Cardiac Sodium Current Research Articles

Larger late sodium current density as well as greater sensitivities to ATX II and ranolazine in rabbit left atrial than left ventricular myocytes

An increase of cardiac late sodium current (INa.L) is arrhythmogenic in atrial and ventricular tissues, but the densities of INa.L and thus the potential relative contributions of this current to sodium ion (Na(+)) influx and arrhythmogenesis in atria and ventricles are unclear. In this study, whole-cell and cell-attached patch-clamp techniques were used to measure INa.L in rabbit left atrial and ventricular myocytes under identical conditions. The density of INa.L was 67% greater in left atrial (0.50 ± 0.09 pA/pF, n = 20) than in left ventricular cells (0.30 ± 0.07 pA/pF, n = 27, P < 0.01) when elicited by step pulses from -120 to -20 mV at a rate of 0.2 Hz. Similar results were obtained using step pulses from -90 to -20 mV. Anemone toxin II (ATX II) increased INa.L with an EC50 value of 14 ± 2 nM and a Hill slope of 1.4 ± 0.1 (n = 9) in atrial myocytes and with an EC50 of 21 ± 5 nM and a Hill slope of 1.2 ± 0.1 (n = 12) in ventricular myocytes. Na(+) channel open probability (but not mean open time) was greater in atrial than in ventricular cells in the absence and presence of ATX II. The INa.L inhibitor ranolazine (3, 6, and 9 μM) reduced INa.L more in atrial than ventricular myocytes in the presence of 40 nM ATX II. In summary, rabbit left atrial myocytes have a greater density of INa.L and higher sensitivities to ATX II and ranolazine than rabbit left ventricular myocytes.

Riluzole reduces arrhythmias and myocardial damage induced by coronary occlusion in anaesthetized pigs

The cardiac persistent sodium current (IN aP ) presents a novel target for cardiac ischaemic protection. Herein we investigated the effects of the IN aP blocker riluzole in a pig model of regional myocardial ischaemia. Landrace or Large White pigs were subjected to 3h ligation of the left anterior descending coronary artery (LAD). Pigs received either saline (500mL/h, i.v.) throughout the experiment (control; n=7) or riluzole (2mg/kg in 2mL propylene glycol in 100mL saline, i.v.; RIL; n=7) between 15 and 5min prior to ligation. The arrhythmia score was calculated in 5min epochs. Myocardial damage was assessed using epicardial image analysis and histological sectioning. In the control group, all seven pigs developed premature ventricular contractions (PVC), seven developed non-sustained arrhythmias and six of seven developed sustained arrhythmias. Of the sustained arrhythmias, 23 of 28 instances were initiated by R-on-T extrasytoles (extrasystoles within the vulnerable period that can trigger re-entrant arrhythmias). In the RIL group, all seven pigs developed PVC, six of seven developed non-sustained arrhythmias and only three developed sustained arrhythmias, of which two of five instances were R-on-T initiated. The riluzole-treated pigs exhibited less myocardial damage than pigs in the control group (65% smaller surface area (P=0.008) on gross epicardial inspection, 51% less oedema (P=0.01), 53% less fibre waviness (P=0.029) assessed by haematoxylin and eosin staining and 79% fewer fragmented nuclei (P=0.009) assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling). In conclusion, riluzole significantly reduced Phase 2 (the period associated with irreversible damage) ischaemic R-on-T triggered and non-R-on-T arrhythmias and myocardial damage occurring during the 3h period of regional ischaemia.

Lasting alterations of the sodium current by short-term hyperlipidemia as a mechanism for initiation of cardiac remodeling

Clinical and animal studies indicate that increased fatty acid delivery to lean tissues induces cardiac electrical remodeling and alterations of cellular calcium homeostasis. Since this may represent a mechanism initiating cardiac dysfunction during establishment of insulin resistance and diabetes or anaerobic cardiac metabolism (ischemia), we sought to determine if short-term exposure to high plasma concentration of fatty acid in vivo was sufficient to alter the cardiac sodium current (INa) in dog ventricular myocytes. Our results show that delivery of triglycerides and nonesterified fatty acids by infusion of Intralipid + heparin (IH) for 8 h increased the amplitude of INa by 43% and shifted its activation threshold by -5 mV, closer to the resting membrane potential. Steady-state inactivation (availability) of the channels was reduced by IH with no changes in recovery from inactivation. As a consequence, INa "window" current, a strong determinant of intracellular Na+ and Ca2+ concentrations, was significantly increased. The results indicate that increased circulating fatty acids alter INa gating in manners consistent with an increased cardiac excitability and augmentation of intracellular calcium. Moreover, these changes could still be measured after the dogs were left to recover for 12 h after IH perfusion, suggesting lasting changes in INa. Our results indicate that fatty acids rapidly induce cardiac remodeling and suggest that this process may be involved in the development of cardiac dysfunctions associated to insulin resistance and diabetes.

Different Atrial and Ventricular Resting Membrane Potentials May Explain the Phenotypical Variability of a Truncating SCN5A Mutation

<h3>Background</h3> The <i>SCN5A</i> gene encodes for the alpha-subunit of the cardiac sodium channel. The cardiac sodium current I_Na is essential for generation and transmission of the action potential, and I_Na dysfunction is responsible for multiple arrhythmias. Here, we characterized a distal truncating <i>SCN5A</i> mutation, R1860GfsX12, which was identified in a family with mixed clinical phenotypes of sick sinus syndrome, atrial fibrillation, atrial flutter, and AV block. <h3>Methods and Results</h3> Patch-clamp analysis performed in HEK 293 cells expressing the mutant channel showed a 70% reduction in I_Na density compared to cells expressing the wild-type (WT) channels, consistent with western blot analysis showing a partial degradation of the mutant. In addition, the R1860Gfs12X mutation drastically altered the steady-state inactivation leading to a –25-mV shift, delayed the fast and slow inactivation, and increased the persistent current. Moreover, when mimicking the heterozygous state of the patients by coexpressing WT with R1860GfsX12, the biophysical properties were still altered. Loss of I_Na density and alteration of the inactivation properties of this mutant channel are associated with a mix of atrial phenotype and AV block within the family. The variety of clinical phenotype seen in this family for the same mutation and the absence of a ventricular phenotype led us to investigate I_Na density in HEK cells at membrane potentials mimicking a ventricular vs an atrial resting membrane potential (–86 and –83 mV, respectively). Surprisingly, despite an only 3-mV hyperpolarizing difference in the resting membrane potential of atria, we observed a further decrease in I_Na density by 40% at the atrial resting membrane potential compared to the resting membrane potential of the ventricles. <h3>Conclusions</h3> Multiple mechanisms underlie functional consequences of <i>SCN5A</i> mutations. Here, we demonstrated that one mutation may have different clinical consequences in the atria or the ventricles due to their difference in resting membrane potential based on its biophysical defects. These results could explain how this mutation led to different atrial arrhythmias and conduction defect phenotype within the same family but to the absence of a Brugada phenotype.

The cardiac sodium current Nav1.5 is functionally expressed in rabbit bronchial smooth muscle cells

A collagenase-proteinase mixture was used to isolate airway smooth muscle cells (ASMC) from rabbit bronchi, and membrane currents were recorded using the whole cell patch-clamp technique. Stepping from -100 mV to a test potential of -40 mV evoked a fast voltage-dependent Na(+) current, sometimes with an amplitude of several nanoamperes. The current disappeared within 15 min of exposure to papain + DTT (n = 6). Comparison of the current in ASMC with current mediated by NaV1.5 α-subunits expressed in human embryonic kidney cells revealed similar voltage dependences of activation (V1/2 = -42 mV for NaV1.5) and sensitivities to TTX (IC50 = 1.1 and 1.2 μM for ASMC and NaV1.5, respectively). The current in ASMC was also blocked by lidocaine (IC50 = 160 μM). Although veratridine, an agonist of voltage-gated Na(+) channels, reduced the peak current by 33%, it slowed inactivation, resulting in a fourfold increase in sustained current (measured at 25 ms after onset). In current-clamp mode, veratridine prolonged evoked action potentials from 37 ± 9 to 1,053 ± 410 ms (n = 8). Primers for NaV1.2-1.9 were used to amplify mRNA from groups of ∼20 isolated ASMC and from whole bronchial tissue by RT-PCR. Transcripts for NaV1.2, NaV1.3, and NaV1.5-1.9 were detected in whole tissue, but only NaV1.2 and NaV1.5 were detected in single cells. We conclude that freshly dispersed rabbit ASMC express a fast voltage-gated Na(+) current that is mediated mainly by the NaV1.5 subtype.

Distribution and function of sodium channel subtypes in human atrial myocardium

Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary β subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and β subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where β1 and β3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with β2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to β3 and β4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and β subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation–contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.

Sudden cardiac arrest associated with use of a non-cardiac drug that reduces cardiac excitability: evidence from bench, bedside, and community

Non-cardiac drugs that impair cardiac repolarization (electrocardiographic QT prolongation) are associated with an increased sudden cardiac arrest (SCA) risk. Emerging evidence suggests that non-cardiac drugs that impair cardiac depolarization and excitability (electrocardiographic QRS prolongation) also increase the risk for SCA. Nortriptyline, which blocks the SCN5A-encoded cardiac sodium channel, may exemplify such drugs. We aimed to study whether nortriptyline increases the risk for SCA, and to establish the underlying mechanisms. We studied QRS durations during rest/exercise in an index patient who experienced ventricular tachycardia during exercise while using nortriptyline, and compared them with those of 55 controls with/without nortriptyline and 24 controls with Brugada syndrome (BrS) without nortriptyline, who carried an SCN5A mutation. We performed molecular-genetic (exon-trapping) and functional (patch-clamp) experiments to unravel the mechanisms of QRS prolongation by nortriptyline and the SCN5A mutation found in the index patient. We conducted a prospective community-based study among 944 victims of ECG-documented SCA and 4354-matched controls to determine the risk for SCA associated with nortriptyline use. Multiple mechanisms may act in concert to increase the risk for SCA during nortriptyline use. Pharmacological (nortriptyline), genetic (loss-of-function SCN5A mutation), and/or functional (sodium channel inactivation at fast heart rates) factors conspire to reduce the cardiac sodium current and increase the risk for SCA. Nortriptyline use in the community was associated with a 4.5-fold increase in the risk for SCA [adjusted OR: 4.5 (95% CI: 1.1-19.5)], particularly when other sodium channel-blocking factors were present. Nortriptyline increases the risk for SCA in the general population, particularly in the presence of genetic and/or non-genetic factors that decrease cardiac excitability by blocking the cardiac sodium channel.

Ion channel complex disease in long QT syndrome

Sixty years have passed since the first report of congenital long syndrome (LQTS). The pathogenesis of LQTS was speculated to be ion channel dysfunction in the early 1990s, 1 and mutations in cardiac ion channels were found one after another at the end of the 20th century. 2‐4 Since mutations in cardiac ion channels induced various diseases, including LQTS, Brugada syndrome, and progressive cardiac conduction disease, these inherited arrhythmic diseases were named “ion channel diseases” or “channelopathies.” 5 At first, mutations found in inherited arrhythmia diseases were all mutations in a-subunits of the channels, which are the main components of the ion channels, the mutations in the b-subunit, an auxiliary subunit of the channel, were later reported in Jervell and Lange-Nielsen syndrome, a phenotype of LQTS. 6 It was then shown that the regulation of channels depends not only on its a- and b-subunits but also on multiple cytoskeletal and membrane proteins and that mutations in these protein genes can also cause inherited arrhythmia syndromes. For example, the a-subunit of the cardiac sodium channel (NaV1.5) encoded by the SCN5A gene composes a main channel protein and conducts cardiac sodium current. Some auxiliary proteins interact with NaV1.5 and control its transport to the sarcolemma, cellular location, and gating properties by phosphorylation or dephosphorylation, and anchor NaV1.5 to the cytoskeleton. Mutations in some of these auxiliary proteins showed clinical phenotypes similar to type 3 LQTS (gain of function) or Brugada syndrome (loss of function). Thus, the recent concept of “channelopathy” should not be limited to mutations in the channel protein itself but should be expanded to mutations in the adaptor proteins. Cardiac potassium currents that contribute to ventricular repolarization consist of a rapid activation component (IKr) and a slow activation component (IKs). IKs is activated at phases 2 and 3 of action potential, and it terminates the

Epilepsy Alters the Activity and Adrenergic Response of the Cardiac Sodium Current

Abnormalities in cardiac rhythm or repolarization are observed in epilepsy. They appear during or shortly after seizure and include atrioventricular (AV) block, atrial fibrillation, supraventricular tachycardia, ventricular premature depolarization and bundle-branch block. The ECG recordings often show altered heart rate dynamics, indicative of changes in sympathovagal balance in epileptic patients. It has been suggested that autonomic imbalance induced remodelling of the heart and altered cardiac sympathetic response. To test this hypothesis we measured the response of the cardiac sodium current INa to isoproterenol in the rat epilepsy model (kainic acid). Using patch clamp technique on isolated ventricular myocytes we found that epilepsy by itself increased maximum INa density by 76% from 59±7 pA/pF to 104±20 pA/pF. Isoproterenol increased INa more importantly in epileptic rats than in control animals with respective changes of 93% and 127%. Mid-potential for steady-state activation was shifted from −42.9±0,3 mV in control to −55±1 mV in epileptic rats. Steady state mid-inactivation went from −79±1mV to −84±1 mV. Isoproterenol attenuated the differences in steady state parameters by bringing the activation and inactivation mid potential to −63±1 mV and −89±1mV respectively in sham and epileptic rats. We conclude that epilepsy modifies INa activity in a manner consistent with an increase in cardiac excitability. Moreover, the changes in gating parameters increase sodium entry into ventricular myocytes and may lead to calcium overload and associated delayed afterdepolarizations. These mechanisms may explain in part the arrhythmias observed in epileptic patients.

A Novel, Potent, and Selective Inhibitor of Cardiac Late Sodium Current Suppresses Experimental Arrhythmias

Inhibition of cardiac late sodium current (late I(Na)) is a strategy to suppress arrhythmias and sodium-dependent calcium overload associated with myocardial ischemia and heart failure. Current inhibitors of late I(Na) are unselective and can be proarrhythmic. This study introduces GS967 (6-[4-(trifluoromethoxy)phenyl]-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine), a potent and selective inhibitor of late I(Na), and demonstrates its effectiveness to suppress ventricular arrhythmias. The effects of GS967 on rabbit ventricular myocyte ion channel currents and action potentials were determined. Anti-arrhythmic actions of GS967 were characterized in ex vivo and in vivo rabbit models of reduced repolarization reserve and ischemia. GS967 inhibited Anemonia sulcata toxin II (ATX-II)-induced late I(Na) in ventricular myocytes and isolated hearts with IC(50) values of 0.13 and 0.21 µM, respectively. Reduction of peak I(Na) by GS967 was minimal at a holding potential of -120 mV but increased at -80 mV. GS967 did not prolong action potential duration or the QRS interval. GS967 prevented and reversed proarrhythmic effects (afterdepolarizations and torsades de pointes) of the late I(Na) enhancer ATX-II and the I(Kr) inhibitor E-4031 in isolated ventricular myocytes and hearts. GS967 significantly attenuated the proarrhythmic effects of methoxamine+clofilium and suppressed ischemia-induced arrhythmias. GS967 was more potent and effective to reduce late I(Na) and arrhythmias than either flecainide or ranolazine. Results of all studies and assays of binding and activity of GS967 at numerous receptors, transporters, and enzymes indicated that GS967 selectively inhibited late I(Na). In summary, GS967 selectively suppressed late I(Na) and prevented and/or reduced the incidence of experimentally induced arrhythmias in rabbit myocytes and hearts.

Postinfarct cardiac remodeling and the substrate for sudden cardiac death: role of oxidative stress and myocardial fibrosis

“…in a heart with well-coupled myocytes, a greater number of myocytes with simultaneous early afterdepolarization and delayed afterdepolarization are required to overcome the electrotonic sink effect…” The biological changes that occur after myocardial infarction (MI) produce gross structural abnormalities in the heart, such as scar tissue, hypertrophy and chamber dilatation, which may be followed by functional deterioration and the clinical symptoms of heart failure or ventricular arrhythmia. Heart failure is a well-known risk factor for ventricular arrhythmia that is associated with significantly higher car diovascular mortality. It may be too late to cure a dysfunctional heart. Therefore, understanding the molecular mechanisms and signaling pathways involved in postMI remodeling can provide useful insights for the very important period of time that follows infarction (the ‘golden therapeutic window’). Infarcted hearts show several important pathological changes that are arrhythmogenic, such as nerve sprouting [1], action potential duration (APD) alternans [2] and a decreased cardiac sodium current [3]. Among the underlying pathophysiologic changes, oxidative stress and myocardial fibrosis are particularly impor tant for several reasons. First, there is strong experimental and clinical evidence for their arrhythmogenicity. Second, there are potential therapeutic options to prevent them. Also, oxidative stress promotes cardiac fibrosis. Finally, oxidative stress and cardiac fibrosis potentiate the arrhyth mogenic effect of each other. This article highlights the role of oxidative stress and myocardial fibrosis in providing the sub strate for sudden cardiac death in infarcted hearts.

Atrial Fibrillation Substrate in Heart Failure: Expression Levels of Cardiac Sodium Channel, Connexin 43 and Connexin 40 in the Left Atrium

Introduction: Reduction in cardiac sodium current and gap junctional proteins (connexin43 and 40) may play important role in reduction of conduction velocity of the propagating wave and providing substrate for Atrial Fibrillation (AF). We sought to determine the changes in the protein levels of SCNA5, Cx43 and Cx40 in the left atrial tissue in a dog model of heart failure.Method: In five dogs, rapid right ventricular pacing was performed at 240bpm for three weeks to induce heart failure. Then AF inducibility was assessed by rapid atrial pacing. In five control dogs, AF inducibility was assessed similarly, followed by removal of the atria. The levels of SCNA5, Cx43, and Cx40 were detected in the Posterior Left Atrium (PLA) and Left Atrial Appendage (LAA) tissues of the CHF and control dogs using western blot technique.Results: All animals with heart failure showed sustained inducible AF (i.e. >5 minutes) in the electrophysiology study. No AF was induced in the control dogs (p<0.05). Cardiac SCNA5 protein levels were decreased in both PLA and LAA tissues compared to the control (56% and 37%, P < 0.05). Cx43 and Cx40 levels were significantly decreased only in LAA tissues of the heart failure animals compared to the control (12% and 53%, P<0.05). The changes in Cx43 and Cx40 levels in PLA tissue of heart failure dogs were not statistically significant.Conclusion: Decreased level of SCNA5 protein which may result in decreased sodium current can provide substrate for AF in heart failure. In addition, significantly decreased levels of gap junctional proteins (CX43 and 40) in LAA provide additional substrate for impaired conduction and arrhythmia as well as impairing contractility and predisposing to thrombus formation in LAA.

Contribution of Local Anesthetic Binding Site Residues F1760 and Y1767 to Block of the Cardiac Na+ Channel, hNaV1.5, by Ranolazine

Amino acid residues F1760 and Y1767 in S6/Domain IV of voltage gated Na+ channels are part of the local anesthetic binding site in the inner mouth of the pore. Mutagenesis experiments have shown that use-dependent block (UDB) of the cardiac hNaV1.5 Na+ channel by the Class Ia anti-arrhythmic drug lidocaine is significantly reduced by alanine substitutions at these two sites. The antianginal drug ranolazine, which has structural similarity to lidocaine, was shown previously to block the cardiac late sodium current (INa) and to block peak and late INa in a use-dependent manner (Rajamani et al., 2009). As with lidocaine, UDB of peak INa by ranolazine was reduced in the F1760A mutant channel (Fredj et al., 2006; Wang et al., 2008). We used the F1760A and Y1767A mutants to investigate further the block by ranolazine of the closed, inactivated, and open states of the channel. To measure affinity to the open state, we used an inactivation-deficient hNaV1.5 channel (L409C/A410W, Edrich et al., 2005) in the presence and absence of the F1760A or Y1767A mutations. We found that both mutations (F1760A and Y1767A) significantly decreased UDB of peak INa, block of the open state, and block of late INa by ranolazine compared to WT channels (14-24 fold shift of the value of IC50 for ranolazine). The effect of the mutations on block by ranolazine of the resting and inactivated states was modest (1.3-1.9 fold shift of the value of IC50 for ranolazine). The results indicate that both the F1760A and Y1767A mutations reduce the effect of ranolazine to cause UDB and open state block of hNaV1.5.

A Novel Frequency Analysis Method for Assessing Kir2.1 and Nav1.5 Currents

Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, Kir2.1, and the channel encoding the cardiac fast sodium current, Nav1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

The ‘overly-sensitive’ heart: sodium channel block and QRS interval prolongation

Cardiac safety remains of paramount importance in the development of successful clinical drug candidates. Great progress has been made recently in understanding liabilities associated with delayed ventricular repolarization (manifest as QT prolongation) and in predicting (thus avoiding) drugs that delay repolarization based on application of strategic preclinical assays. Following the advances made in clinical electrophysiological monitoring and conduct of thorough QT studies, focus is now shifting towards monitoring of additional drug-induced effects, particularly on ventricular conduction (measured as changes in the QRS interval on the ECG) as part of evolving clinical thorough ECG studies. In this issue of the British Journal of Pharmacology, a study by Harmer et al. proposes provisional safety margins for QRS prolongation in man based on retrospective clinical data and a single in vitro approach to assess potency of block of cardiac sodium current (hNav1.5), the ionic current responsible for ventricular conduction (observed as QRS prolongation). The present commentary places their study in context with evolving preclinical cardiac electrophysiological safety assessments, along with discussions focused on ensuring the proper 'translation' of preclinical findings with potential clinical concerns. Given the extant limitations and uncertainties of presently available data, as well as our limited understanding of the pro-arrhythmic potential associated with these changes, due caution should be applied when considering the proposed in vitro-based margins for drug-induced QRS prolongation measured clinically. Additional validation with multiple preclinical models and more rigorous clinical safety studies will be necessary to substantiate these recommended margins. This article is a commentary on Harmer et al., pp. 260-273 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2011.01415.x.

Striking In Vivo Phenotype of a Disease-Associated Human SCN5A Mutation Producing Minimal Changes in Vitro

The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose-dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at -30 mV; DN/H, 19.2±3.1 pA/pF, P<0.001 vs. H/H; DN/DN, 9.3±1.1 pA/pF, P<0.001 versus H/H). Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.

The Persistent Sodium Current Blocker Riluzole Is Antiarrhythmic and Anti-Ischaemic in a Pig Model of Acute Myocardial Infarction

BackgroundThe potential of the cardiac persistent sodium current as a target for protection of the myocardium from ischaemia and reperfusion injury is gaining increasing interest. We have investigated the anti-ischaemic and antiarrhythmic effects of riluzole, a selective INaP blocker, in an open chest pig model of infarction.Methods and Principal FindingsThe left anterior descending coronary artery (LAD) was ligated in 27 anesthetised pigs (landrace or large white, either sex, 20–35 kg) which had received riluzole (8 mg/kg IP; n = 6), lidocaine (2.5–12 mg/kg bolus plus 0.05–0.24 mg/kg/min; n = 11) or vehicle (n = 10) 50 min prior. Arrhythmias could be delineated into phase 1a (0 to 20 min), phase 1b (20 to 50 min) and phase 2 (from 50 min to termination at 180 min) and were classified as premature ventricular contractions (PVCs), non-sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) (spontaneously reverting within 15 s) or sustained VT or VF (ie. requiring cardioversion at 15 s). Riluzole reduced the average number of all arrhythmias in Phase 2 (PVCs from 484+/−119 to 32+/−13; non sustained arrhythmias from 8.9+/−4.4 to 0.7+/−0.5; sustained arrhythmias from 3.9+/−2.2 to 0.5+/−0.4); lidocaine reduced the average number of non-sustained and sustained arrhythmias (to 0.4+/−0.3 and 0.4+/−0.3 respectively) but not PVCs (to 390+/−234). Riluzole and lidocaine reduced the average number of sustained arrhythmias in phase 1b (from 1.8+/−0.4 to 0.17+/−0.13 (p<0.02) and to 0.55+/−0.26 (p = ns) respectively). Neither lidocaine or riluzole changed the ECG intervals: there was no statistical significance between groups at time zero (just before ligation) for any ECG measure. During the course of the 3 hour period of the ischaemia R-R, and P-R intervals shortened slightly in control and riluzole groups (not significantly different from each other) but not in the lidocaine group (significantly different from control). QRS and QTc did not change appreciably in any group Riluzole reduced the degree of histopathological tissue damage across the infarct zone considerably more than did lidocaine.ConclusionsAt the doses used, riluzole was at least as effective as lidocaine at reducing the number of episodes of ischaemic VT or VF in pigs, and much more effective at reducing the number of PVCs. We propose that this is related to the ability of riluzole to block cardiac persistent sodium current.

ST segment elevation by current-to-load mismatch: an experimental and computational study

Recently, we demonstrated that ajmaline caused ST segment elevation in the heart of an SCN5A mutation carrier by excitation failure in structurally discontinuous myocardium. In patients with Brugada syndrome, ST segment elevation is modulated by cardiac sodium (I(Na)), transient outward (I(to)), and L-type calcium currents (I(CaL)). To establish experimentally whether excitation failure by current-to-load mismatch causes ST segment elevation and is modulated by I(to) and I(CaL). In porcine epicardial shavings, isthmuses of 0.9, 1.1, or 1.3 mm in width were created parallel to the fiber orientation. Local activation was recorded electrically or optically (di-4-ANEPPS) simultaneously with a pseudo-electrocardiogram (ECG) before and after ajmaline application. Intra- and extracellular potentials and ECGs were simulated in a computer model of the heart and thorax before and after introduction of right ventricular structural discontinuities and during varying levels of I(Na), I(to), and I(CaL). In epicardial shavings, conduction blocked after ajmaline in a frequency-dependent manner in all preparations with isthmuses ≤ 1.1 mm width. Total conduction block occurred in three of four preparations with isthmuses of 0.9 mm versus one of seven with isthmuses ≥ 1.1 mm (P<.05). Excitation failure resulted in ST segment elevation on the pseudo-ECG. In computer simulations, subepicardial structural discontinuities caused local activation delay and made the success of conduction sensitive to I(Na), I(to), and I(CaL). Reduction of I(to) and increase of I(CaL) resulted in a higher excitatory current, overcame subepicardial excitation failure, and reduced the ST segment elevation. Excitation failure by current-to-load mismatch causes ST segment elevation and, like ST segment elevation in Brugada patients, is modulated by I(to) and I(CaL).