Inhibition of EPAC1 signaling pathway alters atrial electrophysiology and prevents atrial fibrillation.

Introduction: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with increased mortality and morbidity. The Exchange Protein directly Activated by cAMP (EPAC), has been implicated in pro-arrhythmic signaling pathways in the atria, but the underlying mechanisms remain unknown. Methods: In this study, we investigated the involvement of EPAC1 and EPAC2 isoforms in the genesis of AF in wild type (WT) mice and knockout (KO) mice for EPAC1 or EPAC2. We also employed EPAC pharmacological modulators to selectively activate EPAC proteins (8-CPT-AM; 10 µM), or inhibit either EPAC1 (AM-001; 20 µM) or EPAC2 (ESI-05; 25 µM). Transesophageal stimulation was used to characterize the induction of AF in vivo in mice. Optical mapping experiments were performed on isolated mouse atria and cellular electrophysiology was examined by whole-cell patch-clamp technique. Results: In wild type mice, we found 8-CPT-AM slightly increased AF susceptibility and that this was blocked by the EPAC1 inhibitor AM-001 but not the EPAC2 inhibitor ESI-05. Consistent with this, in EPAC1 KO mice, occurrence of AF was observed in 3/12 (vs. 4/10 WT littermates) and 4/10 in EPAC2 KO (vs. 5/10 WT littermates). In wild type animals, optical mapping experiments revealed that 8-CPT-AM perfusion increased action potential duration even in the presence of AM-001 or ESI-05. Interestingly, 8-CPT-AM perfusion decreased conduction velocity, an effect blunted by AM-001 but not ESI-05. Patch-clamp experiments demonstrated action potential prolongation after 8-CPT-AM perfusion in both wild type and EPAC1 KO mice and this effect was partially prevented by AM-001 in WT. Conclusion: Together, these results indicate that EPAC1 and EPAC2 signaling pathways differentially alter atrial electrophysiology but only the EPAC1 isoform is involved in the genesis of AF. Selective blockade of EPAC1 with AM-001 prevents AF in mice.

The antimalarial MMV688533 provides potential for single-dose cures with a high barrier to Plasmodium falciparum parasite resistance.

The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.

TRPM4 Participates in Aldosterone-Salt-Induced Electrical Atrial Remodeling in Mice.

Aldosterone plays a major role in atrial structural and electrical remodeling, in particular through Ca2+-transient perturbations and shortening of the action potential. The Ca2+-activated non-selective cation channel Transient Receptor Potential Melastatin 4 (TRPM4) participates in atrial action potential. The aim of our study was to elucidate the interactions between aldosterone and TRPM4 in atrial remodeling and arrhythmias susceptibility. Hyperaldosteronemia, combined with a high salt diet, was induced in mice by subcutaneously implanted osmotic pumps during 4 weeks, delivering aldosterone or physiological serum for control animals. The experiments were conducted in wild type animals (Trpm4+/+) as well as Trpm4 knock-out animals (Trpm4-/-). The atrial diameter measured by echocardiography was higher in Trpm4-/- compared to Trpm4+/+ animals, and hyperaldosteronemia-salt produced a dilatation in both groups. Action potentials duration and triggered arrhythmias were measured using intracellular microelectrodes on the isolated left atrium. Hyperaldosteronemia-salt prolong action potential in Trpm4-/- mice but had no effect on Trpm4+/+ mice. In the control group (no aldosterone-salt treatment), no triggered arrythmias were recorded in Trpm4+/+ mice, but a high level was detected in Trpm4-/- mice. Hyperaldosteronemia-salt enhanced the occurrence of arrhythmias (early as well as delayed-afterdepolarization) in Trpm4+/+ mice but decreased it in Trpm4-/- animals. Atrial connexin43 immunolabelling indicated their disorganization at the intercalated disks and a redistribution at the lateral side induced by hyperaldosteronemia-salt but also by Trpm4 disruption. In addition, hyperaldosteronemia-salt produced pronounced atrial endothelial thickening in both groups. Altogether, our results indicated that hyperaldosteronemia-salt and TRPM4 participate in atrial electrical and structural remodeling. It appears that TRPM4 is involved in aldosterone-induced atrial action potential shortening. In addition, TRPM4 may promote aldosterone-induced atrial arrhythmias, however, the underlying mechanisms remain to be explored.

TRPM4 non-selective cation channel in human atrial fibroblast growth.

Cardiac fibroblasts play an important role in cardiac matrix turnover and are involved in cardiac fibrosis development. Ca2+ is a driving belt in this phenomenon. This study evaluates the functional expression and contribution of the Ca2+-activated channel TRPM4 in atrial fibroblast phenotype. Molecular and electrophysiological investigations were conducted in human atrial fibroblasts in primary culture and in atrial fibroblasts obtained from wild-type and transgenic mice with disrupted Trpm4 gene (Trpm4-/-). A typical TRPM4 current was recorded on human cells (equal selectivity for Na+ and K+, activation by internal Ca2+, voltage sensitivity, conductance of 23.2 pS, inhibition by 9-phenanthrol (IC50 = 6.1 × 10-6 mol L-1)). Its detection rate was 13% on patches at days 2-4 in culture but raised to 100% on patches at day 28. By the same time, a cell growth was observed. This growth was smaller when cells were maintained in the presence of 9-phenanthrol. Similar cell growth was measured on wild-type mice atrial fibroblasts during culture. However, this growth was minimized on Trpm4-/- mice fibroblasts compared to control animals. In addition, the expression of alpha smooth muscle actin increased during culture of atrial fibroblasts from wild-type mice. This was not observed in Trpm4-/- mice fibroblasts. It is concluded that TRPM4 participates in fibroblast growth and could thus be involved in cardiac fibrosis.

Transient receptor potential channels in cardiac health and disease.

Transient receptor potential (TRP) channels are nonselective cationic channels that are generally Ca2+ permeable and have a heterogeneous expression in the heart. In the myocardium, TRP channels participate in several physiological functions, such as modulation of action potential waveform, pacemaking, conduction, inotropy, lusitropy, Ca2+ and Mg2+ handling, store-operated Ca2+ entry, embryonic development, mitochondrial function and adaptive remodelling. Moreover, TRP channels are also involved in various pathological mechanisms, such as arrhythmias, ischaemia-reperfusion injuries, Ca2+-handling defects, fibrosis, maladaptive remodelling, inherited cardiopathies and cell death. In this Review, we present the current knowledge of the roles of TRP channels in different cardiac regions (sinus node, atria, ventricles and Purkinje fibres) and cells types (cardiomyocytes and fibroblasts) and discuss their contribution to pathophysiological mechanisms, which will help to identify the best candidates for new therapeutic targets among the cardiac TRP family.

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.

TRPM4 non-selective cation channel variants in long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is an inherited arrhythmic disorder characterized by prolongation of the QT interval, a risk of syncope, and sudden death. There are already a number of causal genes in LQTS, but not all LQTS patients have an identified mutation, which suggests LQTS unknown genes. METHODS: A cohort of 178 LQTS patients, with no mutations in the 3 major LQTS genes (KCNQ1, KCNH2, and SCN5A), was screened for mutations in the transient potential melastatin 4 gene (TRPM4). RESULTS: Four TRPM4 variants (2.2% of the cohort) were found to change highly conserved amino-acids and were either very rare or absent from control populations. Therefore, these four TRPM4 variants were predicted to be disease causing. Furthermore, no mutations were found in the DNA of these TRPM4 variant carriers in any of the 13 major long QT syndrome genes. Two of these variants were further studied by electrophysiology (p.Val441Met and p.Arg499Pro). Both variants showed a classical TRPM4 outward rectifying current, but the current was reduced by 61 and 90% respectively, compared to wild type TRPM4 current. CONCLUSIONS: This study supports the view that TRPM4 could account for a small percentage of LQTS patients. TRPM4 contribution to the QT interval might be multifactorial by modulating whole cell current but also, as shown in Trpm4-/- mice, by modulating cardiomyocyte proliferation. TRPM4 enlarges the subgroup of LQT genes (KCNJ2 in Andersen syndrome and CACNA1C in Timothy syndrome) known to increase the QT interval through a more complex pleiotropic effect than merely action potential alteration.

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.

TRPM4 in cardiac electrical activity.

TRPM4 forms a non-selective cation channel activated by internal Ca(2+). Its functional expression was demonstrated in cardiomyocytes of several mammalian species including humans, but the channel is also present in many other tissues. The recent characterization of the TRPM4 inhibitor 9-phenanthrol, and the availability of transgenic mice have helped to clarify the role of TRPM4 in cardiac electrical activity, including diastolic depolarization from the sino-atrial node cells in mouse, rat, and rabbit, as well as action potential duration in mouse cardiomyocytes. In rat and mouse, pharmacological inhibition of TRPM4 prevents cardiac ischaemia-reperfusion injuries and decreases the occurrence of arrhythmias. Several studies have identified TRPM4 mutations in patients with inherited cardiac diseases including conduction blocks and Brugada syndrome. This review identifies TRPM4 as a significant actor in cardiac electrophysiology.

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.

The calcium stored in the sarcoplasmic reticulum acts as a safety mechanism in rainbow trout heart.

Cardiomyocyte contraction depends on rapid changes in intracellular Ca(2+). In mammals, Ca(2+) influx as L-type Ca(2+) current (ICa) triggers the release of Ca(2+) from sarcoplasmic reticulum (SR) and Ca(2+)-induced Ca(2+) release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca(2+) is unclear. Here, we characterized the role of ICa to trigger SR Ca(2+) release in rainbow trout ventricular myocytes using ICa regulation by Ca(2+) as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca(2+) chelator in control and isoproterenol conditions. In the absence of ß-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 µM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca(2+), while with isoproterenol, inactivation was Ca(2+)-dependent (∼65%) and highly reliant on SR Ca(2+) (∼46%). Thus, SR Ca(2+) is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca(2+) is an important source of cytosolic Ca(2+). This was not attributed to differences in SR Ca(2+) load because caffeine-induced transients were not different in both conditions. Therefore, Ca(2+) stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.

Current recordings at the single channel level in adult mammalian isolated cardiomyocytes.

This chapter describes appropriate methods to investigate mammalian cardiac channels properties at the single channel level. Cell isolation is performed from new born or adult heart by enzymatic digestion on minced tissue or using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. In addition, isolation of human atrial cardiomyocytes is described. Such freshly isolated cells or cells maintained in primary culture are suitable for patch-clamp studies. Here we describe the single channel variants of the patch-clamp technique (cell-attached, inside-out, outside-out) used to investigate channel properties. Proceedings for the evaluation of biophysical properties such as conductance, ionic selectivity, regulations by extracellular and intracellular mechanisms are described. To illustrate the study, we provide an example by the characterization of a calcium-activated non-selective cation channel (TRPM4).

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.

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.

Epac activator critically regulates action potential duration by decreasing potassium current in rat adult ventricle.

Sympathetic stimulation is an important modulator of cardiac function via the classic cAMP-dependent signaling pathway, PKA. Recently, this paradigm has been challenged by the discovery of a family of guanine nucleotide exchange proteins directly activated by cAMP (Epac), acting in parallel to the classic signaling pathway. In cardiac myocytes, Epac activation is known to modulate Ca(2+) cycling yet their actions on cardiac ionic currents remain poorly characterized. This study attempts to address this paucity of information using the patch clamp technique to record action potential (AP) and ionic currents on rat ventricular myocytes. Epac was selectively activated by 8-CPT-AM (acetoxymethyl ester form of 8-CPT). AP amplitude, maximum depolarization rate and resting membrane amplitude were unaltered by 8-CPT-AM, strongly suggesting that Na(+) current and inward rectifier K(+) current are not regulated by Epac. In contrast, AP duration was significantly increased by 8-CPT-AM (prolongation of duration at 50% and 90% of repolarization by 41±10% and 43±8% respectively, n=11). L-type Ca(2+) current density was unaltered by 8-CPT-AM (n=16) so this cannot explain the action potential lengthening. However, the steady state component of K(+) current was significantly inhibited by 8-CPT-AM (-38±6%, n=15), while the transient outward K(+) current was unaffected by 8-CPT-AM. These effects were PKA-independent since they were observed in the presence of PKA inhibitor KT5720. Isoprenaline (100nM) induced a significant prolongation of AP duration, even in the presence of KT5720. This study provides the first evidence that the cAMP-binding protein Epac critically modulates cardiac AP duration by decreasing steady state K(+) current. These observations may be relevant to diseases in which Epac is upregulated, like cardiac hypertrophy.

Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel.

Brugada syndrome (BrS) is a condition defined by ST-segment alteration in right precordial leads and a risk of sudden death. Because BrS is often associated with right bundle branch block and the TRPM4 gene is involved in conduction blocks, we screened TRPM4 for anomalies in BrS cases. The DNA of 248 BrS cases with no SCN5A mutations were screened for TRPM4 mutations. Among this cohort, 20 patients had 11 TRPM4 mutations. Two mutations were previously associated with cardiac conduction blocks and 9 were new mutations (5 absent from ~14'000 control alleles and 4 statistically more prevalent in this BrS cohort than in control alleles). In addition to Brugada, three patients had a bifascicular block and 2 had a complete right bundle branch block. Functional and biochemical studies of 4 selected mutants revealed that these mutations resulted in either a decreased expression (p.Pro779Arg and p.Lys914X) or an increased expression (p.Thr873Ile and p.Leu1075Pro) of TRPM4 channel. TRPM4 mutations account for about 6% of BrS. Consequences of these mutations are diverse on channel electrophysiological and cellular expression. Because of its effect on the resting membrane potential, reduction or increase of TRPM4 channel function may both reduce the availability of sodium channel and thus lead to BrS.

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.

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.

The non-selective monovalent cationic channels TRPM4 and TRPM5.

Transient Receptor Potential (TRP) proteins are non-selective cationic channels with a consistent Ca(2+)-permeability, except for TRPM4 and TRPM5 that are not permeable to this ion. However, Ca(2+) is a major regulator of their activity since both channels are activated by a rise in internal Ca(2+). Thus TRPM4 and TRPM5 are responsible for most of the Ca(2+)-activated non-selective cationic currents (NSC(Ca)) recorded in a large variety of tissues. Their activation induces cell-membrane depolarization that modifies the driving force for ions as well as activity of voltage gated channels and thereby strongly impacts cell physiology. In the last few years, the ubiquitously expressed TRPM4 channel has been implicated in insulin secretion, the immune response, constriction of cerebral arteries, the activity of inspiratory neurons and cardiac dysfunction. Conversely, TRPM5 whose expression is more restricted, has until now been mainly implicated in taste transduction.