Lena Rubi

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 licenced 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 hearts electrophysiology. Here, to assess the drugs 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 drugs calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaines 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.

Enhanced currents through L-type calcium channels in cardiomyocytes disturb the electrophysiology of the dystrophic heart

Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for the cytoskeletal protein dystrophin, is an inherited disease characterized by progressive muscle weakness. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with cardiac complications. These include cardiomyopathy development and cardiac arrhythmias. The current understanding of the pathomechanisms in the heart is very limited, but recent research indicates that dysfunctional ion channels in dystrophic cardiomyocytes play a role. The aim of the present study was to characterize abnormalities in L-type calcium channel function in adult dystrophic ventricular cardiomyocytes. By using the whole cell patch-clamp technique, the properties of currents through calcium channels in ventricular cardiomyocytes isolated from the hearts of normal and dystrophic adult mice were compared. Besides the commonly used dystrophin-deficient mdx mouse model for human DMD, we also used mdx-utr mice, which are both dystrophin- and utrophin-deficient. We found that calcium channel currents were significantly increased, and channel inactivation was reduced in dystrophic cardiomyocytes. Both effects enhance the calcium influx during an action potential (AP). Whereas the AP in dystrophic mouse cardiomyocytes was nearly normal, implementation of the enhanced dystrophic calcium conductance in a computer model of a human ventricular cardiomyocyte considerably prolonged the AP. Finally, the described dystrophic calcium channel abnormalities entailed alterations in the electrocardiograms of dystrophic mice. We conclude that gain of function in cardiac L-type calcium channels may disturb the electrophysiology of the dystrophic heart and thereby cause arrhythmias.

Raised Activity of L-Type Calcium Channels Renders Neurons Prone to Form Paroxysmal Depolarization Shifts

Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca2+ influx to Ca2+-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca2+ homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.

Exploring the Structure of the Voltage-Gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

Background: Currently the structure of eukaryotic voltage-gated Na+ channels (VGSCs) is predicted from available prokaryotic VGSC crystals. Results: In a mammalian VGSC, the predicted position of a multifunctional tryptophan in the external vestibule enabled design of a second conduction pathway. Conclusion: External parts of pro- and eukaryotic VGSCs are structurally similar. Significance: Engineered external drug access pathways may allow development of novel VGSC modulators. Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels.

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 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.

Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes

ABSTRACT Kir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current IK1, which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential IK1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that IK1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

Anti-addiction Drug Ibogaine Prolongs the Action Potential in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Ibogaine is a plant alkaloid used as anti-addiction drug in dozens of alternative medicine clinics worldwide. Recently, alarming reports of life-threatening cardiac arrhythmias and cases of sudden death associated with the ingestion of ibogaine have accumulated. Using whole-cell patch clamp recordings, we assessed the effects of ibogaine and its main metabolite noribogaine on action potentials in human ventricular-like cardiomyocytes derived from induced pluripotent stem cells. Therapeutic concentrations of ibogaine and its long-lived active metabolite noribogaine significantly retarded action potential repolarization in human cardiomyocytes. These findings represent the first experimental proof that ibogaine application entails a cardiac arrhythmia risk for humans. In addition, they explain the clinically observed delayed incidence of cardiac adverse events several days after ibogaine intake. We conclude that therapeutic concentrations of ibogaine retard action potential repolarization in the human heart. This may give rise to a prolongation of the QT interval in the electrocardiogram and cardiac arrhythmias.

Calcium current properties in dystrophin-deficient ventricular cardiomyocytes from aged mdx mice

Duchenne muscular dystrophy (DMD), caused by mutations in the gene encoding for the cytoskeletal protein dystrophin, is linked with severe cardiac complications including cardiomyopathy development and cardiac arrhythmias. We and others recently reported that currents through L‐type calcium (Ca) channels were significantly increased, and channel inactivation was reduced in dystrophin‐deficient ventricular cardiomyocytes derived from the mdx mouse, the most commonly used animal model for human DMD. These gain‐of‐function Ca channel abnormalities may enhance the risk of Ca‐dependent arrhythmias and cellular Ca overload in the dystrophic heart. All studies, which have so far investigated L‐type Ca channel properties in dystrophic cardiomyocytes, have used hearts from either neonatal or young adult mdx mice as cell source. In consequence, the dimension of the Ca channel abnormalities present in the severely‐diseased aged dystrophic heart has remained unknown. Here, we have studied potential abnormalities in Ca currents and intracellular Ca transients in ventricular cardiomyocytes derived from aged dystrophic mdx mice. We found that both the L‐type and T‐type Ca current properties of mdx cardiomyocytes were similar to those of myocytes derived from aged wild‐type mice. Accordingly, Ca release from the sarcoplasmic reticulum was normal in cardiomyocytes from aged mdx mice. This suggests that, irrespective of the presence of a pronounced cardiomyopathy in aged mdx mice, Ca currents and Ca release in dystrophic cardiomyocytes are normal. Finally, our data imply that dystrophin‐ regulation of L‐type Ca channel function in the heart is lost during aging.

Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes

Background/Aims: Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B). Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. Methods and Results: By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf) mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Conclusion: Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types.