René Cervenka

Voltage-Gated Ion Channel Dysfunction Precedes Cardiomyopathy Development in the Dystrophic Heart

Background Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is associated with severe cardiac complications including cardiomyopathy and cardiac arrhythmias. Recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. It is, however, unknown if the ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiac pathology. Methodology/Principal Findings To address this question, we first investigated sodium channel impairments in cardiomyocytes derived from dystrophic neonatal mice prior to cardiomyopahty development, by using the whole cell patch clamp technique. Besides the most common model for DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying an utrophin mutation. In neonatal cardiomyocytes, dystrophin-deficiency generated a 25% reduction in sodium current density. In addition, extra utrophin-deficiency significantly altered sodium channel gating parameters. Moreover, also calcium channel inactivation was considerably reduced in dystrophic neonatal cardiomyocytes, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. To assess developmental changes, we also studied sodium channel impairments in cardiomyocytes derived from dystrophic adult mice, and compared them with the respective abnormalities in dystrophic neonatal cells. Here, we found a much stronger sodium current reduction in adult cardiomyocytes. The described sodium channel impairments slowed the upstroke of the action potential in adult cardiomyocytes, and only in dystrophic adult mice, the QRS interval of the electrocardiogram was prolonged. Conclusions/Significance Ion channel impairments precede pathology development in the dystrophic heart, and may thus be considered potential cardiomyopathy triggers.

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 (rNaV1.4) 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,” IUS) 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 IUS. The fact that mutations at site 1575 modulate IUS 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.

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.

Sodium current properties of primary skeletal myocytes and cardiomyocytes derived from different mouse strains

The mouse has become the preferred animal for genetic manipulations. Because of the diverse genetic backgrounds of various mouse strains, these can manifest strikingly different characteristics. Here, we studied the functional properties of currents through voltage-gated sodium channels in primary cultures of skeletal myocytes and cardiomyocytes derived from the three commonly used mouse strains BL6, 129/Sv, and FVB, by using the whole-cell patch-clamp technique. We found strain-specific sodium current function in skeletal myocytes, which could partly be explained by differences in sodium channel isoform expression. In addition, we found significant effects of cell source (neonatal or adult animal-derived) and variation of the differentiation time period. In contrast to skeletal myocytes, sodium current function in cardiomyocytes was similar in all strains. Our findings are relevant for the design and proper interpretation of electrophysiological studies, which use excitable cells in primary culture as a model system.

The Outer Vestibule of the Na+ Channel–Toxin Receptor and Modulator of Permeation as Well as Gating

The outer vestibule of voltage-gated Na+ channels is formed by extracellular loops connecting the S5 and S6 segments of all four domains (“P-loops”), which fold back into the membrane. Classically, this structure has been implicated in the control of ion permeation and in toxin blockage. However, conformational changes of the outer vestibule may also result in alterations in gating, as suggested by several P-loop mutations that gave rise to gating changes. Moreover, partial pore block by mutated toxins may reverse gating changes induced by mutations. Therefore, toxins that bind to the outer vestibule can be used to modulate channel gating.

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.

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.

The anti-addictive drug ibogaine modulates voltage-gated ion channels and may trigger cardiac arrhythmias

Background Ibogaine is an alkaloid derived from the African shrub Tabernanthe iboga. Psychoactive properties of ibogaine have been known for decades, but more recently the drug has received much attention because of its promising “anti-addictive” actions. Thus, ibogaine and its derivatives are being studied as potential treatment for opioid and stimulant abuse, as well as for alcoholism and smoking. Because ibogaine has a complex pharmacology and is known to interact with numerous different cellular targets, its potential to generate adverse effects is significant. Besides the expected neurotoxic actions, ibogaine may e.g. also affect the heart. Thus, several cases of sudden death after ibogaine use were reported, which have been hypothesised to be related to cardiac arrhythmias. In accordance, a severely prolonged QT interval of the electrocardiogram and ventricular tachyarrhythmias were observed in a woman after she had taken ibogaine.

Impaired L-type Ca2+ channel function in the dystrophic heart

Background Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited disease characterized by progressive muscle weakness and degeneration. Besides the relatively well described skeletal muscle degenerative processes, DMD is associated with cardiovascular complications including cardiomyopathy and cardiac arrhythmias. The current understanding of the patho-mechanisms is still very limited, but recent research suggests, that dysfunctional ion channels in dystrophic cardiomyocytes considerably contribute to the cardiovascular complications.

Altered sodium channel function in dystrophin/utrophin-deficient cardiomyocytes

Background Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited disease characterized by progressive muscle weakness and degeneration. Besides the relatively well-described skeletal muscle degenerative processes, DMD and some other muscular dystrophy types are also associated with cardiovascular complications including cardiomyopathy and cardiac arrhythmias. The current understanding of the patho-mechanisms underlying these cardiovascular complications is still very limited, but recent research points to a contribution of dysfunctional ion channels in dystrophic cardiomyocytes. Materials and methods By using the whole cell patch-clamp technique, the functional properties of voltage-gated sodium channels were studied in cardiomyocytes derived from normal and dystrophic mice. In addition, a computer model was used to simulate the effects of altered sodium channel properties on the cardiac action potential. Besides the most common mouse model for human DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying a mutation in the utrophin gene. The mdx-utr double mutant mouse exhibits a more severe cardiac disease phenotype than the mdx mouse, and is thought to represent a more suitable animal model for human DMD. Results We found that dystrophic cardiomyocytes show a reduced sodium current density compared to wild-type cardiomyocytes. In addition, extra utrophin deficiency significantly shifted both the sodium channel activation and inactivation curve to more depolarised potentials, which was not observed in only dystrophin-deficient mdx cardiomyocytes. Computer modelling revealed that the described sodium channel impairments in dystrophic cardiomyocytes suffice to affect the action potential. Conclusions Sodium channel dysfunction may perturb electrical impulse propagation in the dystrophic heart, and thus contribute to cardiac complications associated with the muscular dystrophies.