Oral batyl alcohol supplementation rescues decreased cardiac conduction in ether phospholipid-deficient mice.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In-vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age- and sex-matched wild-type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age-matched wild-type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

Distinct modulation of inactivation by a residue in the pore domain of voltage-gated Na+ channels: mechanistic insights from recent crystal structures.

Inactivation of voltage-gated Na+ channels (VGSC) is essential for the regulation of cellular excitability. The molecular rearrangement underlying inactivation is thought to involve the intracellular linker between domains III and IV serving as inactivation lid, the receptor for the lid (domain III S4-S5 linker) and the pore-lining S6 segements. To better understand the role of the domain IV S6 segment in inactivation we performed a cysteine scanning mutagenesis of this region in rNav 1.4 channels and screened the constructs for perturbations in the voltage-dependence of steady state inactivation. This screen was performed in the background of wild-type channels and in channels carrying the mutation K1237E, which profoundly alters both permeation and gating-properties. Of all tested constructs the mutation I1581C was unique in that the mutation-induced gating changes were strongly influenced by the mutational background. This suggests that I1581 is involved in specific short-range interactions during inactivation. In recently published crystal structures VGSCs the respective amino acids homologous to I1581 appear to control a bend of the S6 segment which is critical to the gating process. Furthermore, I1581 may be involved in the transmission of the movement of the DIII voltage-sensor to the domain IV S6 segment.

Mechanism of Modification, by Lidocaine, of Fast and Slow Recovery from Inactivation of Voltage-Gated Na⁺ Channels.

The clinically important suppression of high-frequency discharges of excitable cells by local anesthetics (LA) is largely determined by drug-induced prolongation of the time course of repriming (recovery from inactivation) of voltage-gated Na(+) channels. This prolongation may result from periodic drug-binding to a high-affinity binding site during the action potentials and subsequent slow dissociation from the site between action potentials ("dissociation hypothesis"). For many drugs it has been suggested that the fast inactivated state represents the high-affinity binding state. Alternatively, LAs may bind with high affinity to a native slow-inactivated state, thereby accelerating the development of this state during action potentials ("stabilization hypothesis"). In this case, slow recovery between action potentials occurs from enhanced native slow inactivation. To test these two hypotheses we produced serial cysteine mutations of domain IV segment 6 in rNav1.4 that resulted in constructs with varying propensities to enter fast- and slow-inactivated states. We tested the effect of the LA lidocaine on the time course of recovery from short and long depolarizing prepulses, which, under drug-free conditions, recruited mainly fast- and slow-inactivated states, respectively. Among the tested constructs the mutation-induced changes in native slow recovery induced by long depolarizations were not correlated with the respective lidocaine-induced slow recovery after short depolarizations. On the other hand, for long depolarizations the mutation-induced alterations in native slow recovery were significantly correlated with the kinetics of lidocaine-induced slow recovery. These results favor the "dissociation hypothesis" for short depolarizations but the "stabilization hypothesis" for long depolarizations.

Small molecule cardiogenol C upregulates cardiac markers and induces cardiac functional properties in lineage-committed progenitor cells.

BACKGROUND/AIMS: Cell transplantation into the heart is a new therapy after myocardial infarction. Its success, however, is impeded by poor donor cell survival and by limited transdifferentiation of the transplanted cells into functional cardiomyocytes. A promising strategy to overcome these problems is the induction of cardiomyogenic properties in donor cells by small molecules. METHODS: Here we studied cardiomyogenic effects of the small molecule compound cardiogenol C (CgC), and structural derivatives thereof, on lineage-committed progenitor cells by various molecular biological, biochemical, and functional assays. RESULTS: Treatment with CgC up-regulated cardiac marker expression in skeletal myoblasts. Importantly, the compound also induced cardiac functional properties: first, cardiac-like sodium currents in skeletal myoblasts, and secondly, spontaneous contractions in cardiovascular progenitor cell-derived cardiac bodies. CONCLUSION: CgC induces cardiomyogenic function in lineage-committed progenitor cells, and can thus be considered a promising tool to improve cardiac repair by cell therapy.

Mechanism of hERG channel block by the psychoactive indole alkaloid ibogaine.

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

Anti-addiction drug ibogaine inhibits hERG channels: a cardiac arrhythmia risk.

Ibogaine, an alkaloid derived from the African shrub Tabernanthe iboga, has shown promising anti-addictive properties in animals. Anecdotal evidence suggests that ibogaine is also anti-addictive in humans. Thus, it alleviates drug craving and impedes relapse of drug use. Although not licensed as therapeutic drug, and despite evidence that ibogaine may disturb the rhythm of the heart, this alkaloid is currently used as an anti-addiction drug in alternative medicine. Here, we report that therapeutic concentrations of ibogaine reduce currents through human ether-a-go-go-related gene potassium channels. Thereby, we provide a mechanism by which ibogaine may generate life-threatening cardiac arrhythmias.

Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: a study to assess the drug's cardiac ion channel profile.

The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licensed as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Nav1.5 sodium and Cav1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 µM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias.

VUT-MK142 : a new cardiomyogenic small molecule promoting the differentiation of pre-cardiac mesoderm into cardiomyocytes.

Intra-cardiac cell transplantation is a new therapy after myocardial infarction. Its success, however, is impeded by the limited capacity of donor cells to differentiate into functional cardiomyocytes in the heart. A strategy to overcome this problem is the induction of cardiomyogenic function in cells prior to transplantation. Among other approaches, recently, synthetic small molecules were identified, which promote differentiation of stem cells of various origins into cardiac-like cells or cardiomyocytes. The aim of this study was to develop and characterise new promising cardiomyogenic synthetic low-molecular weight compounds. Therefore, the structure of the known cardiomyogenic molecule cardiogenol C was selectively modified, and the effects of the resulting compounds were tested on various cell types. From this study, VUT-MK142 was identified as the most promising candidate with respect to cardiomyogenic activity. Treatment using this novel agent induced the strongest up-regulation of expression of the cardiac marker ANF in both P19 embryonic carcinoma cells and C2C12 skeletal myoblasts. The activity of VUT-MK142 on this marker superseded CgC; moreover, the novel compound significantly up-regulated the expression of other cardiac markers, and promoted the development of beating cardiomyocytes from cardiovascular progenitor cells. We conclude that VUT-MK142 is a potent new cardiomyogenic synthetic agent promoting the differentiation of pre-cardiac mesoderm into cardiomyocytes, which may be useful to differentiate stem cells into cardiomyocytes for cardiac repair. Additionally, an efficient synthesis of VUT-MK142 is reported taking advantage of continuous flow techniques superior to classical batch reactions both in yield and reaction time.

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel.

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K(+) channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na(+) channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNa(V)1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state ("ultra-slow inactivation," I(US)) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of I(US). The fact that mutations at site 1575 modulate I(US) produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.

Effects of duramycin on cardiac voltage-gated ion channels.

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 microM did not generate any effects on these currents. Concentrations >or=0.3 microM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (>or=3.3 microM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias.

Speeding the recovery from ultraslow inactivation of voltage-gated Na+ channels by metal ion binding to the selectivity filter: a foot-on-the-door?

Slow inactivated states in voltage-gated ion channels can be modulated by binding molecules both to the outside and to the inside of the pore. Thus, external K(+) inhibits C-type inactivation in Shaker K(+) channels by a "foot-in-the-door" mechanism. Here, we explore the modulation of a very long-lived inactivated state, ultraslow inactivation (I(US)), by ligand binding to the outer vestibule in voltage-gated Na(+) channels. Blocking the outer vestibule by a mutant mu-conotoxin GIIIA substantially accelerated recovery from I(US). A similar effect was observed if Cd(2+) was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 microM Cd(2+), the time constant of recovery from I(US) was decreased from 145.0 +/- 10.2 s to 32.5 +/- 3.3 s (P < 0.001). Recovery from I(US) was only accelerated if Cd(2+) was added to the bath solution during recovery (V = -120 mV) from I(US), but not when the channels were selectively exposed to Cd(2+) during the development of I(US) (-20 mV). These data could be explained by a kinetic model in which Cd(2+) binds with high affinity to a slow inactivated state (I(S)), which is transiently occupied during recovery from I(US). A total of 50 microM Cd(2+) produced an approximately 8 mV hyperpolarizing shift of the steady-state inactivation curve of I(S), supporting this kinetic model. Binding of lidocaine to the internal vestibule significantly reduced the number of channels entering I(US), suggesting that I(US) is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (I(S)) occurs by a closure of the outer vestibule, whereas I(US) arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd(2+) to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I(US). Thus, Cd(2+) ions may act like a foot-on-the-door, kicking the I(S) gate to close.

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells.

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na+ channels in the C(2)C(12) murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca2+ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na+ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na+ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na+ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na+ channel isoform Na(v)1.5 compared with the skeletal muscle isoform Na(v)1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties.