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Rationale: Loss-of-function of the cardiac sodium channel Na V 1.5 causes conduction slowing and arrhythmias. Na V 1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current ( I Na ) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I Na reduction, but the mechanisms underlying microdomain-specific targeting of Na V 1.5 remain largely unknown. Objective: To investigate the role of the microtubule plus-end tracking proteins EB1 (end-binding protein 1) and CLASP2 (cytoplasmic linker associated protein 2) in mediating Na V 1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human-induced pluripotent stem cell-derived cardiomyocytes resulted in enhanced whole-cell I Na , increased action potential upstroke velocity ( V max ), and enhanced Na V 1.5 localization at the plasma membrane as detected by multicolor stochastic optical reconstruction microscopy. Fluorescence recovery after photobleaching experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na V 1.5 forward trafficking. Knockout of MAPRE1 in human induced pluripotent stem cell-derived cardiomyocytes led to reduced whole-cell I Na , decreased V max , and action potential duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish ( mapre1b ) resulted in decreased ventricular conduction velocity and V max as well as increased APD. Stochastic optical reconstruction microscopy imaging and macropatch I Na measurements showed that subacute treatment (2–3 hours) with SB216763 (SB2), a GSK3β (glycogen synthase kinase 3β) inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na V 1.5 and I Na preferentially at the ID region of wild-type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I Na or Na V 1.5 localization in cardiomyocytes from Clasp2 -deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of Na V 1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the microtubule plus-end tracking protein EB1 on Na V 1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I Na within the ID region of cardiomyocytes.

Background Patients with long QT syndrome (LQTS) are predisposed to life-threatening arrhythmias. A delay in cardiac repolarization is characteristic of the disease. Pharmacotherapy, implantable cardioverter-defibrillators, and left cardiac sympathetic denervation are part of the current treatment options, but no targeted therapy for LQTS exists to date. Previous studies indicate that induced autoimmunity against the voltage-gated KCNQ1 K+ channels accelerates cardiac repolarization. Objectives However, a causative relationship between KCNQ1 antibodies and the observed electrophysiological effects has never been demonstrated, and thus presents the aim of this study. Methods The authors purified KCNQ1 antibodies and performed whole-cell patch clamp experiments as well as single-channel recordings on Chinese hamster ovary cells overexpressing IKs channels. The effect of purified KCNQ1 antibodies on human cardiomyocytes derived from induced pluripotent stem cells was then studied. Results The study demonstrated that KCNQ1 antibodies underlie the previously observed increase in repolarizing IKs current. The antibodies shift the voltage dependence of activation and slow the deactivation of IKs. At the single-channel level, KCNQ1 antibodies increase the open time and probability of the channel. In models of LQTS type 2 (LQTS2) using human induced pluripotent stem cell-derived cardiomyocytes, KCNQ1 antibodies reverse the prolonged cardiac repolarization and abolish arrhythmic activities. Conclusions Here, the authors provide the first direct evidence that KCNQ1 antibodies act as agonists on IKs channels. Moreover, KCNQ1 antibodies were able to restore alterations in cardiac repolarization and most importantly to suppress arrhythmias in LQTS2. KCNQ1 antibody therapy may thus present a novel promising therapeutic approach for LQTS2.

Aims: several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results: using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion: altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases., National Research Agency; European Union (EU); Horizon 2020; Marie Curie Actions International Incoming Fellowship FP7-PEOPLE-2012-IIF; La Fédération Française de Cardiologie; Fondation LefoulonDelalande; Eiffel Scholarship Programme of Excellence (Campus France), Doctoral School of Science and Technology-Lebanese University and The Fondation Genavie

Aims Brugada syndrome (BrS) is characterized by a unique electrocardiogram (ECG) pattern and life-threatening arrhythmias. However, the Type 1 Brugada ECG pattern is often transient, and a genetic cause is only identified in Methods and results For antibody (Ab) discovery, normal human ventricular myocardial proteins were solubilized and separated by isoelectric focusing (IEF) and molecular weight on two-dimensional (2D) gels and used to discover Abs by plating with sera from patients with BrS and control subjects. Target proteins were identified by mass spectrometry (MS). Brugada syndrome subjects were defined based on a consensus clinical scoring system. We assessed discovery and validation cohorts by 2D gels, western blots, and ELISA. We performed immunohistochemistry on myocardium from BrS subjects (vs. control). All (3/3) 2D gels exposed to sera from BrS patients demonstrated specific Abs to four proteins, confirmed by MS to be α-cardiac actin, α-skeletal actin, keratin, and connexin-43, vs. 0/8 control subjects. All (18/18) BrS subjects from our validation cohorts demonstrated the same Abs, confirmed by western blots, vs. 0/24 additional controls. ELISA optical densities for all Abs were elevated in all BrS subjects compared to controls. In myocardium obtained from BrS subjects, each protein, as well as SCN5A, demonstrated abnormal protein expression in aggregates. Conclusion A biomarker profile of autoantibodies against four cardiac proteins, namely α-cardiac actin, α-skeletal actin, keratin, and connexin-43, can be identified from sera of BrS patients and is highly sensitive and specific, irrespective of genetic cause for BrS. The four involved proteins, along with the SCN5A-encoded Nav1.5 alpha subunit are expressed abnormally in the myocardium of patients with BrS.

International audience; Patients with HIV present with a higher prevalence of QT prolongation, of which molecularbases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulatesviral genes expression and is required for efficient HIV replication. Tat is actively secretedinto the blood by infected T-cells and affects organs such as the heart. Tat has been shown toalter cardiac repolarization in animal models but how this is mediated and whether this is alsothe case in human cells is unknown. In the present study, we show that Tat transfection inheterologous expression systems led to a decrease in hERG (underlying cardiac IKr) andhuman KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of theirdeactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observedeffects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive,further confirming the hypothesis. Twenty four-hour incubation of human induced pluripotentstem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated itsdeactivation. Concordantly, this Tat incubation led to a prolongation of the action potential(AP) duration. Events of AP alternans were also recorded in the presence of Tat, and wereexacerbated at a low pacing cycle length. Altogether, these data obtained on human K+channels both in heterologous expression systems and in human cardiomyocytes stronglysuggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecularmechanism for QT prolongation in HIV-infected patients.Key

In cardiomyocytes, the voltage-gated transient outward potassium current (Ito) is responsible for the phase-1 repolarization of the action potential (AP). Gain-of-function mutations in KCND3, the gene encoding the Ito carrying KV4.3 channel, have been associated with Brugada syndrome (BrS). While the role of Ito in the pro-arrhythmic mechanism of BrS has been debated, recent studies have suggested that an increased Ito may directly affect cardiac conduction. However, the effects of an increased Ito on AP upstroke velocity or sodium current at the cellular level remain unknown. We here investigated the consequences of KV4.3 overexpression on NaV1.5 current and consequent sodium channel availability. We found that overexpression of KV4.3 protein in HEK293 cells stably expressing NaV1.5 (HEK293-NaV1.5 cells) significantly reduced NaV1.5 current density without affecting its kinetic properties. In addition, KV4.3 overexpression decreased AP upstroke velocity in HEK293-NaV1.5 cells, as measured with the alternating voltage/current clamp technique. These effects of KV4.3 could not be explained by alterations in total NaV1.5 protein expression. Using computer simulations employing a multicellular in silico model, we furthermore demonstrate that the experimentally observed increase in KV4.3 current and concurrent decrease in NaV1.5 current may result in a loss of conduction, underlining the potential functional relevance of our findings. This study gives the first proof of concept that KV4.3 directly impacts on NaV1.5 current. Future studies employing appropriate disease models should explore the potential electrophysiological implications in (patho)physiological conditions, including BrS associated with KCND3 gain-of-function mutations.

International audience; Voltage-gated Na(+) (NaV) channels are key regulators of myocardial excitability, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na(+) current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

International audience; Aims: Loss-of-function mutations in SCN5A, the gene encoding NaV1.5 channel, have been associated with inherited progressive cardiac conduction disease (PCCD). We have proposed that Scn5a heterozygous knock-out (Scn5a+/-) mice, which are characterized by ventricular fibrotic remodelling with ageing, represent a model for PCCD. Our objectives were to identify the molecular pathway involved in fibrosis development and prevent its activation. Methods and results: Our study shows that myocardial interstitial fibrosis occurred in Scn5a+/- mice only after 45 weeks of age. Fibrosis was triggered by transforming growth factor β (TGF-β) pathway activation. Younger Scn5a+/- mice were characterized by a higher connexin 43 expression than wild-type (WT) mice. After the age of 45 weeks, connexin 43 expression decreased in both WT and Scn5a+/- mice, although the decrease was larger in Scn5a+/- mice. Chronic inhibition of cardiac sodium current with flecainide (50 mg/kg/day p.o) in WT mice from the age of 6 weeks to the age of 60 weeks did not lead to TGF-β pathway activation and fibrosis. Chronic inhibition of TGF-β receptors with GW788388 (5 mg/kg/day p.o.) in Scn5a+/- mice from the age of 45 weeks to the age of 60 weeks prevented the occurrence of fibrosis. However, current data could not detect reduction in QRS duration with GW788388. Conclusion: Myocardial fibrosis secondary to a loss of NaV1.5 is triggered by TGF-β signalling pathway. Those events are more likely secondary to the decreased NaV1.5 sarcolemmal expression rather than the decreased Na+ current per se. TGF-β receptor inhibition prevents age-dependent development of ventricular fibrosis in Scn5a+/- mouse.

Aims Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, determining depolarization, duration, waveform and refractoriness of action potentials. In heart failure, action potentials are prolonged, and evidence suggests that this arrhythmogenic abnormality is associated with Ca2+/ Calmodulin-dependent protein Kinase II (CaMKII) and altered phosphorylation of the main ventricular NaV channel subunit, NaV1.5. The global native phosphorylation patterns of NaV1.5 channels in failing, compared with nonfailing, ventricles, and the associated channel regulatory defects, however, remain unknown. Methods and results Phosphoproteomic analyses were undertaken to identify and quantify in situ the phosphorylation sites on the NaV1.5 proteins purified from adult wild-type (WT) and failing CaMKIIdc-overexpressing (CaMKIIdc-Tg) mouse ventricles. Of the nineteen native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIdc-Tg, compared with the WT, ventricles. Because these two phosphoserines are located in close proximity to the binding site for Fibroblast Growth Factor 13 (FGF13), we tested the hypothesis that phosphorylation at these sites impairs the FGF13- mediated regulation of channel inactivation. Whole cell voltage-clamp analyses demonstrated that FGF13 increases NaV1.5 channel availability, accelerates recovery from inactivation and decreases the late Na+ current in HEK293 cells, three effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Further co-immunoprecipitation experiments revealed that phosphorylation at both sites decreases the interaction of FGF13 and of calmodulin with NaV1.5. Conclusions These analyses provide two novel native phosphorylation sites in the C-terminus of NaV1.5 which show increased phosphorylation in failing ventricles and impair FGF13-dependent inactivation. The author hereby declares no conflict of interest

Complete atrioventricular block (CAVB) and related ventricular bradycardia are known to induce ventricular hypertrophy and arrhythmias. Different animal models of CAVB have been established with the most common being the dog model. Related studies were mainly focused on the consequences on the main repolarizing currents in these species, i.e. IKr and IKs, with a limited time point kinetics post-AVB. In order to explore at a genomic scale the electrical remodeling induced by AVB and its chronology, we have developed a novel model of CAVB in the mouse using a radiofrequency-mediated ablation procedure. We investigated transcriptional changes in ion channels and contractile proteins in the left ventricles as a function of time (12h, 1, 2 and 5 days after CAVB), using high-throughput real-time RT-PCR. ECG in conscious and anesthetized mice, left ventricular pressure recordings and patch-clamp were used for characterization of this new mouse model. As expected, CAVB was associated with a lengthening of the QT interval. Moreover, polymorphic ventricular tachycardia was recorded in 6/9 freely-moving mice during the first 24h post-ablation. Remarkably, myocardial hypertrophy was only evident 48 h post-ablation and was associated with increased heart weight and altered expression of contractile proteins. During the first 24 hours post-CAVB, genes encoding ion channel subunits were either up-regulated (such as Nav1.5, +74%) or down-regulated (Kv4.2, -43%; KChIP2, -47%; Navβ1, -31%; Cx43, -29%). Consistent with the transient alteration of Kv4.2 expression, I(to) was reduced at day 1, but restored at day 5. In conclusion, CAVB induces two waves of molecular remodeling: an early one (≤24 h) leading to arrhythmias, a later one related to hypertrophy. These results provide new molecular basis for ventricular tachycardia induced by AV block.

KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and cardiac cells, but the mechanism involved remains elusive. In COS-7 cells expressing the KCNE1–KCNQ1 fusion protein, extracellular hypoosmolarity and hyperosmolarity modify the channel biophysical parameters. These changes are consistent with hypoosmolarity increasing the level of membrane phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn upregulates KCNE1–KCNQ1 channels. We showed that increasing PIP2 levels with a water-soluble PIP2 analogue prevented channel upregulation in hypoosmotic condition, suggesting a variation of the channel–PIP2 interaction during channel osmoregulation. Furthermore, we showed that polyamines and Mg2+, already known to tonically inhibit KCNQ channels by screening PIP2 negative charges, are involved in the osmoregulatory process. Indeed, intracellular Mg2+ removal and polyamines chelation inhibited the channel osmoregulation. Thus, the dilution of those cations during cell swelling might decrease channel inhibition and explain the channel upregulation by hypoosmolarity. To support this idea, we quantified the role of Mg2+ in the osmodependent channel activity. Direct measurement of intracellular [Mg2+] variations during osmotic changes and characterization of the channel Mg2+ sensitivity showed that Mg2+ participates significantly to the osmoregulation. Using intracellular solutions that mimic the variation of Mg2+ and polyamines, we were able to recapitulate the current amplitude variations in response to extracellular osmolarity changes. Altogether, these results support the idea of a modulation of the channel–PIP2 interactions by Mg2+ and polyamines during cell volume changes. It is likely that this mechanism applies to other channels that are sensitive to both osmolarity and PIP2.

The two components of the cardiac delayed rectifier current have been the subject of numerous studies since firstly described. This current controls the action potential duration and is highly regulated. After identification of the channel subunits underlying IKs, KCNQ1 associated with KCNE1, and IKr, HERG, their involvement in human cardiac channelopathies have provided various models allowing the description of the molecular mechanisms of the KCNQ1 and HERG channels trafficking, activity and regulation. More recently, studies have been focusing on the unveiling of different partners of the pore-forming proteins that contribute to their maturation, trafficking, activity and/or degradation, on one side, and on their respective expression in the heterogeneous cardiac tissue, on the other side. The aim of this review is to report and discuss the major works on IKs and IKr and the most recent ones that help to understand the precise function of these currents in the heart.

KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function.We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers.Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed.KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes.The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially explaining the malignant clinical phenotype in affected patients.

Background Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes ( CM s) are extremely limited, the use of urine samples to generate induced pluripotent stem cell–derived CM s would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine‐derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. Methods and Results Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal‐based method. Uhi PS cells were then differentiated into CM s using the matrix sandwich method. Uhi PS ‐ CM s showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal‐, atrial‐ and ventricular‐like action potentials recorded using high‐throughput optical and patch‐clamp techniques. Comparison of HERG expression from the patient's Uhi PS ‐ CM s to the mother's Uhi PS ‐ CM s showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K + current (I Kr ). This phenotype gave rise to action potential prolongation and arrhythmias. Conclusions UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.

Objective: The K + channel encoded by the human ether-a`-go-go-related gene (HERG) is crucial for repolarization in the human heart. In order to investigate the impact of HERG current (IKr) on the incidence of cardiac arrhythmias, we generated a transgenic mouse expressing HERG specifically in the heart. Methods and results: ECG recordings at baseline showed no obvious difference between transgenic and wild-type (WT) mice with the exception of the T wave, which was more negative in transgenic mice than in WT mice. E4031 (20 mg/kg) prolonged the QTc interval and flattened the Twave in transgenic mice, but not in WT mice. Injection of BaCl2 (25 mg/kg) induced short runs of ventricular tachycardia in 9/ 10 WT mice, but not in transgenic animals. Atrial pacing reproducibly induced atrial tachyarrhythmias in 11/15 WT mice. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice. When pretreated with dofetilide (10 mg/kg), transgenic mice were as sensitive to experimental arrhythmias as WT mice. Microelectrode studies showed that atrial action potentials have a steeper slope of duration-rate adaptation in WT than in transgenic mice. Transgenic mice were also characterized by a post-repolarization refractoriness, which could result from the substantial amount of IKr subsisting after repolarization as assessed with action potential-clamp experiments and simulations with a model of the transgenic mouse action potential. Conclusion: HERG expression in the mouse heart can protect against experimental induction of arrhythmias. This is the first report of such a protective effect of HERG in vivo.

Voltage-gated Na+ (Nav) channels are key determinants of myocardial excitability and defects in Nav channel functioning or regulation, associated with inherited and acquired cardiac disease, increase the risk of life-threatening arrhythmias. In heart failure, the inactivation gating properties of Nav1.5 channels are altered, resulting in decreased channel availability and increased late Na+ current. Although previous studies have suggested roles for CaMKII- and SGK1-dependent Nav1.5 phosphorylation sites, the global native phosphorylation pattern of Nav1.5 channels associated with these pathophysiological alterations is unknown. Mass spectrometric (MS)-based phosphoproteomic analyses were undertaken to identify in situ the native phosphorylation sites on the Nav1.5 protein purified from ventricles isolated from WT and CaMKIIdc- Tg mice overexpressing CaMKIIdc in the heart. Quantitative analyses of Nav1.5 phosphopeptides allowed comparing the relative abundances of Nav1.5 phosphorylation sites in CaMKIIdc-Tg, versus WT, ventricles. A total of seventeen Nav1.5 phosphorylation sites were identified, seven of which are novel as compared with those reported in our previous MS analyses. Thirteen of these sites are located in the first loop, one in the second loop, one in the N-terminus and two in the Cterminus of Nav1.5. Interestingly, out of these seventeen phosphorylation sites, the C-terminal phosphoserine pS1938 is present in the CaMKIIdc-Tg IPs (n=3/4) and absent in the WT IPs (n=0/4), and pS1989 is 9-fold more represented (p

Objective The acquired long QT syndrome results most often from the action of I(Kr) blocking-drugs on cardiac repolarization. We have evaluated a transgenic (TG) mouse (FVB) overexpressing a dominant-negative KvLQT1 isoform, as an in vivo screening model for I(Kr) blocking drugs. Results In TG mice, six-lead ECGs demonstrated sinus bradycardia, atrioventricular block, and QTc prolongation. Various drugs were injected intraperitoneally after blockade of the autonomic nervous system and serial ECGs were recorded. The end of the initial rapid phase of the T wave corrected for heart rate using a formula for mouse heart (QTrc), was used as a surrogate for the QT interval. Dofetilide, a specific I(Kr) blocker, did not prolong the QTrc interval either in TG or in wild-type (WT) mice but dose-dependently lengthened the sinus period in TG mice but not in WT mice. Other I(Kr) blockers including E 4031, haloperidol, sultopride, astemizole, cisapride and terikalant behaved similarly to dofetilide. Tedisamil, a blocker of the transient outward current, dose-dependently prolonged the QTrc in WT mice but not in TG mice and also reduced the sinus rhythm in both WT and TG mice. Lidocaine dose-dependently shortened the QTrc interval in TG mice and also lengthened the P wave duration. Nicardipine dose-dependently shortened QTrc and also produced sinus arrest in both WT and TG mice. Conclusions We conclude that KvLQT1-invalidated TG mice discriminates in vivo drugs that blocks I(Kr) from drugs that block the transient outward current, the sodium current or the calcium current.

Objective: The present study was designed to examine the effects of chronic amiodarone on the different ventricular cell subtypes in situ and to evaluate its interactions with sotalol. Methods: Three groups of dogs were studied. Group I ( n =8) received no treatment. Group II ( n =7) and group III ( n =8) received, respectively, 100 and 200 mg amiodarone orally twice a day for 6 weeks to 8 months. In vivo studies were performed under halothane anesthesia 14 h after the last administration of amiodarone. Three leads ECG, femoral blood pressure and left ventricular intramural monophasic action potentials (MAP) were continuously recorded. Bradycardia was obtained by clamping the sinus node and β-blockade and the heart was driven by atrial pacing. Three weeks before the in vivo experiments, the cellular electrophysiologic properties of right ventricular tissues obtained by cardiac biopsy in six treated and six control dogs were studied with standard microelectrodes. Results: Amiodarone produced a dose-dependent decrease in plasma levels of triiodothyronine (T3; 5.9±0.4 pM in control dogs, 3.1±0.2 pM in group III, P

Rationale Human genetically inherited cardiac diseases have mainly been studied in heterologous systems or animal models, independently of the patients’ genetic background. Because sources for human cardiomyocytes are extremely limited, the use of urine samples to derive cardiomyocytes would be a non-invasive method to identify cardiac dysfunctions that lead to pathologies within the patients’ specific genetic background. Objective Cardiomyocytes differentiated from urine-derived pluripotent stem cells (UhiPS-CMs) were obtained from a patient with long QT syndrome and a mutation in hERG KCNH2 gene (p. A561P), and were characterized. Methods and Results Cells obtained from urine samples from the A561P patient and his asymptomatic mother carrying no hERG mutation were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into cardiomyocytes using a modified matrix sandwich method. UhiPS-CMs showed proper expression of ventricular cytoskeletal proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials (APs) recorded using both high-throughput CellOptiq and patch-clamp techniques. Application of ajmaline, 4-aminopyridine, nifedipine, chromanol 293B or E-4031 to the UhiPS-CMs confirmed that I Na , I to , I Ca , I Ks and I Kr currents, respectively, contributed to the APs. Comparing hERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in a reduced I Kr current. This phenotype led to APs prolongation that sometimes resulted in arrhythmias (early afterdepolarizations). Conclusion Urine-derived pluripotent stem cells from patients carrying ion channels mutations can be used as novel models to differentiate functional cardiomyocytes that recapitulate cardiac arrhythmia phenotypes.

Mutations in the KvLQT1 gene are the cause of the long QT syndrome 1. KvLQT1 gene product is associated with the regulator protein IsK to produce a component of the delayed rectifier K+ current in cardiac myocytes. We identified an N-terminal truncated isoform of the KvLQT1 gene product, referred to as isoform 2. In RNase protection assays, isoform 2 represented 28.1 +/- 0.6% of the total KvLQT1 expression in the human adult ventricle. COS-7 cells injected intranuclearly with KvLQT1 isoform 1 cDNA exhibited a fast-activating K+ current, whereas those injected with a KvLQT1 isoform 1 plus IsK cDNA showed a slow-activating K+ current. Cells injected with KvLQT1 isoform 2 plasmid showed no detectable K+ current. Those injected with a 1/1 isoform 2/isoform 1 ratio showed no detectable K+ current. Those injected with 1/5 and 2/5 ratios showed a K+ current with markedly reduced amplitude. Coexpression of the IsK regulator consistently reduced the dominant negative effects of isoform 2. Our results indicate that KvLQT1 isoform 2 exerts a pronounced negative dominance on isoform 1 channels and that the cardiac KvLQT1 K+ channel complex is composed of at least three different proteins as follows: isoform 1, isoform 2, and IsK.

Beta3-adrenoceptors are involved in metabolism, gut relaxation, and vascular vasodilation. However, their existence and role in the human heart have not been documented. We investigated the effects of several beta-adrenoceptor agonists and antagonists on the mechanical properties of ventricular endomyocardial biopsies. In the presence of nadolol, a beta1- and beta2-adrenoceptor antagonist, isoprenaline produced consistent negative inotropic effects. Similar negative inotropic effects also resulted from the action of beta3-adrenoceptor agonists with an order of potency: BRL 37344 > SR 58611 approximately CL 316243 > CGP 12177. The dose-response curve to BRL 37344-decreasing myocardial contractility was not modified by pretreatment with nadolol, but was shifted to the right by bupranolol, a nonselective beta-adrenoceptor antagonist. Beta3-adrenoceptor agonists also induced a reduction in the amplitude and an acceleration in the repolarization phase of the human action potential. Beta3-adrenoceptor transcripts were detected in human ventricle by a polymerase chain reaction assay. These results indicate that: (a) beta3-adrenoceptors are present and functional in the human heart; and (b) these receptors are responsible for the unexpected negative inotropic effects of catecholamines and may be involved in pathophysiological mechanisms leading to heart failure.

Compared to the general population, individuals with HIV have a 4.5 fold higher risk of sudden cardiac death, and up to 20% of them present with a long QT syndrome (LQTS). Notably, torsades de pointes arrhythmias have been described even in the absence of drug therapy, suggesting a rather direct implication of HIV in LQTS. The HIV-encoded Tat protein appears as a potential candidate for inducing ventricular arrhythmias, since it was shown to prolong the action potential (AP) of guinea-pig ventricular cardiomyocytes by reducing IKr. Tat protein serves as a transactivator of transcription required for HIV replication. Tat is released from infected cells and is found circulating in the blood of HIV-infected patients.The aim of this study was to evaluate the human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) as a model to study the cellular mechanisms involved in the Tat-dependent alteration of cardiac electrical activity in human.Our data show that Tat incubation reduces IKr in hiPS-CMs while it does not alter IhERG in transfected COS-7 and HEK293 cellular models. hERG protein expression was reduced only in Tat-incubated hiPS-CMs; such reduction likely contributes to IKr reduction. Ventricular AP durations (APD70 and APD90) were significantly increased in hiPS-CMs incubated with Tat compared to buffer incubation. In addition, most Tat-treated cells showed a higher APD90 dispersion which resulted from AP duration and amplitude alternans. Alternans were exacerbated at faster stimulation rates.This work highlights that Tat-treated hiPS-CMs recapitulate alterations of the cardiomyocyte electrical activity, consistent with the arrhythmias observed in HIV patients. hiPS-CMs represent a relevant model for further investigations of the cellular mechanisms involved in AIDS, and, more generally, in cardiac non-genetic diseases.

Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca2+-induced Ca2+ release via phosphorylation of L-type Ca2+ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca2+ current (ICa,L) is increased only in Pde4b–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca2+ transients, cell contraction, and spontaneous Ca2+ release events were increased in Pde4b–/– and Pde4d–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b–/– mice but not in WT mice. These results identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca2+-induced Ca2+ release in cardiomyocytes.

International audience; Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

International audience; Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a phospholipid that has been shown to modulate several ion channels, including some voltage-gated channels like Kv11.1 (hERG). From a biophysical perspective, the mechanisms underlying this regulation are not well characterized. From a physiological perspective, it is critical to establish whether the PIP(2) effect is within the physiological concentration range. Using the giant-patch configuration of the patch-clamp technique on COS-7 cells expressing hERG, we confirmed the activating effect of PIP(2). PIP(2) increased the hERG maximal current and concomitantly slowed deactivation. Regarding the molecular mechanism, these increased amplitude and slowed deactivation suggest that PIP(2) stabilizes the channel open state, as it does in KCNE1-KCNQ1. We used kinetic models of hERG to simulate the effects of the phosphoinositide. Simulations strengthened the hypothesis that PIP(2) is more likely stabilizing the channel open state than affecting the voltage sensors. From the physiological aspect, we established that the sensitivity of hERG to PIP(2) comes close to that of KCNE1-KCNQ1 channels, which lies in the range of physiological PIP(2) variations.

Background— Inflammatory processes play a critical role in myocarditis, dilated cardiomyopathy, and heart failure. The expression of the inflammatory chemokine osteopontin (OPN) is dramatically increased in cardiomyocytes and inflammatory cells during myocarditis and heart failure in human and animals. However, its role in the development of heart diseases is not known. Methods and Results— To understand whether OPN is involved in cardiomyopathies, we generated a transgenic mouse (MHC-OPN) that specifically overexpresses OPN in cardiomyocytes with cardiac-specific promoter-directed OPN expression. Young MHC-OPN mice were phenotypically indistinguishable from their control littermates, but most of them died prematurely with a half-life of 12 weeks of age. Electrocardiography revealed conduction defects. Echocardiography showed left ventricular dilation and systolic dysfunction. Histological analysis revealed cardiomyocyte loss, severe fibrosis, and inflammatory cell infiltration. Most of these inflammatory cells were activated T cells with Th1 polarization and cytotoxic activity. Autoantibodies against OPN, cardiac myosin, or troponin I, were not found in the serum of MHC-OPN mice. Conclusions— These data show that OPN expression in the heart induces in vivo T-cell recruitment and activation leading to chronic myocarditis, the consequence of which is myocyte destruction and hence, dilated cardiomyopathy. Thus, OPN might therefore constitute a potential therapeutic target to limit heart failure.

Cell-based therapies have great potential for the treatment of cardiovascular diseases. Recently, using a transgenic mouse model Roell et al. reported that cardiac engraftment of connexin43 (Cx43)-overexpressing myoblasts in vivo prevents post-infarct arrhythmia, a common cause of death in patients following heart attack. We carried out a similar study but in a clinically relevant context via transplantation of autologous connexin43-overexpressing myoblasts in infarcted rats. Seven days after coronary ligation, rats were randomized into three groups: a control group injected with myoblasts, a null group injected with myoblasts transduced with an empty lentivirus vector (null) and a Cx43 group injected with myoblasts transduced with a lentivirus vector encoding connexin43. In contrast to Roell's report, arrhythmia occurrence was not statistically different between groups (58%, 64% and 48% for the control (n= 12), null (n= 14) and Cx43 (n= 23) groups, respectively, P= 0.92). Using ex vivo intramural monophasic action potential recordings synchronous electrical activity was observed between connexin43-overexpressing myoblasts and host cardiomyocytes, whereas such synchrony did not occur in the null-transduced group. This suggests that ex vivo connexin43 gene transfer and expression in myoblasts improved intercellular electrical coupling between myoblasts and cardiomyocytes. However, in our model such electrical coupling was not sufficient to decrease arrhythmia induction. Therefore, we would suggest a note of caution on the use of combined Cx43 gene and cell therapy to prevent post-infarct arrhythmias in heart failure patients.

International audience; We hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB.

International audience; Both gain- and loss-of-function mutations in the SCN5A gene, which encodes the alpha-subunit of the cardiac voltage-gated Na(+) channel Na(v)1.5, are well established to underlie hereditary arrhythmic syndromes (cardiac channelopathies) such as the type 3 long QT syndrome, cardiac conduction diseases, Brugada syndrome, sick sinus syndrome, atrial standstill and numerous overlap syndromes. Although patch-clamp studies in heterologous expression systems have provided important information to understand the genotype-phenotype relationships of these diseases, they could not clarify how mutations can be responsible for such a large spectrum of diseases, the late age of onset or the progressiveness of some of them, and for the overlapping syndromes. Genetically modified mice rapidly appeared as promising tools for understanding the pathophysiological sequence of cardiac SCN5A-related channelopathies and several mouse models have been established. Here, we review the results obtained on these models that, for most of them, convincingly recapitulate the clinical phenotypes of the patients but that also have their own limitations. Mouse models turn out to be powerful tools to elucidate the pathophysiological mechanisms of SCN5A-related diseases and offer the opportunity to investigate the cellular consequences of SCN5A mutations such as the remodelling of other gene expression that might participate in the overall phenotype and explain some of the differences among patients. Finally, they also constitute useful tools for future studies addressing as yet unanswered questions, such as the role of genetic and environmental modifiers on cardiac conduction and repolarisation.

The voltage-gated cardiac sodium channel Nav1.5, is a critical regulator of cardiac electrical excitability, responsible for the rapid upstroke of the action potential (AP) in both atrial and ventricular cardiomyocytes. In addition to regulating single cell electrophysiology Nav1.5 plays a critical role in conjunction with connexin43, in ventricular conductance. Mutations in the SCN5A gene encoding Nav1.5 are linked with several inherited arrhythmic disorders, including Brugada syndrome and long QT syndrome. Heterozygous mutant mouse models demonstrate severe conduction disturbances and intramural fibrosis, preferentially in the right ventricle (RV). Despite the observation that single cardiomyocyte upstroke velocity is significantly reduced, it has thus far been difficult to separate the relative contribution of reduced Nav1.5 conductance and intramural fibrosis to the appearance of lethal ventricular arrhythmias. We address this issue by characterizing in greater detail the transmural electrophysiological behavior of ventricular tissue in a mouse model of SCN5A downregulation. Heterozygous SCN5A+/− mutant mice (SCN5A+/−; n=7) and wild type (WT; n=6) littermates’ hearts were langendorff perfused beneath a combined 2P and epifluorescence microscope system and loaded with voltage (di-4 ANEPPS) and intracellular calcium (Fura-2AM) sensitive dyes. Under physiological endo-epicardial activation, no significant difference between groups was apparent from optical map recordings of the RV surface. Transmural 2P line scanning revealed significantly slower AP upstroke times within the subepicardium, but not close to the surface, in SCN5A+/− vs WT. Conduction velocity was slower in SCN5A+/− vs WT mice, and was preferentially slow in the midmyocardium vs epi/endocardium. Beat-to-beat activation was also significantly higher in SCN5A+/− vs WT hearts. These data suggest significant conduction abnormalities within the midmyocardial wall which affect the rate and path of conduction preferentially within this transmural region.

We investigated the effects of chronic and moderate heart rate (HR) reduction on ion channel expression in the mouse sinoatrial node (SAN) and ventricle. Ten-week-old male C57BL/6 mice were treated twice daily with either vehicle or ivabradine at 5 mg/kg given orally during 3 wk. The effects of HR reduction on cardiac electrical activity were investigated in anesthetized mice with serial ECGs and in freely moving mice with telemetric recordings. With the use of high-throughput real-time RT-PCR, the expression of 68 ion channel subunits was evaluated in the SAN and ventricle at the end of the treatment period. In conscious mice, ivabradine induced a mean 16% HR reduction over a 24-h period that was sustained over the 3-wk administration. Other ECG parameters were not modified. Two-way hierarchical clustering analysis of gene expression revealed a separation of ventricles from SANs but no discrimination between treated and untreated ventricles, indicating that HR reduction per se induced limited remodeling in this tissue. In contrast, SAN samples clustered in two groups depending on the treatment. In the SAN from ivabradine-treated mice, the expression of nine ion channel subunits, including Navβ1 (−25%), Cav3.1 (−29%), Kir6.1 (−28%), Kvβ2 (−41%), and Kvβ3 (−30%), was significantly decreased. Eight genes were significantly upregulated, including K+ channel α-subunits (Kv1.1, +30%; Kir2.1, +29%; Kir3.1, +41%), hyperpolarization-activated cation channels (HCN2, +24%; HCN4, +52%), and connexin 43 (+26%). We conclude that reducing HR induces a complex remodeling of ion channel expression in the SAN but has little impact on ion channel transcripts in the ventricle.

Ion channels are an ensemble of specialized membrane proteins that act in concert to create and modulate the electrical activity of many excitable cells, including cardiac myocytes. Following completion of the sequencing of various genomes, including that of the human, the complete repertoire of ion channel genes has been elucidated for different species. How transcripts issued from this gene collection are expressed and modulated in relation to variable physiological and pathological situations is the subject of functional or physiological genomics. Specialized microarrays (IonChips) comprising probes for the ensemble of ion channel and regulatory genes were developed as an alternative to whole-genome DNA chips. Physiological genomics of cardiac ion channel genes is a growing field that, in combination with genetics, should markedly increase our comprehension of the molecular mechanisms leading to arrhythmias.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits (“IonChips”) were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I to,f and I K,slow densities, and increased I Ks density in hypothyroid myocytes. In addition to effects on K + channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the α1C Ca 2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na + , Cl − , and inwardly rectifying K + channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel α- and β-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.

OBJECTIVES: The KCNQ1 gene encodes the KvLQT1 potassium channel, which generates in the human heart the slow component of the cardiac delayed rectifier current, I(Ks). Mutations in KCNQ1 are the most frequent cause of the congenital long QT syndrome. We have previously cloned a cardiac KCNQ1 human isoform, which exerts a strong dominant-negative effect on KvLQT1 channels. We took advantage of this dominant-negative isoform to engineer an in vivo model of KvLQT1 disruption, obtained by overexpressing the dominant-negative subunit under the control of the alpha-myosin heavy chain promoter. RESULTS: Three different transgenic lines demonstrated a phenotype with increasing severity. Functional suppression of KvLQT1 in transgenic mice led to a markedly prolonged QT interval associated with sinus node dysfunction. Transgenic mice also demonstrated atrio-ventricular block leading to occasional Wenckebach phenomenon. The atrio-ventricular block was associated with prolonged AH but normal HV interval in His recordings. Prolonged QT interval correlated with prolonged action potential duration and with reduced K(+) current density in patch-clamp experiments. RNase protection assay revealed remodeling of K(+) channel expression in transgenic mice. CONCLUSIONS: Our transgenic mouse model suggests a role for KvLQT1 channels not only in the mouse cardiac repolarisation but also in the sinus node automaticity and in the propagation of the impulse through the AV node

The KCNE1 gene encodes a channel regulator IsK which in association with the KvLQT1 K + channel protein determines the slow component of the cardiac delayed rectifier current. We have investigated the cellular electrophysiological characteristics of adult KCNE1 -knockout mouse hearts by means of the standard microelectrode technique. Action potential parameters from the ventricular endocardium of KCNE1 −/− mice were indistinguishable from those of KCNE1 +/+ animals. In particular, KCNE1 −/− hearts did not exhibit prolonged repolarization. E-4031, a specific blocker of erg K+ channels consistently prolonged repolarization in KCNE1 +/+ but not in KCNE1 −/− hearts. By contrast, the chromanol compound 293B, a specific blocker of KvLQT1 K+ channel produced comparable effects on repolarization in KCNE1 −/− and KCNE1 +/+ mice. We conclude that invalidation of the mouse KCNE1 gene by homologous recombination leads to a mild cardiac phenotype at the cellular level.

Mutations in the KvLQT1 gene are the cause for the long QT syndrome [Circulation 94:1996-2012 (1996)]. Coexpression of KvLQT1 in association with the channel regulator protein IsK produces a K+ current with characteristics reminiscent of the slow component of the delayed rectifier in cardiac myocytes. We explored the pharmacological properties of trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dime thyl- chromane (293B), a chromanol compound, on the K+ current produced by direct intranuclear injection of KvLQT1 and IsK cDNA plasmids in COS-7 cells. Injected cells were recorded by means of the whole-cell and cell-attached patch-clamp configurations under chloride-free conditions. Cells injected with KvLQT1 cDNA alone exhibited a fast-activating outward K+ current, whereas cells coinjected with KvLQT1 plus IsK cDNAs exhibited a time-dependent outward current with slower activation kinetics. The chromanol 293B blocked the K+ current related to KvLQT1 expression in both the absence or presence of IsK. The IC50 value for 293B to block KvLQT1-related current was not significantly modified by the presence of IsK (9.9 microM in the absence of IsK versus 9.8 microM in its presence). The block produced by 293B was strongly voltage-dependent inasmuch as it was close to 0 at -80 mV and occurred during a depolarizing voltage step. The time constants for the drug to block the current were in the same order of magnitude as activation kinetics of the current. Kinetics for drug unblock at the holding potential were much faster, in the order of a few tenths of a msec. KvLQT1 currents recorded in the cell-attached configuration were also blocked by externally applied 293B, suggesting that the compound penetrated the cell to block the channel. Cromakalim, another chromanol compound, also blocked KvLQT1 currents. Our results show that the chromanol compound 293B is targeted to KvLQT1 channels but not to the IsK regulator.

We used standard microelectrode techniques to study the developmental changes and beta-adrenergic modulation of membrane potential and of Na-K pump activity in adult (> 1 yr of age) and neonatal (2-10 days) canine Purkinje fibers. Isoproterenol (10(-7) M) increased the rate of development and magnitude of pacing-induced hyperpolarization of adult fibers driven at a 1-s cycle length. This effect of isoproterenol was attenuated by treating dogs with pertussis toxin (PTX), (30 micrograms/kg). Other adult and neonatal fibers were superfused with a Tyrode solution containing Ba2+ 0.2 mM, Cs+ 2 mM, and 10(-6) M verapamil, thus leading to depolarization and cessation of spontaneous activity. The Na-K pump was studied by alternating solutions containing [K] at 0 mM (inhibiting the pump) and 4 mM (reactivating the pump). Although the kinetics of the Na-K pump appeared faster in neonatal fibers than in adult fibers, measurement of cell surface-to-volume ratio compensated for the difference. We therefore conclude that 1) the apparent age-related changes in Na-K pump activity in canine Purkinje fibers in fact reflect cell surface-to-volume ratio and, 2) the beta-adrenergic agonist-induced hyperpolarization in adults requires the presence of a PTX-sensitive G protein for its occurrence.

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.

Long Q-T mutant (KvLQT1) K+ channels associate with their regulatory subunit IsK to produce the slow component of the delayed rectifier potassium ( I Ks) cardiac current. The amplitude of KvLQT1 current depends on the expression of a KvLQT1 splice variant (isoform 2) that exerts strong dominant negative effects on the full-length KvLQT1 protein (isoform 1). We used RNase protection assays to determine the relative expression of KvLQT1 isoforms 1 and 2 and IsK mRNAs in human ventricular layers. Overall expression of KvLQT1 and IsK genes was similar in the three layers. However, there was a significant difference in the ratio between KvLQT1 isoforms 1 and 2. Isoform 2 represented 25.2 ± 2.3%, 31.7 ± 1.2%, and 24.9 ± 1.7% of total KvLQT1 expression in left ventricular endocardial, midmyocardial, and epicardial tissues, respectively. Similar data were obtained from right ventricular samples. COS-7 cells were intranuclearly injected with KvLQT1 isoforms 1 or 2 plus IsK cDNAs, using two different isoform 2-to-isoform 1 ratios. Cells injected with an isoform 2-to-isoform 1 ratio mimicking that in the midmyocardium showed a K+current with ∼75% reduced amplitude compared with those injected with a ratio mimicking that in the epicardium. Our results suggest that differential expression of KvLQT1 isoform 2 in endocardial, midmyocardial, and epicardial tissues is responsible for differential I Ks amplitude and contributes to the regional action potential heterogeneity observed across the ventricular wall.

Point mutation of nearby residues in ion channels can be associated with diametrically opposed clinical phenotypes despite the mutant channels exhibit similar biophysical characteristics. Hence a characterization of the channel structure/function at the amino-acid scale is required for better understanding of channel genotype/phenotype relationship. R555C and R539W KCNQ1 mutant channels are a good illustration of this idea: R555C mutation is associated with a fruste form of type 1 long QT syndrome, whereas R539W mutation is associated with sudden death. Puzzling enough, the genotype/phenotype relationship is difficult to understand because the mutated residues are in the same helix C module, they both concern arginine residues, both channels have the same biophysical properties, and the same sensitivity to short chain phosphatidylinositol-4,5-bisphosphate (PIP2).To better understand the genotype/phenotype relationship in the context of these mutations, we performed several tests in COS-7 cells expressing the WT or mutant channels and used tail-currents amplitudes as readout. We show that R539W is very peculiar: As opposed to WT and R555C channels, the R539W channel current is barely running down when available PIP2 is decreased, either by wortmannin application in whole-cell, or by magnesium application in inside-out configuration. Consistent with that, the R539W channel is also insensitive to extracellular osmolarity, known to modulate the channel activity via PIP2. These results suggest that KCNQ1-R539W mutation shortcuts PIP2 in the channel open pore stabilization. Both structural model prediction and functional analysis implicate membrane cholesterol in this effect. Indeed, structural model prediction suggests that the introduced tryptophan in R539W interacts with cholesterol. Both cyclodextrin application on R539W and substitution of R539 by residues other than tryptophan restore channel rundown, consistent with the supposed tryptophan-cholesterol interaction. We conclude that the R539W/cholesterol interaction substitutes for R539/PIP2 interaction in the channel open pore stabilization.

Voltage-gated Na+ (Nav) channels are key determinants of myocardial excitability and defects in Nav channel functioning or regulation, associated with inherited and acquired cardiac disease, increase the risk of life-threatening arrhythmias. In heart failure, the inactivation gating properties of Nav1.5 channels are altered, resulting in decreased channel availability and increased late Na+ current. Although previous studies have suggested roles for CaMKII and CaMKII-dependent Nav1.5 phosphorylation sites, the global native phosphorylation pattern of Nav1.5 channels associated with these pathophysiological alterations is unknown. Mass spectrometric (MS)-based phosphoproteomic analyses were undertaken to identify in situ the native phosphorylation sites on the Nav1.5 protein purified from ventricles isolated from mice (CaMKIIdc-Tg) overexpressing CaMKIIdc in the heart. Quantitative analyses of Nav1.5 phosphopeptides allowed comparing the relative abundances of Nav1.5 phosphorylation sites in CaMKIIdc-Tg, versus wild-type, ventricles. A total of eighteen Nav1.5 phosphorylation sites were identified, seven of which are novel as compared with those reported in our previous LC-MS/MS analyses. Fourteen of these sites are located in the first loop, one in the second loop, one in the N-terminus and two in the C-terminus of Nav1.5. Interestingly, out of these eighteen phosphorylation sites, the C-terminal phosphoserine pS1938 is present in the CaMKIIdc-Tg IPs (n=3/4) and absent in the WT IPs (n=0/4), and pS1989 is 9-fold more represented (p