TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.

KEY POINTS: The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells. ABSTRACT: Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate cardiac conduction and be involved in triggering arrhythmias.

Dronedarone versus amiodarone in preventing premature ventricular contractions in an in vitro model of "border zone".

OBJECTIVE: To compare the acute ability of amiodarone and dronedarone (a noniodinated benzofuran derivative with a pharmacologic profile similar to amiodarone) to prevent premature ventricular contractions (PVCs) occurrence. METHODS: We used an in vitro model of rabbit right ventricle mimicking the "border zone" existing between normal and ischemic/reperfused regions to test acute electrophysiological effects of dronedarone and amiodarone both at 1 and 10 µmol/L. RESULTS: Similar to amiodarone, dronedarone affected action potential parameters with multichannel blocking properties. Dronedarone at both concentrations was equivalent to amiodarone regarding PVCs occurrence, except regarding extrasystoles occurrence during the reperfusion period (dronedarone, 10 µmol/L, was superior to amiodarone with extrasystoles incidence at 33% and 50%, respectively vs. 92% in controls, P < 0.05). Both dronedarone and amiodarone systematically induced conduction blocks during simulated ischemia (in 100% of preparations vs. 42% in controls, P < 0.05) and a marked decrease of Vmax (to 24 and 23 V/s at 10 minutes of ischemia with 1 and 10 µmol/L dronedarone versus 65 V/s in controls, P < 0.05), thus indicating class 1 antiarrhythmic effects. Both dronedarone and amiodarone at 10 µmol/L induced an increase of APD90 dispersion between normal and ischemic regions, without pro-arrhythmic effects. CONCLUSIONS: Dronedarone and amiodarone have very similar electrophysiological effects in this in vitro model of border zone and were both efficient in preventing PVCs occurrence particularly through a class 1 antiarrhythmic effect.

Proarrhythmic effects of aldosterone during myocardial ischemia-reperfusion: implication of the sarcolemmal-KATP channels.

OBJECTIVE: To assess the electrophysiological impact of aldosterone during myocardial ischemia-reperfusion. METHODS: We used an in vitro model of "border zone" using rabbit right ventricle and standard microelectrodes. RESULTS: Aldosterone (10 and 100 nmol/L) shortened ischemic action potential [action potential duration at 90% of repolarization (APD90), from 55 ± 3 to 39 ± 1 ms and 36 ± 3 ms, respectively, P < 0.05] and induced resting membrane potential (RMP) hyperpolarization in the nonischemic zone (from -83 ± 1 to -93 ± 7 mV and -94 ± 3 mV, respectively, P < 0.05) and in the ischemic zone during reperfusion (from -81 ± 2 to -88 ± 2 mV and -91 ± 2 mV, respectively, P < 0.05). Bimakalim, an ATP-sensitive potassium (K(ATP)) channel opener, also induced RMP hyperpolarization and APD90 shortening. Aldosterone (10 and 100 nmol/L) increased APD90 dispersion between ischemic and nonischemic zones (from 96 ± 3 to 117 ± 5 ms and 131 ± 6 ms, respectively, P < 0.05) and reperfusion-induced severe premature ventricular contraction occurrence (from 18% to 67% and 75%, respectively, P < 0.05). Adding glibenclamide, a nonspecific K(ATP) antagonist, to aldosterone superfusion abolished these effects different to sodium 5-hydroxydecanoate, a mitochondrial-K(ATP) antagonist. CONCLUSIONS: In this in vitro rabbit model of border zone, aldosterone induced RMP hyperpolarization and decreased ischemic APD90 evoking the modulation of K currents. Glibenclamide prevented these effects different to 5-hydroxydecanoate, suggesting that sarcolemmal-K(ATP) channels may be involved in this context.

Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species.

BACKGROUND: Desflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium. METHODS: We recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz). After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyl)theophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- standard deviation) between the groups by a variance analysis and post hoc test. RESULTS: Desflurane 6% (84 +/- 6% of baseline) enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 +/- 8% of baseline, P < 0.0001). N-mercaptopropionylglycine (54 +/- 3% of baseline), 8-(p-Sulfophenyl)theophylline (62 +/- 9% of baseline), HOE140 (58 +/- 6% of baseline) abolished desflurane-induced postconditioning. Adenosine (80 +/- 9% of baseline) and bradykinin (83 +/- 4% of baseline) induced postconditioning (P < 0.0001 vs control), N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 +/- 8 and 58 +/- 5% of baseline, respectively). CONCLUSIONS: In vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin administered at the beginning of reoxygenation, was mediated, at least in part, through ROS production.

[How to measure high V(max) values by systems aimed at action potential recording?]. / Systèmes d'acquisition de potentiels d'action : comment mesurer les V(max) élevées ?

This investigation was aimed at increasing both accuracy and performance of systems used to obtain and measure V(max) (dV/dt(max)), an important yet underevaluated physiological parameter. A method is presented to correct measured V(max) (V(mes)) based on an algorythm adapted to 2 tested systems: IOX and DataPac. We also investigated 89 rabbit Purkinje fibres (before and 30 min following drugs effective on ventricular repolarization) to derive experimental electrophysiological correlations. In fact, no method may be reliable without knowing its accuracy over a large scale of representative physiological values. This is why it is essential to estimate accuracy, precision and fidelity of systems aimed at action potential recording before pharmacological or pathophysiological investigations are performed, even more if therapeutical consequences might ensue. A formula is presented to obtain real V(max), based on V(mes) [V(max)=V(mes)/1 - (tau.V(mes)/APA)(2.p)], where tau=49.64 µs, p=0.72 and APA=action potential amplitude. This formula is reliable up to V(max) values of 1000 V/s which may be seen in rabbit Purkinje fibres, a classical model for in vitro studies. Using this formula may have practical implications in cellular electrophysiology which may impact on safety pharmacology and therapeutics.

The electrophysiological effects of racemic ketamine and etomidate in an in vitro model of "border zone" between normal and ischemic/reperfused guinea pig myocardium.

BACKGROUND: Etomidate and ketamine are used during induction of anesthesia in high-risk patients. However, their effects on action potential (AP) variables and ischemia/reperfusion-induced arrhythmias and conduction blocks are unknown. METHODS: Guinea pig right ventricular muscle strips were mounted in a 5-mL double chamber bath with the strips separated into two zones by an impermeable latex membrane. One-half (normal zone) was exposed to normal perfusate while the other half (altered zone) was exposed to hypoxia, hyperkalemia, acidosis, and lack of glucose. AP variables were recorded continuously in the normal and altered zones. Spontaneous arrhythmias and conduction blocks were noted. Etomidate (10(-7), 10(-6), and 10(-5) M) and ketamine (10(-6), 10(-5), and 10(-4) M) were superfused into the bath throughout the experiment and the electrophysiologic effects compared with the control group. RESULTS: We found that under control conditions, etomidate and ketamine did not modify resting membrane potential, maximal upstroke velocity, AP amplitude, or AP duration at 90% of repolarization (APD90). Ketamine (10(-4) M), but not weaker concentrations and none of the concentration of etomidate, reversed the ischemia-induced shortening of APD90 and APD dispersion. Etomidate and ketamine did not modify the occurrence of conduction block during simulated ischemia. In contrast, ketamine (25% at 10(-6) M, 13% at 10(-5) M, and 13% at 10(-4) M vs 90% in the control group, P < 0.05) but not etomidate (38% at 10(-7) M, 63% at 10(-6) M, and 63% at 10(-5) M vs 90% in the control group, NS) decreased the incidence of reperfusion-induced spontaneous arrhythmias. CONCLUSIONS: In guinea pig myocardium, our data suggest that ketamine, in clinically relevant concentrations, decreases ischemia-induced AP shortening and spontaneous reperfusion-induced ventricular arrhythmias. Further study is required to precisely determine the effect of etomidate on reperfusion-induced arrhythmias.

PAR1 contribution in acute electrophysiological properties of oral anticoagulants in rabbit pulmonary vein sleeve preparations.

Whether oral anticoagulants, vitamin K antagonists (VKAs), and nonvitamin K oral anticoagulant (NOACs) frequently prescribed to atrial fibrillation (AF) patients, do themselves have a pro- or anti-arrhythmic effect have never been addressed. Transmembrane action potentials were recorded in an acute rabbit model of superfused pulmonary veins (PVs) sleeves preparations using standard microelectrode technique. Fluindione 10 µm (n = 6) increased the AP (action potential) duration (APD), induced a significantly Vmax depression (from 95 ± 14 to 53 ± 5 V/s, P < 0.05), and 2 : 1 blocks during rapid atrial pacing thus evoking class I anti-arrhythmic properties, and prevented spontaneous trigger APs. Apixaban 10 µm (n = 6) increased the APD, significantly prolonged the effective refractory period (from 56.3 ± 4.2 to 72.0 ± 8.6 ms, P < 0.05), and prevented triggered APs occurrence. Fluindione and apixaban effects were suppressed with the addition of the protease-activated receptors 1 (PAR 1) agonist SFLLR-NH2 . Warfarin 10 µm (n = 6) significantly abbreviated the early refractory period (from 56.3 ± 4.2 to 45.0 ± 2.2 ms, P < 0.05) and increased triggered APs occurrence that were successfully prevented by nifedipine but not by the addition of the protease-activated receptors 1 agonist SFLLR-NH2 . In this acute rabbit PVs model, VKAs and NOACs, at physiological concentrations, exhibited very different pharmacological properties that influence PVs electrophysiology, implying PAR1, with fluindione and apixaban which exhibited more anti-arrhythmic properties, whereas warfarin exhibited more pro-arrhythmic properties.

Argon Exposure Induces Postconditioning in Myocardial Ischemia-Reperfusion.

BACKGROUND AND PURPOSE: Cardioprotection against ischemia-reperfusion (I/R) damages remains a major concern during prehospital management of acute myocardial infarction. Noble gases have shown beneficial effects in preconditioning studies. Because emergency proceedings in the context of myocardial infarction require postconditioning strategies, we evaluated the effects of argon in such protocols on mammalian cardiac tissue. EXPERIMENTAL APPROACHES: In rat, cardiac I/R was induced in vivo by transient coronary artery ligature and cardiac functions were evaluated by magnetic resonance imaging. Hypoxia-reoxygenation (H/R)-induced arrhythmias were evaluated in vitro using intracellular microelectrodes on both rat-isolated ventricle and a model of border zone in guinea pig ventricle. Hypoxia-reoxygenation loss of contractile force was assessed in human atrial appendages. In those models, postconditioning was induced by 5 minutes application of argon at the time of reperfusion. KEY RESULTS: In the in vivo model, I/R produced left ventricular ejection fraction decrease (24%) and wall motion score increase (36%) which was prevented when argon was applied in postconditioning. In vitro, argon postconditioning abolished H/R-induced arrhythmias such as early after depolarizations, conduction blocks, and reentries. Recovery of contractile force in human atrial appendages after H/R was enhanced in the argon group, increasing from 51% ± 2% in the nonconditioned group to 83% ± 7% in the argon-treated group ( P < .001). This effect of argon was abolished in the presence of wortmannin and PD98059 which inhibit prosurvival phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and MEK/extracellular receptor kinase 1/2 (ERK 1/2), respectively, or in the presence of the mitochondrial permeability transition pore opener atractyloside, suggesting the involvement of the reperfusion injury salvage kinase pathway. CONCLUSION AND IMPLICATIONS: Argon has strong cardioprotective properties when applied in conditions of postconditioning and thus appears as a potential therapeutic tool in I/R situations.

Class III effects of dofetilide and arrhythmias are modulated by [K+]o in an in vitro model of simulated-ischemia and reperfusion in guinea-pig ventricular myocardium.

To evaluate class III effects of clinically relevant concentrations of dofetilide (5 and 10 nmol/l) and the effects of extracellular potassium [K+]o modulation of arrhythmias onset at the level of the "border zone," we used a previously reported in vitro model whereby normoxic and ischemic/reperfused zones were studied. Guinea-pig right ventricular strips (driven at 1 Hz at 36.5+/-0.5 degrees C) were superfused with Tyrode's solution in oxygenated (HCO3- 25 mmol/l, K+ 4 mmol/l, pH 7.35+/-0.05, glucose 5.5 mmol/l: normal zone) and ischemia-simulating conditions (HCO3- 9 mmol/l, pH 6.90+/-0.05, no oxygen and no glucose: altered zone) having either [K+]o 4 (n=20), 8 (n=20) or 12 (n=20) mmol/l. Action potentials in normal and altered zones were recorded simultaneously during 30 min of simulated-ischemia and after 30 min of reperfusion with oxygenated Tyrode's solution. Each preparation served as control for successive phases of dofetilide studies (at 5 and 10 nmol/l) and action potential values were normalized to those present at the beginning of the experiment. During simulated-ischemia, the higher the [K+]o the worse were action potential changes, although full recovery was seen upon 30 min of reperfusion in all [K+]o groups. A high incidence of ischemia/reperfusion arrhythmias was observed in 4 and 12 mmol/l [K+]o groups as opposed to a low incidence of arrhythmias in 8 mmol/l [K+]o group. Dofetilide at 5 and 10 nmol/l with all [K+]o explored: (i) exhibited class III effects, (ii) was effective (or neutral) against ventricular arrhythmias during both simulated-ischemia and reperfusion, and (iii) did not globally increase the dispersion of action potential durations between normal and altered zones. Different arrhythmogenic mechanisms are involved in this model at different [K+]o with 8 mmol/l providing relative protection. Class III effects of dofetilide are evident in the normal zone when in the ischemic-like zone [K+]o ranges from 4 to 12 mmol/l. Thus dofetilide did not increase dispersion of repolarization and had either an antiarrhythmic or a neutral effect during ischemia/reperfusion.

Electrophysiological effects of azimilide in an in vitro model of simulated-ischemia and reperfusion in guinea-pig ventricular myocardium.

There are few investigations on azimilide effects during ischemia/reperfusion. We have therefore investigated low concentrations of azimilide (0.1 and 0.5 micromol/l) versus Controls on action potential parameters and occurrence of repetitive responses during simulated ischemia and reperfusion. An in vitro model of "border zone" in guinea-pig ventricular myocardium (n=30) was used. Azimilide 0.5 micromol/l lengthened action potential duration in normoxic but not in ischemic-like conditions. Therefore an increased dispersion of action potential duration at 90% of repolarization during simulated ischemia in presence of azimilide was seen. Upon reperfusion, both normal and reperfused myocardium showed azimilide-induced action potential duration increase. There was a neutral effect on the occurrence of arrhythmias during simulated ischemia; however azimilide showed significant (P=0.033) antiarrhythmic properties following reperfusion. To mimic I(Kr) and I(Ks) blocking properties of azimilide we further used dofetilide 10 nmol/l with HMR 1556 1 nmol/l (N=9), which was accompanied by less severe shortening (P<0.05) of action potential duration at 90% of repolarization at 30 min of ischemic-like conditions (-43+/-9%), as compared with azimilide 0.5 micromol/l (-64+/-5%) but similar to what seen with azimilide 0.1 micromol/l (-53+/-5%) and Controls (-52+/-6%). During reperfusion, 2/9 (22%) preparations had sustained activities, which was less than what observed in Controls (5/10, 50%) and with azimilide 0.5 micromol/l (0/10, 0%), although not statistically different (respectively, P=0.35 and P=0.21). Lack versus homogenous class III effects of azimilide in respectively simulated ischemia and reperfusion may explain its different efficacy on arrhythmias, although prevention of reperfusion arrhythmias calls for other than just its I(Kr) and I(Ks) blocking properties.

Cardiotoxic Electrophysiological Effects of the Herbicide Roundup(®) in Rat and Rabbit Ventricular Myocardium In Vitro.

Roundup (R), a glyphosate (G)-based herbicide (GBH), containing unknown adjuvants is widely dispersed around the world. Used principally by farmers, intoxications have increasingly been reported. We have studied R effects (containing 36 % of G) on right ventricular tissues (male Sprague-Dawley rats, up to 20,000 ppm and female New Zealand rabbits, at 25 and 50 ppm), to investigate R cardiac electrophysiological actions in vitro. We tested the reduced Ca(++) intracellular uptake mechanism as one potential cause of the electrical abnormalities after GBH superfusion, using the Na(+)/K(+)-ATPase inhibitor ouabain or the 1,4-dihydropyridine L-type calcium channel agonist BAY K 8644 which increases I Ca. R concentrations were selected based on human blood ranges found after acute intoxication. The study showed dose-dependent V max, APD50 and APD90 variations during 45 min of R superfusion. At the highest concentrations tested, there was a high incidence of conduction blocks, and 30-min washout with normal Tyrode solution did not restore excitability. We also observed an increased incidence of arrhythmias at different doses of R. Ouabain and BAY K 8644 prevented V max decrease, APD90 increase and the cardiac inexcitability induced by R 50 ppm. Glyphosate alone (18 and 180 ppm) had no significant electrophysiological effects. Thus, the action potential prolonging effect of R pointing to I Ca interference might explain both conduction blocks and proarrhythmia in vitro. These mechanisms may well be causative of QT prolongation, atrioventricular conduction blocks and arrhythmias in man after GBH acute intoxications as reported in retrospective hospital records.

Electrophysiological and antiarrhythmic properties of potassium canrenoate during myocardial ischemia-reperfusion.

INTRODUCTION: Recent clinical studies have reported the potential benefit of an early mineralocorticoid receptor (MR) blockade with potassium canrenoate (PC) on ventricular arrhythmias (VAs) occurrence in patients experiencing an ST-segment elevation myocardial infarction (STEMI). However, most of the electrophysiological properties of PC demonstrated to date have been investigated in normoxic conditions, and therefore, in vitro experiments during an acute myocardial ischemia-reperfusion were lacking. MATERIALS AND METHODS: We used rabbit in vitro models and standard microelectrode technique to assess the electrophysiological impact of PC during myocardial ischemia-reperfusion, including right ventricle mimicking the "border zone" existing between normal and ischemic/reperfused areas (1 µmol/L, 10 and 100 nmol/L), isolated right ventricle, and sinoatrial node (SAN) experiments (1 µmol/L, respectively). RESULTS: During ischemia-reperfusion, acute superfusion of PC 100 nmol/L prevented the increase in action potential (AP) duration at 90% of repolarization (APD90) dispersion between ischemic and nonischemic areas and in VAs occurrence induced by aldosterone 10 nmol/L (86 ± 3 vs 114 ± 4 milliseconds for aldosterone alone, P < .05). Potassium canrenoate also induced conduction blocks and significantly decreased Vmax during simulated ischemia (from 25 ± 5 to 12 ± 4, 14 ± 3, and 14 ± 5 V/s, respectively, for PC 1 µmol/L, 100, and 10 nmol/L, P < .05). Potassium canrenoate 1 µmol/L demonstrated cycle length (CL)-dependent effects on APD90 and on Vmax, and it also reduced SAN beating CL (from 446 ± 28 to 529 ± 24 millisecond, P < .05). CONCLUSION: Our experimental study highlights new evidence for an antiarrhythmic impact of PC during myocardial ischemia-reperfusion via multiple channels modulation. These results are in line with recent clinical trials suggesting that an early MR blockade in STEMI may be preventive of VAs.

Rapid and MR-Independent IK1 Activation by Aldosterone during Ischemia-Reperfusion.

In ST elevation myocardial infarction (STEMI) context, clinical studies have shown the deleterious effect of high aldosterone levels on ventricular arrhythmia occurrence and cardiac mortality. Previous in vitro reports showed that during ischemia-reperfusion, aldosterone modulates K+ currents involved in the holding of the resting membrane potential (RMP). The aim of this study was to assess the electrophysiological impact of aldosterone on IK1 current during myocardial ischemia-reperfusion. We used an in vitro model of "border zone" using right rabbit ventricle and standard microelectrode technique followed by cell-attached recordings from freshly isolated rabbit ventricular cardiomyocytes. In microelectrode experiments, aldosterone (10 and 100 nmol/L, n=7 respectively) increased the action potential duration (APD) dispersion at 90% between ischemic and normoxic zones (from 95±4 ms to 116±6 ms and 127±5 ms respectively, P<0.05) and reperfusion-induced sustained premature ventricular contractions occurrence (from 2/12 to 5/7 preparations, P<0.05). Conversely, potassium canrenoate 100 nmol/L and RU 28318 1 µmol/l alone did not affect AP parameters and premature ventricular contractions occurrence (except Vmax which was decreased by potassium canrenoate during simulated-ischemia). Furthermore, aldosterone induced a RMP hyperpolarization, evoking an implication of a K+ current involved in the holding of the RMP. Cell-attached recordings showed that aldosterone 10 nmol/L quickly activated (within 6.2±0.4 min) a 30 pS K+-selective current, inward rectifier, with pharmacological and biophysical properties consistent with the IK1 current (NPo =1.9±0.4 in control vs NPo=3.0±0.4, n=10, P<0.05). These deleterious effects persisted in presence of RU 28318, a specific MR antagonist, and were successfully prevented by potassium canrenoate, a non specific MR antagonist, in both microelectrode and patch-clamp recordings, thus indicating a MR-independent IK1 activation. In this ischemia-reperfusion context, aldosterone induced rapid and MR-independent deleterious effects including an arrhythmia substrate (increased APD90 dispersion) and triggered activities (increased premature ventricular contractions occurrence on reperfusion) possibly related to direct IK1 activation.

Aldosterone and testosterone: two steroid hormones structurally related but with opposite electrophysiological properties during myocardial ischemia-reperfusion.

Steroid hormones appear to be a key factor in the gender differences in the rates and severity of cardiovascular diseases. Aldosterone and testosterone have typical steroid ring structure, but despite this, they demonstrate very different properties. During acute myocardial ischemia-reperfusion, the deleterious impact of aldosterone is now well established. Conversely, the electrophysiological impact of testosterone in this context remained unknown. We used female rabbit in vitro models and standard microelectrode technique including right ventricle mimicking the 'border zone' existing between normal and ischemic/reperfused areas and isolated right ventricle experiments to assess the acute electrophysiological impact of both aldosterone and testosterone. During ischemia-reperfusion, acute superfusion of 10 and 100 nmol/L testosterone decreased normoxic and reperfused action potential duration at 90% (APD90 ), systematically induced conduction blocks, and decreased APD90 dispersion between ischemic and nonischemic areas (from 98 ± 4 to 57 ± 7 ms and 66 ± 3 ms, for, respectively, testosterone 10 and 100 nmol/L, P < 0.05). Testosterone 10 and 100 nmol/L concomitantly decreased sustained premature ventricular contraction (PVC) occurrence (from 55 to 0%, P < 0.05). Conversely, aldosterone 10 and 100 nmol/L increased normoxic and reperfused APD90 , APD90 dispersion, and reperfusion-induced PVCs. Furthermore, testosterone demonstrated cycle length-dependent effects on APD90 for high heart rate, whereas aldosterone did not exhibit any significant effect compared with controls. During acute myocardial ischemia-reperfusion, acute superfusion of physiological concentrations of testosterone seemed to be anti-arrhythmic by removing a pro-arrhythmic substrate (APD90 dispersion), inducing conduction blocks and decreasing triggered activities (PVC occurrence). Further experiments are warranted to confirm our results.

Effect of long-chain triglyceride lipid emulsion on bupivacaine-induced changes in electrophysiological parameters of rabbit Purkinje cells.

Lipid emulsions are used in the reversal of local anesthetic toxicity. The aim of this study was to investigate the cellular electrophysiological effects of long-chain triglyceride lipid emulsion (LCTE) on cardiac action potential characteristics and conduction disturbances induced by bupivacaine. Purkinje fibers were dissected from the left ventricle of New Zealand white rabbit hearts and superfused with either Tyrode's solution during 30 min (control group), with bupivacaine 10(-6) M, 10(-5) M, and 5.10(-5) M alone, or in the presence of LCTE 0.5%, in addition, LCTE at 0.1%, 0.5%, and 1% was perfused alone. Electrophysiological parameters were recorded using the conventional microelectrode technique (37 °C, 1 Hz frequency). Bupivacaine 5.10(-5) M-induced conduction blocks (8/8 preparations): LCTE 0.5% suppressed the bupivacaine 5.10(-5) M-induced conduction blocks (1/8 preparations). Exposure to bupivacaine 10(-6) M, 10(-5) M, and 5.10(-5) M resulted in a significant decrease in the maximal rate of depolarization (Vmax) (respectively, 25%, 55%, 75%; P < 0.002 vs. control group). In the presence of LCTE 0.5%, bupivacaine 10(-6) M did not significantly decreased Vmax (13%; P = 0.10 vs. control group). The decrease in Vmax resulting from bupivacaine 10(-5) M alone was significantly less in the presence of LCTE 0.5% (P < 0.01 vs. bupivacaine 10(-5) M alone). Exposure to bupivacaine 10(-6) M, 10(-5) M, and 5.10(-5) M alone or in the presence of LCTE 0.5% resulted in a significant decrease in action potential duration measured at 50% and 90% repolarization (APD50 and APD90; P < 0.01 vs. control group). LCTE inhibited the Purkinje fibers conduction blocks induced by bupivacaine. Moreover, LCTE 0.5% attenuates the decrease in Vmax induced by bupivacaine 10(-6) M and 10(-5) M.

Rabbit ventricular myocardium undergoing simulated ischemia and reperfusion in a double compartment tissue bath: a model to investigate both antiarrhythmic and arrhythmogenic likelihood.

An ischemia/reperfusion-simulating model in rabbit tissue should be right oriented and clinically relevant to provide a non expensive approach for manipulations of currents involved in the repolarization process. Standard right ventricular guinea-pig (N=18) and newly investigated rabbit (N=12) myocardial strips were placed in a special perfusion chamber allowing partition into two segments independently superfused with oxygenated Tyrode's solution or a modified Tyrode's solution mimicking ischemia by: 1) increased extracellular potassium concentration (12 mmol/L), 2) decreased HCO3 (-) concentration (9 mmol/L), leading to a decrease in pH (6.90 ± 0.05), 3) decreased pO2 by replacement of 95% O2 and 5% CO2 by 95% N2 and 5% CO2 gas mixture, and 4) complete withdrawal of glucose. There were significant differences in rabbit as compared to guinea-pig preparations in baseline (p<0.02) and post-ischemic-like (p<0.01) APA and RMP with lower values in the formers, and lower post-ischemic Vmax in rabbit preparations (25±15 versus 97±83 V/s, p<0.01) but neither baseline nor post-ischemic-like or absolute changes in APD50, APD90 were different. In ischemia- and reperfusion-like phases, there were high proportions of single spontaneous repetitive responses, both in guinea-pig (respectively 50 and 89%) and rabbit preparations (respectively 67 and 92%). Guinea-pig preparations showed higher incidence of severe spontaneous repetitive responses (61 versus 17%, p<0.02). This rabbit model is proposed to investigate both anti- and pro-arrhythmic effects of drugs acting at various levels electrophysiologically, which may be obtained with great power and relatively few (around 10 per group) preparations. This model should now be tested pharmacologically.

Implication of the TRPM4 nonselective cation channel in mammalian sinus rhythm.

BACKGROUND: The transient receptor potential melastatin 4 (TRPM4) channel is expressed in the sinoatrial node, but its physiologic roles in this tissue with cardiac pacemaker properties remain unknown. This Ca(2+)-activated nonselective cation channel (NSCCa) induces cell depolarization at negative potentials. It is implicated in burst generation in neurons and participates in induction of ectopic beating in cardiac ventricular preparations submitted to hypoxia/reoxygenation. Accordingly, TRPM4 may participate in action potential (AP) triggering in the sinoatrial node. OBJECTIVE: The purpose of this study was to investigate the influence of TRPM4 on spontaneous heart beating. METHODS: Spontaneous APs were recorded using intracellular microelectrodes in mouse, rat, and rabbit isolated right atria. RESULTS: In the spontaneously beating mouse atrium, superfusion of the TRPM4-specific inhibitor 9-phenanthrol produced a concentration-dependent reduction in AP rate (maximal reduction = 62% that of control; EC50 = 8 × 10(-6) mol●L(-1)) without affecting other AP parameters. These effects were absent in TRPM4(-/-) mice. 9-Phenanthrol exerted a rate-dependent reduction with a higher effect at low rates. Similar results were obtained in rat. Moreover, application of 9-phenanthrol produced a reduction in diastolic depolarization slope in rabbit sinus node pacemaker cells. CONCLUSION: These data showed that TRPM4 modulates beating rate. Pacemaker activity in the sinoatrial node results from the slow diastolic depolarization slope due to the "funny" current, Na/Ca exchange, and a Ca(2+)-activated nonselective cation current, which can be attributable in part to TRPM4 that may act against bradycardia.

Transient receptor potential melastatin 4 inhibitor 9-phenanthrol abolishes arrhythmias induced by hypoxia and re-oxygenation in mouse ventricle.

BACKGROUND AND PURPOSE: Hypoxia and subsequent re-oxygenation are associated with cardiac arrhythmias such as early afterdepolarizations (EADs), which may be partly explained by perturbations in cytosolic calcium concentration. Transient receptor potential melastatin 4 (TRPM4), a calcium-activated non-selective cation channel, is functionally expressed in the heart. Based on its biophysical properties, it is likely to participate in EADs. Hence, modulators of TRPM4 activity may influence arrhythmias. The aim of this study was to investigate the possible anti-arrhythmic effect of 9-phenanthrol, a TRPM4 inhibitor in a murine heart model of hypoxia and re-oxygenation-induced EADs. EXPERIMENTAL APPROACH: Mouse heart was removed, and the right ventricle was pinned in a superfusion chamber. After a period of normoxia, the preparation was superfused for 2 h with a hypoxic solution and then re-oxygenated. Spontaneous electrical activity was investigated by intracellular microelectrode recordings. KEY RESULTS: In normoxic conditions, the ventricle exhibited spontaneous action potentials. Application of the hypoxia and re-oxygenation protocol unmasked hypoxia-induced EADs, the occurrence of which increased under re-oxygenation. The frequency of these EADs was reduced by superfusion with either flufenamic acid, a blocker of Ca(2+) -dependent cation channels or with 9-phenanthrol. Superfusion with 9-phenanthrol (10(-5) or 10(-4) mol·L(-1) ) caused a dramatic dose-dependent abolition of EADs. CONCLUSIONS AND IMPLICATIONS: Hypoxia and re-oxygenation-induced EADs can be generated in the mouse heart model. 9-Phenanthrol abolished EADs, which strongly suggests the involvement of TRPM4 in the generation of EAD. This identifies non-selective cation channels inhibitors as new pharmacological candidates in the treatment of arrhythmias.

I(Ks) blockade in border zone arrhythmias from guinea-pig ventricular myocardium submitted to simulated ischemia and reperfusion.

I(Ks) blockade might be a promising way to treat tachyarrhythmia because of the accumulation of activated potassium channels. However, I(Ks) blockade during ischemia/reperfusion has not been investigated. Thus, the electrophysiological effects of two I(Ks) blockers, chromanol 293B (10 µm) and HMR 1556 (1 µm), were assessed in an in vitro model of border zone between normal and ischemic/reperfused right ventricular myocardium from guinea-pigs, and classic electrophysiological parameters and the incidence of arrhythmias were studied. HMR 1556 and chromanol 293B exhibited slight conventional class III effects on action potential duration in the normal zone (NZ) (APD(90) : -2 ± 5%, not significant (NS); +6 ± 3%, NS; and +5 ± 1%, P < 0.05, respectively, in control, HMR 1556, and chromanol 293B groups) but failed to oppose its decrease after 30 min of simulated ischemic superfusion (APD(90) : -52 ± 5%, P < 0.01; -64 ± 5%, P < 0.01; and -61 ± 3%, P < 0.01, respectively, in control, HMR 1556, and chromanol 293B groups), leading to repolarization dispersion between normal and ischemic zones. Chromanol 293B and HMR 1556 prolonged APD(90) during reperfusion, respectively, by +11 ± 1%, P < 0.01 and +25 ± 4%, P < 0.01 in the NZ and by +13 ± 3%, NS and +31 ± 2%, P < 0.01 in the simulated ischemic zone. Both compounds exhibited neutral arrhythmogenic effects during ischemia or reperfusion. Thus, I(Ks) blockade was neutral on the occurrence of ventricular arrhythmias during ischemia and reperfusion in guinea-pig ventricular tissue.

Nifedipine blocks ondansetron electrophysiological effects in rabbit purkinje fibers and decreases early after depolarization incidence.

We hypothesized that a high concentration of nifedipine (1 µM), known to inhibit at least 75%of L-type Ca++ current, might counteract proarrhythmic dose-dependent effects of ondansetron (0.1 to 10 µM) in rabbit Purkinje fibers. Ondansetron is a 5-HT3 receptor antagonist commonly prescribed to prevent nausea and vomiting caused by cancer chemotherapy, radiation therapy, and surgery but may increase the risk of developing prolongation of the QT interval of the electrocardiogram, which can lead to an abnormal and potentially fatal heart rhythm and recently raised FDA concerns and warnings. Neostigmine, a quaternary nitrogen agent that was also used clinically concomitant to antiemetics after anesthesia was further investigated dose-dependently (0.1 to 10 µM) and at fixed concentration (10 µM) with 0.1 to 10 µM ondansetron. The protocol included use-dependent (1 to 0.33 Hz) studies. APD durations, triangulation and early after depolarization (EAD) incidence were assessed. Ondansetron increased APD50, APD70 and APD90 (0.01 > p < 0.05) dose-dependently. APD90 averaged 102�1%of baseline to 302�49%dose-dependently (p < 0.001) and, at the highest dose, increased to 511�73%reverse use-dependently (p < 0.001). EAD were seen at top concentrations (33%) which were increased at lower rates (50%). Neostigmine induced reverse use-dependent APD changes (p < 0.05) but no EAD. In preparations treated by nifedipine and ondansetron, APD90 changes averaged 101�2%of baseline to 151�8%dose-dependently (p < 0.01) and to 193�13%reverse use-dependently (p < 0.05) and no EAD were seen. Thus nifedipine significantly shortened ondansetron-induced APD changes (p < 0.01), whereas neostigmine only slightly shortened ondansetron-induced APD changes (p < 0.05). There was a tendency for increased incidence of EAD (p < 0.06) in the ondansetron and neostigmine group vs. neostigmine alone. It is concluded that inhibition of L-type Ca++ current by high concentration nifedipine may counteract the ondansetron effects on APD changes.