Laurent Sallé

Differential Modulation of L-type Ca2+ Current by SR Ca2+ Release at the T-Tubules and Surface Membrane of Rat Ventricular Myocytes

We have characterized modulation of ICa by Ca2+ at the t-tubules (ie, in control cells) and surface sarcolemma (ie, in detubulated cells) of cardiac ventricular myocytes, using the whole-cell patch clamp technique to record ICa. ICa inactivation was significantly slower in detubulated cells than in control cells (27.1±7.8 ms, n=22, versus 16.4±7.9 ms, n=22; P <0.05). In atrial myocytes, which lack t-tubules, ICa inactivation was not changed by the treatment used to produce detubulation. In the presence of ryanodine or BAPTA, or when Ba2+ was used as the charge carrier, the rate of inactivation was not significantly different in control and detubulated cells. Frequency-dependent facilitation occurred in control cells but not in detubulated cells, and was abolished by ryanodine. These results suggest that Ca2+ released from the SR has a greater effect on ICa in the t-tubules than at the surface sarcolemma. This does not appear to be due to differences in local Ca2+ release from the SR, because the gain of Ca2+ release was not significantly different in control and detubulated cells. These data suggest that the t-tubules are a key site for the regulation of transsarcolemmal Ca2+ flux by Ca2+ release from the SR; this could play a role in altered Ca2+ homeostasis in pathological conditions. The full text of this article is available online at http://circres.ahajournals.org.

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.

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.

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.

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.

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

Cardiomyocyte contraction depends on rapid changes in intracellular Ca2+. In mammals, Ca2+ influx as L-type Ca2+ current (ICa) triggers the release of Ca2+ from sarcoplasmic reticulum (SR) and Ca2+-induced Ca2+ release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca2+ is unclear. Here, we characterized the role of ICa to trigger SR Ca2+ release in rainbow trout ventricular myocytes using ICa regulation by Ca2+ as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca2+ chelator in control and isoproterenol conditions. In the absence of β-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 Ca2+, while with isoproterenol, inactivation was Ca2+-dependent (∼65%) and highly reliant on SR Ca2+ (∼46%). Thus, SR Ca2+ is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca2+ is an important source of cytosolic Ca2+. This was not attributed to differences in SR Ca2+ load because caffeine-induced transients were not different in both conditions. Therefore, Ca2+ 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.

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

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.

IKr vs. IKs blockade and arrhythmogenicity in normoxic rabbit Purkinje fibers: does it really make a difference?

The electrophysiological (standard intracellular microelectrode technique) and pro‐arrhythmic (occurrence of early after‐depolarization) effects of five class III agents acting on delayed rectifier current (IK), rapid (IKr), and/or slow (IKs) components have been studied in rabbit Purkinje fibers taken near the septum and submitted in vitro to reduced stimulation rate (from 1 to 0.5 Hz) in the absence or presence of epinephrine (10 nm) during normoxic conditions. There were two IKr blockers (d‐sotalol and dofetilide), two IKs blockers (chromanol 293B and HMR 1556), and a non‐selective IK blocker (azimilide). d‐sotalol, dofetilide, and azimilide lengthened APD60 and APD90 in a concentration‐dependent manner. Both d‐sotalol and dofetilide showed pro‐arrhythmia at highest concentrations and in the presence of epinephrine and lower stimulation rate. Despite azimilide markedly lengthened APD90, it was globally less pro‐arrhythmic than dofetilide. Thus, in normoxic rabbit Purkinje fibers, IKr blockade prolonged action potential duration (APD) and increased the incidence of early after‐depolarizations, particularly so in the presence of adrenergic stimulation and bradycardia, IKs blockade did neither, and non‐selective IK blockade (by azimilide) behaved principally as IKr blockade. It is concluded that in normoxic rabbit Purkinje fibers, IKs blockade was neutral, whereas IKr blockade was pro‐arrhythmic, which may make a difference worth exploration in more complex models.

Nifedipine Blocks Ondansetron Electrophysiological Effects in Rabbit Purkinje Fibers and Decreases Early Afterdepolarization 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.