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The black mamba (Dendroaspis polylepis) is, due to its extremely toxic venom, one of the most dangerous snake species in Sub-Saharan Africa. A D. polylepis bite is a medical emergency and requires adequate action to prevent severe complications. However, there are no comprehensive reviews available based on clinical cases, and no readily accessible guidelines for standardized treatment. Therefore, we aim to provide an overview regarding the currently available clinical literature on D. polylepis envenomations; in order to promote knowledge on symptomatology and treatment options. We searched for cases reporting humans bitten by D. polylepis in PubMed, Embase, Scopus, and Sabinet. We searched the reference lists of all eligible articles for additional articles. After quality assessment, 29 cases were included in this review. We used descriptive analysis to create an overview of the collected parameters. Among the included case reports and case series, D. polylepis envenomations most frequently resulted in decreased respiratory function, sweating and paralysis. The onset of symptoms usually occurred within 60 minutes. Neurological symptoms occurred more often than symptoms of autonomic dysfunction. In the reported cases most patients (26/29) received antivenom and most survived (25/29). We recommend the reporting of additional structured case reports to improve future analyses on the clinical course of envenomations, in order to improve public health response to D. polylepis envenomations.

Publications baring falsified and fabricated images appear frequently in the primary literature. Industrialized forms of image forgery as practiced by the so-called paper mills worsen the current situation even further. Good education and awareness within the scientific society are essential to create an environment in which honesty and trust are the prime values in experimental research. Here I focus on the detection of publication fraud and provide some examples and advice. Finally, my views on the future of fraud detection and prevention are given.

Cantú syndrome (CS) is caused by dominant gain‐of‐function mutation in ATP‐dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg‐ATP, glibenclamide and HMR1098 were measured by inside‐out patch‐clamp electrophysiology. Results were interpreted in view of cryo‐EM IKATP channel structures. Mg‐ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild‐type (0.15 ± 0.01). HMR1098 dose‐dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg‐ATP, reaching, at 30 μmol/L, current levels displayed by wild‐type and H60Y channels in the presence of 0.15 mmol/L Mg‐ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg‐ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.

This work presents drug-likeness and the cardiotoxicity profiles of six potent pentamidine analogs 1–6 and three new compounds 7–9 as chemotherapeutics for therapy of Pneumocystis jiroveci pneumonia. A combination of experimental and computational approaches was used in the cardiotoxicity examination. The hERG trafficking and functionality of the hERG currents were tested by western blot analyses, immunofluorescent staining procedures, and patch-clamp electrophysiological assays. Cardiotoxicity combined with blocking the hERG K+ channel was predicted, and then simulated by docking to the CSM-TM model 732 protein. Location of pentamidines in the proximity of Leu622, Thr623, Ser649, Tyr652, Ala653, and Phe656, and the high energies of interactions were in accordance with probable blocking of the hERG channel. However, in the biochemical experiments, no significant changes in IhERG densities and a minor effect on hERG maturation were observed. Predicted metabolic transformation of pentamidines with S atoms in the aliphatic linker leads to oxidation of one S atom, but those with the phenyl sulfanilide moiety can be oxidized to chinones. The tested pentamidines characterized by the presence of sulfur atoms or sulfanilide groups, have favorable drug-likeness parameters and are promising lead structures in the development of new potent chemotherapeutics against PJP.

The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiCMs) complicates their use for therapeutic and pharmacological purposes. An insufficient inward rectifying current (IK1) and the presence of a funny current (if) cause spontaneous electrical activity. This study tests the hypothesis that the co-culturing of hiCMs with a human embryonic kidney (HEK) cell-line expressing the Kir2.1 channel (HEK-IK1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-IK1:hiCM ratios was compared with co-cultures using wildtype (HEK–WT:hiCM) or hiCM alone on days 3–8 after plating. Only ratios of 1:3 and 1:1 showed a significant reduction in spontaneous rate at days 4 and 6, suggesting that IK1 was influencing the electrophysiology. Detailed analysis at day 4 revealed an increased incidence of quiescent wells or sub-areas. Electrical activity showed a decreased action potential duration (APD) at 20% and 50%, but not at 90%, alongside a reduced amplitude of the aggregate AP signal. A computational model of the 1:1 co-culture replicates the electrophysiological effects of HEK–WT. The addition of the IK1 conductance reduced the spontaneous rate and APD20, 50 and 90, and minor variation in the intercellular conductance caused quiescence. In conclusion, a 1:1 co-culture HEK-IK1:hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of IK1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient IK1 was integrated into the syncytium.

IntroductionTorsade de pointes arrhythmias (TdP) in the chronic atrioventricular block (CAVB) dog model result from proarrhythmic factors, which trigger TdP and/or reinforce the arrhythmic substrate. This study investigated electrophysiological and arrhythmogenic consequences of severe bradycardia for TdP.MethodsDofetilide (25 μg/kg per 5 min) was administered to eight anesthetized, idioventricular rhythm (IVR) remodeled CAVB dogs in two serial experiments: once under 60 beats per minute (bpm), right ventricular apex paced (RVA60) conditions, once under more bradycardic IVR conditions. Recordings included surface electrocardiogram and short-term variability (STV) of repolarization from endocardial unipolar electrograms. TdP inducibility (three or more episodes within 10 min after start of dofetilide) and arrhythmic activity scores (AS) were established. Mapping experiments in 10 additional dogs determined the effect of lowering rate on STV and spatial dispersion of repolarization (SDR) in baseline.ResultsIVR-tested animals had longer baseline RR-interval (1,403 ± 271 ms) and repolarization intervals than RVA60 animals. Dofetilide increased STV similarly under both rhythm strategies. Nevertheless, TdP inducibility and AS were higher under IVR conditions (6/8 and 37 ± 27 vs. 1/8 and 8 ± 12 in RVA60, respectively, both p < 0.05). Mapping: Pacing from high (128 ± 10 bpm) to middle (88 ± 10 bpm) to experimental rate (61 ± 3 bpm) increased all electrophysiological parameters, including interventricular dispersion, due to steeper left ventricular restitution curves, and intraventricular SDR: maximal cubic dispersion from 60 ± 14 (high) to 69 ± 17 (middle) to 84 ± 22 ms (p < 0.05 vs. high and middle rate).ConclusionIn CAVB dogs, severe bradycardia increases the probability and severity of arrhythmic events by heterogeneously causing electrophysiological instability, which is mainly reflected in an increased spatial, and to a lesser extent temporal, dispersion of repolarization.

Ventricular cardiac arrhythmia is a life threating condition arising from abnormal functioning of many factors in concert. Animal models mirroring human electrophysiology are essential to predict and understand the rare pro- and anti-arrhythmic effects of drugs. This is very well accomplished by the canine chronic atrioventricular block (CAVB) model. Here we summarize canine models for cardiovascular research, and describe the development of the CAVB model from its beginning. Understanding of the structural, contractile and electrical remodelling processes following atrioventricular (AV) block provides insight in the many factors contributing to drug-induced arrhythmia. We also review all safety pharmacology studies, efficacy and mechanistic studies on anti-arrhythmic drugs in CAVB dogs. Finally, we compare pros and cons with other in vivo preclinical animal models. In view of the tremendous amount of data obtained over the last 100 years from the CAVB dog model, it can be considered as man's best friend in preclinical drug research. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g. dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use due to their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue WT or G601S Kv11.1 trafficking as shown by western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 μM dofetilide or 10 μM dofetilide + 5 μM LUF7244. In trafficking defective G601S Kv11.1 cells, dofetilide (10 μM) or dofetilide+LUF7244 (10+5 μM) restored Kv11.1 trafficking also, as demonstrated by western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased IKv11.1 despite the presence of dofetilide (1 μM) in WT Kv11.1 cells. In G601S expressing cells, long-term treatment (24-48 h) with LUF7244 (10 μM) and dofetilide (1 μM) increased IKv11.1 compared to non-treated, or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1. Thus, combined administration of LUF7244 and an IKV11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects.

Background: Electronic cigarettes (e-cigarettes), the smokeless alternative to conventional tobacco cigarettes, have become increasingly popular. E-cigarettes vaporise e-liquid, a solution of highly concentrated nicotine, propylene glycol (PG) and vegetable glycerine (VG). With the popularity of e-cigarettes, e-liquid refills have become easily accessible and several cases of intoxication due to the ingestion of e-liquid have been reported. We provide an overview of these cases, their pathophysiology and patients' characteristics. Methods: We carried out a retrospective evaluation of the scientific literature reporting on cases of liquid nicotine intoxication, using the following inclusion criteria: (1) the article is or contains a case report, (2) describes an intoxication with e-liquid, (3) the substance contains nicotine, and (4) intake is oral, intravenous or subcutaneous. Results: We found 26 case reports describing a total of 31 patients who suffered from e-liquid intoxication. All intoxications up to the age of six were reported as unintentional, whereas nearly all cases from ages 13 to 53 were due to suicide attempts. The three most prevalent symptoms of e-liquid intoxication were tachycardia, altered mental status and vomiting. Eleven cases resulted in the death of the patient. In the survivors, the highest plasma concentration of nicotine was 800 µg L-1, while the lowest concentration in the non-survivors was 1600 µg L-1. Conclusions: There is a mismatch between the generally accepted lethal oral nicotine dose of 60 mg, resulting in approximately 180 µg L-1 plasma concentration, and the 4.4- to 8.9-fold higher lethal plasma concentrations we found in cases of e-liquid intoxication. In these severe intoxications, plasma cotinine concentration does not act as a more reliable indicator of nicotine intoxication than nicotine itself. The ages of the patients display a bimodal distribution. In patients above the age of 10, intoxication results mainly from suicide attempts rather than accidental ingestion. The role of PG and VG in e-liquid intoxications is remarkably unclear. However, the similarity across nicotine and PG toxicity symptoms leads us to believe a cumulative effect cannot be excluded.

© 2018 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

Preclinical drug screens are not based on human physiology, possibly complicating predictions on cardiotoxicity. Drug screening can be humanised with in vitro assays using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, in contrast to adult ventricular cardiomyocytes, iPSC-CMs beat spontaneously due to presence of the pacemaking current If and reduced densities of the hyperpolarising current IK1. In adult cardiomyocytes, IK1 finalises repolarisation by stabilising the resting membrane potential while also maintaining excitability. The reduced IK1 density contributes to proarrhythmic traits in iPSC-CMs, which leads to an electrophysiological phenotype that might bias drug responses. The proarrhythmic traits can be suppressed by increasing IK1 in a balanced manner. We systematically evaluated all studies that report strategies to mature iPSC-CMs and found that only few studies report IK1 current densities. Furthermore, these studies did not succeed in establishing sufficient IK1 levels as they either added too little or too much IK1. We conclude that reduced densities of IK1 remain a major flaw in iPSC-CMs, which hampers their use for in vitro drug screening.

Background and Purpose Inward rectifier current (IK1) density increases in atrial fibrillation (AF), shortening effective refractory period and thus promotes atrial reentry. Pentamidine-Analogue 6 (PA-6) is a selective and potent IK1 inhibitor. We tested PA-6 for anti-AF efficacy and potential proarrhythmia, in established large animal models. Experimental Approach PA-6 was applied i.v. in anaesthetized goats with rapid pacing induced AF and anaesthetized chronic AV block dogs. Electrophysiological and pharmacological parameters were determined. Key Results PA-6 (2.5 mg.kg-1.10min-1) induced cardioversion to sinus rhythm (SR) in 5/6 goats. PA-6 prolonged AF cycle length (LA:139.0±23.2 vs. 152.1±22.1ms, p

Supplemental Digital Content is Available in the Text., Short-term variability (STV), to quantify beat-to-beat variability of repolarization, is a surrogate parameter that reliably identifies proarrhythmic risk in preclinical models. Examples include not only the use in the chronic atrioventricular block (CAVB) dog model whereby it was developed but also in vulnerable patients with heart failure or drug-induced long QT syndrome. In the CAVB dog model, STV can specifically distinguish between safe and unsafe drugs in proarrhythmic screening. Conversely, this dog model also offers the possibility to evaluate antiarrhythmic strategies in a setting of Torsades de Pointes (TdP) induction with a standard IKr inhibitor. The different antiarrhythmic interventions studied in suppression and prevention of drug-induced TdP in vivo in the CAVB dog model and in vitro in canine ventricular cardiomyocytes are described in this overview. We provide evidence that STV predicts the magnitude of antiarrhythmic effect against TdP better than other repolarization parameters in both suppression and prevention conditions. Moreover, suppression and prevention experiments revealed the same level of antiarrhythmic efficacy, whereas cellular experiments seem more sensitive in comparison with drug testing in vivo. Together, these observations suggest that STV could be used as a consistent indicator to rank efficacy of antiarrhythmic interventions in a number of conditions.

The ubiquitously expressed family of inward rectifier potassium (KIR) channels, encoded by KCNJ genes, is primarily involved in cell excitability and potassium homeostasis. Channel mutations associate with a variety of severe human diseases and syndromes, affecting many organ systems including the central and peripheral neural system, heart, kidney, pancreas, and skeletal muscle. A number of mutations associate with altered ion channel expression at the plasma membrane, which might result from defective channel trafficking. Trafficking involves cellular processes that transport ion channels to and from their place of function. By alignment of all KIR channels, and depicting the trafficking associated mutations, three mutational hotspots were identified. One localized in the transmembrane-domain 1 and immediately adjacent sequences, one was found in the G-loop and Golgi-export domain, and the third one was detected at the immunoglobulin-like domain. Surprisingly, only few mutations were observed in experimentally determined Endoplasmic Reticulum (ER)exit-, export-, or ER-retention motifs. Structural mapping of the trafficking defect causing mutations provided a 3D framework, which indicates that trafficking deficient mutations form clusters. These “mutation clusters” affect trafficking by different mechanisms, including protein stability.

K IR2.1 potassium channels, producing inward rectifier potassium current (I K1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal K IR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. K IR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within K IR2.1 sequences using the "epestfind" webtool. WT and ΔPEST K IR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. K IR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine K IR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the K IR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in K IR2.2, K IR2.3, and K IR2.6 proteins. Deletion of the PEST domain in California kingsnake and human K IR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T1/2 from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of K IR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels.

KATP channels consist of four Kir6.x pore–forming subunits and four regulatory sulfonylurea receptor (SUR) subunits. These channels couple the metabolic state of the cell to membrane excitability and play a key role in physiological processes such as insulin secretion in the pancreas, protection of cardiac muscle during ischemia and hypoxic vasodilation of arterial smooth muscle cells. Abnormal channel function resulting from inherited gain or loss-of-function mutations in either the Kir6.x and/or SUR subunits are associated with severe diseases such as neonatal diabetes, congenital hyperinsulinism or Cantú syndrome (CS). CS is an ultra-rare genetic autosomal dominant disorder, caused by dominant gain-of-function mutations in SUR2A or Kir6.1 subunits. No specific pharmacotherapeutic treatment options are currently available for Cantú syndrome. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for Cantú syndrome, particular for mutations, which lack high affinity for sulfonylurea inhibitor glibenclamide. By applying a combination of computational methods including atomistic MD simulations, free energy calculations and pharmacophore modelling, we identified several novel Kir6.1 inhibitors, which might be possible candidates for drug repurposing. The in silico predictions were confirmed using inside/out patch-clamp analysis. Importantly, Cantú mutation C176S in Kir6.1 and S1020P in SUR2A, retained high affinity towards the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point towards developing novel therapies for Cantú disease.

Hyperkalemia is a metabolic disturbance of the potassium balance that can cause potentially fatal cardiac arrhythmias. Kidney dysfunction and renin-angiotensin-aldosterone system inhibiting drugs are notorious for their tendency to induce hyperkalemia by decreasing the excretion of potassium. The role of dietary potassium intake in inducing hyperkalemia is less clear. We review and analyze the common presentation, laboratory, and electrocardiogram (ECG) findings and therapeutic options associated with dietary-induced hyperkalemia, and find evidence for hyperkalemia development in non-renal impaired patients. Thirty-five case reports including 44 incidences of oral intake-induced hyperkalemia were assessed, 17 patients did not suffer from kidney dysfunction. Mean age was 49 ± 20 years. Mean potassium concentration was 8.2 ± 1.4 mEq/l, most frequently caused by abundant intake of fruit and vegetables (n = 17) or salt substitutes (n = 12). In patients with normal kidney function, intake of salt substitutes or supplements was the main cause of hyperkalemia. Main symptoms encompassed muscle weakness (29.5%), vomiting (20.4%), and dyspnea (15.9%). When ECGs were performed (n = 30), abnormalities were present in 86.7% of cases. Treatment involved administration of insulin (n = 22), sodium/calcium polystyrene sulfonate (n = 14), and/or calcium gluconate (n = 14). Forty patients fully recovered. Three, non-renal impaired, patients passed away. These results offer insight into the clinical aspects of dietary-induced hyperkalemia and suggest that the common assumption that dietary-induced hyperkalemia is a condition of renal impaired patients might be incorrect.

Ventricular arrhythmias compose a heterogeneous group of cardiac complications that can lead to sudden cardiac death. An increased susceptibility to (prolonged) repolarization associated arrhythmias is suggested to be the result of a reduced 'repolarization reserve', associated with an increase in the beat-to-beat variability in repolarization (BVR). Multiple parameters, related to ventricular remodelling as occurs in the chronic, complete atrioventricular block (CAVB) dog model or in patients in heart failure have already been demonstrated to increase BVR in baseline, which further increases upon infusion with action potential duration (APD) prolonging drugs. We demonstrate that the beat-to-beat variability in preload, as a result of atrioventricular dyssynchrony, can assist to determine the status of repolarization reserve. The increase in BVR, induced by varying the preload, could be diminished by infusion with streptomycin, an inhibitor of the stretch-activated channels (SACs). We therefore identified the SACs as important components of the process that contributes to the beat-to-beat variability in preload to increase the BVR. The preload variability (PLV) induced increase in BVR was however not associated with an increase in the incidence of ventricular arrhythmias. Therefore, the effect of variability in preload is able to unmask a latent decrease of repolarization reserve. The identification of a new method to determine repolarization reserve status opens numerous doors for future research in the field of arrhythmias. For instance, PLV could be used in the optimization of the BVR as a predictor of arrhythmias. Furthermore, clinical applicability and anti-arrhythmic efficacy of diminishing effects of variable preload on SACs in cardiac patients as (part of) an anti-arrhythmic therapy could also be further explored.

Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological, and arrhythmogenic evaluation. SkMBs were magnetically labeled with magnetite nanoparticles and co-cultured with neonatal rat ventricular myocytes (NRVMs) on multi-electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca(2+) imaging using a gene-encoded Ca(2+) indicator, G-CaMP2. In the monoculture of NRVMs (control), conduction was well-organized. In the randomly-mixed co-culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the "patterned" co-culture where an en bloc SKMB-layer was inserted into the NRVM-layer, excitation homogenously propagated although conduction was distorted by the SkMB-area. The 4-mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM-area did not change (25 ± 3 ms), although CT through the SkMB-area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat-to-beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca(2+) transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. J. Cell. Physiol. 231: 2249-2256, 2016. © 2016 Wiley Periodicals, Inc.

Acute excessive alcohol consumption is associated with heart rhythm disorders like atrial fibrillation but also premature ventricular contractions, collectively known as the "holiday heart syndrome". More rarely but clinically significant are reports of atrioventricular (AV) conduction disturbances in binge drinkers with no underlying heart disease or chronic alcohol consumption. To obtain better insights into common denominators and the potential underlying mechanisms we collected and compared individual case reports of AV block following acute alcohol intoxication in otherwise healthy people. By screening PubMed, Google Scholar, Scopus and JSTOR, fifteen cases were found of which eight were sufficiently documented for full analysis. Blood alcohol levels ranged from 90 to 958 mg/dl (19 to 205 mM). Second and third degree AV block was observed most (6/8) albeit that in two of these patients a vagal stimulus led to deterioration from first into higher order AV block. In all cases, patients reverted to normal sinus rhythm upon becoming sober again. Mildly lowered body temperature (35.9 ± 0.5°C) was observed but can be excluded as a major cause of conduction blockade. We hypothesize that ethanol induced partial inhibition of calcium and potentially also sodium currents in conductive tissue structures may be one of the mechanisms of conduction slowing and block that may become exaggerated upon increased vagal tone. An impairment of gap junction function cannot be excluded as a contributing factor. In conclusion, cases of documented alcohol induced AV block are very rare but events can occur at relatively low serum alcohol levels which should prompt to awareness of this phenomenon in alcohol intoxicated patients.

Inward rectifier potassium ion channels (IK1-channels) of the Kir2.x family are responsible for maintaining a stable negative resting membrane potential in excitable cells, but also play a role in processes of non-excitable tissues, such as bone development. IK1-channel loss-of-function, either congenital or acquired, has been associated with cardiac disease. Currently, basic research and specific treatment are hindered by the absence of specific and efficient Kir2.x channel activators. However, twelve different compounds, including approved drugs, show off-target IK1 activation. Therefore, these compounds contain valuable information towards the development of agonists of Kir channels, AgoKirs. We reviewed the mechanism of IK1 channel activation of these compounds, which can be classified as direct or indirect activators. Subsequently, we examined the most viable starting points for rationalized drug development and possible safety concerns with emphasis on cardiac and skeletal muscle adverse effects of AgoKirs. Finally, the potential value of AgoKirs is discussed in view of the current clinical applications of potentiators and activators in cystic fibrosis therapy.

Current inotropic agents in heart failure therapy associate with low benefit and significant adverse effects, including ventricular arrhythmias. Istaroxime, a novel Na+/K+-transporting ATPase inhibitor, also stimulates SERCA2a activity, which would confer improved inotropic and lusitropic properties with less proarrhythmic effects. We investigated hemodynamic, electrophysiological and potential proarrhythmic and antiarrhythmic effects of istaroxime in control and chronic atrioventricular block (CAVB) dogs sensitive to drug-induced Torsades de Pointes arrhythmias (TdP). In isolated normal canine ventricular cardiomyocytes, istaroxime (0.3-10 μM) evoked no afterdepolarizations and significantly shortened action potential duration (APD) at 3 and 10 μM. Istaroxime at 3 μg/kg/min significantly increased left ventricular (LV) contractility (dP/dt+) and relaxation (dP/dt-) respectively by 81 and 94% in anesthetized control dogs (n = 6) and by 61 and 49% in anesthetized CAVB dogs (n = 7) sensitive to dofetilide-induced TdP. While istaroxime induced no ventricular arrhythmias in control conditions, only single ectopic beats occurred in 2/7 CAVB dogs, which were preceded by increase of short-term variability of repolarization (STV) and T wave alternans in LV unipolar electrograms. Istaroxime pre-treatment (3 μg/kg/min for 60 min) did not alleviate dofetilide-induced increase in repolarization and STV, and mildly reduced incidence of TdP from 6/6 to 4/6 CAVB dogs. In six CAVB dogs with dofetilide-induced TdP, administration of istaroxime (90 μg/kg/5 min) suppressed arrhythmic episodes in two animals. Taken together, inotropic and lusitropic properties of istaroxime in CAVB dogs were devoid of significant proarrhythmic effects in sensitive CAVB dogs, and istaroxime provides a moderate antiarrhythmic efficacy in prevention and suppression of dofetilide-induced TdP.

Objectives This study investigated the arrhythmogenic mechanisms responsible for torsade de pointes (TdP) in the chronic atrioventricular block dog model, known for its high susceptibility for TdP. Background The mechanism of TdP arrhythmias has been under debate for many years. Focal activity as well as re-entry have both been mentioned in the initiation and the perpetuation of TdP. Methods In 5 TdP-sensitive chronic atrioventricular block dogs, 56 needle electrodes were evenly distributed transmurally to record 240 unipolar local electrograms simultaneously. Nonterminating (NT) episodes were defibrillated after 10 s. Software was developed to automatically detect activation times and to create 3-dimensional visualizations of the arrhythmia. For each episode of ectopic activity (ranging from 2 beats to NT episodes), a novel methodology was created to construct directed graphs of the wave propagation and detect re-entry loops by using an iterative depth-first-search algorithm. Results Depending on the TdP definition (number of consecutive ectopic beats), we analyzed 29 to 54 TdP: 29 were longer than 5 beats. In the total group, 9 were NT and 45 were self-terminating. Initiation and termination were always based on focal activity. Re-entry becomes more important in the longer-lasting episodes (>14 beats), whereas in all NT TdP, re-entry was the last active mechanism. During re-entry, excitation fronts were constantly present in the heart, while during focal TdP, there was always a silent interval between 2 consecutive waves (142 ms) during which excitation fronts were absent. Interbeat intervals were significantly smaller for re-entry episodes—220 versus 310 ms in focal. Electrograms recorded in particular areas during NT TdP episodes had significantly smaller amplitude (0.38) than during focal episodes (0.59). Conclusions TdP can be driven by focal activity as well as by re-entry depending on the duration of the episode. NT episodes are always maintained by re-entry, which can be identified in local unipolar electrograms by shorter interbeat intervals and smaller deflection amplitude.

Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.

Background The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. Methods Molecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. Results PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM). Conclusions 1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

CONTEXT: Zinc chloride (ZnCl2)-based smoke bombs and screens are in use since the Second World War (1939-1945). Many case descriptions on ZnCl2 smoke inhalation incidents appeared since 1945. OBJECTIVE: We provide a comprehensive overview of the clinical symptoms and underlying pathophysiology due to exposure to fumes from ZnCl2 smoke producing bombs. In addition, we give a historical overview of treatment regimens and their outcomes. METHODOLOGY: We performed a literature search on Medline, Scopus and Google Scholar databases using combinations of the following search terms "smoke bomb", "smoke screen", "ZnCl2", "intoxication", "poisoning", "case report", "HE smoke", "hexachloroethane smoke", "smoke inhalation" and "white smoke". We retrieved additional reports based on the primary hits. We collected 30 case reports from the last seven decades encompassing 376 patients, 23 of whom died. Of all the patient descriptions, 31 were of sufficient detail for prudent analysis. RESULTS AND CONCLUSIONS: Intoxication with clinical signs mainly took place in war situations and in military and fire emergency training sessions in enclosed spaces. Symptoms follow a biphasic course mainly characterised by dyspnoea, coughing and lacrimation, related to irritation of the airways in the first six hours, followed by reappearance of early signs complemented with inflammation related signs and tachycardia from 24 h onwards. Acute respiratory stress syndrome developed in severely affected individuals. Chest radiographs did not always correspond with clinical symptoms. Common therapy comprises corticosteroids, antibiotics and supplemental oxygen or positive pressure ventilation in 64% of the cases. Of the 31 patients included, eight died, three had permanent lung damage and 15 showed complete recovery, whereas in five patients outcome was not reported. Early signs likely relate to caustic reactions in the airway lining, whereas inhaled ZnCl2 particles may trigger an inflammatory response and associated delayed fibrotic lung damage. Smoke bomb poisoning is a potentially lethal condition that can occur in large cohorts of victims simultaneously.

Potassium inward rectifier K IR 2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the K IR 2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of K IR 2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. K IR 2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian K IR 2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of K IR 2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed K IR 2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle K IR 2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 K IR 2.1 sequences showed that all disease causing K IR 2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake K IR 2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC 50 ∼6 μM) inward rectifier currents.

INTRODUCTION: The methoxamine-sensitized rabbit model is widely used to screen drugs for proarrhythmic properties, especially repolarization-dependent TdP arrhythmias. With the change of anesthesia and/or sensitizing agent, conduction disturbances have been reported as well. Therefore, we compared currently available in-house anesthetics in order to preserve arrhythmia sensitivity and preclude conduction disturbances. METHODS AND RESULTS: Rabbits were randomly assigned to 3 groups: (1) 35 mg/kg ketamine + 5 mg/kg xylazine; (2) 0.5 mL/kg hypnorm + 3 mg/kg midazolam; (3) 35 mg/kg ketamine + 20 mg/kg propofol. Anesthesia was maintained by 1.5% isoflurane. Concomitant infusion of methoxamine (17 μg/kg/min for 40 minutes) and dofetilide (10 μg/kg/min for 30 minutes) was used to induce arrhythmias. Sole methoxamine infusion exclusively decreased HR in groups 1 and 3. Dofetilide lengthened repolarization, followed in time by PQ/QRS prolongation, second-degree AV block, and subsequently TdP arrhythmias. TdP was seen in 80%, 0%, and 33% of the rabbits in groups 1, 2, and 3, respectively. Decreasing the dose of dofetilide to 5 μg/kg/min in ketamine/xylazine anesthetized rabbits resulted in a drop in TdP incidence (25%) while conduction disturbances persisted. Flunarizine (n = 6) suppressed all TdP arrhythmias while conduction disturbances remained present. CONCLUSION: TdP incidence in the methoxamine-sensitized rabbit could be dramatically influenced by anesthesia, drug dose, and flunarizine, while conduction slowing remained present. Thus, conduction slowing seems to be the integral outcome in this model.

We read with great interest the excellent paper by Varghese (2016) who describes an in silico approach to study the consequences of reciprocal regulation of expression of the sodium current (INa) and the inward rectifier potassium current (IK1) in the ventricle for cardiac excitability and conduction. The in silico approach follows the experimental results obtained by Milstein et al. (2012) who demonstrated functional co-regulation of the sodium and inward rectifier currents, and their underlying channel proteins Nav1.5 and KIR2.1 respectively, and electrophysiological consequences upon overexpression in rodent cardiomyocytes with respect to action potential duration and re-entry based arrhythmia propensity. Varghese adapted the guinea pig ventricular cardiomyocyte model, developed by Noble et al. (1998), that is extrapolated to simulations for one dimensional cardiac fibers, in which the fiber is represented as a linear cable model. Varghese changed either the conductance for INa and IK1 individually or in tandem, to assess their influence on the excitability of mammalian ventricular cardiomyocytes. One of the most interesting findings in this paper is the dominance of IK1 over INa in regulation of cardiac excitability, which yields important questions about the significance of the inward rectifier in this process (Varghese, 2016). This commentary will put these results in a broader context in order to provide a framework for future research questions.

Background and PurposeKv11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of Kv11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model.Experimental ApproachLUF7244 was tested in vitro for (a) increasing human IKv11.1 and canine IKr and (b) decreasing dofetilide‐induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block.Key ResultsLUF7244 (0.5–10 μM) concentration dependently increased IKv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on IKIR2.1, INav1.5, ICa‐L, and IKs, doubled IKr, shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide‐induced early afterdepolarizations. LUF7244 (2.5 mg·kg−1·15 min−1) in dogs with sinus rhythm was not proarrhythmic and shortened, non‐significantly, repolarization parameters (QTc: −6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide‐induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block.Conclusions and ImplicationsLUF7244 counteracted dofetilide‐induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of Kv11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.

Aims In excitable cells, KIR2.x ion-channel-carried inward rectifier current ( I K1) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific I K1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of I K1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits I K1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific I K1 inhibitors based on the P structure. Methods and results We analysed seven pentamidine analogues (PA-1 to PA-7) for I K1 blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC50 values (12–15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (Δ G −44.1 kJ/Mol) than for P (Δ G −31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect I Nav1.5, I Ca-L, I Kv4.3, I Kv11.1, and I Kv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of I K1 in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 ( n = 9), 26 ( n = 5), and 34% ( n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. Conclusion PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete I K1 block.

The cardiac action potential arises and spreads throughout the myocardium as a consequence of highly organized spatial and temporal expression of ion channels conducting Na(+), Ca(2+) or K(+) currents. The cardiac Na(+) current is responsible for the initiation and progression of the action potential. Altered Na(+) current has been found implicated in a number of different arrhythmias, including atrial fibrillation. In the atrium, the resting membrane potential is more depolarized than in the ventricles, and as cardiac Na(+) channels undergo voltage-dependent inactivation close to this potential, minor changes in the membrane potential have a relatively large impact on the atrial Na(+) current. The atrial resting membrane potential is established following ionic currents through the inwardly rectifying K(+) currents IK1, IK,ACh and IK,Ca and to a lesser extent by other ion channels as well as by exchangers and pumps. This review will focus on the relative and regulated contribution of IK1, IK,ACh and IK,Ca, and on pharmacological modification of the channels underlying these currents in respect to the resting membrane potential, Na(+) channel availability and atrial electrophysiology in health and disease.

Beat-to-beat variability in ventricular repolarization (BVR) associates with increased arrhythmic risk. Proarrhythmic remodeling in the dog with chronic AV-block (CAVB) compromises repolarization reserve and associates with increased BVR, which further increases upon dofetilide infusion and correlates with Torsade de Pointes (TdP) arrhythmias. It was hypothesized that these pro-arrhythmia-associated increases in BVR are induced by beat-to-beat variability in preload.Left ventricular monophasic action potential duration (LVMAPD) was recorded in acute (AAVB) and CAVB dogs, before and after dofetilide infusion. BVR was quantified as short-term variability of LVMAPD. The PQ-interval was controlled by pacing: either a constant or an alternating preload pattern was established, verified by PV-loop. The effect of the stretch-activated channel blocker, streptomycin, on BVR was evaluated in a second CAVB group. At alternating preload only, BVR was increased after proarrhythmic remodeling (0.45±0.14 ms AAVB vs. 2.2±1.1 ms CAVB, P0.01). At CAVB, but not at AAVB, dofetilide induced significant proarrhythmia. Preload variability augmented the dofetilide-induced BVR increase at CAVB (+1.5±0.8 ms vs. +0.9±0.9 ms, P=0.058). In the second group, the increase in baseline BVR by alternating preload (0.3±0.03 ms to 1.0±0.8 ms, P0.01) was abolished by streptomycin (0.5±0.2 ms, P0.05).In CAVB dogs, the inverse relation between BVR and repolarization reserve originates from an augmented sensitivity of ventricular repolarization to beat-to-beat preload changes. Stretch-activated channels appear to be involved in the mechanism of BVR. (Circ J 2016; 80: 1336-1345).

Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K(d) = 7 nM) over PKA-RII (K(d) = 53 nM) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RIα/β in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.

The rapid component of the delayed rectifier potassium current (I(Kr)), encoded by the ether-a-go-go-related gene (ERG1, officially denominated as KCNH2), is a major contributor to repolarization in the mammalian heart. Acute (e.g. drug-induced) and chronic (e.g. inherited genetic disorder) disruptions of this current can lead to prolongation of the action potential and potentiate occurrence of lethal arrhythmias. Many cardiac and non-cardiac drugs show high affinity for the I(Kr) channel and it is therefore extensively studied during safety pharmacology. The unique biophysical and pharmacological properties of the I(Kr) channel are largely recapitulated by expressing the human variant (hERG1a) in overexpressing systems. hERG1a channels are tetramers consisting of four 1159 amino acid long proteins and have electrophysiological properties similar, but not identical, to native I(Kr). In the search for an explanation to the discrepancies between I(Kr) and hERG1a channels, two alternative hERG1 proteins have been found. Alternative transcription of hERG1 leads to a protein with a 56 amino acid shorter N-terminus, known as hERG1b. hERG1b can form channels alone or coassemble with hERG1a. Alternative splicing leads to an alternate C-terminus and a protein known as hERGuso. hERGuso and hERG1b regulate hERG1a channel trafficking, functional expression and channel kinetics. Expression of hERGuso leads to a reduced number of channels at the plasma membrane and thereby reduces current density. On the contrary, co-assembly with hERG1b alters channel kinetics resulting in more available channels and a larger current. These findings have implication for understanding mechanisms of disease, acute and chronic drug effects, and potential gender differences.

Here we reveal that the characterization of large-scale re-arrangements of signaling scaffolds induced by heart failure can serve as a novel concept to identify more specific therapeutic targets. In the mammalian heart, the cAMP pathway, with the cAMP-dependent protein kinase (PKA) in a central role, acts directly downstream of adrenergic receptors to mediate cardiac contractility and rhythm. Heart failure, characterized by severe alterations in adrenergic stimulation is, amongst other interventions, often treated with β-blockers. Contrasting results, however, have shown both beneficial and detrimental effects of decreased cAMP levels in failing hearts. We hypothesize that the origin of this behavior lies in the complex spatiotemporal organization of the regulatory subunit of PKA (PKA-R), which associates tightly with various A-kinase anchoring proteins (AKAPs) to specifically localize PKA's activity. Using chemical proteomics directly applied to human patient and control heart tissue we demonstrate that the association profile of PKA-R with several AKAPs is severely altered in the failing heart, for instance effecting the interaction between PKA and the novel AKAP SPHKAP was 6-fold upregulated upon failing heart conditions. Also a significant increase in captured cGMP-dependent protein kinase (PKG) and phosphodiesterase 2 (PDE2) was observed. The observed altered profiles can already explain many aspects of the aberrant cAMP-response in the failing human heart, validating that this dataset may provide a resource for several novel, more specific, treatment options. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

Drug-induced Torsade de Pointes arrhythmia is a life-threatening adverse effect feared by pharmaceutical companies. For the last decade, the cardiac safety guidelines have imposed human ether-a-go-go-related gene channel blockade and prolongation of QT interval as surrogates for proarrhythmic risk propensity of a new chemical entity. Suffering from a lack of specificity, this assessment strategy led to a great amount of false positive outcomes. Therefore, this review will discuss new pharmaceutical strategies: the cardiac safety proposal that recently emerged, the Comprehensive in vitro Proarrhythmia Assay, combining in vitro assays that integrate effects on main cardiac ion channels, with computational models of human ventricular action potential as well as assays using human stem cell-derived cardiomyocytes for an improved prediction of drug’s proarrhythmic liability, alternative pharmacological perspectives as well as the current treatment of drug-induced long QT syndrome.

BACKGROUND AND PURPOSE Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) could be used to identify pro-arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro-arrhythmic properties of IKr blockers, using moxifloxacin (safe compound) and dofetilide or E-4031 (unsafe compounds). EXPERIMENTAL APPROACH Assays included the anaesthetized remodelled chronic complete AV block (CAVB) dog, the anaesthetized methoxamine-sensitized unremodelled rabbit, multi-cellular hESC-CM clusters, isolated CM obtained from CAVB dogs and isolated CM obtained from the normal rabbit. Arrhythmic outcome was defined as Torsade de Pointes (TdP) in the animal models and early afterdepolarizations (EADs) in the cell models. KEY RESULTS At clinically relevant concentrations (5–12 µM), moxifloxacin was free of pro-arrhythmic properties in all assays with the exception of the isolated CM, in which 10 µM induced EADs in 35% of the CAVB CM and in 23% of the rabbit CM. At supra-therapeutic concentrations (≥100 µM), moxifloxacin was pro-arrhythmic in the isolated rabbit CM (33%), in the hESC-CM clusters (18%), and in the methoxamine rabbit (17%). Dofetilide and E-4031 induced EADs or TdP in all assays (50–83%), and the induction correlated with a significant increase in beat-to-beat variability of repolarization. CONCLUSION AND IMPLICATIONS Isolated cardiomyocytes lack specificity to discriminate between TdP liability of the IKr blocking drugs moxifloxacin and dofetilide or E4031.

Carvedilol, a β- and α-adrenoceptor blocker, is used to treat congestive heart failure, mild to moderate hypertension, and myocardial infarction. It has been proposed to block K ATP channels by binding to the bundle crossing region at a domain including cysteine at position 166, and thereby plugging the pore region. However, carvedilol was reported not to affect Kir2.1 channels, which lack 166 Cys. Here, we demonstrate that carvedilol inhibits Kir2.3 carried current by an alternative mechanism. Carvedilol inhibited Kir2.3 channels with at least 100 fold higher potency (IC 50 = 0.49 μM) compared to that for Kir2.1 (IC 50 > 50 μM). Kir2.3 channel inhibition was concentration-dependent and voltage-independent. Increasing Kir2.3 channel affinity for PIP 2 , by a I213L point mutation, decreased the inhibitory effect of carvedilol more than twentyfold (IC 50 = 11.1 μM). In the presence of exogenous PIP 2 , Kir2.3 channel inhibition by carvedilol was strongly reduced (80 vs. 2% current inhibition). These results suggest that carvedilol, as other cationic amphiphilic drugs, inhibits Kir2.3 channels by interfering with the PIP 2 -channel interaction.