Optical mapping of ventricular arrhythmias in LQTS mice with SCN5A mutation N1325S

Transgenic expression of SCN5A mutation N1325S creates a mouse model for type-3 long QT syndrome (LQT3), TG-NS/LQT3. Optical mapping is a high temporal and spatial resolution fluorescence mapping system that records 256 action potentials simultaneously in a Langendorff-perfused heart. Here for the first-time, we provide a spatial view of VT in a genetic LQT3 model using optical mapping. Spontaneous VT was detected in TG-NS/LQT3 hearts, but not in littermate control hearts. VT was initiated primarily by activation of a new firing focus as well as functional conduction block of new activation waves. New firing was initiated at many different Loci in the heart, suggesting that "increased automaticity" is a key mechanism for initiation of VT. The sustained VT was maintained by a reentry mechanism. Nifedipine, an L-type calcium channel blocker, decreased the frequency of VT, indicating the involvement of abnormalities of the calcium homeostasis in the genesis of VT in TG-NS/LQT3 mice.     

A novel mutation in the SCN5A gene is associated with Brugada syndrome

Brugada syndrome (BS) is an inherited cardiac disorder associated with a high risk of sudden cardiac death and is caused by mutations in the SCN5A gene encoding the cardiac sodium channel alpha-subunit (Na(v)1.5). The aim of this study was to identify the genetic cause of familial BS and characterize the electrophysiological properties of a novel SCN5A mutation (W1191X). Four families and one patient with BS were screened for SCN5A mutations by PCR and direct sequencing. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in tsA201 cells, and the sodium currents (I(Na)) were analyzed using the whole-cell patch-clamp technique. A novel mutation, W1191X, was identified in a family with BS. Expression of the WT or the mutant channel (Na(v)1.5/W1191X) co-transfected with the beta(1)-subunit in tsA201 cells resulted in a loss of function of Na(v)1.5 channels. While voltage-clamp recordings of the WT channel showed a distinct acceleration of Na(v)1.5 activation and fast inactivation kinetics, the Na(v)1.5/W1191X mutant failed to generate any currents. Co-expression of the WT channel and the mutant channel resulted in a 50% reduction in I(Na). No effect on activation and inactivation were observed with this heterozygous expression. The W1191X mutation is associated with BS and resulted in the loss of function of the cardiac sodium channel.     

Cardiac-specific overexpression of SCN5A gene leads to shorter P wave duration and PR interval in transgenic mice

The Cardiac sodium channel gene SCN5A plays a critical role in cardiac electrophysiology and its mutations, either gain- or loss-of-functions, are associated with lethal arrhythmias. In this study, we investigated the effect of overexpression of SCN5A on the cardiac phenotype in a transgenic mouse model (TG-WT L10). Compared to NTG mice, heart rate, QRS duration, and QT intervals remained unchanged in TG-WT mice. Moreover, no spontaneous ventricular arrhythmias were detected in TG-WT hearts. Despite these results, a mild, irregular cardiac phenotype was observed in TG-WT mice. The P wave and PR interval were significantly shorter in TG-WT compared with NTG mice (P, 8.8+/-0.8 ms vs. 12.6+/-0.9 ms; PR, 12.5+/-2 ms vs. 33.5+/-0.7 ms). Furthermore, spontaneous premature atrial contractions were often detected in TG-WT mice. These results suggest that the expression level of the SCN5A gene is a determinant for the length of the P wave duration and PR interval on electrocardiograms (ECG).     

Mexiletine rescues a mixed biophysical phenotype of the cardiac sodium channel arising from the SCN5A mutation,N406K,found in LQT3 patients

Introduction: Individual mutations in the SCN5A-encoding cardiac sodium channel α-subunit usually cause a single cardiac arrhythmia disorder, some cause mixed biophysical or clinical phenotypes. Here we report an infant, female patient harboring a N406K mutation in SCN5A with a marked and mixed biophysical phenotype and assess pathogenic mechanisms. Methods and Results: A patient suffered from recurrent seizures during sleep and torsades de pointes with a QTc of 530 ms. Mutational analysis identified a N406K mutation in SCN5A. The mutation was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells. After 48 hours incubation with and without mexiletine, macroscopic voltage-gated sodium current (I_Na) was measured with standard whole-cell patch clamp techniques. SCN5A-N406K elicited both a significantly decreased peak I_Na and a significantly increased late I_Na compared to wide-type (WT) channels. Furthermore, mexiletine both restored the decreased peak I_Na of the mutant channel and inhibited the increased late I_Na of the mutant channel. Conclusion: SCN5A-N406K channel displays both “gain-of-function” in late I_Na and “loss-of-function” in peak I_Na density contributing to a mixed biophysical phenotype. Moreover, our finding may provide the first example that mexiletine exerts a dual rescue of both “gain-of-function” and “loss-of-function” of the mutant sodium channel.     

Analysis of the T-type calcium channel in embryonic chick ventricular myocytes

Summary T-type calcium channels (I _T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca²⁺ with Na⁺ (120mm) as the charge carrier.I _T channels were distinguished from L-type calcium channels (I _L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times ₀ was fit by a single-exponential curve while that of closed times _c was biexponeintial. _o was the same atR+40 mV andR+60 mV whereas _c was shortened atR+60 mV. The open-state probability (P _o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP _o at depolarized potentials could be accounted for by the decrease in _c.     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

Biophysical characterization of a new SCN5A mutation S1333Y in a SIDS infant linked to long QT syndrome

Various entities and genetic etiologies, including inherited long QT syndrome type 3 (LQT3), contribute to sudden infant death syndrome (SIDS). The goal of our research was to biophysically characterize a new SCN5A mutation (S1333Y) in a SIDS infant. S1333Y channels showed the gain of Na⁺ channel function characteristic of LQT3, including a persistent inward Na⁺ current and an enhanced window current that was generated by a −8 mV shift in activation and a +7 mV shift in inactivation. The correlation between the biophysical data and arrhythmia susceptibility suggested that the SIDS was secondary to the LQT3-associated S1333Y mutation.     

Differential Ca2+ sensitivity of RyR2 mutations reveals distinct mechanisms of channel dysfunction in sudden cardiac death

Arrhythmogenic point mutations in RyR2 result in abnormal Ca(2+) release following cardiac stimulation, leading to sudden cardiac death (SCD). Recently, we have demonstrated that significant functional differences exist between SCD-linked RyR2 mutations. Here, we investigated the molecular basis of this heterogeneity and determined the sensitivity of mutant RyR2 channels to cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in living cells. Using streptolysin-O permeabilised human embryonic kidney cells, [Ca(2+)](c) was clamped in cells expressing GFP-tagged wild-type (WT) or SCD-linked RyR2 mutants (L(433)P, N(2386)I, and R(176)Q/T(2504)M). Although resting [Ca(2+)](c) was comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca(2+)-dependent inhibition following caffeine stimulation when compared with WT channels. The ER Ca(2+) store was not perturbed in these experiments. Our findings support the hypothesis that SCD-linked mutational loci may be an important mechanistic determinant of RyR2 dysfunction and indicate that there is unlikely to be a unifying mechanism for channel dysfunction in SCD.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Modeling the structural and dynamical changes of the epithelial calcium channel TRPV5 caused by the A563T variation based on the structure of TRPV6

TRPV5, transient receptor potential cation channel vanilloid subfamily member 5, is an epithelial Ca²⁺ channel that plays a key role in the active Ca²⁺ reabsorption process in the kidney. A single nucleotide polymorphism (SNP) rs4252499 in the TRPV5 gene results in an A563T variation in the sixth transmembrane (TM) domain of TRPV5. Our previous study indicated that this variation increases the Ca²⁺ transport function of TRPV5. To understand the molecular mechanism, a model of TRPV5 was established based on the newly deposited structure of TRPV6 that has 83.1% amino acid identity with TRPV5 in the modeled region. Computational simulations were performed to study the structural and dynamical differences between the TRPV5 variants with A563 and T563. Consistent with the TRPV1-based simulation, the results indicate that the A563T variation increases the contacts between residues 563 and V540, which is one residue away from the key residue D542 in the Ca²⁺-selective filter. The variation enhanced the stability of the secondary structure of the pore region, decreased the fluctuation of residues around residue 563, and reduced correlated and anti-correlated motion between monomers. Furthermore, the variation increases the pore radius at the selective filter. These findings were confirmed using simulations based on the recently determined structure of rabbit TRPV5. The simulation results provide an explanation for the observation of enhanced Ca²⁺ influx in TRPV5 caused by the A563T variation. The A563T variation is an interesting example of how a residue distant from the Ca²⁺-selective filter influences the Ca²⁺ transport function of the TRPV5 channel.

Communicated by Ramaswamy H. Sarma     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

The monoclonal antibody mAB 1A binds to the excitation--contraction coupling domain in the II-III loop of the skeletal muscle calcium channel alpha(1S) subunit

Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.     

Characterization of the novel heterozygous SCN5A genetic variant Y739D associated with Brugada syndrome

Genetic variants in SCN5A gene were identified in patients with various arrhythmogenic conditions including Brugada syndrome. Despite significant progress of last decades in studying the molecular mechanism of arrhythmia-associated SCN5A mutations, the understanding of relationship between genetics, electrophysiological consequences and clinical phenotype is lacking. We have found a novel genetic variant Y739D in the SCN5A-encoded sodium channel Na_v1.5 of a male patient with Brugada syndrome (BrS). The objective of the study was to characterize the biophysical properties of Na_v1.5-Y739D and provide possible explanation of the phenotype observed in the patient. The WT and Y739D channels were heterologously expressed in the HEK-293T cells and the whole-cell sodium currents were recorded. Substitution Y739D reduced the sodium current density by 47 ± 2% at ?20 mV, positively shifted voltage-dependent activation, accelerated both fast and slow inactivation, and decelerated recovery from the slow inactivation. The Y739D loss-of-function phenotype likely causes the BrS manifestation. In the hNa_v1.5 homology models, which are based on the cryo-EM structure of rat Na_v1.5 channel, Y739 in the extracellular loop IIS1-S2 forms H-bonds with K1381 and E1435 and pi-cation contacts with K1397 (all in loop IIIS5-P1). In contrast, Y739D accepts H-bonds from K1397 and Y1434. Substantially different contacts of Y739 and Y739D with loop IIIS5-P1 would differently transmit allosteric signals from VSD-II to the fast-inactivation gate at the N-end of helix IIIS5 and slow-inactivation gate at the C-end of helix IIIP1. This may underlie the atomic mechanism of the Y739D channel dysfunction.