Epigenetic Changes Governing Scn5a Expression in Denervated Skeletal Muscle.

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.

CRISPR -Mediated Expression of the Fetal Scn5a Isoform in Adult Mice Causes Conduction Defects and Arrhythmias.

Background The sodium channel, Nav1.5, encoded by SCN 5A, undergoes developmentally regulated splicing from inclusion of exon 6A in the fetal heart to exon 6B in adults. These mutually exclusive exons differ in 7 amino acids altering the electrophysiological properties of the Nav1.5 channel. In myotonic dystrophy type 1, SCN 5A is mis-spliced such that the fetal pattern of exon 6A inclusion is detected in adult hearts. Cardiac manifestations of myotonic dystrophy type 1 include conduction defects and arrhythmias and are the second-leading cause of death. Methods and Results This work aimed to determine the impact of SCN 5A mis-splicing on cardiac function. We used clustered regularly interspaced short palindromic repeat ( CRISPR) /CRISPR-associated protein 9 (Cas9) to delete Scn5a exon 6B in mice, thereby redirecting splicing toward exon 6A. These mice exhibit prolonged PR and QRS intervals, slowed conduction velocity, extended action potential duration, and are highly susceptible to arrhythmias. Conclusions Our findings highlight a nonmutational pathological mechanism of arrhythmias and conduction defects as a result of mis-splicing of the predominant cardiac sodium channel. Animals homozygous for the deleted exon express only the fetal isoform and have more-severe phenotypes than heterozygotes that also express the adult isoform. This observation is directly relevant to myotonic dystrophy type 1, and possibly pathological arrhythmias, in which individuals differ with regard to the ratios of the isoforms expressed.

Participation of voltage-gated sodium and calcium channels in the acute cardiac effects of toluene.

Inhaling solvents can lead to occurrence of cardiac arrhythmias and sudden sniffing death. Mechanisms related to this phenomenon are not fully understood. The purpose of this study was to investigate the effect of acute toluene exposure on heart reactivity to epinephrine and the participation of voltage-gated sodium and calcium channels. We found that acute toluene exposure increased perfusion pressure, left ventricular developed pressure, and heart rate. These actions were inhibited by lidocaine and nifedipine. Our results suggest that acute toluene exposure modify voltage-gated sodium and calcium channel function and expression likely due to a cardiac adrenergic mechanism and these effects could be participating, at least in part, in the presence of cardiac arrhythmias. To our best knowledge, this is the first report to establish a direct participation of voltage-gated Na+ and Ca2+ channels, toluene and epinephrine on cardiac function in rats.

Microtubular remodeling and decreased expression of Nav1.5 with enhanced EHD4 in cells from the infarcted heart.

Cardiac Na+ channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 cytoskeletal- and endosomal-based membrane trafficking and function are linked to tubulin, microtubular (MT) networks, and Eps15 homology domain containing proteins like EHD4. AIM: Our objective is to understand the relation of tubulin and EHD4 to Nav1.5 channel protein remodeling observed in border zone cells (IZs) when arrhythmias are known to occur; that is, 3-h, 48-h and 5-day post coronary occlusion. MATERIALS METHODS FINDINGS: Our voltage clamp and immunostaining data show that INa density is decreased in the epicardial border zone cells of the 48 h infarcted heart (IZ48h). Immunostaining studies reveal that in post MI cells the cell surface staining of Nav1.5 was reduced and Nav1.5 distribution changed. However, intense co-staining of Nav1.5 and tubulin occurs in core planes and the perinuclear areas in post MI cells. At the same time, there were marked changes in the subcellular location of the EHD4 protein. EHD4 is co-localized with tubulin protein in discrete intracellular "highway" structures. SIGNIFICANCE: The distribution and expression of the three proteins are altered dynamically in post MI cells. In sum, our work illustrates the spatiotemporal complexity of remodeling mechanisms in the post-infarct myocyte. It will be important in future experiments to further explore direct links between MT, EHD proteins, and cell proteins involved in forward trafficking.

HuR-mediated SCN5A messenger RNA stability reduces arrhythmic risk in heart failure.

BACKGROUND: Downregulated sodium currents in heart failure (HF) have been linked to increased arrhythmic risk. Reduced expression of the messenger RNA (mRNA)-stabilizing protein HuR (also known as ELAVL1) may be responsible for the downregulation of sodium channel gene SCN5A mRNA. OBJECTIVE: The purpose of this article was to investigate whether HuR regulates SCN5A mRNA expression and whether manipulation of HuR benefits arrhythmia control in HF. METHODS: Quantitative real-time reverse-transcriptase polymerase chain reaction was used to investigate the expression of SCN5A. Optical mapping of the intact heart was adopted to study the effects of HuR on the conduction velocity and action potential upstroke in mice with myocardial infarct and HF after injection of AAV9 viral particles carrying HuR. RESULTS: HuR was associated with SCN5A mRNA in cardiomyocytes, and expression of HuR was downregulated in failing hearts. The association of HuR and SCN5A mRNA protected SCN5A mRNA from decay. Injection of AAV9 viral particles carrying HuR increased SCN5A expression in mouse heart tissues after MI. Optical mapping of the intact heart demonstrated that overexpression of HuR improved action potential upstroke and conduction velocity in the infarct border zone, which reduced the risk of reentrant arrhythmia after MI. CONCLUSION: Our data indicate that HuR is an important RNA-binding protein in maintaining SCN5A mRNA abundance in cardiomyocytes. Reduced expression of HuR may be at least partially responsible for the downregulation of SCN5A mRNA expression in ischemic HF. Overexpression of HuR may rescue decreased SCN5A expression and reduce arrhythmic risk in HF. Increasing mRNA stability to increase ion channel currents may correct a fundamental defect in HF and represent a new paradigm in antiarrhythmic therapy.

Neonatal Nav1.5 protein expression in normal adult human tissues and breast cancer.

Expression of the neonatal splice variant of the voltage-gated sodium channel α-subunit (VGSC) subtype Nav1.5 (nNav1.5), encoded by the gene SCN5A, was shown earlier to be upregulated in human breast cancer (BCa), both in vitro and in vivo. Channel activity promoted BCa invasion of Matrigel®in vitro and metastasis in vivo. Consequently, expression of nNav1.5 has been proposed as a functional biomarker of BCa cells with metastatic potential. Here, we have determined immunohistochemically both nNav1.5 and total VGSC (tVGSC) protein expression in a range of adult human tissues. Some VGSC protein was expressed in normal colon, small intestine, stomach, prostate, bladder and breast. As expected, high levels of VGSC protein were expressed in brain, skeletal muscle and cardiac muscle. On the other hand, nNav1.5 protein was not expressed in any of the normal tissues tested except breast where a low-level of protein was present. In comparison to normal breast, nNav1.5 protein expression in BCa was consistently widespread and occurred at a significantly higher level. We also questioned whether there was any relationship between the nNav1.5 protein expression and the estrogen receptor (ERα) status of BCa and obtained the following results. First, all cases lacking nNav1.5 were positive for ERα. Second, in all ERα-negative tissues, nNav1.5 protein was expressed in plasma membrane. Third, however, in ERα-positive cases, nNav1.5 protein expression was observed in both plasma membrane and cytoplasm. In conclusion, nNav1.5 protein has a restricted expression pattern among human tissues. High level expression occurs in BCa and associates with ERα status. These results further support the proposition that nNav1.5 is a novel biomarker of metastatic BCa.

Cardiotoxic effect of levofloxacin and ciprofloxacin in rats with/without acute myocardial infarction: Impact on cardiac rhythm and cardiac expression of Kv4.3, Kv1.2 and Nav1.5 channels.

Prolongation of QT interval is possible with fluoroquinolones, yet the underlying contributing factors have not been elucidated. Two widely used fluoroquinolone drugs were at the focus of this study in rats with/without acute myocardial dysfunction (AMI) induced by isoproterenol. The effects of levofloxacin and ciprofloxacin on the cardiac mRNA expression of rat Kv4.3, Kv1.2 and Nav1.5 mRNAs were determined. Administration of the two antibiotics produced dose-dependent changes in ECG parameters that were more prominent in rats with AMI than healthy rats; this was accompanied by elevations in serum lactate dehydrogenase and creatine kinase-MB. Histopathological examination indicated some loss of striations, edema and fibrotic changes in rats with AMI; however the two antibiotics did not further exacerbate the cardiac histopathology. mRNA expression of the ion channels was altered in rats with AMI and healthy rats. In conclusion, long-term administration of levofloxacin and ciprofloxacin produced deleterious effects on the ECG pattern of rats with/without AMI. The effect was generally baseline-dependent and therefore, rats with AMI showed greater ECG disturbances and increases in cardiac enzymes. Taken together, these data make it advisable to monitor patients with a history of acute AMI requiring treatment with these antibiotics until data from human studies are available.

Patient-Specific and Genome-Edited Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Brugada Syndrome.

BACKGROUND: Brugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder. OBJECTIVES: The objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). METHODS: This study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs. RESULTS: BrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression. CONCLUSIONS: Patient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.

Inhibitory effects of neferine on Nav1.5 channels expressed in HEK293 cells.

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition (IC50) being 26.15 µmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 µmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

Prolongation of action potential duration and QT interval during epilepsy linked to increased contribution of neuronal sodium channels to cardiac late Na+ current: potential mechanism for sudden death in epilepsy.

BACKGROUND: Arrhythmias associated with QT prolongation on the ECG often lead to sudden unexpected death in epilepsy. The mechanism causing a prolongation of the QT interval during epilepsy remains unknown. Based on observations showing an upregulation of neuronal sodium channels in the brain during epilepsy, we tested the hypothesis that a similar phenomenon occurs in the heart and contributes to QT prolongation by altering cardiac sodium current properties (INa). METHODS AND RESULTS: We used the patch clamp technique to assess the effects of epilepsy on the cardiac action potential and INa in rat ventricular myocytes. Consistent with QT prolongation, epileptic rats had longer ventricular action potential durations attributable to a sustained component of INa (INaL). The increase in INaL was because of a larger contribution of neuronal Na channels characterized by their high sensitivity to tetrodotoxin. As in the brain, epilepsy was associated with an enhanced expression of the neuronal isoform NaV1.1 in cardiomyocyte. Epilepsy was also associated with a lower INa activation threshold resulting in increased cell excitability. CONCLUSIONS: This is the first study correlating increased expression of neuronal sodium channels within the heart to epilepsy-related cardiac arrhythmias. This represents a new paradigm in our understanding of cardiac complications related to epilepsy.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Expression of neonatal Nav1.5 in human brain astrocytoma and its effect on proliferation, invasion and apoptosis of astrocytoma cells.

In the present study, we designed and conducted a series of assays to determine the expression of voltage-gated sodium channel (VGSC) neonatal isoform Nav1.5 (nNav1.5) in human brain astrocytoma and its effect on the proliferation, migration, invasion and apoptosis of astrocytoma U251 cells. The results showed that nNav1.5 mRNA and protein were expressed in both human brain astrocytoma and normal brain tissues, but their expression levels in astrocytoma were significantly higher (P<0.05). In astrocytomas, nNav1.5 mRNA and protein levels were also different (P<0.05) and were correlated with pathological grades. The immunofluorescence confocal microscopy observations demonstrated that nNav1.5 protein was expressed in the nucleus, cytoplasm and membrane of the astrocytoma cells. After transfection, the small interfering RNA (siRNA) targeted to nNav1.5 significantly reduced the expression levels of SCN5A/nNav1.5 mRNA and protein by 57.2% (P<0.05) and 66.6% (P<0.05), respectively. The MTT, wound healing, Matrigel invasion and flow cytometric assays confirmed that following siRNA downregulation of the expression of the SCN5A/nNav1.5 gene, the in vitro proliferation and in vitro invasiveness of the U251 cells were significantly reduced (P<0.05 for both comparisons), and the apoptosis rate was significantly increased (P<0.05). These results revealed that nNav1.5 expression in human brain astrocytoma was upregulated, and its expression was positively correlated with the degree of malignancy. Additionally, reduced nNav1.5 expression significantly suppressed the proliferation and invasiveness of astrocytoma cells, indicating a new target in the molecular diagnosis and therapy of astrocytoma.

The Xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

Nicorandil improves electrical remodelling, leading to the prevention of electrically induced ventricular tachyarrhythmia in a mouse model of desmin-related cardiomyopathy.

1. Transgenic (TG) mice overexpressing an arg120gly missense mutation in heat shock protein B5 (HSPB5; i.e. R120G TG mice) exhibit desmin-related cardiomyopathy. Recently, the cardioprotective effect of nicorandil has been shown to prolong the survival of R120G TG mice. However, whether the TG mice exhibit ventricular arrhythmias and whether nicorandil can inhibit these arrhythmias remain unknown. In the present study we examined the effects of chronic nicorandil administration on ventricular electrical remodelling and arrhythmias in R120G TG mice. 2. Mice were administered nicorandil (15 mg/kg per day) or vehicle (water) orally from 5 to 30 weeks of age. Electrocardiograms (ECG) and optical action potentials were recorded from R120G TG mouse hearts. In addition, the expression of ventricular connexin 43 and the cardiac Na(+) channel Nav1.5 was examined in TG mice. 3. All ECG parameters tested were prolonged in R120G TG compared with non-transgenic (NTG) mice. Nicorandil improved the prolonged P, PQ and QRS intervals in R120G TG mice. Interestingly, impulse conduction slowing and increases in the expression of total and phosphorylated connexin 43 and Nav1.5 were observed in ventricles from R120G TG compared with NTG mice. Nicorandil improved ventricular impulse conduction slowing and normalized the increased protein expression levels of total and phosphorylated connexin 43, but not of Nav1.5, in R120G TG mouse hearts. Electrical rapid pacing at the ventricle induced ventricular tachyarrhythmias (VT) in six of eight R120G TG mouse hearts, but not in any of the eight nicorandil-treated R120G TG mouse hearts (P < 0.05). 4. These findings demonstrate that nicorandil inhibits cardiac electrical remodelling and that the prevention of VT by nicorandil is associated with normalization of connexin 43 expression in this model.

Effect of testosterone replacement therapy on cardiac performance and oxidative stress in orchidectomized rats.

AIM: To investigate the effects of testosterone on myocardial contractility, oxidative stress status and expression of sodium channel protein (Nav1.5) and inward rectifying K channels (Kir 2.x) in normal and orchidectomized (ORX) rats. METHODS: One hundred four rats were randomly assigned into four groups (n = 26, each) as follows: (i) untreated controls, (ii) testosterone treated, (iii) orchidectomized rats and (iv) orchidectomized, testosterone-treated rats. Treatments with the vehicle or testosterone were carried out for 12 weeks, three times per week. At the end of treatment, surface ECG, isolated heart, tissue oxidative stress and lipid peroxidation experiments were carried out on the cardiac tissues. Also, immunohistochemical examination for Nav1.5 and PCR detection of mRNA of Kir2.1, Kir2.2 and Kir2.4 subunits of K channels were carried out. RESULTS: Orchidectomy impaired cardiac contractile function parameters left ventricular developed pressure (LVDP) and the peaks of the positive and negative pressure derivatives (dP/dtmax and -dP/dtmax respectively), increased heart rate and prolonged QT and QTc intervals, elevated pro-oxidant state in rat's hearts and decreased the expression of Kir 2.1 but not Kir2.2, Kir 2.4 and Nav1.5 channels. Exogenous testosterone administration to orchidectomized rats restored heart contractility and shortened QT and QTc intervals to their normal values, ameliorated the generated pro-oxidant state and improved the expression of Nav1.5 and Kir2.1, but not Kir2.2 or Kir2.4 channels. CONCLUSION: Testosterone improved cardiac contractility and shortened QT and QTc intervals in ORX rats. An effect that might be dependent of reduction in oxidative stress and enhancement of Kir2.1 channels but independent of Nav1.5 channel protein.

A transgenic zebrafish model of a human cardiac sodium channel mutation exhibits bradycardia, conduction-system abnormalities and early death.

The recent exponential increase in human genetic studies due to the advances of next generation sequencing has generated unprecedented numbers of new gene variants. Determining which of these are causative of human disease is a major challenge. In-vitro studies and murine models have been used to study inherited cardiac arrhythmias but have several limitations. Zebrafish models provide an attractive alternative for modeling human heart disease due to similarities in cardiac electrophysiology and contraction, together with ease of genetic manipulation, external development and optical transparency. Although zebrafish cardiac mutants and morphants have been widely used to study loss and knockdown of zebrafish gene function, the phenotypic effects of human dominant-negative gene mutations expressed in transgenic zebrafish have not been evaluated. The aim of this study was to generate and characterize a transgenic zebrafish arrhythmia model harboring the pathogenic human cardiac sodium channel mutation SCN5A-D1275N, that has been robustly associated with a range of cardiac phenotypes, including conduction disease, sinus node dysfunction, atrial and ventricular arrhythmias, and dilated cardiomyopathy in humans and in mice. Stable transgenic fish with cardiac expression of human SCN5A were generated using Tol2-mediated transgenesis and cardiac phenotypes were analyzed using video microscopy and ECG. Here we show that transgenic zebrafish expressing the SCN5A-D1275N mutation, but not wild-type SCN5A, exhibit bradycardia, conduction-system abnormalities and premature death. We furthermore show that SCN5A-WT, and to a lesser degree SCN5A-D1275N, are able to compensate the loss of endogenous zebrafish cardiac sodium channels, indicating that the basic pathways, through which SCN5A acts, are conserved in teleosts. This proof-of-principle study suggests that zebrafish may be highly useful in vivo models to differentiate functional from benign human genetic variants in cardiac ion channel genes in a time- and cost-efficient manner. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.

Multiple sclerosis (MS) is the most common nontraumatic cause of neurologic disability in young adults. Despite treatment, progressive tissue injury leads to accumulation of disability in many patients. Here, our goal was to develop an immune-mediated strategy to promote tissue repair and clinical recovery in an MS animal model. We previously demonstrated that a variant of the voltage-gated sodium channel NaV1.5 is expressed intracellularly in human macrophages, and that it regulates cellular signaling. This channel is not expressed in mouse macrophages, which has limited the study of its functions. To overcome this obstacle, we developed a novel transgenic mouse model (C57BL6), in which the human macrophage NaV1.5 splice variant is expressed in vivo in mouse macrophages. These mice were protected from experimental autoimmune encephalomyelitis, the mouse model of MS. During active inflammatory disease, NaV1.5-positive macrophages were found in spinal cord lesions where they formed phagocytic cell clusters; they expressed markers of alternative activation during recovery. NaV1.5-positive macrophages that were adoptively transferred into wild-type recipients with established experimental autoimmune encephalomyelitis homed to lesions and promoted recovery. These results suggest that NaV1.5-positive macrophages enhance recovery from CNS inflammatory disease and could potentially be developed as a cell-based therapy for the treatment of MS.

The cardiomyocyte molecular clock, regulation of Scn5a, and arrhythmia susceptibility.

The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop. Bmal1 encodes a core molecular clock transcription factor. Germline Bmal1 knockout mice show a loss of circadian variation in heart rate and blood pressure, and they develop dilated cardiomyopathy. We tested the role of the molecular clock in adult cardiomyocytes by generating mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1). ECG telemetry showed that cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1(-/-)) in adult mice slowed heart rate, prolonged RR and QRS intervals, and increased episodes of arrhythmia. Moreover, isolated iCSΔBmal1(-/-) hearts were more susceptible to arrhythmia during electromechanical stimulation. Examination of candidate cardiac ion channel genes showed that Scn5a, which encodes the principle cardiac voltage-gated Na(+) channel (Na(V)1.5), was circadianly expressed in control mouse and rat hearts but not in iCSΔBmal1(-/-) hearts. In vitro studies confirmed circadian expression of a human Scn5a promoter-luciferase reporter construct and determined that overexpression of clock factors transactivated the Scn5a promoter. Loss of Scn5a circadian expression in iCSΔBmal1(-/-) hearts was associated with decreased levels of Na(V)1.5 and Na(+) current in ventricular myocytes. We conclude that disruption of the molecular clock in the adult heart slows heart rate, increases arrhythmias, and decreases the functional expression of Scn5a. These findings suggest a potential link between environmental factors that alter the cardiomyocyte molecular clock and factors that influence arrhythmia susceptibility in humans.