Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.

25 years of basic and translational science in EP Europace: novel insights into arrhythmia mechanisms and therapeutic strategies.

In the last 25 years, EP Europace has published more than 300 basic and translational science articles covering different arrhythmia types (ranging from atrial fibrillation to ventricular tachyarrhythmias), different diseases predisposing to arrhythmia formation (such as genetic arrhythmia disorders and heart failure), and different interventional and pharmacological anti-arrhythmic treatment strategies (ranging from pacing and defibrillation to different ablation approaches and novel drug-therapies). These studies have been conducted in cellular models, small and large animal models, and in the last couple of years increasingly in silico using computational approaches. In sum, these articles have contributed substantially to our pathophysiological understanding of arrhythmia mechanisms and treatment options; many of which have made their way into clinical applications. This review discusses a representative selection of EP Europace manuscripts covering the topics of pacing and ablation, atrial fibrillation, heart failure and pro-arrhythmic ventricular remodelling, ion channel (dys)function and pharmacology, inherited arrhythmia syndromes, and arrhythmogenic cardiomyopathies, highlighting some of the advances of the past 25 years. Given the increasingly recognized complexity and multidisciplinary nature of arrhythmogenesis and continued technological developments, basic and translational electrophysiological research is key advancing the field. EP Europace aims to further increase its contribution to the discovery of arrhythmia mechanisms and the implementation of mechanism-based precision therapy approaches in arrhythmia management.

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.

ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.

Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition". AIM: To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093). METHODS: Ca2+ transients (CaT) were recorded at 36 °C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN where immunolabeled in single hiPSC-CMs. Mutant preparations (MUT) were compared to isogenic wild-type ones (WT), obtained by mutation reversal. RESULTS: WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) and half-time of CaT decay were shorter in MUT; (2) several CaT profiles were identified in WT, "hyperdynamic" ones largely prevailed in MUT; (3) whereas tpeak rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 turned all the above properties to resemble those of MUT; when applied to MUT, PST3093 had a smaller or negligible effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. CONCLUSIONS: Functional data converge to argue for PLN p.Arg14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, possibly including dysregulation of Ca2+-dependent transcription.

Repolarization instability and arrhythmia by IKr block in single human-induced pluripotent stem cell-derived cardiomyocytes and 2D monolayers.

AIMS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. METHODS AND RESULTS: Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation-contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. CONCLUSION: Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.

Role of the ß3-adrenergic receptor subtype in catecholamine-induced myocardial remodeling.

ß3-Adrenoceptors (AR) stimulate cardiac Na+/K+ pump in healthy hearts. ß3-ARs are upregulated by persistent sympathetic hyperactivity; however, their effect on Na+/K+ ATPase activity and ventricular function in this condition is still unknown. Here, we investigate preventive effects of additional ß3-AR activation (BRL) on Na+/K+ ATPase activity and in vivo hemodynamics in a model of noradrenaline-induced hypertrophy. Rats received NA or NA plus simultaneously administered BRL in vivo infusion for 14 days; their cardiac function was investigated by left ventricular pressure-volume analysis. Moreover, fibrosis and apoptosis were also assessed histologically. NA induced an hypertrophic pattern, as detected by morphological, histological, and biochemical markers. Additional BRL exposure reversed the hypertrophic pattern and restored Na+/K+ ATPase activity. NA treatment increased systolic function and depressed diastolic function (slowed relaxation). Additional BRL treatment reversed most NA-induced hemodynamic changes. NA decreased Na+/K+ pump α2 subunit expression selectively, a change also reversed by additional BRL treatment. Increasing ß3-AR stimulation may prevent the consequences of chronic NA exposure on Na+/K+ pump and in vivo hemodynamics. ß3-AR agonism may thus represent a new therapeutic strategy for pharmacological modulation of hypertrophy under conditions of chronically enhanced sympathetic activity.

Action potential contour contributes to species differences in repolarization response to ß-adrenergic stimulation.

Aim: Repolarization response to ß-adrenergic (ß-AR) stimulation differs between guinea-pig and canine myocytes and, within the latter, between myocardial layers. Correlative analysis suggests that this may be due to differences in action potential (AP) contour. Here we tested whether AP contour may set the response of current and of repolarization to ß-AR stimulation (10 nM isoproterenol, ISO). Methods and results: The responses of AP and current to ISO were measured under I-clamp and "AP-clamp" in guinea-pig (GP), dog epicardial (DEPI) and dog subendocardial (DENDO) myocytes. Dynamic-clamp (DC) was used to evaluate the impact of AP features on AP response to ISO. ISO prolonged AP duration (APD) in GP myocytes, did not affect it in DENDO and shortened it in DEPI ones. The current induced by ISO (IISO) sharply differed between GP and canine myocytes and, to a lesser extent, between DENDO and DEPI ones. Differences in IISO profile likely important in setting APD response (time-to-peak, time-to-reversal), were minimized when canine myocytes where clamped with GP AP-waveforms and vice versa. Introduction of a "notch" in GP AP (by DC) was alone insufficient to affect the APD response to ISO; nevertheless, when incorporated in a GP AP-waveform, the main "canine" AP features ("notch" and low plateau potential) caused IISO of GP myocytes to acquire canine features. Conclusion: Early repolarization contour and level of plateau potential contribute to species-specificity of IISO profile. Changes in AP contour, also when generated by modulation of ISO-insensitive currents, may be crucial in setting APD response to ß-AR stimulation.

Peptidomimetic Targeting of Cavß2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

Late sodium current and intracellular ionic homeostasis in acute ischemia.

Blockade of the late Na+ current (I NaL) protects from ischemia/reperfusion damage; nevertheless, information on changes in I NaL during acute ischemia and their effect on intracellular milieu is missing. I NaL, cytosolic Na+ and Ca2+ activities (Nacyt, Cacyt) were measured in isolated rat ventricular myocytes during 7 min of simulated ischemia (ISC); in all the conditions tested, effects consistently exerted by ranolazine (RAN) and tetrodotoxin (TTX) were interpreted as due to I NaL blockade. The results indicate that I NaL was enhanced during ISC in spite of changes in action potential (AP) contour; I NaL significantly contributed to Nacyt rise, but only marginally to Cacyt rise. The impact of I NaL on Cacyt was markedly enhanced by blockade of the sarcolemmal(s) Na+/Ca2+ exchanger (NCX) and was due to the presence of (Na+-sensitive) Ca2+ efflux through mitochondrial NCX (mNCX). sNCX blockade increased Cacyt and decreased Nacyt, thus indicating that, throughout ISC, sNCX operated in the forward mode, in spite of the substantial Nacyt increment. Thus, a robust Ca2+ source, other than sNCX and including mitochondria, contributed to Cacyt during ISC. Most, but not all, of RAN effects were shared by TTX. (1) The paradigm that attributes Cacyt accumulation during acute ischemia to decrease/reversal of sNCX transport may not be of general applicability; (2) I NaL is enhanced during ISC, when the effect of Nacyt on mitochondrial Ca2+ transport may substantially contribute to I NaL impact on Cacyt; (3) RAN may act mostly, but not exclusively, through I NaL blockade during ISC.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.

Combined action potential- and dynamic-clamp for accurate computational modelling of the cardiac IKr current.

In the present work Action-Potential clamp (APC) and Dynamic clamp (DC) were used in combination in order to optimize the Luo-Rudy (LRd) mathematical formulation of the guinea-pig rapid delayed rectifier K(+) current (IKr), and to validate the optimized model. To this end, IKr model parameters were adjusted to fit the experimental E4031-sensitive current (IE4031) recorded under APC in guinea-pig myocytes. Currents generated by LRd model (ILRd) and the optimized one (IOpt) were then compared by testing their suitability to replace IE4031 under DC. Under APC, ILRd was significantly larger than IE4031 (mean current densities 0.51±0.01 vs 0.21±0.05pA/pF; p<0.001), mainly because of different rectification. IOpt mean density (0.17±0.01pA/pF) was similar to the IE4031 one (NS); moreover, IOpt accurately reproduced IE4031 distribution along the different AP phases. Models were then compared under DC by blocking native IKr (5µM E4031) and replacing it with ILRd or IOpt. Whereas injection of ILRd overshortened AP duration (APD90) (by 25% of its pre-block value), IOpt injection restored AP morphology and duration to overlap pre-block values. This study highlights the power of APC and DC for the identification of reliable formulations of ionic current models. An optimized model of IKr has been obtained which fully reversed E4031 effects on the AP. The model strongly diverged from the widely used Luo-Rudy formulation; this can be particularly relevant to the in silico analysis of AP prolongation caused by IKr blocking or alterations.

Deranged sodium to sudden death.

In February 2014, a group of scientists convened as part of the University of California Davis Cardiovascular Symposium to bring together experimental and mathematical modelling perspectives and discuss points of consensus and controversy on the topic of sodium in the heart. This paper summarizes the topics of presentation and discussion from the symposium, with a focus on the role of aberrant sodium channels and abnormal sodium homeostasis in cardiac arrhythmias and pharmacotherapy from the subcellular scale to the whole heart. Two following papers focus on Na(+) channel structure, function and regulation, and Na(+)/Ca(2+) exchange and Na(+)/K(+) ATPase. The UC Davis Cardiovascular Symposium is a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The focus on Na(+) in the 2014 symposium stemmed from the multitude of recent studies that point to the importance of maintaining Na(+) homeostasis in the heart, as disruption of homeostatic processes are increasingly identified in cardiac disease states. Understanding how disruption in cardiac Na(+)-based processes leads to derangement in multiple cardiac components at the level of the cell and to then connect these perturbations to emergent behaviour in the heart to cause disease is a critical area of research. The ubiquity of disruption of Na(+) channels and Na(+) homeostasis in cardiac disorders of excitability and mechanics emphasizes the importance of a fundamental understanding of the associated mechanisms and disease processes to ultimately reveal new targets for human therapy.

Late sodium current (INaL) in pancreatic ß-cells.

Recent evidence of beneficial effects of ranolazine (RAN) in type II diabetes motivates interest in the role of the late sodium current (INaL) in glucose-stimulated insulin secretion. In the present work, we characterize INaL and its function in rat INS-1E cells and human islets cells. INaL was identified as steady-state current blocked by 10 µM RAN (IRAN) or 0.5 µM tetrodotoxin (TTX) (ITTX). Veratridine (VERA, 40 µM) was used as INaL enhancer. Baseline INaL was similar between INS-1E and human islet cells. In INS-1E cells, activated by glucose or tolbutamide, TTX or RAN hyperpolarized membrane potential (V m). VERA-induced depolarization was countered by TTX or RAN. ITTX and IRAN reversal potentials were negative to Na(+) equilibrium one, but they approached it after Na(+) substitution with Li(+) or when K(+) channels were blocked. This revealed INaL coupling with Na(+)-activated K(+) current (IKNa); expression of IKNa channels (Slick/Slack) was confirmed by transcript analysis and Western blot. RAN or TTX blunted cytosolic Ca(2+) response to depolarization. Long-term incubation in high (33 mM) glucose (CHG) constitutively enhanced INaL. VERA immediately increased glucose-stimulated insulin secretion. CHG increased glucose-independent secretion instead and abolished the secretory response to glucose. RAN or TTX countered VERA- and CHG-induced changes in insulin secretion. Our study demonstrated that (1) INaL was expressed in insulin-secreting cells and coupled to IKNa; INaL affected cytosolic Ca(2+) but, unless enhanced, barely contributed to glucose-stimulated insulin secretion (GSIS); and (2) sustained hyperglycemic stress enhanced INaL, which contributed to the attending increase of glucose-independent insulin "leak" and GSIS impairment.

Opportunities and challenges of current electrophysiology research: a plea to establish 'translational electrophysiology' curricula.

Cardiac electrophysiology has evolved into an important subspecialty in cardiovascular medicine. This is in part due to the significant advances made in our understanding and treatment of heart rhythm disorders following more than a century of scientific discoveries and research. More recently, the rapid development of technology in cellular electrophysiology, molecular biology, genetics, computer modelling, and imaging have led to the exponential growth of knowledge in basic cardiac electrophysiology. The paradigm of evidence-based medicine has led to a more comprehensive decision-making process and most likely to improved outcomes in many patients. However, implementing relevant basic research knowledge in a system of evidence-based medicine appears to be challenging. Furthermore, the current economic climate and the restricted nature of research funding call for improved efficiency of translation from basic discoveries to healthcare delivery. Here, we aim to (i) appraise the broad challenges of translational research in cardiac electrophysiology, (ii) highlight the need for improved strategies in the training of translational electrophysiologists, and (iii) discuss steps towards building a favourable translational research environment and culture.

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

Determinants of beat-to-beat variability of repolarization duration in the canine ventricular myocyte: a computational analysis.

Beat-to-beat variability of repolarization duration (BVR) is an intrinsic characteristic of cardiac function and a better marker of proarrhythmia than repolarization prolongation alone. The ionic mechanisms underlying baseline BVR in physiological conditions, its rate dependence, and the factors contributing to increased BVR in pathologies remain incompletely understood. Here, we employed computer modeling to provide novel insights into the subcellular mechanisms of BVR under physiological conditions and during simulated drug-induced repolarization prolongation, mimicking long-QT syndromes type 1, 2, and 3. We developed stochastic implementations of 13 major ionic currents and fluxes in a model of canine ventricular-myocyte electrophysiology. Combined stochastic gating of these components resulted in short- and long-term variability, consistent with experimental data from isolated canine ventricular myocytes. The model indicated that the magnitude of stochastic fluctuations is rate dependent due to the rate dependence of action-potential (AP) duration (APD). This process (the "active" component) and the intrinsic nonlinear relationship between membrane current and APD ("intrinsic component") contribute to the rate dependence of BVR. We identified a major role in physiological BVR for stochastic gating of the persistent Na(+) current (INa) and rapidly activating delayed-rectifier K(+) current (IKr). Inhibition of IKr or augmentation of INa significantly increased BVR, whereas subsequent ß-adrenergic receptor stimulation reduced it, similar to experimental findings in isolated myocytes. In contrast, ß-adrenergic stimulation increased BVR in simulated long-QT syndrome type 1. In addition to stochastic channel gating, AP morphology, APD, and beat-to-beat variations in Ca(2+) were found to modulate single-cell BVR. Cell-to-cell coupling decreased BVR and this was more pronounced when a model cell with increased BVR was coupled to a model cell with normal BVR. In conclusion, our results provide new insights into the ionic mechanisms underlying BVR and suggest that BVR reflects multiple potentially proarrhythmic parameters, including increased ion-channel stochasticity, prolonged APD, and abnormal Ca(2+) handling.