDE2 and PDE3, but there was no interaction between PDE2 and PDE3. These results suggest that PDE2 and the L-type Ca 2þ channel colocalizes at the caveolae for the non-genomic regulation of I Ca,L by P 4 . Background: The cardiac nervous system plays an important role in the initiation and maintenance of atrial fibrillation (AF). Besides the classical neurotransmitters, the nerves innervating the atria also release a wide range of neuropeptides that may play a role in the pathophysiology of AF. Since the direct effect of these neuropeptides on atrial myocytes is largely unknown, we investigated the electrophysiological response of atrial myocytes to several neuropeptides, importantly substance-P (Sub-P). Methods and Results: Single left atrial myocytes from rabbit hearts were obtained by enzymatic dissociation and the effects of Sub-P, neuropeptide-Y (NPY), somatostatin-14 (SOM-14) and vasoactive intestinal peptide (VIP), were studied using patch-clamp and indo-1 fluorescence methodologies. NPY, SOM-14 and VIP (1 mM) did not affect atrial action potential (AP) characteristics. Sub-P however, reduced both resting membrane potential (RMP) and action potential amplitude, and increased action potential duration at 90% of repolarisation (APD 90 ) by 40%. The effects on APD 90 were dose-dependent and occurred from a concentration of 10 nM. Voltageclamp analysis revealed that Sub-P significantly diminished L-type calcium current, inward rectifier K þ current (I K1 ) and an unknown steady-state outward current. Transient outward current, Ca 2þ -activated chloride current and Na þ -Ca 2þ -exchanger current were unaffected, as was the intracellular calcium handling. The reduction in RMP and increase in APD 90 are likely due to the decrease in I K1 and steady-state outward current, respectively. Our experiments indicate that the latter is probably carried by backgroundlike potassium channels. Conclusion: Atrial myocyte electrophysiology is significantly modulated by the neuropeptide Sub-P, mainly characterized by a substantial AP prolongation due to inhibition of a background potassium current. Since lengthening of atrial repolarization is potentially anti-arrhythmic, we hypothesize that release of Sub-P may be beneficial to the prevention and/or termination of AF. Inherited dilated cardiomyopathy (DCM), characterized by dilation and impaired systolic function of ventricle, is a progressive disease often resulting in death with congestive heart failure (CHF) or sudden death (SD). It remains unclear how electrical remodeling proceed in inherited DCM. Furthermore, although angiotensin II receptor blockers (ARBs) is known to have beneficial effects on HF in general, little is known about the effects of ARB on the electrical remodeling in inherited DCM. The aim of our study was to investigate effects of ARB on the remodeling in inherited DCM hearts using a knock-in mouse model of DCM (TNNT2 DK210). In DCM mice at 2 months of age when mortality rate abruptly increased, myocardium showed a significant reduction in K þ current density with substantial decreases in expression of Kv4.2, Kv1.5 and KChIP2. In contrast, at 1 month, down-regulation of Kv1.5 or KChIP2 was not observed, but up-regulation of Cav3.1 was detected. At 3 months, some of DCM mice showed a lung edema, a sign of CHF. CHF myocardium showed further decrease in expression of the various Kþ channels. Treatment with ARB, candesartan, started at 1 months, considerably extended lifespan of DCM mice. Interestingly, expression changes that occurred in later phase were inhibited by ARB whereas changes occurred at early stage were not affected. Our results indicate that the electrical remodeling at later stage of DCM is critical for survival of this mouse model and the remodeling is controllable by ARB. . Recently, roles for nitric oxide (NO) signalling pathways have been linked with abnormalities in cardiac repolarisation and cardiac arrhythmias. Studies from isolated heart preparations have demonstrated an antifibrillatory effect following vagus nerve stimulation, which is NO dependent. The cellular and molecular basis for this protective effect is unknown. In this study, cGMP dependent regulation of repolarisation was investigated using BAY 60-2770, a novel NO and haem independent soluble guanylyl cyclase (sGC) activator. Action potentials and currents were recorded from isolated guinea pig ventricular myocytes using the perforated patch clamp technique. Cellular cGMP was quantified by radioimmunoassay and expressed as a fold change relative to basal levels. 1 mM BAY 60-2770 increased cellular cGMP by 2.2 fold and by 7.1 fold in the presence of 100 mM IBMX (3-Isobutyl-1methylxanthine), a non-selective phosphodiesterase (PDE) inhibitor. In vitro assays on purified sGC have shown that ODQ (1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one) potentiates the action of BAY 60-2770. This was corroborated in our experiments, BAY 60-2770 þ 10 mM ODQ increased cGMP by 27.9 fold. Despite substantial increases in cellular cGMP, changes in times to 90% repolarisation (APD 90 ) were modest. BAY 60-2770 shortened APD 90 by 11.3 ms and adding ODQ to BAY 60-2770 caused no further APD 90 shortening. In contrast, when cGMP hydrolysis was blocked by IBMX, BAY 60-2770 lengthened APD 90 by 15.7 ms. Preliminary findings indicate that delayed rectifier potassium currents are modulated by these pathways. Overall, these findings demonstrate the complex interplay between cGMP and cAMP mediated effects on the ion channels regulating cardiac repolarisation. The modest effect of BAY 60-2770 þ ODQ on APD 90 , despite a substantial increase in cellular cGMP, highlights that PDEs limit cGMP accumulation close to ion channels in the sarcolemma, thus compartmentalising cGMP signalling. The universal second messenger 3',5'-cyclic adenosine monophosphate (cAMP) has been considered for many years the main regulator of several cellular physiopathological process uniquely via protein kinase A (PKA) activation. However, in the last years the discovery of a new sensor for cAMP named Epac (exchange protein directly activated by cAMP) has profoundly changed this cAMP-PKA dogma. We have recently shown that Epac activation activates excitation-transcription coupling and, as a result, in few hours has a positive inotropic effect, which differs from acute effects. These data suggests that Epac modulates the expression of different proteins involved in excitation-contraction coupling. We analyzed the possible modulator effect of Epac on ionic currents in rat cardiac myocytes incubated or not in the presence of the selective Epac activator, 8-pCPT. We analyzed L-type Ca 2þ current (ICa) and background currents by patch-clamp in the whole cell configuration. We found that while the peak of L-type Ca 2þ current was not significantly altered by Epac activation, there was a shift in the voltagedependent activation curve towards more hyperpolarized potentials. However, the voltage-dependent inactivation curve was not significantly modified by Epac. As a result, the ICa window current was higher in the Epac-activated group, which is associated with calmodulin upregulation and could contribute to the positive-inotropic effect but also induce arrhythmia. The background current was increased by Epac activation and is currently under investigation. We conclude that Epac sustained activation induce electrophysiological changes in the cardiac myocyte. 282a Monday, February 4, 2013

Autonomic Neuroscience, 2012

The heart receives both a left and right sympathetic innervation. Currently there is no descripti... more 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 (Pb 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 mm Hg, by 22± 5 mm Hg (LSS, 10 Hz) and 4 ± 1 mm Hg (RSS, 10 Hz) respectively (Pb 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.

Background Ivabradine is a specific bradycardic agent used in coronary artery disease and heart f... more 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.