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The cardiac conduction system (CCS) is a network of specialized cardiomyocytes that coordinates electrical impulse generation and propagation for synchronized heart contractions. Although the components of the CCS, including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers, were anatomically discovered more than 100 years ago, their molecular constituents and regulatory mechanisms remain incompletely understood. Here, we demonstrate the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution with spatial information. Integration of single-cell and spatial transcriptomics uncover region-specific markers and zonation patterns of expression. Network inference shows heterogeneous gene regulatory networks across the CCS. Notably, region-specific gene regulation is recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing CCS-specific transcription factors, Tbx3 and/or Irx3. This finding is supported by ATAC-seq of different CCS regions, Tbx3 ChIP-seq, and Irx motifs. Overall, this study provides comprehensive molecular profiles of the postnatal CCS and elucidates gene regulatory mechanisms contributing to its heterogeneity., (© 2024. The Author(s).)

The T618I KCNH2-encoded hERG mutation is the most frequently observed mutation in genotyped cases of the congenital short QT syndrome (SQTS), a cardiac condition associated with ventricular fibrillation and sudden death. Most T618I hERG carriers exhibit a pronounced U wave on the electrocardiogram and appear vulnerable to ventricular, but not atrial fibrillation (AF). The basis for these effects is unclear. This study used the action potential (AP) voltage clamp technique to determine effects of the T618I mutation on hERG current (I hERG ) elicited by APs from different cardiac regions. Whole-cell patch-clamp recordings were made at 37 °C of I hERG from hERG-transfected HEK-293 cells. Maximal I hERG during a ventricular AP command was increased ∼4-fold for T618I I hERG and occurred much earlier during AP repolarization. The mutation also increased peak repolarizing currents elicited by Purkinje fibre (PF) APs. Maximal wild-type (WT) I hERG current during the PF waveform was 87.2 ± 4.5% of maximal ventricular repolarizing current whilst for the T618I mutant, the comparable value was 47.7 ± 2.7%. Thus, the T618I mutation exacerbated differences in repolarizing I hERG between PF and ventricular APs; this could contribute to heterogeneity of ventricular-PF repolarization and consequently to the U waves seen in T618I carriers. The comparatively shorter duration and lack of pronounced plateau of the atrial AP led to a smaller effect of the T618I mutation during the atrial AP, which may help account for the lack of reported AF in T618I carriers. Use of a paired ventricular AP protocol revealed an alteration to protective I hERG transients that affect susceptibility to premature excitation late in AP repolarization/early in diastole. These observations may help explain altered arrhythmia susceptibility in this form of the SQTS., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations., Methods and Results: We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase., Conclusion: Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms., Methods: Mouse lines heterozygous for the Calm1 N98S allele (Calm1 N98S/+ ) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1 +/+ ) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca 2+ ] i , as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers., Results: Two biologically independent knock-in mouse lines heterozygous for the Calm1 N98S allele were generated. Calm1 N98S/+ mice of either sex and line exhibited sinus bradycardia, QT c interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1 N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of I Ca.L significantly more in Calm1 N98S/+ versus Calm1 +/+ ventricular myocytes, increasing late I Ca.L in the former. Rapidly paced Calm1 N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1 N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis., Conclusions: Heterozygosity for the Calm1 N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced I Ca.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1 N98S/+ hearts.

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a degenerative muscle disease triggered by mutations in the gene encoding for the intracellular protein dystrophin. A major source for the arrhythmias in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant mechanisms. The reason for ventricular conduction impairments and the associated arrhythmias in the dystrophic heart has remained unidentified. In the present study, we explored the hypothesis that dystrophin-deficient cardiac Purkinje fibers have reduced Na + currents ( I Na ), which would represent a potential mechanism underlying slowed ventricular conduction in the dystrophic heart. Therefore, by using a Langendorff perfusion system, we isolated Purkinje fibers from the hearts of adult wild-type control and dystrophin-deficient mdx mice. Enhanced green fluorescent protein (eGFP) expression under control of the connexin 40 gene allowed us to discriminate Purkinje fibers from eGFP-negative ventricular working cardiomyocytes after cell isolation. Finally, we recorded I Na from wild-type and dystrophic mdx Purkinje fibers for comparison by means of the whole cell patch clamp technique. We found substantially reduced I Na densities in mdx compared with wild-type Purkinje fibers, suggesting that dystrophin deficiency diminishes I Na . Because Na + channels in the Purkinje fiber membrane represent key determinants of ventricular conduction velocity, we propose that reduced I Na in Purkinje fibers at least partly explains impaired ventricular conduction and the associated arrhythmias in the dystrophic heart. NEW & NOTEWORTHY Dystrophic cardiac Purkinje fibers have abnormally reduced Na + current densities. This explains impaired ventricular conduction in the dystrophic heart.

Cardiac Purkinje cells (PCs) are implicated in lethal arrhythmias caused by cardiac diseases, mutations, and drug action. However, the pro-arrhythmic mechanisms in PCs are not entirely understood, particularly in humans, as most investigations are conducted in animals. The aims of this study are to present a novel human PCs electrophysiology biophysically-detailed computational model, and to disentangle ionic mechanisms of human Purkinje-related electrophysiology, pacemaker activity and arrhythmogenicity. The new Trovato2020 model incorporates detailed Purkinje-specific ionic currents and Ca 2+ handling, and was developed, calibrated and validated using human experimental data acquired at multiple frequencies, both in control conditions and following drug application. Multiscale investigations were performed in a Purkinje cell, in fibre and using an experimentally-calibrated population of PCs to evaluate biological variability. Simulations demonstrate the human Purkinje Trovato2020 model is the first one to yield: (i) all key AP features consistent with human Purkinje recordings; (ii) Automaticity with funny current up-regulation (iii) EADs at slow pacing and with 85% hERG block; (iv) DADs following fast pacing; (v) conduction velocity of 160 cm/s in a Purkinje fibre, as reported in human. The human in silico PCs population highlights that: (1) EADs are caused by I CaL reactivation in PCs with large inward currents; (2) DADs and triggered APs occur in PCs experiencing Ca 2+ accumulation, at fast pacing, caused by large L-type calcium current and small Na + /Ca 2+ exchanger. The novel human Purkinje model unlocks further investigations into the role of cardiac Purkinje in ventricular arrhythmias through computer modeling and multiscale simulations., Competing Interests: Declaration of Competing Interest CT, EP, NN, AV, SS and BR declare no conflicts of interest. NAG is an employee of AnaBios Corporation., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recently, four SCN5A mutations have been associated with Multifocal Ectopic Purkinje-related Premature Contractions (MEPPC), a rare cardiac syndrome combining polymorphic ventricular arrhythmia with dilated cardiomyopathy (DCM). Here, we identified a novel heterozygous mutation in SCN5A (c.611C>A, pAla204Glu) in a young woman presenting with polymorphic premature ventricular contractions (PVCs) and DCM. After failure of antiarrhythmic drugs and an attempt of radiofrequency catheter ablation showing three exit-sites of PVCs, all with presystolic Purkinje potentials, a treatment by hydroquinidine was tried, leading to an immediate and spectacular disappearance of all PVCs and normalization of cardiac function. Electrophysiological studies showed that Na v 1.5-A204E mutant channels exhibited a significant leftward shift of 8 mV of the activation curve, leading to a larger hyperpolarized window current when compared to wild-type. Action potential modeling using Purkinje fiber and ventricular cell models predicted an arrhythmogenic effect predominant in Purkinje fibers for the A204E mutation. Comparison with other MEPPC-associated Na v 1.5 mutations revealed a common electrophysiological pattern of abnormal voltage-dependence of activation leading to a larger hyperpolarized window current as a shared biophysical mechanism of this syndrome. These features of the mutant sodium channels are likely to be responsible for the hyperexcitability of the fascicular-Purkinje system observed in patients with MEPPC., (© 2020 Wiley Periodicals, Inc.)

Transient receptor potential (TRP) channels are nonselective cationic channels that are generally Ca 2+ permeable and have a heterogeneous expression in the heart. In the myocardium, TRP channels participate in several physiological functions, such as modulation of action potential waveform, pacemaking, conduction, inotropy, lusitropy, Ca 2+ and Mg 2+ handling, store-operated Ca 2+ entry, embryonic development, mitochondrial function and adaptive remodelling. Moreover, TRP channels are also involved in various pathological mechanisms, such as arrhythmias, ischaemia-reperfusion injuries, Ca 2+ -handling defects, fibrosis, maladaptive remodelling, inherited cardiopathies and cell death. In this Review, we present the current knowledge of the roles of TRP channels in different cardiac regions (sinus node, atria, ventricles and Purkinje fibres) and cells types (cardiomyocytes and fibroblasts) and discuss their contribution to pathophysiological mechanisms, which will help to identify the best candidates for new therapeutic targets among the cardiac TRP family.

Transient receptor potential melastatin member 4 (TRPM4) channels are nonselective monovalent cationic channels found in human atria and conduction system. Overexpression of TRPM4 channels has been found in families suffering from inherited cardiac arrhythmias, notably heart block. In this study, we integrate a mathematical formulation of the TRPM4 channel into a Purkinje cell model (Pan-Rudy model). Instead of simply adding the channel to the model, a combination of existing currents equivalent to the TRPM4 current was constructed, based on TRPM4 current dynamics. The equivalent current was then replaced by the TRPM4 current to preserve the model action potential. Single-cell behavior showed early afterdepolarizations for increases in TRPM4 channel expression above twofold. In a homogeneous strand of tissue, propagation conducted faithfully for lower expression levels but failed completely for more than a doubling of TRPM4 channel expression. Only with a heterogeneous distribution of channel expression was intermittent heart block seen. This study suggests that in Purkinje fibers, TRPM4 channels may account for sodium background current (I Nab ), and that a heterogeneous expression of TRPM4 channels in the His/Purkinje system is required for type II heart block, as seen clinically., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mammals and birds have a specialized cardiac atrioventricular conduction system enabling rapid activation of both ventricles. This system may have evolved together with high heart rates to support their endothermic state (warm-bloodedness) and is seemingly lacking in ectothermic vertebrates from which first mammals then birds independently evolved. Here, we studied the conduction system in crocodiles ( Alligator mississippiensis ), the only ectothermic vertebrates with a full ventricular septum. We identified homologues of mammalian conduction system markers ( Tbx3-Tbx5, Scn5a, Gja5, Nppa-Nppb ) and show the presence of a functional atrioventricular bundle. The ventricular Purkinje network, however, was absent and slow ventricular conduction relied on trabecular myocardium, as it does in other ectothermic vertebrates. We propose the evolution of the atrioventricular bundle followed full ventricular septum formation prior to the development of high heart rates and endothermy. In contrast, the evolution of the ventricular Purkinje network is strongly associated with high heart rates and endothermy., Competing Interests: BJ, BB, DC, JC, RM, Kv, AP, CG, RE, DS, IE, VC No competing interests declared

Background and Purpose: We evaluated the concordance of results from two sets of nonclinical cardiovascular safety studies on pitolisant., Experimental Approach: Nonclinical studies envisaged both in the International Conference on Harmonization (ICH) S7B guideline and Comprehensive in vitro Pro-arrhythmia Assay (CiPA) initiative were undertaken. The CiPA initiative included in vitro ion channels, stem cell-derived human ventricular myocytes, and in silico modelling to simulate human ventricular electrophysiology. ICH S7B-recommended assays included in vitro hERG (K V 11.1) channels, in vivo dog studies with follow-up investigations in rabbit Purkinje fibres and the in vivo Carlsson rabbit pro-arrhythmia model., Key Results: Both sets of nonclinical data consistently excluded pitolisant from having clinically relevant QT-liability or pro-arrhythmic potential. CiPA studies revealed pitolisant to have modest calcium channel blocking and late I Na reducing activities at high concentrations, which resulted in pitolisant reducing dofetilide-induced early after-depolarizations (EADs) in the ICH S7B studies. Studies in stem cell-derived human cardiomyocytes with dofetilide or E-4031 given alone and in combination with pitolisant confirmed these properties. In silico modelling confirmed that the ion channel effects measured are consistent with results from both the stem cell-derived cardiomyocytes and rabbit Purkinje fibres and categorized pitolisant as a drug with low torsadogenic potential. Results from the two sets of nonclinical studies correlated well with those from two clinical QT studies., Conclusions and Implications: Our findings support the CiPA initiative but suggest that sponsors should consider investigating drug effects on EADs and the use of pro-arrhythmia models when the results from CiPA studies are ambiguous., (© 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Surviving Purkinje fibers in myocardial infarct are regarded as an important substrate in arrhythmogenesis. However, poorly understood are functional properties of Purkinje fibers in the infarcted heart. We sought to visualize intracellular Ca 2+ ([Ca 2+ ] i ) dynamics of Purkinje fiber networks in the mouse myocardial infarct. Using 3- to 4-day-old or 7- to 9-day-old infarcted hearts after the left coronary-artery ligation corresponding, respectively, to acute or healing phase, we conducted rapid fluo4-fluorescence imaging on the endocardial surface of the left ventricular septum by macro-zoom fluorescence microscopy and rapid-scanning confocal microscopy. In contrast with the intact heart, where uniform Ca 2+ transients propagated rapidly, the infarcted heart exhibited slow, non-uniform impulse propagations. On confocal microscopy, Purkinje fibers in the peri-infarct zone exhibited non-uniform [Ca 2+ ] i dynamics: beat-to-beat alternans of the Ca 2+ transient amplitude in and among the individual fibers, whereas the intact fibers exhibited uniform Ca 2+ transients. Such non-uniform [Ca 2+ ] i dynamics were more conspicuous in the acute infarcted hearts than in the healing ones. In accordance with [Ca 2+ ] i dynamics, fixed fluo4-loaded heart preparations exhibited definitive connexin-40 plaques in the peri-infarct Purkinje fibers, whereas the subjacent myocardium presented coagulative necrosis and granulation tissues, respectively. The surviving Purkinje fibers in the peri-infarct zone exhibited non-uniform [Ca 2+ ] i dynamics, which may lead to arrhythmogenesis.

Background: Purkinje cells (PCs) are important in cardiac arrhythmogenesis. Whether small-conductance calcium-activated potassium (SK) channels are present in PCs remains unclear. We tested the hypotheses that subtype 2 SK (SK2) channel proteins and apamin-sensitive SK currents are abundantly present in PCs., Methods and Results: We studied 25 normal rabbit ventricles, including 13 patch-clamp studies, 4 for Western blotting, and 8 for immunohistochemical staining. Transmembrane action potentials were recorded in current-clamp mode using the perforated-patch technique. For PCs, the apamin (100 nmol/L) significantly prolonged action potential duration measured to 80% repolarization by an average of 10.4 ms (95% CI, 0.11-20.72) (n=9, P =0.047). Voltage-clamp study showed that apamin-sensitive SK current density was significantly larger in PCs compared with ventricular myocytes at potentials ≥0 mV. Western blotting of SK2 expression showed that the SK2 protein expression in the midmyocardium was 58% ( P =0.028) and the epicardium was 50% ( P =0.018) of that in the pseudotendons. Immunostaining of SK2 protein showed that PCs stained stronger than ventricular myocytes. Confocal microscope study showed SK2 protein was distributed to the periphery of the PCs., Conclusions: SK2 proteins are more abundantly present in the PCs than in the ventricular myocytes of normal rabbit ventricles. Apamin-sensitive SK current is important in ventricular repolarization of normal PCs., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Background: Three distinct Ca 2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca 2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca 2+ cycling of P-cells is unclear. Recent modeling of Ca 2+ mobilization in P-cells suggested that Ca 2+ sensitivity of Ca 2+ induced Ca 2+ release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca 2+ release exists in live P-cells. We compared the regional Ca 2+ dynamics with the arrangement of intracellular Ca 2+ release (CR) channels., Methods: Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca 2+ activity and electrically-evoked Ca 2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca 2+ events. We used the Ca 2+ propagation velocities as indicators of channel Ca 2+ sensitivity., Results: RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP 3 R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca 2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca 2+ -transients: (1) large asymmetrical Ca 2+ sparks under the membrane, and (2) typical Ca 2+ -wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca 2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3μM) and xestospongin C (XeC; 50μM) dramatically reduced their frequency. The Ca 2+ wavelets propagated in a 5-10μm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10μM). The velocity of wavelets was two times larger (86±16μm/s; n=14) compared to CWWs' (46±10μm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca 2+ concentration under the membrane which preceded the propagation of Ca 2+ into the interior of the cell. Elevated Ca i propagated at 150μm/s (147±34μm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24μm/s; n=5) in the central region wherein predominant RyR2 expression was detected., Conclusion: We identified two layers of distinct Ca 2+ release channels in the periphery of Pcell: an outer layer of InsP 3 Rs under the membrane and an inner layer of RyR3s. The propagation of Ca 2+ events in these layers revealed that Ca 2+ sensitivity of Ca 2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Key Points: The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells., Abstract: Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate cardiac conduction and be involved in triggering arrhythmias., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300-1,800 working cardiomyocytes or 90-160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia.

Glucocorticoids are known to be involved in myocardial regeneration and destruction. Cardiomyocytes are mostly devoid of nuclear glucocorticoid receptors (GRs) and it is generally assumed that effects of adrenal steroids in heart are mediated through the mineralocorticoid receptor (MR). Here we used immunocytochemistry to study localization of corticosteroid binding globulin (CBG) in semithin sections of human cardiac tissue samples. With staining of consecutive sections we examined colocalization with GR and MR immunoreactivities. While GR staining was almost undetectable, a portion of myocytes with MR immunostained nuclei was found. Almost all cardiomyocytes exhibited CBG immunostaining in cytoplasm and on the cell membrane. Most pronounced CBG immunoreactivities were found in Purkinje fibers and in smooth muscle cells of arterial walls. With RT-PCR, we found in homogenates of cardiac tissue detectable levels of CBG encoding mRNA. Our findings indicate that CBG is expressed in human heart. Known cardiac effects of adrenal steroids may in part be mediated through the binding globulin and its putative membrane receptor in addition to nuclear steroid receptors and direct genomic action. Highlights of our study: Human cardiomyocytes express mineralocorticoid receptors, but are mostly free of nuclear glucocorticoid receptors. CBG is expressed in myocardium and in Purkinje fibers. CBG in heart is colocalized with mineralocorticoid receptor. Endothelia and smooth muscle cells of arterial walls show colocalization of CBG and MR., (© Georg Thieme Verlag KG Stuttgart · New York.)

Optogenetics provides an alternative to electrical stimulation to manipulate membrane voltage, and trigger or modify action potentials (APs) in excitable cells. We compare biophysically and energetically the cellular responses to direct electrical current injection versus optical stimulation mediated by genetically expressed light-sensitive ion channels, e.g., Channelrhodopsin-2 (ChR2). Using a computational model of ChR2(H134R mutant), we show that both stimulation modalities produce similar-in-morphology APs in human cardiomyocytes, and that electrical and optical excitability vary with cell type in a similar fashion. However, whereas the strength-duration curves for electrical excitation in ventricular and atrial cardiomyocytes closely follow the theoretical exponential relationship for an equivalent RC circuit, the respective optical strength-duration curves significantly deviate, exhibiting higher nonlinearity. We trace the origin of this deviation to the waveform of the excitatory current-a nonrectangular self-terminating inward current produced in optical stimulation due to ChR2 kinetics and voltage-dependent rectification. Using a unifying charge measure to compare energy needed for electrical and optical stimulation, we reveal that direct electrical current injection (rectangular pulse) is more efficient at short pulses, whereas voltage-mediated negative feedback leads to self-termination of ChR2 current and renders optical stimulation more efficient for long low-intensity pulses. This applies to cardiomyocytes but not to neuronal cells (with much shorter APs). Furthermore, we demonstrate the cell-specific use of ChR2 current as a unique modulator of intrinsic activity, allowing for optical control of AP duration in atrial and, to a lesser degree, in ventricular myocytes. For self-oscillatory cells, such as Purkinje, constant light at extremely low irradiance can be used for fine control of oscillatory frequency, whereas constant electrical stimulation is not feasible due to electrochemical limitations. Our analysis offers insights for designing future new energy-efficient stimulation strategies in heart or brain., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Augmented Na(+) /Ca(2+) exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca(2+) i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca(2+) i rise in conditions when [Ca(2+) ]i was augmented via activation of the late sodium current (INaL ) or inhibition of the Na(+) /K(+) pump., Experimental Approach: Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX ) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca(2+) i transients (CaTs) were monitored with a Ca(2+) -sensitive fluorescent dye, Fluo-4., Key Results: Enhanced INaL increased the Ca(2+) load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca(2+) ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca(2+) release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion., Conclusions and Implications: Selective NCX inhibition - presumably by blocking rev INCX (reverse mode NCX current) - is effective against arrhythmogenesis caused by [Na(+) ]i -induced [Ca(2+) ]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca(2+) i handling, should be considered as a promising anti-arrhythmic therapeutic strategy., (© 2014 The British Pharmacological Society.)

Background: The cardiac conduction system consists of the sinus node, nodal extensions, atrioventricular (AV) node, penetrating bundle, bundle branches, and Purkinje fibers. Node-like AV ring tissue also exists at the AV junctions, and the right and left rings unite at the retroaortic node. The study aims were to (1) construct a 3-dimensional anatomical model of the AV rings and retroaortic node, (2) map electrical activation in the right ring and study its action potential characteristics, and (3) examine gene expression in the right ring and retroaortic node., Methods and Results: Three-dimensional reconstruction (based on magnetic resonance imaging, histology, and immunohistochemistry) showed the extent and organization of the specialized tissues (eg, how the AV rings form the right and left nodal extensions into the AV node). Multiextracellular electrode array and microelectrode mapping of isolated right ring preparations revealed robust spontaneous activity with characteristic diastolic depolarization. Using laser microdissection gene expression measured at the mRNA level (using quantitative PCR) and protein level (using immunohistochemistry and Western blotting) showed that the right ring and retroaortic node, like the sinus node and AV node but, unlike ventricular muscle, had statistically significant higher expression of key transcription factors (including Tbx3, Msx2, and Id2) and ion channels (including HCN4, Cav3.1, Cav3.2, Kv1.5, SK1, Kir3.1, and Kir3.4) and lower expression of other key ion channels (Nav1.5 and Kir2.1)., Conclusions: The AV rings and retroaortic node possess gene expression profiles similar to that of the AV node. Ion channel expression and electrophysiological recordings show the AV rings could act as ectopic pacemakers and a source of atrial tachycardia.

Background and Purpose: At present there are no small molecule inhibitors that show strong selectivity for the Na(+) /Ca(2+) exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM-10103, a new NCX inhibitor, on the NCX and L-type Ca(2+) currents and on the formation of early and delayed afterdepolarizations., Experimental Approach: Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea pig ventricular preparations with the use of microelectrodes., Key Results: ORM-10103 significantly reduced both the inward and outward NCX currents. Even at a high concentration (10 μM), ORM-10103 did not significantly change the L-type Ca(2+) current or the maximum rate of depolarization (dV/dtmax ), indicative of the fast inward Na(+) current. At 10 μM ORM-10103 did not affect the amplitude or the dV/dtmax of the slow response action potentials recorded from guinea pig papillary muscles, which suggests it had no effect on the L-type Ca(2+) current. ORM-10103 did not influence the Na(+) /K(+) pump or the main K(+) currents of canine ventricular myocytes, except the rapid delayed rectifier K(+) current, which was slightly diminished by the drug at 3 μM. The amplitudes of pharmacologically- induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 μM) in a concentration-dependent manner., Conclusions and Implications: ORM-10103 is a selective inhibitor of the NCX current and can abolish triggered arrhythmias. Hence, it has the potential to be used to prevent arrhythmogenic events., (© 2013 The British Pharmacological Society.)

Despite strong suspicion that abnormal Ca(2+) handling in Purkinje cells (P-cells) is implicated in life-threatening forms of ventricular tachycardias, the mechanism underlying the Ca(2+) cycling of these cells under normal conditions is still unclear. There is mounting evidence that P-cells have a unique Ca(2+) handling system. Notably complex spontaneous Ca(2+) activity was previously recorded in canine P-cells and was explained by a mechanistic hypothesis involving a triple layered system of Ca(2+) release channels. Here we examined the validity of this hypothesis for the electrically evoked Ca(2+) transient which was shown, in the dog and rabbit, to occur progressively from the periphery to the interior of the cell. To do so, the hypothesis was incorporated in a model of intracellular Ca(2+) dynamics which was then used to reproduce numerically the Ca(2+) activity of P-cells under stimulated conditions. The modelling was thus performed through a 2D computational array that encompassed three distinct Ca(2+) release nodes arranged, respectively, into three consecutive adjacent regions. A system of partial differential equations (PDEs) expressed numerically the principal cellular functions that modulate the local cytosolic Ca(2+) concentration (Cai). The apparent node-to-node progression of elevated Cai was obtained by combining Ca(2+) diffusion and 'Ca(2+)-induced Ca(2+) release'. To provide the modelling with a reliable experimental reference, we first re-examined the Ca(2+) mobilization in swine stimulated P-cells by 2D confocal microscopy. As reported earlier for the dog and rabbit, a centripetal Ca(2+) transient was readily visible in 22 stimulated P-cells from six adult Yucatan swine hearts (pacing rate: 0.1 Hz; pulse duration: 25 ms, pulse amplitude: 10% above threshold; 1 mm Ca(2+); 35°C; pH 7.3). An accurate replication of the observed centripetal Ca(2+) propagation was generated by the model for four representative cell examples and confirmed by statistical comparisons of simulations against cell data. Selective inactivation of Ca(2+) release regions of the computational array showed that an intermediate layer of Ca(2+) release nodes with an ~30-40% lower Ca(2+) activation threshold was required to reproduce the phenomenon. Our computational analysis was therefore fully consistent with the activation of a triple layered system of Ca(2+) release channels as a mechanism of centripetal Ca(2+) signalling in P-cells. Moreover, the model clearly indicated that the intermediate Ca(2+) release layer with increased sensitivity for Ca(2+) plays an important role in the specific intracellular Ca(2+) mobilization of Purkinje fibres and could therefore be a relevant determinant of cardiac conduction.

We have previously found that modest chronic increases in maternal cortisol result in an enlarged fetal heart. To explore the mechanisms of this effect, we used intrapericardial infusions of a mineralocorticoid receptor (MR) antagonist (canrenoate) or of a glucocorticoid receptor (GR) antagonist (mifepristone) in the fetus during maternal infusion of cortisol (1 mg·kg⁻¹·day⁻¹). We have shown that the MR antagonist blocked the increase in fetal heart weight and in wall thickness resulting from maternal cortisol infusion. In the current study we extended those studies and found that cortisol increased Ki67 staining in both ventricles, indicating cell proliferation, but also increased active caspase-3 staining in cells of the conduction pathway in the septum and subendocardial layers of the left ventricle, suggesting increased apoptosis in Purkinje fibers. The MR antagonist blocked the increase in cell proliferation, whereas the GR antagonist blocked the increased apoptosis in Purkinje fibers. We also found evidence of activation of caspase-3 in c-kit-positive cells, suggesting apoptosis in stem cell populations in the ventricle. These studies suggest a potentially important role of corticosteroids in the terminal remodeling of the late gestation fetal heart and suggest a mechanism for the cardiac enlargement with excess corticosteroid exposure.

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.

Introduction: It is generally accepted that at least 2 major mechanisms contribute to sinus node (SN) pacemaking: a membrane voltage (mainly I(f) ) clock and a calcium (Ca) clock (localized submembrane sarcoplasmic reticulum Ca(2+) release during late diastolic depolarization). The aim of this study was to compare the contributions of each mechanism to pacemaker activity in SN and Purkinje fibers (PFs) exhibiting normal or abnormal automaticity., Methods and Results: Conventional microelectrodes were used to record action potentials in isolated spontaneously beating canine SN and free running PF in control and in the presence of 0.1 μM isoproterenol. Ryanodine (0.1-3 μM) and ivabradine (3 μM) were used to inhibit sarcoplasmic reticulum Ca(2+) release or I(f), respectively. To induce automaticity at low membrane potentials, PFs were superfused with BaCl(2). In SN, ivabradine reduced the rate whereas ryanodine had no effect. Isoproterenol significantly accelerated automatic rate, which was decreased by ivabradine and ryanodine. In normally polarized PFs, ryanodine had no effects on the automatic rate in the absence or presence of isoproterenol, whereas ivabradine inhibited both control and isoproterenol-accelerated automaticity. In PF depolarized with BaCl(2), ivabradine decreased BaCl(2) -induced automatic rate while ryanodine had no effect., Conclusion: In canine SN, I(f) contributes to both basal automaticity and β-adrenergic-induced rate acceleration while the ryanodine-inhibited Ca clock appears more involved in β-adrenergic regulation of pacemaker rate. In PF, normal automaticity depends mainly on I(f). Inhibition of basal potassium conductance results in high automatic rates at depolarized membrane potentials with SN-like responses to inhibition of membrane and Ca clocks., (© 2012 Wiley Periodicals, Inc.)

The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Given its critical role in coordinating cardiac performance, a detailed analysis of the molecular mechanisms underlying conduction system formation should inform our understanding of arrhythmia pathophysiology and affect the development of novel therapeutic strategies. Historically, the ability to distinguish cells of the conduction system from neighboring working myocytes presented a major technical challenge for performing comprehensive mechanistic studies. Early lineage tracing experiments suggested that conduction cells derive from cardiomyocyte precursors, and these claims have been substantiated by using more contemporary approaches. However, regional specialization of conduction cells adds an additional layer of complexity to this system, and it appears that different components of the conduction system utilize unique modes of developmental formation. The identification of numerous transcription factors and their downstream target genes involved in regional differentiation of the conduction system has provided insight into how lineage commitment is achieved. Furthermore, by adopting cutting-edge genetic techniques in combination with sophisticated phenotyping capabilities, investigators have made substantial progress in delineating the regulatory networks that orchestrate conduction system formation and their role in cardiac rhythm and physiology. This review describes the connectivity of these gene regulatory networks in cardiac conduction system development and discusses how they provide a foundation for understanding normal and pathological human cardiac rhythms.

Rationale: β-Adrenergic receptor stimulation produces sarcoplasmic reticulum Ca(2+) overload and delayed afterdepolarizations in isolated ventricular myocytes. How delayed afterdepolarizations are synchronized to overcome the source-sink mismatch and produce focal arrhythmia in the intact heart remains unknown., Objective: To determine whether local β-adrenergic receptor stimulation produces spatiotemporal synchronization of delayed afterdepolarizations and to examine the effects of tissue geometry and cell-cell coupling on the induction of focal arrhythmia., Methods and Results: Simultaneous optical mapping of transmembrane potential and Ca(2+) transients was performed in normal rabbit hearts during subepicardial injections (50 μL) of norepinephrine (NE) or control (normal Tyrode's solution). Local NE produced premature ventricular complexes (PVCs) from the injection site that were dose-dependent (low-dose [30-60 μmol/L], 0.45±0.62 PVCs per injection; high-dose [125-250 μmol/L], 1.33±1.46 PVCs per injection; P<0.0001) and were inhibited by propranolol. NE-induced PVCs exhibited abnormal voltage-Ca(2+) delay at the initiation site and were inhibited by either sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibition or reduced perfusate [Ca(2+)], which indicates a Ca(2+)-mediated mechanism. NE-induced PVCs were more common at right ventricular than at left ventricular sites (1.48±1.50 versus 0.55±0.89, P<0.01), and this was unchanged after chemical ablation of endocardial Purkinje fibers, which suggests that source-sink interactions may contribute to the greater propensity to right ventricular PVCs. Partial gap junction uncoupling with carbenoxolone (25 μmol/L) increased focal activity (2.18±1.43 versus 1.33±1.46 PVCs per injection, P<0.05), which further supports source-sink balance as a critical mediator of Ca(2+)-induced PVCs., Conclusions: These data provide the first experimental demonstration that localized β-adrenergic receptor stimulation produces spatiotemporal synchronization of sarcoplasmic reticulum Ca(2+) overload and release in the intact heart and highlight the critical nature of source-sink balance in initiating focal arrhythmias.

Background and Purpose: Action potential (AP) recordings in ex vivo heart preparations constitute an important component of the preclinical cardiac safety assessment according to the ICH S7B guideline. Most AP measurement models are sensitive, predictive and informative but suffer from a low throughput. Here, effects of selected anti-arrhythmics (flecainide, quinidine, atenolol, sotalol, dofetilide, nifedipine, verapamil) on field/action potentials (FP/AP) of guinea pig and rabbit ventricular slices are presented and compared with data from established in vitro and in vivo models., Experimental Approach: Data from measurements of membrane currents (hERG, I(Na) ), AP/FP (guinea pig and rabbit ventricular slices), AP (rabbit Purkinje fibre), haemodynamic/ECG parameters (conscious, telemetered dog) were collected, compared and correlated to complementary published data (focused literature search)., Key Results: The selected anti-arrhythmics, flecainide, quinidine, atenolol, sotalol, dofetilide, nifedipine and verapamil, influenced the shape of AP/FP of guinea pig and rabbit ventricular slices in a manner similar to that observed for rabbit PF. The findings obtained from slice preparations are in line with measurements of membrane currents in vitro, papillary muscle AP in vitro and haemodynamic/ECG parameters from conscious dogs in vivo, and were also corroborated by published data., Conclusion and Implications: FP and AP recordings from heart slices correlated well with established in vitro and in vivo models in terms of pharmacology and predictability. Heart slice preparations yield similar results as papillary muscle but offer enhanced throughput for mechanistic investigations and may substantially reduce the use of laboratory animals., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

The cerebellum plays a fundamental, but as yet poorly understood, role in the control of locomotion. Recently, mice with gene mutations or knockouts have been used to investigate various aspects of cerebellar function with regard to locomotion. Although many of the mutant mice exhibit severe gait ataxia, kinematic analyses of limb movements have been performed in only a few cases. Here, we investigated locomotion in ho15J mice that have a mutation of the δ2 glutamate receptor. The cerebellum of ho15J mice shows a severe reduction in the number of parallel fiber-Purkinje synapses compared with wild-type mice. Analysis of hindlimb kinematics during treadmill locomotion showed abnormal hindlimb movements characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles in ho15J mice. The great trochanter heights in ho15J mice were lower than in wild-type mice throughout the step cycle. However, there were no significant differences in various temporal parameters between ho15J and wild-type mice. We suggest that dysfunction of the cerebellar neuronal circuits underlies the observed characteristic kinematic abnormality of hindlimb movements during locomotion of ho15J mice.

Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia.

Functioning of the cardiac conduction system depends critically on its structure and its complement of ion channels. Therefore, the aim of this study was to document both the structure and ion channel expression of the left and right ventricular His-Purkinje networks, as we have previously done for the sinoatrial and atrioventricular nodes. A three-dimensional (3D) anatomical computer model of the His-Purkinje network of the rabbit heart was constructed after staining the network by immunoenzyme labelling of a marker protein, middle neurofilament. The bundle of His is a ribbon-like structure and the architecture of the His-Purkinje network differs between the left and right ventricles. The 3D model is able to explain the breakthrough points of the action potential on the ventricular epicardium during sinus rhythm. Using quantitative PCR, the expression levels of the major ion channels were measured in the free running left and right Purkinje fibres of the rabbit heart. Expression of ion channels differs from that of the working myocardium and can explain the specialised electrical activity of the Purkinje fibres as suggested by computer simulations; the expression profile of the left Purkinje fibres is more specialised than that of the right Purkinje fibres. The structure and ion channel expression of the Purkinje fibres are highly specialised and tailored to the functioning of the system. The His-Purkinje network in the left ventricle is more developed than that in the right ventricle and this may explain its greater clinical importance., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Histiocytoid cardiomyopathy (HC) is a rare but distinctive arrhythmogenic disorder characterized by incessant ventricular tachycardia, cardiomegaly, and often sudden death by age 2 years. The underlying genetic mechanism of HC has eluded researchers for decades. To further identify the potential molecular-genetic bases of HC, molecular analyses of HC hearts and hearts of age-matched controls were performed. Total RNA and genomic DNA were prepared from formalin-fixed, paraffin-embedded cardiac tissue from 12 cases of HC and 12 age-matched controls. To identify genes differentially expressed in HC, whole genome cDNA-mediated annealing, selection, extension, and ligation profiling was performed. TaqMan quantitative polymerase chain reaction confirmed changes in RNA expression. DNA copy number changes were measured by TaqMan copy number variant analysis. Analysis of differential gene expression in HC cases identified 2 significantly downregulated gene sets aligned sequentially along the genome. The 1st gene cluster consisted of genes S100A8 , S100A9 , and S100A12 at 1q21.3c, and the 2nd cluster consisted of genes IL1RL1 ( ST2 ), IL18R1 , and IL18RAP at 2q12.1a. Strong decreases in interleukin 33 expression were also observed. Decreases in copy number of the S100A genes were confirmed by TaqMan copy number variant assays. S100A genes are downstream of the p38-MAPK pathway that can be activated by interleukin 33 signaling. These data suggest a model in which the interleukin 33-IL1RL1/p38-MAPK/ S100A8-S100A9 axis is downregulated in HC cardiac tissue and provide several candidate genes on 1q21.3c and 2q12.1a for inherited mutations that may predispose individuals to HC.

The electrophysiological (standard intracellular microelectrode technique) and pro-arrhythmic (occurrence of early after-depolarization) effects of five class III agents acting on delayed rectifier current (I(K)), rapid (I(Kr)), and/or slow (I(Ks)) components have been studied in rabbit Purkinje fibers taken near the septum and submitted in vitro to reduced stimulation rate (from 1 to 0.5 Hz) in the absence or presence of epinephrine (10 nm) during normoxic conditions. There were two I(Kr) blockers (d-sotalol and dofetilide), two I(Ks) blockers (chromanol 293B and HMR 1556), and a non-selective I(K) blocker (azimilide). d-sotalol, dofetilide, and azimilide lengthened APD(60) and APD(90) in a concentration-dependent manner. Both d-sotalol and dofetilide showed pro-arrhythmia at highest concentrations and in the presence of epinephrine and lower stimulation rate. Despite azimilide markedly lengthened APD(90), it was globally less pro-arrhythmic than dofetilide. Thus, in normoxic rabbit Purkinje fibers, I(Kr) blockade prolonged action potential duration (APD) and increased the incidence of early after-depolarizations, particularly so in the presence of adrenergic stimulation and bradycardia, I(Ks) blockade did neither, and non-selective I(K) blockade (by azimilide) behaved principally as I(Kr) blockade. It is concluded that in normoxic rabbit Purkinje fibers, I(Ks) blockade was neutral, whereas I(Kr) blockade was pro-arrhythmic, which may make a difference worth exploration in more complex models., (© 2010 The Authors Fundamental and Clinical Pharmacology © 2010 Société Française de Pharmacologie et de Thérapeutique.)

This investigation was aimed at increasing both accuracy and performance of systems used to obtain and measure V(max) (dV/dt(max)), an important yet underevaluated physiological parameter. A method is presented to correct measured V(max) (V(mes)) based on an algorythm adapted to 2 tested systems: IOX and DataPac. We also investigated 89 rabbit Purkinje fibres (before and 30 min following drugs effective on ventricular repolarization) to derive experimental electrophysiological correlations. In fact, no method may be reliable without knowing its accuracy over a large scale of representative physiological values. This is why it is essential to estimate accuracy, precision and fidelity of systems aimed at action potential recording before pharmacological or pathophysiological investigations are performed, even more if therapeutical consequences might ensue. A formula is presented to obtain real V(max), based on V(mes) [V(max)=V(mes)/1 - (tau.V(mes)/APA)(2.p)], where tau=49.64 µs, p=0.72 and APA=action potential amplitude. This formula is reliable up to V(max) values of 1000 V/s which may be seen in rabbit Purkinje fibres, a classical model for in vitro studies. Using this formula may have practical implications in cellular electrophysiology which may impact on safety pharmacology and therapeutics.

Rationale: Recurrent ventricular arrhythmias after initial successful defibrillation are associated with poor clinical outcome., Objective: We tested the hypothesis that postshock arrhythmias occur because of spontaneous sarcoplasmic reticulum Ca release, delayed afterdepolarization (DAD), and triggered activity (TA) from tissues with high sensitivity of resting membrane voltage (V(m)) to elevated intracellular calcium (Ca(i)) (high diastolic Ca(i)-voltage coupling gains)., Methods and Results: We simultaneously mapped Ca(i) and V(m) on epicardial (n=14) or endocardial (n=14) surfaces of Langendorff-perfused rabbit ventricles. Spontaneous Ca(i) elevation (SCaE) was noted after defibrillation in 32% of ventricular tachycardia/ventricular fibrillation at baseline and in 81% during isoproterenol infusion (0.01 to 1 micromol/L). SCaE was reproducibly induced by rapid ventricular pacing and inhibited by 3 mumol/L of ryanodine. The SCaE amplitude and slope increased with increasing pacing rate, duration, and dose of isoproterenol. We found TAs originating from 6 of 14 endocardial surfaces but none from epicardial surfaces, despite similar amplitudes and slopes of SCaEs between epicardial and endocardial surfaces. This was because DADs were larger on endocardial surfaces as a result of higher diastolic Ca(i)-voltage coupling gain, compared to those of epicardial surfaces. Purkinje-like potentials preceded TAs in all hearts studied (n=7). I(K1) suppression with CsCl (5 mmol/L, n=3), BaCl(2) (3 micromol/L, n=3), and low extracellular potassium (1 mmol/L, n=2) enhanced diastolic Ca(i)-voltage coupling gain and enabled epicardium to also generate TAs., Conclusions: Higher diastolic Ca(i)-voltage coupling gain is essential for genesis of TAs and may underlie postshock arrhythmias arising from Purkinje fibers. I(K)(1) is a major factor that determines the diastolic Ca(i)-voltage coupling gain.

Background and Purpose: Evaluation of the potential for delayed ventricular repolarization and proarrhythmia by new drugs is essential. We investigated if dog left ventricular midmyocardial myocytes (LVMMs) that can be used as a preclinical model to assess drug effects on action potential duration (APD) and whether in these cells, short-term variability (STV) or triangulation could predict proarrhythmic potential., Experimental Approach: Beagle LVMMs and Purkinje fibres (PFs) were used to record APs. Effects of six reference drugs were assessed on APD at 50% (APD(50)) and 90% (APD(90)) of repolarization, STV(APD), triangulation (ratio APD(90)/APD(50)) and incidence of early afterdepolarizations (EADs) at 1 and 0.5 Hz., Key Results: LVMMs provided stable recordings of AP, which were not affected by four sequential additions of dimethyl sulphoxide. Effects of dofetilide, d-sotalol, cisapride, pinacidil and diltiazem, but not of terfenadine, on APD in LVMMs were found to be comparable with those recorded in PFs. LVMMs, but not PFs, exhibited a proarrhythmic response to I(Kr) blockers. Incidence of EADs was not related to differences in AP prolongation or triangulation, but corresponded to beat-to-beat variability of repolarization, here quantified as STV of APD., Conclusions and Implications: LVMMs provide a suitable preclinical model to assess the effects of new drugs on APD and also yield additional information about putative indicators of proarrhythmia that add value to an integrated QT/TdP risk assessment. Our findings support the concept that increased STV(APD) may predict drug-induced proarrhythmia.

Ischemic focal ventricular tachycardia (VT) occurs in animals and humans. Angiotensin-converting enzyme inhibitors and receptor blockers reduce sudden death in patients with ischemic heart disease. In our dog model of coronary artery occlusion (CAO), we tested the hypothesis that angiotensin II (AGII) will selectively promote focal VT and that the specific AT(2) blocker PD-123319 (PD), or AT(1) blocker losartan, will affect this VT. Anesthetized dogs (n = 90) underwent CAO, followed by three-dimensional activation mapping of inducible VT. Dogs without VT in 1-3 h after CAO received AGII, and those with VT received either PD or losartan. Focal endocardium excised from ischemic sites was studied in vitro with standard microelectrode. Of 33 dogs with no inducible VT, AGII infusion resulted in sustained VT of only focal Purkinje origin in 13 (39%) compared with 0 of 20 dogs with saline. Of 26 dogs with inducible VT at baseline, given PD, reinduction was blocked in 8 of 10 (P < 0.05) focal VT, but only 1 of 15 with reentry. In contrast, of 11 dogs given losartan, reinduction of either mechanism was not blocked. In vitro triggered activity in Purkinje was blocked by PD in 13 of 19 (P < 0.05), but not by losartan in 8. Also, triggered activity was promoted by AGII, losartan, or the combination in 9 of 12 tissues. AGII promotes only focal, mainly Purkinje ischemic VT. PD, but not losartan, preferentially blocked focal VT, which is likely due to triggered activity due to delayed afterdepolarizations in Purkinje.

Progressive familial heart block type I (PFHBI) is a progressive cardiac bundle branch disease in the His-Purkinje system that exhibits autosomal-dominant inheritance. In 3 branches of a large South African Afrikaner pedigree with an autosomal-dominant form of PFHBI, we identified the mutation c.19G-->A in the transient receptor potential cation channel, subfamily M, member 4 gene (TRPM4) at chromosomal locus 19q13.3. This mutation predicted the amino acid substitution p.E7K in the TRPM4 amino terminus. TRPM4 encodes a Ca2+-activated nonselective cation (CAN) channel that belongs to the transient receptor potential melastatin ion channel family. Quantitative analysis of TRPM4 mRNA content in human cardiac tissue showed the highest expression level in Purkinje fibers. Cellular expression studies showed that the c.19G-->A missense mutation attenuated deSUMOylation of the TRPM4 channel. The resulting constitutive SUMOylation of the mutant TRPM4 channel impaired endocytosis and led to elevated TRPM4 channel density at the cell surface. Our data therefore revealed a gain-of-function mechanism underlying this type of familial heart block.

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Na(v)1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Na(v)1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Na(v)1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Na(v)1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean +/- SEM 309 +/- 32 V/s; n = 14) compared to subendocardial myocytes (394 +/- 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Na(v)1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.

Purkinje fibers (PFs) play key roles in cardiac conduction and arrhythmogenesis. Congestive heart failure (CHF) causes well-characterized atrial and ventricular ion channel subunit expression changes, but effects on PF ion channel subunits are unknown. This study assessed changes in PF ion channel subunit expression (real-time PCR, immunoblot, immunohistochemistry), action potential properties, and conduction in dogs with ventricular tachypacing-induced CHF. CHF downregulated mRNA expression of subunits involved in action potential propagation (Nav1.5, by 56%; connexin [Cx]40, 66%; Cx43, 56%) and repolarization (Kv4.3, 43%, Kv3.4, 46%). No significant changes occurred in KChIP2, KvLQT1, ERG, or Kir3.1/3.4 mRNA. At the protein level, downregulation was seen for Nav1.5 (by 38%), Kv4.3 (42%), Kv3.4 (57%), Kir2.1 (26%), Cx40 (53%), and Cx43 (30%). Cx43 dephosphorylation was indicated by decreased larger molecular mass bands (pan-Cx43 antibody) and a 57% decrease in Ser368-phosphorylated Cx43 (phospho-specific antibody). Immunohistochemistry revealed reduced Cx40, Cx43, and phospho-Cx43 expression at intercalated disks. Action potential changes were consistent with observed decreases in ion channel subunits: CHF decreased phase 1 slope (by 56%), overshoot (by 32%), and phase 0 dV/dt(max) (by 35%). Impulse propagation was slowed in PF false tendons: conduction velocity decreased significantly from 2.2+/-0.1 m/s (control) to 1.5+/-0.1 m/s (CHF). His-Purkinje conduction also slowed in vivo, with HV interval increasing from 35.5+/-1.2 (control) to 49.3+/-3.4 ms (CHF). These results indicate important effects of CHF on PF ion channel subunit expression. Alterations in subunits governing conduction properties may be particularly important, because CHF-induced impairments in Purkinje tissue conduction, which this study is the first to describe, could contribute significantly to dyssynchronous ventricular activation, a major determinant of prognosis in CHF-patients.

Activators of the human ether-a-go-go-related gene (hERG) K(+) channel have been reported recently to enhance hERG current amplitude (five synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound A-935142 ({4-[4-(5-trifluoromethyl-1H-pyrazol-3-yl)-phenyl]-cyclohexyl}-acetic acid) on guinea-pig atrial and canine ventricular action potentials (microelectrode techniques) and hERG channels expressed in HEK-293 cells (whole-cell patch clamp techniques). A-935142 shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current-voltage relationship revealed that this compound (60 microM) increased both outward and inward K(+) current as well as the slope conductance of the linear portion of the fully activated I-V relation. A-935142 significantly reduced the time constants (tau) of hERG channel activation at two example voltages (-10 mV: tau=100+/-17 ms vs. 164+/-24 ms, n=6, P<0.01; +30 mV: tau=16.7+/-1.8 ms vs. 18.9+/-1.8 ms, n=5, P<0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9 mV. The time course of hERG channel deactivation was slowed at multiple potentials (-120 to -70 mV). A-935142 also reduced the rate of inactivation and shifted the voltage-dependence of inactivation in the depolarizing direction by 15 mV. Recovery of hERG channel from inactivation was not affected by A-935142. In conclusion, A-935142 enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.

The short QT syndrome (SQTS) is a cardiac repolarisation disorder characterised by abbreviated QT intervals on the electrocardiogram and by an increased risk of atrial and ventricular arrhythmias and sudden death. The SQT1 variant involves a gain-of-function mutation (N588K) that impairs inactivation of the hERG (human ether-a-go-go-related gene) potassium channel and, thereby, increases current mediated by the rapid delayed rectifier potassium current (I(Kr)) in the heart. Here, the action potential voltage clamp (AP clamp) technique was applied to Chinese Hamster Ovary cells expressing wild-type or N588K-hERG at 37 degrees C, to compare effects of the N588K mutation on hERG current (I(hERG)) during ventricular, atrial and Purkinje fibre APs. The N588K mutation altered the I(hERG) profile during each AP type; increased maximal repolarising current occurred earlier during AP repolarisation (with shifts of +60 mV, +30 mV and +15 mV respectively for ventricular, Purkinje fibre and atrial APs). Thus SQT1 may influence repolarising I(hERG) for each cell type, with AP clamp experiments and simulation data indicating the greatest effect during ventricular APs. Changes in the timing of outward I(hERG) transients elicited by premature stimuli following AP commands indicate that SQT1 may alter the protection that hERG provides cardiac tissue against premature arrhythmogenic stimuli.

Purpose of Review: Ventricular fibrillation is a challenging arrhythmia for physicians, with only a limited number of treatment options available. Implantable cardioverter defibrillator therapy is the treatment of choice for patients for both primary and secondary prevention with the role of antiarrhythmic therapy aimed at reducing the number of recurrences. Catheter ablation is an accepted treatment for a number of atrial and ventricular arrhythmias, but until recently ventricular fibrillation was thought to be a bridge too far. The recent advances in ventricular fibrillation ablation will be reviewed., Recent Findings: Recent work has demonstrated that the Purkinje network is critical in the triggering and maintenance of ventricular fibrillation. Catheter ablation targeting the ventricular ectopic(s) or Purkinje potentials responsible for triggering ventricular fibrillation or both has been shown to be both possible and efficacious in a number of conditions, ranging from the Brugada syndrome to ischaemic ventricular fibrillation to idiopathic ventricular fibrillation. Although there are relatively few reports of catheter ablation of ventricular fibrillation in the literature, the method appears robust as it is being repeated by a number of different groups around the world., Summary: Catheter ablation targeting ventricular ectopics and Purkinje potentials is a successful treatment for ventricular fibrillation. Although the long-term results of such procedures are awaited, the short-term results are very encouraging.

The purpose of this study was to determine whether IP(3)Rs contribute to the generation of wide long lasting perinuclear Ca(2+) release events in canine Purkinje cells. Spontaneous Ca(2+) release events (elevations of basal [Ca(2+)] equivalent to F/F(0) 3.4SD over F(0)) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca(2+) waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 microm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035+/-0.0007 events (ev)/microm(2)/s, N=34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022+/-0.00005 ev/microm(2)/s, N=41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0+/-3.9 vs. TE 9.0+/-0.3 microm(2), P<0.01) and higher amplitude (F/F(0) 1.38+/-0.02 vs. TE 1.20+/-0.003, P<0.01). WLE event rate was increased by phenylephrine (10 microM, P<0.01), inhibited by 2APB and U73122 (P<0.05), and abolished by tetracaine (1 mM) and ryanodine (100 microM). While SSL WLEs were scattered randomly, Core WLEs (n=69 events) were predominantly distributed longitudinally 18.2+/-1.6 microm from the center of nuclei. Immunocytochemistry showed that IP(3)R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca(2+) release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP(3)R1s evoked Ca(2+) release and may play a role in Ca(2+) dependent nuclear processes.

The spatiotemporal distribution of the endothelin-converting enzyme (ECE) protein in the embryonic chick heart and the association of this polypeptide with the developing cardiac conduction system is described here for the first time. Further, we show how cardiac hemodynamic load directly affects ECE level and distribution. Endothelin (ET) is a cytokine involved in the inductive recruitment of Purkinje fibers. ET is produced by proteolytic cleavage of Big-ET by ECE. We generated an antibody against chick ECE recognizing a single band at approximately 70 kD to correlate the cardiac expression of this protein with that reported previously for its mRNA. ECE protein expression was more widespread compared to its mRNA, being present in endothelial cells, mesenchymal cells, and myocytes, and particularly enriched in the trabeculae and nascent ventricular conduction system. The myocardial expression was significantly modified under experimentally altered hemodynamic loading. In vivo, ET receptor blockade with bosentan delayed activation sequence maturation. These data support a role for ECE in avian cardiac conduction system differentiation and maturation.

Brugada syndrome is a genetic disease associated with sudden cardiac death that is characterized by ventricular fibrillation and right precordial ST segment elevation on ECG. Loss-of-function mutations in SCN5A, which encodes the predominant cardiac sodium channel alpha subunit NaV1.5, can cause Brugada syndrome and cardiac conduction disease. However, SCN5A mutations are not detected in the majority of patients with these syndromes, suggesting that other genes can cause or modify presentation of these disorders. Here, we investigated SCN1B, which encodes the function-modifying sodium channel beta1 subunit, in 282 probands with Brugada syndrome and in 44 patients with conduction disease, none of whom had SCN5A mutations. We identified 3 mutations segregating with arrhythmia in 3 kindreds. Two of these mutations were located in a newly described alternately processed transcript, beta1B. Both the canonical and alternately processed transcripts were expressed in the human heart and were expressed to a greater degree in Purkinje fibers than in heart muscle, consistent with the clinical presentation of conduction disease. Sodium current was lower when NaV1.5 was coexpressed with mutant beta1 or beta1B subunits than when it was coexpressed with WT subunits. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.

The aim of our experiments was to investigate by means of a whole cell patch-clamp technique the characteristics of the slowly inactivating sodium current (I(Na2)) found in the plateau range in canine cardiac Purkinje single cells. The I(Na2) was separated from the fast-activating and -inactivating I(Na) (labelled here I(Na1)) by applying a two-step protocol. The first step, from a holding potential (V(h)) of -90 or -80 mV to -50 mV, led to the quick activation and inactivation of I(Na1). The second step consisted of depolarizations of increasing amplitude from -50 mV to less negative values, which led to the quick activation and slow inactivation of I(Na2). The I(Na2) was fitted with a double exponential function with time constants of tens and hundreds milliseconds, respectively. After the activation and inactivation of I(Na1) at -50 mV, the slope conductance was very small and did not change with time. Instead, during I(Na2), the slope conductance was larger and decreased as a function of time. Progressively longer conditioning steps at -50 mV resulted in a progressive decrease in amplitude of I(Na2) during the subsequent test steps. Gradually longer hyperpolarizing steps (increments of 100 ms up to 600 ms) from V(h) -30 mV to -100 mV were followed on return to -30 mV by a progressively larger I(Na2), as were gradually more negative 500 ms steps from V(h) -30 mV to -90 mV. At the end of a ramp to -20 mV, a sudden repolarization to approximately -35 mV fully deactivated I(Na2). The I(Na2) was markedly reduced by lignocaine (lidocaine) and by low extracellular [Na(+)], but it was little affected by low and high extracellular [Ca(2+)]. At negative potentials, the results indicate that there was little overlap between I(Na2) and the transient outward current, I(to), as well as the calcium current, I(Ca). In the absence of I(to) and I(Ca) (blocked by means of 4-aminopyridine and nickel, respectively), I(Na2) reversed at 60 mV. In conclusion, I(Na2) is a sodium current that can be initiated after the inactivation of I(Na1) and has characteristics that are quite distinct from those of I(Na1). The results have a bearing on the mechanisms underlying the long plateau of Purkinje cell action potential and its modifications in different physiological and pathological conditions.