Arrhythmogenic Response Research Articles

Pharmacological inducibility of short-coupled Purkinje ectopy in patients with spontaneous short-coupled ventricular fibrillation

Abstract Funding Acknowledgements Type of funding sources: None. Introduction In a subset of patients with idiopathic ventricular fibrillation (IVF), the cause is identified as short-coupled premature ventricular complexes (ScPVCs), which mostly originate from Purkinje cells, and are often short-living and undetected. There is no consistent test allowing identification of patients vulnerable to this malignant arrhythmia. We aimed at evaluating sodium channel blocker (NaBl) testing in patients who survived IVF associated with SCPVCs. Methods We retrospectively reviewed data of 112 patients at 3 centers, with the diagnosis of IVF associated with spontaneous and documented scPVCs. No patient from the previous report was included. We analyzed all patients who underwent NaBl tests during evaluation of IVF. An arrhythmogenic NaB test was defined as the occurrence of ≥10 scPVCs and/or ≥ 1 repetitive form (couplet, triplet, run) during the test, without spontaneous scPVC at baseline. We defined scPVCs as an R-on-T pattern PVC. We evaluated the occurrence of scPVC during NaB infusion, the arrhythmia pattern and we compared the characteristics of the arrhythmogenic versus non-arrhythmogenic response groups. Data regarding anti-arrhythmic drug treatment and follow-up were collected at each center. Results In total, 48 patients were included (Table). The NaB tests were performed using ajmaline 1mg/kg/5 or 10min, or flecainide 2mg/kg/10min. Overall, an arrhythmogenic NaB test occurred in 23 of 48 (48%) patients. Isolated ScPVCs were observed in 11 patients, couplet/triplets in 10, and runs in 6. The arrhythmogenic response was reproduced in 5/5 patients who had a second NaB test during scPVC mapping before catheter ablation. There were no significant differences between patients with arrhythmogenic or non-arrhythmogenic NaB tests in all baseline clinical variables tested including VF burden (Table). All patients were followed at least 1 year after initial diagnosis and NaB test. Under hydroquinidine (600 or 900mg/day), appropriate defibrillator shocks occurred in 7 of 8 patients with arrhythmogenic NaB test; whereas shocks occurred only in 2 of 11 patients with non-arrhythmogenic NaB test (P=0.02). Conclusion Sodium blockers unravel scPVCs in up to 50% of patients with IVF and may therefore represent a valuable tool to detect a potential cause for sudden cardiac death (SCD) or unexplained syncope. Additionally, the arrhythmogenic response to NaB may be associated with the success of hydroquinidine in preventing IVF recurrence, a finding that suggests the existence of distinct scPVCs phenotypes and that may have important therapeutic implications. Further functional and genetical investigations are warranted to explore the mechanisms underlying this association.

Remodelling of potassium currents underlies arrhythmic action potential prolongation under beta-adrenergic stimulation in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) patients often present an enhanced arrhythmogenicity that can lead to lethal arrhythmias, especially during exercise. Recent studies have indicated an abnormal response of HCM cardiomyocytes to β-adrenergic receptor stimulation (β-ARS), with prolongation of their action potential rather than shortening. The mechanisms underlying this aberrant response to sympathetic stimulation and its possible proarrhythmic role remain unknown. The aims of this study are to investigate the key ionic mechanisms underlying the HCM abnormal response to β-ARS and the resultant repolarisation abnormalities using human-based experimental and computational methodologies. We integrated and calibrated the latest models of human ventricular electrophysiology and β-ARS using experimental measurements of human adult cardiomyocytes from control and HCM patients. Our major findings include: (1) the developed in silico models of β-ARS capture the behaviour observed in the experimental data, including the aberrant response of HCM cardiomyocytes to β-ARS; (2) the reduced increase of potassium currents under β-ARS was identified as the main mechanism of action potential prolongation in HCM, rather than a more sustained inward calcium current; (3) action potential duration differences between healthy and HCM cardiomyocytes were increased upon β-ARS, while endocardial to epicardial differences in HCM cardiomyocytes were reduced; (4) models presenting repolarisation abnormalities were characterised by downregulation of the rapid delayed rectifier potassium current and the sodium‑potassium pump, while inward currents were upregulated. In conclusion, our results identify causal relationships between the HCM phenotype and its arrhythmogenic response to β-ARS through the downregulation of potassium currents.

Effect of icosapent ethyl on susceptibility to ventricular arrhythmias in postinfarcted rat hearts: Role of GPR120-mediated connexin43 phosphorylation.

The ω‐3 fatty acids exert as an antioxidant via the G protein‐coupled receptor 120 (GPR120). Icosapent ethyl, a purified eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connexin43 expression and whether the GPR120 signalling is involved in the protection. Male Wistar rats after ligating coronary artery were assigned to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative‐nitrosative stress. In concert, myocardial connexin43 levels revealed a significant decrease in vehicle‐treated infarcted rats compared with sham. These changes of oxidative‐nitrosative stress and connexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative‐nitrosative stress and connexin43 phosphorylation were abolished by administering AH‐7614, an inhibitor of GPR120. SIN‐1 abolished the Cx43 phosphorylation of icosapent ethyl without affecting GPR120 levels. Taken together, chronic use of icosapent ethyl after infarction is associated with up‐regulation of connexin43 phosphorylation through a GPR120‐dependent antioxidant pathway and thus plays a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

GPR120‐mediated Connexin43 Phosphorylation is Implicated in Antiarrhythmic Effects of Icosapent Ethyl in Post‐infarcted Rat Hearts

AimsThe w‐3 fatty acids exert as an antioxidant via the G protein‐coupled receptor 120 (GPR120). Icosapent ethyl, a highly purified ethyl ester of eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connnexin43 expression and whether the GPR120 signaling is involved in the protection.Methods and resultsMale Wistar rats after ligating coronary artery were randomized to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative‐nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial connnexin43 levels revealed a significant decrease in vehicle‐treated infarcted rats compared with sham. These changes of oxidative‐nitrosative stress and connnexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connnexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative‐nitrosative stress and connnexin43 phosphorylation were abolished by administering AH‐7614, an inhibitor of GPR120. SIN‐1 abolished the Cx43 phosphorylation of IPE without affecting GPR120 levels.ConclusionsChronic use of icosapent ethyl after infarction is associated with up‐regulation of connnexin43 phosphorylation through a GPR120‐dependent antioxidant pathway and thus plays a role in the beneficial effect on arrhythmogenic response to programmed electrical stimulation.Support or Funding InformationThis work was supported by the grants of China Medical University (DMR‐108‐035) and Ministry of Science and Technology (MOST‐108‐2622‐B‐039‐002‐CC3), Taiwan.

Oxidative Stress Remodeling of Zebrafish Cardiac Electrical Gradients

In mammalian hearts, electrical gradients ensure well-orchestrated electrical stability and mechanical efficiency under physiological baseline. Regional heterogeneities of ion channels and/or calcium handling proteins establish gradients of activation, action potential duration (APD), and repolarization that are protective at baseline, but susceptible to disruptive remodeling in myocardial injuries and failure. However, in zebrafish hearts, physiological electrical gradients are unexplored. Considering the important application of zebrafish hearts in high-throughput screening for drug-induced cardiotoxicities (such as QT prolongation) and the role of the apicobasal APD and repolarization gradients in the inscription of the T wave and the QT interval, this study investigates the apicobasal electrical gradients in normal adult zebrafish and the arrhythmogenic impact of acute H2O2-induced oxidative stress. Following epicardial optical mapping of intact zebrafish hearts, apicobasal gradients of activation, repolarization, and APD under normal baseline vs. oxidative stress (H2O2, 100 μmol/L) condition were determined. At baseline, an apicobasal APD90 gradient was evident with longer APD90 in the apex due to earlier activation and later repolarization compared to the base (apex 198 ± 23 > base 121 ± 13 ms, n=5; P<0.0004). Acute H2O2 challenge abolished or reversed the apicobasal APD gradient and prolonged both apical and basal APDs (apex 352 ± 86 < base 401 ± 37 ms, n=4; NS). However, H2O2 remodeling impact was regionally heterogeneous, affecting the base more than apex. H2O2 impaired repolarization and induced occasional early after depolarizations and trigger activity. Given that the apicobasal APD and repolarization gradients in normal zebrafish are reverse those in normal humans, one should exert caution when extrapolating drug-induced QT prolongation in zebrafish to humans. Nonetheless, based on the human-like arrhythmogenic response of the zebrafish heart to acute H2O2 challenge, zebrafish constitutes a relevant model to investigate repolarization disorders associated with oxidative stress.

Anatomical dilatation of the superior vena cava associated with an arrhythmogenic response induced by SVC scan pacing after atrial fibrillation ablation

BackgroundThe relationship between pulmonary vein (PV) arrhythmogenicity and its anatomy has been reported. However, that of the superior vena cava (SVC) has not been well discussed. Arrhythmogenic response induced by pacing stimulation at SVC might help with identifying SVC arrhythmogenicity. The purpose of this study was to investigate the relationship between the anatomical dilatation of SVC and the arrhythmogenic response induced by pacing at SVC. MethodsForty-three patients who underwent atrial fibrillation (AF) ablation were enrolled in this study. After PV isolation, scan pacing (up to triple extra stimulation following intrinsic sinus beats) was performed at SVC. The arrhythmogenic response was defined as following: (1) repetitive atrial responses, (2) non-sustained, and (3) sustained AF/ atrial tachycardia. To assess the dilatation of SVC, we measured the cross-sectional area of the SVC (SVC-area) using multi-planar reconstruction CT imaging. ResultsArrhythmogenic responses were documented in 24 patients (Group 1). No arrhythmogenic responses were documented in the remaining 19 patients (Group 2). The SVC-area was significantly larger in Group 1 than Group 2 (3.1±0.9 vs. 2.2±0.8cm2, P=0.004). A multivariate analysis revealed only SVC-area was associated with arrhythmogenic responses (odds ratio=2.87, CI 1.05–7.82, P=0.04). Furthermore, AF recurrence rate was significantly higher in patients with SVC-area>2.56cm2 than those with SVC-area <2.56cm2 (9 [42.9%] of 21 vs. 3 [13.6%] of 22, P=0.026). ConclusionDilatation of SVC was associated with an arrhythmogenic response, and the AF recurrence rate was significantly higher in patients with large SVC-area. Adjunctive catheter intervention for the SVC might be indicated in patients with a dilated SVC and an arrhythmogenic response.

Effects of urate-lowering agents on arrhythmia vulnerability in post-infarcted rat hearts

Hyperuricemia has been shown to be associated with ventricular arrhythmias. However, the mechanisms remained unknown. We assessed whether different urate-lowering agents can attenuate arrhythmias through lowering urate itself or inhibiting xanthenes oxidize (XO) activity in infarcted rats. Male Wistar rats after ligating coronary artery were randomized to either allopurinol, or febuxostat, chemically unrelated inhibitors of XO, benzbromarone or vehicle for 4 weeks. Post-infarction was associated with increased oxidant stress, as measured by myocardial superoxide, isoprostane, XO activity and dihydroethidine fluorescence staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham-operated rats. Sympathetic hyperinnervation was blunted after administering both XO inhibitors, assessed by immunofluorescent analysis, Western blotting and real-time quantitative RT-PCR. Besides, the XO inhibitors-attenuated nerve growth factor levels were reversed in the presence of peroxynitrite generator. Arrhythmic scores in the XO inhibitors-treated infarcted rats were significantly lower than that in vehicle. For similar levels of urate lowering, the uricosuric agent benzbromarone had no beneficial effects on oxidative stress, sympathetic hyperinnervation or arrhythmia vulnerability. Chronic use of XO inhibitors, but not uricosuric agent, down-regulated sympathetic innervation probably through a superoxide-dependent pathway and plays a role in the beneficial effect on arrhythmogenic response.

Inhibition of glycogen synthase kinase-3β prevents sympathetic hyperinnervation in infarcted rats.

We have demonstrated that nerve growth factor (NGF) expression in the myocardium is selectively increased during chronic stage of myocardial infarction, resulting in sympathetic hyperinnervation. Glycogen synthase kinase-3 (GSK-3) signal has been shown to play key roles in the regulation of cytoskeletal assembly during axon regeneration. We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction through attenuated NGF expression and Tau expression. Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to either LiCl or SB216763, chemically unrelated inhibitors of GSK-3β, a combination of LiCl and SB216763, or vehicle for four weeks. Myocardial norepinephrine levels revealed a significant elevation in vehicle-treated rats compared with sham-operated rats, consistent with excessive sympathetic reinnervation after infarction. Immunohistochemical analysis for sympathetic nerve also confirmed the change of myocardial norepinephrine. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administering either LiCl, SB216763, or combination. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those treated with GSK-3 inhibitors. Addition of SB216763 did not have additional beneficial effects compared with those seen in rats treated with LiCl alone. Furthermore, lithium treatment increased Tau1 and decreased AT8 and AT180 levels. Chronic use of lithium after infarction, resulting in attenuated sympathetic reinnervation by GSK-3 inhibition, may modify the arrhythmogenic response to programmed electrical stimulation.

Increased arrhythmogenesis and expression of left ventricular pS368‐Connexin43 following acute myocardial ischemia (AMI) in female ovariectomized rats

Estrogen has been previously shown to be protective against ventricular arrhythmias following myocardial infarction possibly through changes in connexin 43 (Cx43) expression and phosphorylation. Phosphorylation of Cx43 at S368 is induced with AMI, suggesting that phosphorylation at this site plays a role in arrhythmogenesis. The goal of the current study was to determine if there is a direct correspondence between phosphorylation of left ventricular (LV)‐Cx43 at S368 and the arrhythmogenic response to AMI, and to further examine if this effect is augmented following loss of estrogen. Anesthetized, ventilated female ovariectomized (n=3) and intact (n=3) Sprague‐Dawley rats were subjected to 20‐minutes of AMI produced through ligation of the left coronary artery. The arrhythmic burden in response to AMI was measured using a standard lead II ECG, and quantified through an established scoring method. Total LV‐Cx43 expression and pS368‐Cx43 expression were measured through standard Western blotting techniques. Ovariectomized animals had a greater arrhythmic burden versus intact animals following AMI. Correspondingly, ovariectomized animals also had an increase in the pS368‐Cx43/Total Cx43 ratio. When data were further examined using a linear regression analysis, results show a direct positive correlation between the level of pS368‐Cx43 expression and arrhythmic burden (R2 = 0.94). IOER‐3726.

Lithium promotion of connexin43 protein in infarcted rats by Wnt‐ and PI3K/Akt‐dependent GSK‐3β phosphorylation signaling

The purpose of this study was to determine whether lithium can attenuate arrhythmias through increasing GSK‐3β/β‐catenin‐induced connexin43 by activating Wnt and phosphoinositide‐3 kinase (PI3K)/Akt after myocardial infarction. Male Wistar rats were subjected to coronary artery ligation and then randomized to either saline or lithium treatment for 4 weeks. Myocardial connexin43 levels were significantly decreased in the saline‐treated infarcted rats compared with sham. These attenuated connexin43 levels were blunted after lithium. Arrhythmic scores during programmed stimulation in the lithium‐treated infarcted rats were significantly lower than those treated with the saline‐treated infarcted rats. Lithium increased Akt phosphorylation 4.5‐fold in the border zone compared to saline after infarction. The two downstream targets of Akt, phosphorylated GSK‐3β and nuclear translocated β‐catenin were significantly increased by 11.2‐fold and 3.6‐fold, respectively. Inhibition of Wnt and PI3K/Akt signaling prevented GSK‐3β phosphorylation as well as β‐catenin nuclear translocation, and blocked the beneficial effects of lithium on connexin43 phosphorylation. Chronic use of lithium after infarction, resulting in enhanced connexin43 levels through both Wnt‐ and PI3K/Akt‐dependent GSK‐3β pathways may attenuate the arrhythmogenic response to programmed electrical stimulation.

Cardiac sympathetic hyperinnervation in deoxycorticosterone acetate-salt hypertensive rats

Sympathetic activities are elevated in the central SNSs (sympathetic nervous systems) of hypertensive animals, but it is not known whether sympathetic innervation is also elevated in the heart. Sympathetic hyper-responsiveness in hypertension may result from oxidative stress. The aim of the present study was to investigate sympathetic hyperinnervation in DOCA (deoxycorticosterone acetate)-salt hypertensive rats with established hypertension. At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 8 weeks: vehicle, NAC (N-acetylcysteine) and triple therapy (hydralazine, hydrochlorothiazide and reserpine). DOCA-salt was associated with increased oxidant release. DOCA-salt produced concentric left ventricular hypertrophy and cardiomyocyte hypertrophy. Sympathetic hyperinnervation was observed in DOCA-salt rats, as assessed by myocardial noradrenaline levels, immunofluorescent analysis of tyrosine hydroxylase, growth-associated factor 43 and neurofilament and Western blotting and real-time quantitative RT-PCR (reverse transcription-PCR) of NGF (nerve growth factor). Arrhythmic scores during programmed stimulation in DOCA-salt rats were significantly higher than those in the control rats. Triple therapy, despite being effective on BP (blood pressure), offered neither attenuated cardiomyocyte hypertrophy nor anti-arrhythmia. The effects of DOCA-salt treatment on NGF expression, sympathetic hyperinnervation and arrhythmias were attenuated by NAC. Furthermore, the effects of NAC on NGF were abolished by administering BSO (L-buthionine sulfoximine), an inhibitor of glutamate-cysteine ligase. In conclusion, DOCA-salt treatment contributes to up-regulation of NGF proteins probably through a free radical-dependent pathway in a BP-independent manner. DOCA-salt rats treated with NAC attenuate sympathetic hyperinnervation and thus show a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

KATP channel agonists preserve connexin43 protein in infarcted rats by a protein kinase C‐dependent pathway

Downward remodelling of gap junctional proteins between myocytes may trigger ventricular arrhythmia after myocardial infarction. We have demonstrated that ATP-sensitive potassium (KATP) channel agonists attenuated post-infarction arrhythmias. However, the involved mechanisms remain unclear. The purpose of this study was to determine whether KATP channel agonists can attenuate arrhythmias through preserving protein kinase C (PKC)-–dependent connexin43 level after myocardial infarction. Male Wistar rats after ligating coronary artery were randomized to either vehicle, nicorandil, pinacidil, glibenclamide or a combination of nicorandil and glibenclamide or pinacidil and glibenclamide for 4 weeks. To elucidate the role of PKC in the modulation of connexin43 level, carbachol and myristoylated PKC V1–2 peptide were also assessed. Myocardial connexin43 level was significantly decreased in vehicle-treated infarcted rats compared with sham. Attenuated connexin43 level was blunted after administering KATP channel agonists, assessed by immunofluorescent analysis, Western blotting, and real-time quantitative reverse transcription-PCR of connexin43. Arrhythmic scores during programmed stimulation in the KATP channel agonists-treated rats were significantly lower than those treated with vehicle. The beneficial effects of KATP channel agonists were blocked by either glibenclamide or 5-hydroxydecanoate. Addition of the PKC activator, phorbol 12-myristate 13-acetate and the specific PKC agonist, carbachol, blocked the effects of nicorandil on connexin43 phosphorylation and dye permeability. The specific PKC antagonist, myristoylated PKC V1–2 peptide, did not have additional beneficial effects on connexin43 phosphorylation compared with rats treated with nicorandil alone. Chronic use of KATP channel agonists after infarction, resulting in enhanced connexin43 level through a PKC-dependent pathway, may attenuate the arrhythmogenic response to programmed electrical stimulation.

High‐Rate Pacing Reduces Variability of Repolarization and Prevents Repolarization‐Dependent Arrhythmias in Dogs With Chronic AV Block

high-rate pacing may have an inhibitory effect on the initiation of Torsade de Pointes arrhythmias (TdP). However, permanent pacing is only indicated in high-risk patients. We performed a proof of concept study into automatic overdrive pacing for prevention of drug-induced TdP, using short-term variability of repolarization (STV) as a feedback parameter of arrhythmic risk. the minimal signal sampling frequency required for measuring STV was determined through computer simulation. Arrhythmogenic response to dofetilide (25 microg/kg/5 minutes) was tested at two different paced heart rates (60-65 bpm vs 100-110 bpm) in 7 dogs with chronic atrioventricular block, while recording right and left ventricular (LV) monophasic action potential (MAP) and LV electrogram (EGM). Simulations showed a sampling frequency of 500 Hz is sufficient to capture relevant STV values. High-rate pacing prevented dofetilide-induced TdP seen at the low rate (low: 6/7 vs high: 1/7). At the low rate, STV from LV MAP duration increased before occurrence of spontaneous, ectopic activity and TdP (1.7 ± 0.6-3.0 ± 1.8 ms, P < 0.05), but at the high-rate STV did not change significantly (0.9 ± 0.2-1.5 ± 1.4 ms, NS). Regression analysis showed a close relation between STV calculated from LV MAP and from LV EGM (R(2) = 0.71). high-rate pacing increases repolarization reserve in dogs with chronic atrioventricular block, preventing dofetilide-induced TdP. Changes in repolarization reserve are reflected in values of STV.

Effect of pravastatin on ventricular arrhythmias in infarcted rats: role of connexin43

Epidemiologic studies showed that men treated with statins appear to have a lower incidence of sudden death than men without statins. However, the specific factor for this remained disappointingly elusive. We assessed whether pravastatin enhanced connexin43 expression after myocardial infarction through attenuation of endothelin-1. Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to vehicle, pravastatin, mevalonate, bosentan, or a combination of pravastatin and mevalonate or pravastatin and bosentan for 4 wk. Myocardial endothelin-1 levels were significantly elevated in vehicle-treated rats at the border zone compared with sham-operated rats. Myocardial connexin43 expression at the border zone was significantly decreased in vehicle-treated infarcted rats compared with sham-operated rats. Attenuated connexin43 expression was blunted after administration of pravastatin, as assessed by immunofluorescence analysis, Western blotting, and real-time quantitative RT-PCR of connexin43. Bosentan enhanced connexin43 amount in infarcted rats and did not have additional beneficial effects on pravastatin-treated rats. Arrhythmic scores during programmed stimulation in vehicle-treated rats were significantly higher than scores in those treated with pravastatin. In contrast, the beneficial effects of pravastatin-induced connexin43 were abolished by the addition of mevalonate and a protein kinase C inducer. In addition, the amount of connexin43 showed significant increase after addition of bisindolylmaleimide, implicating that protein kinase C is a relevant target in endothelin-1-mediated connexin43 expression. Thus chronic use of pravastatin after infarction, resulting in enhanced connexin43 amount by attenuation of mevalonate-dependent endothelin-1 through a protein kinase C-dependent pathway, may attenuate the arrhythmogenic response to programmed electrical stimulation.

Effect of Sildenafil on Sympathetic Hyperinnervation in Post‐infarcted Rat Hearts

Sildenafil has been shown to provide cardioprotection against ischemia/reperfusion injury via activation of mitochondrial ATP‐sensitive potassium (KATP) channels. We assessed whether sildenafil attenuates cardiac sympathetic innervation after myocardial infarction through activation of KATP channels. After ligation of the anterior descending artery, male Wistar rats were randomized to either vehicle, nicorandil, pinacidil, sildenafil, glibenclamide, or a combination of nicorandil and glibenclamide, pinacidil and glibenclamide or sildenafil and glibenclamide for 4 weeks. To elucidate the role of mitochondrial KATP channels in modulating nerve growth factor, 5‐hydroxydecanoate was assessed in an in vitro model. Myocardial norepinephrine levels revealed a significant elevation in vehicle‐treated rats at the remote zone compared with sham. Immunohistochemical analysis for tyrosine hydroxylase, growth associated factor 43 and neurofilament also confirmed the change of myocardial norepinephrine. This was paralleled by a significant upregulation of nerve growth factor protein expression and mRNA in the vehicle‐treated rats, which reduced after administering either nicorandil, pinacidil or sildenafil. Arrhythmic scores during programmed stimulation in the vehicle‐treated rats were significantly higher than those treated with sildenafil. In contrast, beneficial effects of sildenafil were reversed by the addition of either glibenclamide or 5‐hydroxydecanoate. Chronic use of sildenafil after infarction, resulting in attenuated sympathetic innervation by activation of mitochondrial KATP channels, may modify the arrhythmogenic response to programmed electrical stimulation.

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the IKr blocker E-4031 (0.03/0.1/0.3/1 μM). Isoproterenol (0.1 μM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n=8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (<50 bpm) and long APD (>200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 μM) prolonged APD (31/31) and induced EADs only in clusters with APD90>300 ms (11/16). EADs were associated with increased T (1.6±0.2 vs 2.0±0.3⁎) and STV (2.7±1.5 vs 6.9±1.9⁎). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3±0.1 vs 1.9±0.4⁎), and STV (1.2±0.9 vs 7.1±5.6⁎). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher⁎ in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher⁎ in ventricular-like cells (⁎P<0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.

Effect of N-acetylcysteine on sympathetic hyperinnervation in post-infarcted rat hearts

The purpose of this study was to determine whether N-acetylcysteine (NAC) attenuates cardiac sympathetic hyperinnervation through replenishment of glutathione in infarcted rats. After ligation of the coronary artery, male Wistar rats were randomized to either vehicle, NAC, or vitamins C + E groups for 4 weeks. Post-infarction was associated with increased oxidant release, as measured by tissue isoprostane and myocardial glutathione. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham-operated rats. Sympathetic hyperinnervation was blunted after administering NAC, as assessed by immunofluorescent analysis of tyrosine hydroxylase and western blotting and real-time quantitative RT-PCR of nerve growth factor. Arrhythmic scores during programmed stimulation in the vehicle-treated infarcted rats were significantly higher than those in animals treated with NAC. Although NAC and vitamins showed similar effects on ventricular remodelling, only NAC demonstrated beneficial effects on sympathetic hyperinnervation. Furthermore, the effects of NAC on nerve growth factor were abolished by administering l-buthionine sulfoximinem, an inhibitor of gamma-glutamylcysteine ligase. Chronic use of NAC, but not vitamins, after infarction is associated with down-regulation of nerve growth factor proteins, probably through a glutathione-dependent pathway, and thus plays a critical role in the beneficial effect on the arrhythmogenic response to programmed electrical stimulation.

Granulocyte colony-stimulating factor increases sympathetic reinnervation and the arrhythmogenic response to programmed electrical stimulation after myocardial infarction in rats

Granulocyte colony-stimulating factor (G-CSF) has been used for the repair of infarcted myocardium, but concerns have been raised regarding its proarrhythmic potential. We analyzed the influence of G-CSF treatment on sympathetic nerve remodeling and the expression of nestin in a rat model of experimental myocardial infarction (MI). Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to receive either saline (MI/C) or G-CSF (MI/G) for 5 days. At 56 days after infarction, MI/G rats had a significantly higher left ventricular ejection fraction accompanied by a significant decrease in the left ventricular end-diastolic dimension than the MI/C group. Myocardial norepinephrine levels revealed a significant elevation in MI/G rats in the border zone compared with MI/C rats. Immunohistochemical analysis for tyrosine hydroxylase, growth-associated protein 43, and neurofilament also confirmed the changes of myocardial norepinephrine. At 5 days after infarction, MI/G rats had increased numbers of tissue-infiltrated CD34(+) cells, although a similar increase in circulating neutrophil counts between sham-operated rats treated with G-CSF and MI/G rats was observed. Compared with MI/C rats, MI/G rats showed an increase of nestin and nerve growth factor expression, as assessed by protein expression and mRNA levels. The arrhythmia scores during programmed stimulation were significantly higher in MI/G rats than in MI/C rats, suggesting proarrhythmic potential. These findings suggest that, although G-CSF administration after infarction improved myocardial function, it resulted in the activation of nestin and nerve growth factor expression and increased sympathetic reinnervation, which may increase the arrhythmogenic response to programmed electrical stimulation.

The arrythmogenic potential of post-myocardial infarction cytokine treatment

a front page story in The New York Times ([22][1]) at the end of March 2001 reported that “the basis of a possible revolution in treating heart attack patients has been laid by three reports of using stem cells from bone marrow to repair heart tissue in animals.” Those three reports ([11][2], [

Effect of ATP-sensitive potassium channel agonists on sympathetic hyperinnervation in postinfarcted rat hearts

Although the acute administration of ATP-sensitive potassium (K(ATP)) channel agonists provides a neuroprotection, it is unclear whether similar benefits are found by modulating sympathetic innervation in chronic settings after myocardial infarction. We assessed whether K(ATP) channel agonists can attenuate the sprouting of cardiac sympathetic nerves after infarction. Male Wistar rats after ligating coronary artery were randomized to either saline, nicorandil, pinacidil, glibenclamide, or a combination of 1) nicorandil and glibenclamide or 2) pinacidil and glibenclamide for 4 wk. To elucidate the role of mitochondrial K(ATP) channels in modulating nerve growth factor, 5-hydroxydecanoate was assessed in an in vitro model. The measurement of myocardial norepinephrine levels revealed a significant elevation in saline-treated infarcted rats compared with sham-operated rats, consistent with excessive sympathetic innervation. Excessive sympathetic innervation was blunted after giving the rats either nicorandil or pinacidil, compared with saline, as assessed by the immunohistochemical analysis of tyrosine hydroxylase, growth associated protein-43, and neurofilament and Western blot analysis and real-time quantitative RT-PCR of nerve growth factor. The arrhythmic scores during programmed stimulation in the saline- or glibenclamide-treated infarcted rats were significantly higher than those of rats treated with K(ATP) channel agonists. In contrast, the beneficial effects of nicorandil and pinacidil were abolished by administering either glibenclamide or 5-hydroxydecanoate. The sympathetic hyperinnervation after infarction is attenuated by the activation of mitochondrial K(ATP) channels. The chronic use of mitochondrial K(ATP) channel agonists after infarction may attenuate the arrhythmogenic response to programmed electrical stimulation.