TRPM4 contribution in mouse uterine contractions.

In brief: Inappropriate uterine contractions are a matter of concern during pregnancy or menses. We identified the transient receptor potential melastatin 4 (TRPM4) ion channel as a new actor in mouse uterine contractions highlighting this protein as a potential pharmacological target for a better control of myometrial activity. Abstract: Control of uterine contractions is of interest in the context of inappropriate myometrial activity during pregnancy and at time of delivery, but it is also a matter for menstrual pain. While several molecular determinants of myometrial contractions have been described, the complete distribution of roles to the various actors is far from understood. A key phenomenon is a variation in cytoplasmic Ca2+ which leads to the activation of calmodulin in smooth muscle and also in the phosphorylation of myosin allowing contraction. The Ca2+ - TRPM4 channel which is known to modulate Ca2+- fluxes in several cell types was shown to participate in vascular as well as detrusor muscle contraction. We thus designed a study to determine whether it also participates in myometrial contraction. Uterine rings were isolated from Trpm4+/+ and Trpm4-/- non-pregnant adult mice and contractions were recorded using an isometric force transducer. In basal conditions, spontaneous contractions were similar in both groups. Application of 9-phenanthrol, a pharmacological TRPM4 inhibitor, dose-dependently reduced contraction parameters in Trpm4+/+ rings with an IC50 around 2.10-6 mol/L. The effect of 9-phenanthrol was significantly reduced in Trpm4-/- rings. The effect of oxytocin was tested and was found to be stronger in Trpm4+/+ rings compared to Trpm4-/-. Under a constant stimulation by oxytocin, 9-phenanthrol still reduced contraction parameters in Trpm4+/+ rings with a smaller effect on Trpm4-/-. Altogether it indicates that TRPM4 participates in uterine contractions in mice and may thus be evaluated as a new target to control such contractions.

Ion Channels in the Development and Remodeling of the Aortic Valve.

The role of ion channels is extensively described in the context of the electrical activity of excitable cells and in excitation-contraction coupling. They are, through this phenomenon, a key element for cardiac activity and its dysfunction. They also participate in cardiac morphological remodeling, in particular in situations of hypertrophy. Alongside this, a new field of exploration concerns the role of ion channels in valve development and remodeling. Cardiac valves are important components in the coordinated functioning of the heart by ensuring unidirectional circulation essential to the good efficiency of the cardiac pump. In this review, we will focus on the ion channels involved in both the development and/or the pathological remodeling of the aortic valve. Regarding valve development, mutations in genes encoding for several ion channels have been observed in patients suffering from malformation, including the bicuspid aortic valve. Ion channels were also reported to be involved in the morphological remodeling of the valve, characterized by the development of fibrosis and calcification of the leaflets leading to aortic stenosis. The final stage of aortic stenosis requires, until now, the replacement of the valve. Thus, understanding the role of ion channels in the progression of aortic stenosis is an essential step in designing new therapeutic approaches in order to avoid valve replacement.

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.

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.

Cardiac 2-D Shear Wave Imaging Using a New Dedicated Clinical Ultrasound System: A Phantom Study.

OBJECTIVE: The purpose of this study was to assess cardiac shear wave imaging implemented in a new MACH 30 ultrasound machine (SuperSonic Imaging, Aix-en-Provence, France) and interfaced with a linear probe and a phased array probe, in comparison with a previously validated Aixplorer system connected to a linear probe (SuperSonic Imaging) using Elasticity QA phantoms (Models 039 and 049, CIRS Inc., Norfolk, VA, USA). METHODS: Quantile-quantile plots were used for distribution agreement. The accuracy of stiffness measurement was assessed by the percentage error and the mean percentage error (MPE), and its homogeneity, by the standard deviation of the MPE. A p value <0.01 was considered to indicate statistical significance. RESULTS: The accuracy of dedicated cardiac sequences for linear probes was similar for the two systems with an MPE of 8 ± 14% versus 20 ± 21% (p = not significant) with the SuperSonic MACH 30 and Aixplorer, respectively, and was influenced by target stiffness and location of the measurement in the field of view, but without drift over time. The optimal transthoracic cardiac probe workspace was located between 4 and 10 cm, with an MPE of 29.5 ± 25% compared with 93.3 ± 130% outside this area (p < 0.0001). In this area, stiffness below 20 kPa was significantly different from the reference (p < 0.0001). The sectorial probe revealed no MPE difference in any of the measurement areas, with no significant lateral or axial gradient. CONCLUSION: The new Supersonic MACH 30 system upgraded with a sectorial probe and specific cardiac settings provided homogenous stiffness measurements, especially when operating at depths between 4 and 10 cm. These phantom results may be useful in designing future in vivo studies.

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.

The TRPM4 non-selective cation channel contributes to the mammalian atrial action potential.

The TRPM4 calcium-activated non-selective monovalent cation channel has been reported in mammalian atrial cardiomyocytes, but its implication in this tissue remains unknown. We used a combination of pharmacological tools and disruption of the Trpm4 gene in mice to investigate the channel implication in atrial action potential (AP). To search for TRPM4 activity, single channel currents were recorded on freshly isolated atrial cardiomyocytes using the patch-clamp technique. To investigate TRPM4 implication in AP, the transmembrane potential was recorded on the multicellular preparation using intracellular microelectrodes after isolating the mouse atrium, under electrical stimulation (rate=5Hz). Isolated atrial cardiomyocytes from the Trpm4(+/+) mouse expressed a typical TRPM4 current while cardiomyocytes from Trpm4(-/-) mouse did not. The Trpm4(+/+) mouse atrium exhibited AP durations at 50, 70 and 90% repolarization of 8.9±0.5ms, 16.0±1.0ms, and 30.2±1.6ms, respectively. The non-selective cation channel inhibitor flufenamic acid (10(-6) and 10(-5)mol·L(-1)) produced a concentration-dependent decrease in AP duration. Similarly, the TRPM4-inhibitor 9-phenanthrol reversibly reduced the duration of AP with an EC50 at 21×10(-6)mol·L(-1), which is similar to that reported for TRPM4 current inhibition in HEK-293 cells. 9-Phenanthrol had no effect on other AP parameters. The effect of 9-phenanthrol is markedly reduced in the mouse ventricle, which displays only weak expression of the channel. Moreover, atria from Trpm4(-/-) mice exhibited an AP that was 20% shorter than that of atria from littermate control mice, and the effect of 9-phenanthrol on AP was abolished in the Trpm4(-/-) mice. Our results showed that TRPM4 is implicated in the waveform of the atrial action potential. It is thus a potential target for pharmacological approaches against atrial arrhythmias.

[18F]FDG PET-MR characterization of aortitis in the IL1rn-/- mouse model of giant-cell arteritis.

BACKGROUND: Metabolic imaging is routinely used to demonstrate aortitis in patients with giant-cell arteritis. We aimed to investigate the preclinical model of aortitis in BALB/c IL1rn-/- mice using [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography-magnetic resonance (PET-MR), gamma counting and immunostaining. We used 15 first-generation specific and opportunistic pathogen-free (SOPF) 9-week-old IL1rn-/- mice, 15 wild-type BALB/cAnN mice and 5 s-generation specific pathogen-free (SPF) 9-week-old IL1rn-/-. Aortic [18F]FDG uptake was assessed as the target-to-background ratio (TBR) using time-of-flight MR angiography as vascular landmarks. RESULTS: [18F]FDG uptake measured by PET or gamma counting was similar in the first-generation SOPF IL1rn-/- mice and the wild-type group (p > 0.05). However, the first-generation IL1rn-/- mice exhibited more interleukin-1ß (p = 0.021)- and interleukin-6 (p = 0.019)-positive cells within the abdominal aorta than the wild-type mice. In addition, the second-generation SPF group exhibited significantly higher TBR (p = 0.0068) than the wild-type mice on the descending thoracic aorta, unlike the first-generation SOPF IL1rn-/- mice. CONCLUSIONS: In addition to the involvement of interleukin-1ß and -6 in IL1rn-/- mouse aortitis, this study seems to validate [18F]FDG PET-MR as a useful tool for noninvasive monitoring of aortitis in this preclinical model.

Targeted Radiation Exposure Induces Accelerated Aortic Valve Remodeling in ApoE-/- Mice.

Thoracic radiation therapy may result in accelerated atherosclerosis and in late aortic valve stenosis (AS). In this study, we assessed the feasibility of inducing radiation-induced AS using a targeted aortic valve irradiation (10 or 20 Grays) in two groups of C57Bl6/J (WT) and ApoE-/- mice compared to a control (no irradiation). Peak aortic jet velocity was evaluated by echocardiography to characterize AS. T2*-weighted magnetic resonance imaging after injection of MPIO-αVCAM-1 was used to examine aortic inflammation resulting from irradiation. A T2* signal void on valve leaflets and aortic sinus was considered positive. Valve remodeling and mineralization were assessed using von Kossa staining. Finally, the impact of radiation on cell viability and cycle from aortic human valvular interstitial cells (hVICs) was also assessed. The targeted aortic valve irradiation in ApoE-/- mice resulted in an AS characterized by an increase in peak aortic jet velocity associated with valve leaflet and aortic sinus remodeling, including mineralization process, at the 3-month follow-up. There was a linear correlation between histological findings and peak aortic jet velocity (r = 0.57, p < 0.01). In addition, irradiation was associated with aortic root inflammation, evidenced by molecular MR imaging (p < 0.01). No significant effect of radiation exposure was detected on WT animals. Radiation exposure did not affect hVICs viability and cell cycle. We conclude that targeted radiation exposure of the aortic valve in mice results in ApoE-/-, but not in WT, mice in an aortic valve remodeling mimicking the human lesions. This preclinical model could be a useful tool for future assessment of therapeutic interventions.

TRPM4 Participates in Irradiation-Induced Aortic Valve Remodeling in Mice.

Thoracic radiotherapy can lead to cardiac remodeling including valvular stenosis due to fibrosis and calcification. The monovalent non-selective cation channel TRPM4 is known to be involved in calcium handling and to participate in fibroblast transition to myofibroblasts, a phenomenon observed during aortic valve stenosis. The goal of this study was to evaluate if TRPM4 is involved in irradiation-induced aortic valve damage. Four-month-old Trpm4+/+ and Trpm4-/- mice received 10 Gy irradiation at the aortic valve. Cardiac parameters were evaluated by echography until 5 months post-irradiation, then hearts were collected for morphological and histological assessments. At the onset of the protocol, Trpm4+/+ and Trpm4-/- mice exhibited similar maximal aortic valve jet velocity and mean pressure gradient. Five months after irradiation, Trpm4+/+ mice exhibited a significant increase in those parameters, compared to the untreated animals while no variation was detected in Trpm4-/- mice. Morphological analysis revealed that irradiated Trpm4+/+ mice exhibited a 53% significant increase in the aortic valve cusp surface while no significant variation was observed in Trpm4-/- animals. Collagen staining revealed aortic valve fibrosis in irradiated Trpm4+/+ mice but not in irradiated Trpm4-/- animals. It indicates that TRPM4 influences irradiation-induced valvular remodeling.

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.

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.

Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity.

Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC50 close to 1 µM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.

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.

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.

Flufenamic acid as an ion channel modulator.

Flufenamic acid has been known since the 1960s to have anti-inflammatory properties attributable to the reduction of prostaglandin synthesis. Thirty years later, flufenamic acid appeared to be an ion channel modulator. Thus, while its use in medicine diminished, its use in ionic channel research expanded. Flufenamic acid commonly not only affects non-selective cation channels and chloride channels, but also modulates potassium, calcium and sodium channels with effective concentrations ranging from 10(-6)M in TRPM4 channel inhibition to 10(-3)M in two-pore outwardly rectifying potassium channel activation. Because flufenamic acid effects develop and reverse rapidly, it is a convenient and widely used tool. However, given the broad spectrum of its targets, experimental results have to be interpreted cautiously. Here we provide an overview of ion channels targeted by flufenamic acid to aid in interpreting its effects at the molecular, cellular, and system levels. If it is used with good practices, flufenamic acid remains a useful tool for ion channel research. Understanding the targets of FFA may help reevaluate its physiological impacts and revive interest in its therapeutic potential.

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.

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.

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.