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The cardiac action potential (AP) is a key species-specific feature of cardiomyocytes that occurs in response to coordinated actions of ion channels. It represents the first step of the cardiac excitation-contraction coupling and it is crucial for cardiomyocyte (CM) physiology. Changes in the cardiac AP may primarily occur as a consequence of diseases or as a direct or unwanted response to drugs. Our ability to quantify these changes defines the reliability of our measurements and its throughput.Cardiac AP parameters are often quantified through manual time-consuming data analysis protocols or custom-made and proprietary data analysis pipelines; to the best of our knowledge, no tools are currently available for automated cardiac AP analysis and AP parameter quantification.Here we introduce a free and open source software tool named Batch Action PoTential Analyser (BAPTA), written in the R language, designed to i) overcome the inherent operator-dependent bias on trace selection affecting reproducibility, ii) vastly improve the throughput of the analyses of large datasets and iii) analyse both spontaneous and triggered APs from CMs of multiple species and origin.We present here four use-cases in which BAPTA can be used at high throughput to investigate the effects of: 1) a disease (cardiomyopathy) on rat CMs, 2) drugs on mouse pacemaker cells, 3) rate-dependency of AP duration in guinea pig CMs and 4) metabolic electrophysiological maturation in human stem-cell-derived CMs. Overall, BAPTA consistently provides faster, more reproducible and scalable readouts which excellently correlate with manual analyses performed by experienced electrophysiologists.

Aims Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. Methods and results Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. Conclusions SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Translational perspective Deficient sarcoplasmic reticulum (SR) Ca2+ uptake has been identified in cardiomyocytes from failing human hearts with impaired diastolic relaxation (e.g. diabetic hearts) and has been associated with a decreased SERCA2a expression and activity and/or with a higher SERCA2a inhibition by phospholamban. Thus, SERCA2a may represent a pharmacological target for interventions aimed at improving cytosolic Ca2+ compartmentalization into the SR to limit diastolic dysfunction pathologies. In this context, istaroxime is the first-in-class luso-inotropic agent targeting SERCA2a that has already demonstrated its efficacy in clinical trials and may be useful to clarify the relevance of SERCA2a stimulation in controlling cytosolic Ca2+ level.

Aims In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. Methods and results We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. Conclusion Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.

BackgroundHeart 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 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.MethodsWe 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.ResultsIstaroxime 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 i.v. toxicity was considerably lower than that of istaroxime and it failed to significantly interact with 50 off-targets.ConclusionsOverall, 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.Graphical abstract

The stimulation of sarcoplasmic reticulum calcium ATPase SERCA2a emerged as a novel therapeutic strategy to efficiently improve overall cardiac function in heart failure (HF) with reduced arrhythmogenic risk. Istaroxime is a clinical-phase IIb compound with a double mechanism of action, Na+/K+ ATPase inhibition and SERCA2a stimulation. Starting from the observation that istaroxime metabolite PST3093 does not inhibit Na+/K+ ATPase while stimulates SERCA2a, we synthesized a series of bioisosteric PST3093 analogues devoid of Na+/K+ ATPase inhibitory activity. Most of them retained SERCA2a stimulatory action with nanomolar potency in cardiac preparations from healthy guinea pigs and streptozotocin (STZ)-treated rats. One compound was further characterized in isolated cardiomyocytes, confirming SERCA2a stimulation and in vivo showing a safety profile and improvement of cardiac performance following acute infusion in STZ rats. We identified a new class of selective SERCA2a activators as first-in-class drug candidates for HF treatment.

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.

Background: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function.Methods: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and by Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. Results: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation and CXCR4/SDF-1α axis activity from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. Conclusion: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.

Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34In cord blood (CB)-derived CD34CD34We provided the first evidence that CD34

Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): This work was supported by CVie Therapeutics Limited (Taipei, Taiwan) and Windtree Therapeutics (Warrington, USA) Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the double property to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). The project aims to characterize istaroxime effects at a concentration (100 nM) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in a model of mild diabetes. Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nM) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Abstract Figure.

Aims NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by IKs blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1-A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced ICaL and INaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed ‘transient inward current’ events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and ICaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes.

In this study we describe the generation and characterization of an human induced pluripotent stem cell (hiPSC) line from a long QT syndrome type 1 (LQT1) patient carrying the KCNQ1 c.940 G > A (p.Gly314Ser) mutation. This patient-specific iPSC line has been obtained by using non-integrational Sendai reprogramming method, expresses pluripotency markers and has the capacity to differentiate into the three germ layers and into spontaneously beating cardiomyocytes (iPSC-CMs).

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues’ side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.

Purpose: Exposure to hypoxia has been suggested to activate multiple adaptive pathways so that muscles are better able to maintain cellular energy homeostasis. However, there is limited research regarding the tissue specificity of this response. The aim of this study was to investigate the influence of tissue specificity on mitochondrial adaptations of rat skeletal and heart muscles after 4 weeks of normobaric hypoxia (FiO2: 0.10). Methods: Twenty male Wistar rats were randomly assigned to either normobaric hypoxia or normoxia. Mitochondrial respiration was determined in permeabilised muscle fibres from left and right ventricles, soleus and extensorum digitorum longus (EDL). Citrate synthase activity and the relative abundance of proteins associated with mitochondrial biogenesis were also analysed. Results: After hypoxia exposure, only the soleus and left ventricle (both predominantly oxidative) presented a greater maximal mass-specific respiration (+48 and +25%, p < 0.05) and mitochondrial-specific respiration (+75 and +28%, p < 0.05). Citrate synthase activity was higher in the EDL (0.63 ± 0.08 vs 0.41 ± 0.10 µmol min− 1 µg− 1) and lower in the soleus (0.65 ± 0.17 vs 0.87 ± 0.20 µmol min− 1 µg− 1) in hypoxia with respect to normoxia. There was a lower relative protein abundance of PGC-1α (−25%, p < 0.05) in the right ventricle and a higher relative protein abundance of PGC-1β (+43%, p < 0.05) in the left ventricle of rats exposed to hypoxia, with few differences for protein abundance in the other muscles. Conclusion: Our results show a muscle-specific response to 4 weeks of normobaric hypoxia. Depending on fibre type, and the presence of ventricular hypertrophy, muscles respond differently to the same degree of environmental hypoxia

beta(3)-Adrenoceptors (AR) stimulate cardiac Na+/K+ pump in healthy hearts. beta(3)-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 beta(3)-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 alpha 2 subunit expression selectively, a change also reversed by additional BRL treatment. Increasing beta(3)-AR stimulation may prevent the consequences of chronic NA exposure on Na+/K+ pump and in vivo hemodynamics. beta(3)-AR agonism may thus represent a new therapeutic strategy for pharmacological modulation of hypertrophy under conditions of chronically enhanced sympathetic activity.

Introduction The addition of anti-human epidermal growth factor receptor 2 (HER2; ErbB2) monoclonal antibody Trastuzumab (TRZ) to Doxorubicin (DOXO) chemotherapy is associated with a synergistic increase in cardiac toxicity. While previous studies have addressed the toxicity of both agents on isolated cardiomyocytes (CMs), little is known regarding this process in vivo, especially with respect to electrophysiological changes. Purpose To investigate electrical and structural changes in LV and RV CMs using an in vivo rat model of DOXO/TRZ cardiotoxicity. Methods Rats received 6 IP injections of either DOXO or TRZ over a 2-week period, or 6 doses of DOXO followed by 6 doses of TRZ (COMBO), or saline as a control. In-vivo echocardiography was performed. Electrical activity and Ca2+ handling were assessed in LV and RV CMs from rat hearts. Single cell patch-clamp and field stimulation experiments were performed. Spontaneous sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks) were recorded at x100 magnification in line-scan mode (sampling rate 0.7 kHz) from 2 μM Fluo4-AM loaded CMs. To assess T-tubular disarray, CMs were incubated with di-3-ANEPPDHQ and periodic component was quantified by Fast Fourier Transform (FFT) analysis of confocal microscopy images. Results DOXO, and to a greater extent COMBO treatment was associated with significant increases in both LV end-systolic and end-diastolic volumes, and decreases in LVEF and fractional shortening. By contrast, TRZ alone merely increased LV end-systolic volume. Electrophysiological studies showed increases in action potential duration (APD), beat-to-beat variability of repolarization (BVR), delayed after depolarizations (DADs), and Ca2+-sparks in both DOXO and COMBO groups. Stimulated intracellular Ca2+ transients (1,2 and 4 Hz) showed significant changes with respect to time to peak, tau decay, amplitude, and fractional release in the DOXO group. These changes were associated with a significant downregulation of sarco/endoplasmic reticulum Ca2+ ATPase pump (SERCA) expression. From a structural viewpoint, these changes were associated with T-tubular disarray in the DOXO and COMBO groups. Conclusions DOXO, and to a greater extent COMBO treatment (but not TRZ alone) cause LV dysfunction in vivo. Moreover, both DOXO and COMBO treatments, but not TRZ alone, induce electrophysiological abnormalities and both structural and functional changes in the sarcoplasmic reticulum. These findings provide novel insights into the cellular mechanisms of CM dysfunction and arrhythmias associated with combined DOXO/TRZ therapy. Acknowledgement/Funding Swiss League against Cancer

Introduction Calcium handling is generally impaired in heart failure (HF). Mechanisms that can restore cardiac relaxation improving cellular Ca2 + cycling, represent a promising therapeutic approach for HF. Istaroxime is a Na-K ATPase (NKA) inhibitor with the property of accelerating Ca2 + re-uptake into sarcoplasmic reticulum (SR) through the SR Ca2 + pump (SERCA) stimulation by displacing the interaction between SERCA and its inhibitor, phospholamban (PLB). Objective To characterize Istaroxime effects in a model of mild diabetes (type 1) with diastolic dysfunction and preserved global systolic function. Istaroxime was tested at a concentration mostly unaffecting NKA to isolate its effects dependent on SERCA only. Method Streptozotocin (STZ)-treated rats were evaluated at 9 weeks after STZ injection in comparison to control (CTR) ones. STZ-induced changes were evaluated in vivo (echocardiography), in isolated left ventricular myocytes and in SERCA2a-enriched microsomes. SERCA and PLB protein levels were measured by western blot and SERCA activity as 32P hydrolysis. Action potential rate-dependency and intracellular Ca2 + handling were evaluated in patch clamped or field-stimulated (2 Hz) cells. Results STZ-induced cardiomyopathy was characterized by impaired diastolic relaxation which was associated to reduced SERCA protein level and activity at the cellular level. Moreover, the monomeric PLB/SERCA ratio was increased. In STZ group, action potentials (AP) were significantly prolonged at each cycle length; Ca2 + transients were characterized by slower decay, delayed onset and increased diastolic Ca2 + . Istaroxime at a concentration of 100 nM significantly stimulated SERCA activity and SR Ca re-uptake after caffeine depletion in STZ group only. Moreover, Istaroxime reduced STZ-induced diastolic Ca2 + enhancement but not affected AP prolongation. Conclusion SERCA stimulation can be considered a promising therapeutic approach for diastolic dysfunction treatment.

Background— Repolarization and its stability are exquisitely sensitive to I Kr features. Information on the relative importance of specific I Kr abnormalities is missing and would assist in the evaluation of arrhythmogenic risk. Methods and Results— In single guinea-pig myocytes, endogenous I Kr was replaced by modeled I Kr (m I Kr ) by dynamic clamp (DC) at a cycle length of 1 s. m I Kr parameters were systematically modified, and the resulting changes in action potential duration (APD) and its short term variability (SD1) were measured. We observed that (1) I Kr blockade increased SD1 more than expected by its dependency on APD; (2) m I Kr completely reversed APD and SD1 changes caused by I Kr blockade; (3) repolarization was most sensitive to inactivation shifts, which affected APD and SD1 concordantly; (4) activation shifts of the same magnitude had marginal impact on APD, but only when reducing m I Kr , they significantly increased SD1; (5) changes in maximal conductance resulted in a pattern similar to that of activation shifts. Conclusions— The largest effect on repolarization and its stability are expected from changes in I Kr inactivation. APD is less sensitive to changes in other I Kr gating parameters, which are better revealed by SD1 changes. SD1 may be more sensitive than APD in detecting I Kr -dependent repolarization abnormalities.

Aims Bradycardic agents are currently used in the treatment of angina and heart failure; direct information on their effects on cardiac sympathetic nerve activity (SNA) may be relevant to their chronic use. The present study evaluates the effect of pacemaker inhibition on SNA; direct nerve recordings and indirect autonomic indexes are compared. Methods and results Experiments were performed in 18 anaesthetized rats. SNA (direct nerve recording) and heart rate variability (HRV) indexes were evaluated in parallel. All parameters were recorded 10 min before to 60 min after administration of the I f blocker ivabradine (IVA; 2 mg/kg, i.v.; n = 8) or vehicle (VEH; n = 5). IVA-induced RR interval (RR) prolongation (at 60 min +15.0 ± 7.1%, P < 0.01) was associated with decreased diastolic arterial pressure (DAP; −17.3 ± 8.4%, P < 0.05) and increased SNA (+51.1 ± 12.3%, P < 0.05). These effects were accompanied by increased RR variance (RR σ 2), which showed strong positive correlation with RR. Frequency-domain HRV indexes (in normalized units) were unchanged by IVA. After baroreceptor reflexes had been eliminated by sino-aortic denervation ( n = 5), similar IVA-induced RR prolongation (at 60 min +14.3 ± 5.9%, NS vs. intact) was associated with a larger DAP reduction (−30.9 ± 4.1%, P < 0.05 vs. intact), but failed to affect SNA. Conclusions (i) IVA-induced bradycardia was associated with increased SNA, resulting from baroreceptor unloading; if this applied to chronic IVA use in humans, it would be of relevance for therapeutic use of the drug. (ii) Whenever mean HR is concomitantly changed, time-domain HRV indexes should not be unequivocally interpreted in terms of autonomic balance.

none 11 si Repolarization response to β-adrenergic (β-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 β-AR stimulation (10 nM isoproterenol, ISO).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. 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 b-AR stimulation. none Sala, Luca; Hegyi, Bence; Bartolucci, Chiara; Altomare, Claudia; Rocchetti, Marcella; Váczi, Krisztina; Mostacciuolo, Gaspare; Szentandrássy, Norbert; Severi, Stefano; Pál Nánási, Péter; Zaza, Antonio Sala, Luca; Hegyi, Bence; Bartolucci, Chiara; Altomare, Claudia; Rocchetti, Marcella; Váczi, Krisztina; Mostacciuolo, Gaspare; Szentandrássy, Norbert; Severi, Stefano; Pál Nánási, Péter; Zaza, Antonio

et al., [Aims]: Polyunsaturated fatty n-3 acids (PUFAs) have been reported to exhibit antiarrhythmic properties. However, the mechanisms of action remain unclear. We studied the electrophysiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on IKs, and on the expression and location of Kv7.1 and KCNE1. [Methods and results]: Experiments were performed using patch-clamp, western blot, and sucrose gradient techniques in COS7 cells transfected with Kv7.1/KCNE1 channels. Acute perfusion with both PUFAs increased Kv7.1/KCNE1 current, this effect being greater for DHA than for EPA. Similar results were found in guinea pig cardiomyocytes. Acute perfusion of either PUFA slowed the activation kinetics and EPA shifted the activation curve to the left. Conversely, chronic EPA did not modify Kv7.1/KCNE1 current magnitude and shifted the activation curve to the right. Chronic PUFAs decreased the expression of Kv7.1, but not of KCNE1, and induced spatial redistribution of Kv7.1 over the cell membrane. Cholesterol depletion with methyl-β-cyclodextrin increased Kv7.1/KCNE1 current magnitude. Under these conditions, acute EPA produced similar effects than those induced in non-cholesterol-depleted cells. A ventricular action potential computational model suggested antiarrhythmic efficacy of acute PUFA application under IKr block. [Conclusions]: We provide evidence that acute application of PUFAs increases Kv7.1/KCNE1 through a probably direct effect, and shows antiarrhythmic efficacy under IKr block. Conversely, chronic EPA application modifies the channel activity through a change in the Kv7.1/KCNE1 voltage-dependence, correlated with a redistribution of Kv7.1 over the cell membrane. This loss of function may be pro-arrhythmic. This shed light on the controversial effects of PUFAs regarding arrhythmias., This work was supported by grants from CICYT (SAF2010-14916 and SAF2013-45800-R to C.V.; BFU2011-23268 and CSD2008-00005 to A.F.) and FIS (PI11/02459, RD06/0014/0006, and RD12/0042/0019 to C.V.). C.M. and M.G. hold FPI grants. N.C. and A.d.l.C. hold Juan de la Cierva and RIC contracts, respectively.

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Aims Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. Methods and Results Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. Conclusion The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to β-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

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.

We evaluated how the increase in lung interstitial pressure correlates with the pulmonary vascular response to chronic hypoxia. In control and hypoxic (30 days; 10% O 2 ) Wistar male rats, we measured: pulmonary interstitial pressure ( P ip ), cardiac and haemodynamic parameters by echocardiography, and performed lung morphometry on tissue specimens fixed in situ . In control animals, mean±sd P ip , air/tissue volume ratio and capillary vascularity index in the air–blood barrier were -12±2.03 cmH 2 O, 3.9 and 0.43, respectively. After hypoxia exposure, the corresponding values of these indices in apparently normal lung regions were 2.6±1.7 cmH 2 O, 3.6, and 0.5, respectively. In oedematous regions, the corresponding values were 12±4 cmH 2 O, 0.4 and 0.3, respectively. Furthermore, in normal regions, the density of pre-capillary vessels (diameter ∼50–200 μm) increased and their thickness/internal diameter ratio decreased, while opposite results were found in oedematous regions. Pulmonary artery pressure increased in chronic hypoxia relative to the control (39.8±5.9 versus 26.2±2.2 mmHg). Heterogeneity in local lung vascular response contributes to developing pulmonary hypertension in chronic hypoxia. In oedematous regions, the decrease in capillary vascularity correlated with the remarkable increase in interstitial pressure and morphometry of the pre-capillary vessels suggested an increase in vascular resistance; the opposite was true in apparently normal regions.

Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage.

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 Ca v β2 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 Ca v α1.2 and the Akt-dependent phosphorylation status of Ca v β2 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 Ca v β2 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 Ca v α1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Ca v α1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Ca v β2, thus facilitating the chaperoning of Ca v α1.2; and promotion of Ca v α1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Ca v β2 to the nucleus, where it limits the transcription of Ca v α1.2 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 Ca v β2, 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 Ca v β2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

β-Adrenergic stimulation modulates ventricular currents and sinus cycle length (CL). We investigated how changes in CL affect the current induced by isoprenaline (Iso) during the action potential (AP) of guinea-pig ventricular myocytes. Action-potential clamp was applied at CLs of 250 and 1000 ms to measure: (1) the net current induced by 0.1 μm Iso (IIso); (2) the L-type Ca2+ current ICaL and slow delayed rectifier current IKs components of IIso (IIsoCa and IIsoK), identified as the Iso-induced current sensitive to nifedipine and HMR1556, respectively; and (3) IIso persisting after inhibition of both ICa and IKs (IisoR). The pause dependency of IKs and its modulation were evaluated in voltage-clamp experiments. The rate dependency of the duration of the action potential at 90% repolarization (APD90) and its modulation by isoprenaline were tested in current-clamp experiments. At a CL of 250 ms IIso was inward during initial repolarization and reversed at 59% of APD90. At a CL of 1000 ms IIso became mostly inward in all cells. Switching to shorter CL did not change IIsoCa and IIsoK amplitudes, but moved their peak amplitudes to earlier repolarization; IIsoR was independent of CL. Acceleration of IIsoK at shorter CL was based on faster pause dependency of IKs activation rate. The ‘restitution’ of activation rates was modulated by isoprenaline. The APD90–CL relation was rotated anticlockwise by isoprenaline and crossed the control curve at a CL of 150 ms (400 beats min−1). We conclude that: (1) isoprenaline induced markedly different current profiles according to pacing rate, involving CL-dependent ICa and IKs modulation; (2) the effect of isoprenaline on APD90 was CL dependent, and negligible during tachycardia; and (3) during sympathetic activation, repolarization stability may involve matched modulation of sinus rate and repolarizing currents.

OBJECTIVE: To gain some insight on the lesser arrhythmogenic properties of PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride] compared with digoxin, we compared modulation of intracellular Ca2+ dynamics by the two agents. METHODS: SERCA (sarcoplasmic reticulum Ca2+-ATPase) activity and Ca2+ leak rate were measured in sarcoplasmic reticulum (SR) vesicles from guinea pig ventricles. Membrane current, intracellular Ca2+, and twitch amplitude were evaluated in guinea pig ventricular myocytes with or without blockade of the Na+/Ca2+ exchanger. RESULTS: In SR vesicles, PST2744 (30-300 nM), but not digoxin, increased SERCA activity; digoxin only (> or =0.1 nM) increased SR Ca2+ leak. In myocytes with blocked Na+/Ca2+ exchanger, Ca2+ reloading of caffeine-depleted SR was enhanced by PST2744 and slightly inhibited by digoxin. In myocytes with functioning Na+/Ca2+ exchanger, both agents increased diastolic Ca2+, SR Ca2+ content, the gain of Ca2+-induced Ca2+ release, the rate of cytosolic Ca2+ decay, twitch amplitude, and relaxation rate. Consistent with the observations in SR vesicles, the effects on SR Ca2+ content and Ca2+ decay rate were significantly larger for PST2744 than for digoxin. CONCLUSIONS: In isolated SR vesicles, PST2744 and digoxin directly affected SR function in opposite ways; this could be reproduced in myocytes during Na+/Ca2+ exchanger blockade. Under physiological conditions (functioning Na+/Ca2+ exchanger), the two agents affected Ca2+ dynamics in the same direction, as expected by their Na+/K+ pump inhibition; however, differential SR modulation was still expressed by quantitative differences. Thus, the more favorable inotropy-to-toxicity ratio previously described for PST2744 appears to be associated with direct SERCA stimulation and/or lack of enhancement of Ca2+ leak.

(E,Z)-3-((2-Aminoethoxy)imino)androstane-6,17-dione hydrochloride (PST2744) is a novel Na(+)/K(+) pump inhibitor with positive inotropic effects. Compared with digoxin in various experimental models, PST2744 was consistently found to be less arrhythmogenic, thus resulting in a significantly higher therapeutic index. The present work compares the electrophysiological effects of PST2744 and digoxin in guinea pig ventricular myocytes, with the aim to identify a mechanism for their different toxicity. The work showed that 1) the action potential was transiently prolonged and then similarly shortened by both agents; 2) the ratio between Na(+)/K(+) pump inhibition and inotropy was somewhat larger for PST2744 than for digoxin; 3) both agents accelerated inactivation of high-threshold Ca(2+) current (I(CaL)), without affecting its peak amplitude; 4) the transient inward current (I(TI)) induced by a Ca(2+) transient in the presence of complete Na(+)/K(+) pump blockade was inhibited (-43%) by PST2744 but not by digoxin; 5) the conductance of Na(+)/Ca(2+) exchanger current (I(NaCa)), recorded under Na(+)/K(+) pump blockade, was only slightly inhibited by PST2744 (-14%) and unaffected by digoxin; and 6) both agents inhibited delayed rectifier current I(Ks) (

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. Stem Cells 2015;33:1187–1199

Voltage dependent L-Type calcium-channels (LTCCs) are located on the cardiomyocyte membrane and regulate cardiac contraction and rhythmicity. In human pathologies, such as heart failure (HF), decreased inward calcium current (I Ca ) is frequently observed. Here, we generated a mimetic peptide (MP) that targets LTCCs and restores impaired intracellular calcium homeostasis through a novel mechanism. Effective delivery of MP, fused with a cell penetrating peptide, was found to correct Ca2+ alterations in a mouse model of HF, in human cardiomyocytes derived from induced pluripotent stem-cells. These data provide a proof-of-concept supporting a therapeutic role for MP to treat human diseases related to LTCC abnormalities. Category: heart failure biology

Cardiac arrhythmias are electrical phenomena; thus, sarcolemmal ion channels have long been considered as targets of antiarrhythmic therapy. The contribution of abnormal intracellular Ca(2+) handling to digitalis-induced arrhythmogenesis is an old concept; however, the role of abnormal Ca(2+) handling as a common cause of arrhythmia, i.e. relevant to all arrhythmogenic mechanisms, has been fully recognized in more recent times. Stability of the intracellular Ca(2+) store (sarcoplasmic reticulum, SR) is crucial to physiological Ca(2+) handling; when it is compromised, Ca(2+) may be released independently from excitation and lead to secondary perturbation of membrane potential. Ca(2+) store stability depends on the interplay between sarcolemmal and SR "effectors" (ion channels and transports), which are mutually linked by Ca(2+)-mediated feed-back control. While instrumental to cell homeostasis, such control makes any attempt to modulate SR stability dauntingly complex. This review discusses current knowledge on the factors leading to SR instability, the mechanisms by which SR instability translates into arrhythmias and which interventions may be best suited to prevent SR instability. Although still at an initial stage of development, such interventions might represent the future of antiarrhythmic drug therapy.

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 Ca2+ 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 Ca2+ 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.

Background During ischemia, enhancement of the "late Na+ current" (INaL) contributes to intracellular Ca2+ overload. Dysregulation of intracellular Ca2+ homeostasis plays a critical role in the pathophysiology of cardiac arrest and cardiopulmonary resuscitation (CPR), leading to ventricular arrhythmias and left ventricle (LV) dysfunction. Objective The purpose of this study was to investigate the effects of the INaL blocker ranolazine on outcome of CPR in a rat model. We hypothesized that ranolazine might reduce postresuscitation arrhythmias and improve survival and recovery. Methods Eighteen rats were assigned to receive intravenous ranolazine 10 mg/kg or vehicle. Ventricular fibrillation was induced and untreated for 8 minutes. CPR then was performed for 8 minutes. ECG and arterial and right atrial pressures were monitored up to 3 hours after CPR. After resuscitation, LV function was monitored by echocardiography, and 72-hour survival with neurologic recovery was evaluated. Plasma was obtained for biomarkers of heart and brain injury. Results All animals in the ranolazine group were resuscitated and survived up to 72 hours, whereas 72% in the vehicle group were resuscitated but 54% survived. The period of postresuscitation arrhythmia with hemodynamic instability was shorter in the ranolazine group compared to vehicle group (P

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether-a-go-go-related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long-QT syndrome and Drosophila ‘seizure’ mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50=16±1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure–function relationships and novel pharmacological tools with potential therapeutic efficacy.—Gurrola, G. B., Rosati, B., Rocchetti, M., Pimienta, G., Zaza, A., Arcangeli, A., Olivotto, M., Possani, L. D., Wanke, E. A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom.

The human ether-a-go-go-related gene (herg) encodes a K+ current (I(HERG)) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that I(HERG), recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the K(ATP) channel blocker glibenclamide (IC50 = 74 microM). The voltage and use dependence of glibenclamide blockade were also evaluated. Another sulfonylurea, glimepiride, had less effective results in blocking I(HERG). The findings of this study are relevant to the interpretation of glibenclamide effects on cellular electrophysiology and suggest that oral antidiabetic therapy with sulfonylureas may contribute to iatrogenic QT prolongation and related arrhythmias.

Abstract —Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca 2+ . However, mostly on the basis of indirect arguments, Ca 2+ -mediated rectification of inward rectifier K + current ( I K1 ) is claimed to play no role in the mammalian heart. The present study investigates Ca 2+ -mediated I K1 rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The action potential waveform was recorded and then applied to reproduce normal excitation under voltage-clamp conditions. Subtraction currents obtained during blockade of K + currents by either 1 mmol/L Ba 2+ ( I Ba ) or K + -free solution ( I 0K ) were used to estimate I K1 . Similar time courses were observed for I Ba and I 0K ; both currents were strongly reduced during depolarization (inward rectification). Blockade of L-type Ca 2+ current by dihydropyridines (DHPs) increased systolic I Ba and I 0K by 50.7% and 254.5%, respectively. β-Adrenergic stimulation, when tested on I 0K , had an opposite effect; ie, it reduced this current by 66.5%. Ryanodine, an inhibitor of sarcoplasmic Ca 2+ release, increased systolic I Ba by 47.7%, with effects similar to those of DHPs. Intracellular Ca 2+ buffering (BAPTA-AM) increased systolic I Ba by 87.7% and blunted the effect of DHPs. Thus, I K1 may be significantly reduced by physiological Ca 2+ transients determined by both Ca 2+ influx and release. Although Ca 2+ -induced effects may represent only a small fraction of total I K1 rectification, they are large enough to affect excitability and repolarization. They may also contribute to facilitation of early afterdepolarizations by conditions increasing Ca 2+ influx.

Aims Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current ( I NaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive I NaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available I NaL blocker, may prevent constitutive I NaL enhancement and PAH-induced myocardial remodelling. Methods and results PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. I NaL was markedly enhanced; diastolic Ca2+ was increased and Ca2+ release was facilitated. K+ currents were down-regulated and APD was prolonged. In the LV, I NaL was enhanced to a lesser extent and cell Ca2+ content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented I NaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. Conclusion PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive I NaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

Background: De novo mutations in calmodulin genes have been recently associated with markedly prolonged QT interval and life-threatening ventricular arrhythmias in infants. Biochemical evidences suggested impaired calcium ion binding to mutant calmodulins. Calcium dependent inactivation (CDI) of L-type calcium channel (ICaL) involves the interaction between Ca2+-calmodulin and the channel protein. Aims: To explore the effects of two CALM1 mutations (F142L and D130G) on electrical activity and ICaL properties in human induced-pluripotent stem cells (iPSC)-derived cardiomyocytes (CM). Methods: Skin fibroblasts of two patients carrying the mutation CALM1-F142L or CALM1-D130G were reprogrammed to generate iPSC and these cells differentiated into CM; a healthy donor was selected as control. Two cell clones for each mutation were analyzed to rule out clone specificity. Patch clamp and micro electrode arrays (MEA) analyses were performed on isolated iPSC-derived CM and beating clusters at about 40 days of differentiation respectively. ICaL was isolated in the presence of calcium or barium ions as charge carriers. Results: CALM1-F142L iPSC-derived CM showed prolonged field potential duration (FPD) with high beta-adrenergic sensitivity; peak ICaL density was unaltered, but CDI was markedly reduced and inactivation incomplete. CALM1-D130G iPSC-derived CM showed higher ICaL density and unaltered CDI. Conclusions: F142L and D130G calmodulin mutations differently affect ICaL properties in human iPSC-derived CM. F142L mutation is consistent with reduced calcium affinity of calmodulin; D130G abnormality suggests ICaL facilitation instead, possibly resulting from upregulation of alternative calmodulin isoforms. Overall, both calmodulin mutations might potentially cause arrhythmogenic calcium overload.