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The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at − 120 mV (73.2 ± 3.7%), − 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

The global reach-scale “ICESat-2 River Surface Slope” (IRIS, https://doi.org/10.5281/zenodo.7098113) dataset comprises average and extreme water surface slopes (WSS) derived from ICESat-2 observations between October 2018 and August 2022 as a supplement to 121,583 reaches from the “SWOT Mission River Database” (SWORD, Altenau et al., 2021). WSS is required to calculate river discharge, which is among the Essential Climate Variables as defined by the Global Climate Observing System. To gain full advantage of ICESat-2’s unique measurement geometry with six parallel lidar beams, the WSS is determined across pairs of beams or along individual beams, depending on the intersection angle of spacecraft orbit and river centerline. The combined results of both approaches are validated against in-situ data in a regional study at 815 reaches in Europe and North America with a median absolute error of 23 mm/km, almost complying with the SWOT science requirements of 17 mm/km (Scherer et al., 2022). IRIS can be used to research river dynamics, estimate river discharge, and correct water level time series from satellite altimetry for shifting ground tracks. Additionally, by referencing SWORD as a common database, IRIS may be used in combination with observations from the recently launched SWOT mission and could be easily compared against WSS measurements from SWOT’s new wide-swath sensor.

Water surface slope (WSS) of rivers is a key parameter in hydrological modelling, which allows for estimation of the transport and erosion capacity of a river, its flow velocity and discharge. On a local scale, WSS can be measured with a GNSS receiver installed on a boat, using remote sensing techniques (e.g. airborne lidar) or from a Digital Elevation Model (DEM). The most accurate method to measure WSS avoiding high-cost field campaigns is based on Water Surface Elevations (WSE) measured at in-situ stations. However, in poorly gauged rivers the neighboring gauges can be up to hundreds of kilometers apart, which inhibits a proper river profile observation. The gap in decreasing number of gauge readings is partially filled with satellite altimetry over rivers. Altimetry based WSE can be used to estimate WSS between neighboring measurements. Here, we present an innovative approach for estimating high-resolution WSS derived from multi-mission satellite altimetry for the largest Polish rivers.In this study, we used measurements from 9 altimetry missions: CryoSat-2, Envisat, ICESat-2, Jason-2/-3, SARAL, Sentinel-3A/-B, and Sentinel-6A. These observations cover the years from 2002 to 2022. We extracted the river centerlines from the global “SWOT Mission River Database” (SWORD). In order to validate the obtained results, we used WSE from 81 gauges, which are maintained by the Institute of Meteorology and Water Management – National Research Institute (Instytut Meteorologii i Gospodarki Wodnej – Państwowy Instytut Badawczy, IMGW-PIB). These measurements are referenced to the Kronsztadt’86 vertical datum and they range from 01.2016 to 05.2022. Additionally, we used the reach-scale “ICESat-2 River Surface Slope” (IRIS) and the DEM-derived WSS values from SWORD.To obtain WSS, we first determined WSE at each satellite pass crossing the studied river. Next, we split rivers into sections without dams and reservoirs. The Support Vector Regression (SVR) has been applied to reject outliers. Then, water levels were assigned to a given river kilometer (bin). For each of them a median WSE has been calculated. Finally, WSS were calculated at river sections between bins, excluding those disrupted by hydraulic structures. Finally, we weighted the section-wise WSS inversely proportional to the length of each section and applied a Least Square Adjustment with an additional Laplace condition to obtain bin-wise WSS for each river kilometer.To assess the accuracy of the proposed approach, we compared the obtained WSS with the slopes between IMGW-PIB gauges. For large rivers (Vistula, Odra, Warta), the multi-mission approach revealed high accuracy with preliminary Root Mean Squared Error (RMSE) below 30 mm/km. For smaller, mountain rivers (San, Dunajec) the preliminary errors were slightly larger (RMSE ~100 mm/km). We also compared our accuracies with those of the slopes based on DEM models, lidar data, ICESat-2 altimetry, and SWORD database. In general, the multi-mission approach revealed the highest accuracy. The research is supported by the National Science Centre, Poland, through the project no. 2020/38/E/ST10/00295.

For nearly three decades, satellite radar altimetry has provided measurements of the water surface elevation (WSE) of rivers. These observations can be used to calculate the water surface slope (WSS), which is an essential parameter for estimating flow velocity and river discharge. In this study, we calculate a high-resolution WSS of 11 Polish rivers based on multi-mission altimetry observations from 11 satellites in the period from 1994 to 2022. The proposed approach is based on a weighted such gauge stations adjustment with an additional Laplace condition and an a priori gradient condition. The processing is divided into river sections not interrupted by dams and reservoirs. After proper determination of the WSE for each river kilometer (bin), the WSS between adjacent bins is calculated. To assess the accuracy of the estimated WSS, it is compared with slopes between gauge stations, which are referenced to a common vertical datum. Such gauge stations are available for 8 investigated rivers. The root mean squared error (RMSE) ranges from 3 mm/km to 80 mm/km, with an average of 26 mm/km. However, the mean RMSE decreases to 10 mm/km when the 2 mountain rivers are excluded. The WSS accuracies are also compared with those of slope datasets based on digital elevation models, ICESat-2 altimetry, and lidar. For 6 rivers the estimated WSS showed the highest accuracy. The improvement was particularly significant for mountain rivers. The proposed approach allows an accurate, high-resolution WSS even for small and medium-sized rivers and can be applied to almost any river worldwide.

The inwardly rectifying potassium current of the cardiomyocyte (I

Geometry is a branch of mathematics that helps us to work with distances, volumes, and areas of the real world. But spatial geometry presents an unusual obstacle: how to understand the elements of a three-dimensional object when they are normally presented in a two-dimensional form. The study was conducted with two classes in the 2nd year of high school in a public school. Class 1 used traditional resources: textbook, blackboard, and chalk. Class 2 used augmented reality (FLARAS) and 3D visualization (Sketchup) and textbook. It was expected that, with the help of augmented reality, the students of Class 2 would perform better than Class 1. A better understanding of the content was achieved, as they would have better visualization of spatial objects. Consequently, better performance was achieved in the exercises and problem questions presented.

Background Postcardiac injury syndrome (PCIS) is an inflammatory complication that derives from injury to the epicardium, myocardium, or endocardium. It occurs after trauma, myocardial infarction, percutaneous coronary intervention, cardiac surgery, intracardiac ablation, and implantation of cardiac implantable electronic device (CIED). In this study we assessed the incidence of PCIS after CIED implantation and its possible risk factors. Material and methods All patients who received CIED implantation at Heidelberg University Hospital between 2000 and 2014 were evaluated (n = 4989 patients). Clinical data including age, sex, underlying cardiac disease, type of implanted CIED, location of electrode implantation, clinical symptoms, time of symptom onset of PCIS, therapy, and outcome were extracted and analyzed. Results We identified 19 cases of PCIS in 4989 patients, yielding an incidence of 0.38%. The age of patients with PCIS ranged from 39 to 86 years. Dilated cardiomyopathy (DCM) as underlying cardiac disease and right atrial (RA) lead implantation had a significant association with occurrence of PCIS (p = 0.045 in DCM and p Conclusion To the best of our knowledge, this is the largest cohort evaluating the incidence of PCIS after CIED implantation. The data show that PCIS is a rare complication after CIED implantation and occurs more frequently in patients with DCM and those with RA lead implantation. Although rare and mostly benign, PCIS can lead to potentially lethal complications and physicians must be aware of its symptoms.

A 23-year-old male patient presented with very mild visual disturbances, but a distinct prominence of the optic discs, more pronounced in the right than in the left eye. The ophthalmic symptoms initially seemed trivial, but a large-scale interdisciplinary workup later identified them as the presenting symptoms of sarcoidosis affecting lung and eyes. A standard steroid monotherapy successfully caused regression of the ophthalmic findings.

Geometry is a branch of Mathematics that helps us to work with distances, volumes, and areas in the real world. However, learning content related to Spatial Geometry, whose visual appeal should be an invitation to study, presents an unusual obstacle for the student: how to understand the elements of a three-dimensional object if they are normally presented in a two-dimensional way (e.g., drawn on the whiteboard or book)? There is several software that can be used in the teaching of Space Geometry. However, in most times, the teacher has no contact with these tools, either due to unavailability of hardware to interact; or due to difficulty in acquiring the software, which in turn may be due to the cost or lack of knowledge of the means to make the acquisition. The focus of this paper is to present the experience report on the use of Augmented Reality using tutorials developed by NIEP group, aiming to generate personalized material for use in the classroom by high school students in Mathematics, in the teaching/learning of Space Geometry. The results found demonstrate improved learning, better understanding of the content and reduced learning time.

Pregnancy is an important phase in women's lives, prenatal care is important to prevent diseases and ensure that women and babies can have a better quality of life. However, in Brazil, many pregnant women still do not perform due prenatal care, there are several factors that contribute to the failure to perform prenatal care among them: geographical factors. Pregnant women who live far from health units are unable to start pregnancy care in time. This work proposes a viable and low-cost alternative for the government or private sector companies. A Microservices architecture is presented, capable of meeting requests from pregnant women via mobile application and allowing health professionals to monitor the pregnancy evolution of a health unit. The architecture was designed to offer satisfactory performance, scalability and simplicity.

Educational Robotics refers to an application of technology in the pedagogical area that offers participants the opportunity to propose and solve difficult problems. So, what is learned by the child’s own effort has much more meaning for him and adapts better to they mental structures. Educational Robotics can be separated according to the apparatus used, which may be the use of commercial kits (e.g., LEGO EV3 or FISCHER TECHNIC) or low-cost kits (e.g., based on Arduino). Commercial kits typically have graphical programming interfaces tightly coupled to their components. Low-cost kits, on the other hand, do not have a defined interface and, therefore, depend on the search for software that allows their programming, but which have no obligation to work perfectly with all the components of the kit. This article shows an analysis of some Visual Programming Platforms (VPP) for educational robotics. This work also presents the design of a robotics VPP based on Arduino, designed for the inclusion of educational robotics to Brazilian children aged 8 to 14 years old.

Mobile devices have become quite popular over the past few years and as a result, the number of applications for the most diverse functions are available for download, many of them free of charge. However, this range of applications does not follow minimum usability parameters, which results in a high level of rejection by users. Within this mobile world, the insertion of devices that aid the health processes, called m-health, has shown an exponential growth in recent years and with this, the warning signal about the efficiency and security of information has been triggered. To remedy this weakness, the introduction of usability techniques during the application development process appears as crucial for minimizing the problem. Numerous studies cite the need for usability in m-health applications, but none of them points out how to obtain it. In this way, this work aims to verify if the design guidelines provided by the Google / Android platform, which is presented as a contribution to mobile software developers, presents guidelines that make it possible to obtain the maximum usability of these applications, considering the proposed user profile and the context of use involved. To this end, the proposal will be based on the comparison between the guidelines contained in the ISO standard (9241) and in consultation with the style guide of the platform called material design.

Background: Pregnancy is an important phase in a woman's life, the gestational period however is accompanied by fears and apprehension, especially for women who are experiencing this phase for the first time. Prenatal care is essential to prevent diseases and reduce risk for pregnant women, thus enabling healthy development for the baby. Nowadays many women find it difficult to make an adequate prenatal care, for the most varied reasons. Taking into account that the world is increasingly connected to the internet, it is important to understand, analyzing a set of health applications, which are the best decisions from the design point of view, usability to become a useful application for pregnant women, another relevant point is to understand how the architecture of these solutions were designed to be scalable. Thus, a well-defined systematic review is needed to identify the impacts of architectural, design and performance decisions on health applications, reducing risks and minimizing human errors.Methods/Design: This study will follow a well-defined methodological structure. The methodology is separated into the following sections: Research question identification; Extraction of relevant studies; Selection criteria; Charting the data; Collating, summarizing, and reporting the results. The research question aims to analyze the approaches used to build health applications aiming at design decisions, architecture and the impacts that are generated for the end user. The researchers will use the following electronic databases for data extraction: PubMed (Medline); IEEE Xplore Digital Library; ACM Digital Library; Web of Science; and Scopus Document Search. The search for research will consist of a set of terms and keywords.Discussion: This work has the interest of presenting a model, a standard, a guide for creating a health software interface, always aiming at the quality of the interface, the user experience and the possible gain with the performance of the application, considering that users will have more security to make decisions.Systematic review registration: Submitted on October 11, 2020 Open Science Framework.

O desenvolvimento de software, em particular, de jogos sempre atraiu a atenção como uma porta de entrada para novos desenvolvedores entrarem no mundo da computação. Inicialmente, era necessário o conhecimento de linguagens de programação altamente complexas (por exemplo, Assembly); depois disso, foi necessário ter conhecimento em uma linguagem de alto nível como C ++ e, finalmente, recentemente o espaço parece ter sido dominado por motores, como Unity e Unreal. É interessante notar que houve uma migração na forma de desenvolvimento, deixando demandas por conhecimentos específicos de programação atingindo situações nas quais o desenvolvimento se baseia quase que exclusivamente no design do jogo desejado. Dessa forma, este trabalho busca analisar os mecanismos de desenvolvimento de jogos, buscando expor suas vantagens e desvantagens, quais recursos, comunidade, suporte e documentação, além de quais requisitos de conhecimento para que o novo desenvolvedor atual possa entrar neste mundo.

A robótica possui quase um atrativo próprio para aglomerar pessoas. Com a possibilidade de movimentar um equipamento, seja via programação ou mesmo via controle (remoto ou via smartphone), a robótica atrai olhares e direciona a vontade de interagir. No caso de alunos, não importando a idade, não é diferente. Infelizmente, no Brasil, o custo para acesso a materiais robóticos, sejam de cunho puramente recreativo ou de potencial educacional, habitualmente, não é convidativo. Neste sentido, tem-se a opção dos materiais de baixo custo e muitas vezes open-source, a exemplo da plataforma Arduino. Entretanto, seu uso, por vezes, demanda conhecimentos para usar e montar as peças e componentes, ter habilidades de eletrônica (para efetivamente construir um robô) até chegar ao ponto da programação do robô criado. Este trabalho apresenta uma visão de uso de robótica de baixo custo baseado na plataforma Arduino, e uma discussão dos motivos que levaram a evolução do robô criado em meados de 2012, custando na época R$228,00, para uma nova versão custando R$145,00.

Despite increasing interest in monitoring the global water cycle, the availability of in-situ discharge time series is decreasing. However, this lack of ground data can be compensated by using remote sensing techniques to observe river discharge.In this contribution, a new approach for estimating the discharge of large rivers by combining various long-term remote sensing data with physical flow equations is presented. For this purpose, water levels derived from multi-mission satellite altimetry and water surface extents extracted from optical satellite images are used, both provided by DGFI-TUM’s “Database of Hydrological Time series of Inland Waters” (DAHITI, https://dahiti.dgfi.tum.de). The datasets are combined by fitting a hypsometric curve in order to describe the stage-width relation, which is then used to derive the water level for each acquisition epoch of the long-term multi-spectral remote sensing missions. In this way, the chance of detecting water level extremes is increased and a bathymetry can be estimated from water surface extent observations. Below the minimum hypsometric water level, the river bed elevation is estimated using an empirical width-to-depth relationship in order to determine the final cross-sectional geometry. The required flow gradient is computed based on a linear adjustment of river surface slope using all altimetry-observed water level differences between synchronous measurements at various virtual stations along the river. The roughness coefficient is set based on geomorphological features quantified by adjustment factors. These are chosen using remote sensing data and a literature decision guide.Within this study, all parameters are estimated purely based on remote sensing data, without using any ground data. In-situ data is only used for the validation of the method at the Lower Mississippi River. It shows that the presented approach yields best results for uniform and straight river sections. The resulting normalized root mean square error for those targets varies between 10% to 35% and is comparable with other studies.

The caged xanthone gambogic acid (GA) is a novel anti-cancer agent which exhibits anti-proliferative, anti-inflammatory and cytotoxic effects in many types of cancer tissues. In a recent phase IIa study, GA exhibits a favourable safety profile. However, limited data are available concerning its interaction with cardiac ion channels. Heteromeric assembly of Kir2.x channels underlies the cardiac inwardly rectifying IK1 current which is responsible for the stabilization of the diastolic resting membrane potential. Inhibition of the cardiac IK1 current may lead to ventricular arrhythmia due to delayed afterdepolarizations. Compared to Kv2.1, hERG and Kir1.1, a slow, delayed inhibition of Kir2.1 channels by GA in a mammalian cell line was reported before but no data exist in literature concerning action of GA on homomeric Kir2.2 and Kir2.3 and heteromeric Kir2.x channels. Therefore, the aim of this study was to provide comparative data on the effect of GA on homomeric and heteromeric Kir2.x channels. Homomeric and heteromeric Kir2.x channels were heterologously expressed in Xenopus oocytes, and the two-microelectrode voltage-clamp technique was used to record Kir2.x currents. To investigate the mechanism of the channel inhibition by GA, alanine-mutated Kir2.x channels with modifications in the channels pore region or at phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sites were employed. GA caused a slow inhibition of homomeric and heteromeric Kir2.x channels at low micromolar concentrations (with IC50 Kir2.1/2.2

The cardiac IK1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of IK1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of IK1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream β-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited IK1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining IK1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from β-adrenoreceptors.

Class IA antiarrhythmic drug quinidine was one of the first clinically used compounds to terminate atrial fibrillation and acts as multichannel inhibitor with well-documented inhibitory effects on several cardiac potassium channels. In the mammalian heart, heteromeric assembly of Kir2.1–2.3 channels underlies IK1 current. Although a low-affinity block of quinidine on Kir2.1 has already been described, a comparative analysis of effects on other Kir2.x channels has not been performed to date. Therefore, we analyzed the effects of quinidine on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Quinidine exerted differential inhibitory effects on Kir2.x channels with the highest affinity toward Kir2.3 subunits. Onset of block was slow and solely reversible in Kir2.2 subunits. Quinidine inhibited Kir2.x currents in a voltage-independent manner. By means of comparative Ala-scanning mutagenesis, we further found that residues E224, F254, D259, and E299 are essential for quinidine block in Kir2.1 subunits. Analogously, quinidine mediated Kir2.3 inhibition by binding corresponding residues E216, D247, D251, and E291. In contrast, Kir2.2 current block merely involved corresponding residue D260. Using channel mutants with altered (phosphatidylinositol 4,5-bisphosphate PIP2) affinities, we were able to demonstrate that high PIP2 affinities (i.e., Kir2.3 I214L) correlate with low quinidine sensitivity. Inversely, mutant channels interacting only weakly with PIP2 (i.e., Kir2.1 K182Q, and L221I) are prone to a higher inhibitory effect. Thus, we conclude that inhibition of Kir2.x channels by quinidine is mediated by joint modes of action involving direct cytoplasmic pore block and an impaired channel stabilization via interference with PIP2.

Pregnancy is an important stage in a woman's life, yet many women still do not get proper prenatal care. Failure to follow prenatal care can cause problems for the woman and the child. An important step in prenatal follow-up is the educational training for the woman, which allows her to overcome fears and get to know herself better. This work proposes a mobile application to support the woman who performs prenatal care in a public health unit and with this receive a better educational training on gestation. In addition, the application should allow a better mapping of pregnancy as well as concentrate data to better analyze the use of public funds and identify needs of public agencies acting based on identified demands.

Educational robotics is one of the ways that educational institutions use to expose that they have curriculum with technological approaches and innovation. Most of the studies that deal with the use of robotics in the classroom approach the student's point of view or even the associated didactics, but without exposing how or even what materials were used. In this work is exposed the report of experience of a teacher in the use of the LEGO EV3 kit, its difficulties in understanding the materials available, assembly and programming of the robot.

IEC ISO 60601-1-6:2015 is the regulatory standard for Medical Device (MD) and directs to ISO IEC 62366:2015 as a technical standard. However, due to the general nature of this standard, it does not provide details about criteria to guide usability testing of MD. In this way, the manufacturer deals with the unpredictability of the user according to the subjective convenience that he deems necessary to the type and class of the device. Objectives: To present a Catalog indicating technologies that can be used to detect the ergonomic or biomechanical behavior of the user. So that the test team has support for the follow-up of the experiment and for assistance in interpreting the test results. Method: A bibliographical review on technologies used to detect human behavior, and a technical survey of the market for products capable of capturing such behavior, were carried out in 2017 to establish and create the Catalog. Results: Findings allowed to list technologies that can be used to analyze the behavior of the user, assisting the execution of usability tests (UT) of the MD, being effective to detect the behavior and generate data to support the analysis and generation of reports. Conclusion: The Catalog classified technologies that can generate information about the usability of the MD, considering the analysis of the user’s behavior. The Catalog contains the association between the technology and behavioral possibilities that can be identified, serving as support to choose the technology most appropriate to the UT.

Left bundle branch block (LBBB) has a predictive value for response to cardiac resynchronization therapy as reported by Zareba et al. (Circulation 123(10):1061–1072, 2011). However, based on ECG criteria, the discrimination between complete LBBB and nonspecific intraventricular conduction delay is challenging. We tested the hypothesis that discrimination can be performed using standard electrophysiological catheters and a simple stimulation protocol. Fifty-nine patients were analyzed retrospectively. Patients were divided into groups of narrow QRS (n = 20), wide QRS of right bundle branch block (RBBB) morphology (n = 14), and wide QRS of LBBB morphology (n = 25). Using a diagnostic catheter placed in the coronary sinus, left ventricular activation was assessed during intrinsic conduction as well as during right ventricular (RV) stimulation. In patients with narrow QRS and RBBB, the Q-LV/QRS ratio was 0.43 ± 0.013 (n = 20) and 0.41 ± 0.026 (n = 14), respectively. In patients with LBBB morphology, the Q-LV/QRS split up into a group of patients with normal (0.43 ± 0.022, n = 7) and a group with delayed left ventricular activation (0.75 ± 0.016, n = 18). By direct comparison of the Q-LV/QRS ratio during intrinsic conduction with the Q-LV/QRS ratio during RV pacing leading to a functional LBBB, a clear distinction between a group of “true LBBB” and another group of “apparent LBBB”/nonspecific intraventricular conduction delay (NICD) could be generated. We present a novel and practical method that might facilitate discrimination between patients with apparent LBBB and true LBBB by comparing Q-LV/QRS ratios during intrinsic activation and during RV stimulation. Although this method can already be directly applied, validation by 3D electrical mapping and prospective correlation to cardiac resynchronization therapy (CRT) response will be required for further translation into clinical practice.

Efficient extraction of valuable products from microalgae and utilization of the residual biomass for biofuel production appear to bring economic benefits for the microalgae biorefineries. The pulsed electric field (PEF) method has been proposed as a promising pre-treatment for microalgae wet extraction. For conversion of microalgae biomass and residues, hydrothermal liquefaction (HTL) is considered a suitable method. Here, a combination of PEF-assisted valuables extraction of microalgae and HTL of the residual biomass is studied for the first time. The microalgae Auxenochlorella protothecoides, Chlorella vulgaris, and Scenedesmus almeriensis were cultivated, harvested, treated by PEF, and then subjected to lipid extraction, protein extraction, or extraction of amino acids after enzymatic protein hydrolysis. The residual biomass obtained from PEF-assisted valuables extraction was subjected to HTL in micro autoclaves at a temperature of 350 °C and pressure of 25 MPa for 15 min holding time. Product yields and analytical results obtained by elemental content measurement, 1H-nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography show that PEF alone has no significant direct influence on microalgae HTL. The harsh HTL conditions decisively determine the product yields and biocrude quality. However, PEF enhances the lipid extraction yield from 4 wt% to 33 wt%. Consequently, the biocrude yield decreases from 58 wt% to 43.2 wt%. Moreover, PEF boosts the protein extraction yield from almost zero to 41.6 wt% of the total protein, resulting in an increased biocrude yield by about 2 wt%. Finally, PEF accelerates the formation of amino acids by enzymatic hydrolysis, improving the extraction efficiency up to 150% in the first 60 min of the extraction. The extracted residue promises to produce a biocrude yield higher by 6 wt% and a better biocrude quality with lower nitrogen content. An overall mass balance of PEF-assisted valuables extraction and HTL is presented.

Pulsed electric field (PEF) treatment has so far been considered as ineffective for protein extraction, but the parameters that influence protein extraction efficiency have not been identified fully. Using Chlorella vulgaris in this study, we show that up to half of the proteins present in the cell can be extracted via PEF treatment and an incubation step for up to 24 h. The extraction efficiency is highly influenced by biomass concentration during the incubation step and the incubation temperature: in our experiments we were able to extract half of the proteins present in the cells at dilute concentrations (5 mg·ml−1), but this efficiency decreases with increasing biomass concentration, indicating that diffusion gradients play a role in the protein release. However, we also show that not only diffusion plays a role, but also a biological process that requires suitable conditions. We observed that PEF treated cells undergo programmed cell death, indicated by DNA laddering. Furthermore cold and hot temperatures as well as protease inhibition impair the release of proteins. In Western blots we can observe that some proteins are not released when the activity of proteases is blocked. We therefore conclude that protein release is facilitated by an enzyme-driven process that occurs after PEF-triggered cell death, and that this process is prone to extreme conditions.

Objectives This study sought to test the hypothesis that inducing an autoimmune response against the cardiac sodium channel (NaV1.5) induces arrhythmias. Background Sporadic evidence supports the concept that autoantibodies may cause cardiac arrhythmias but substantial experimental investigations using in vivo models have been lacking to date. The NaV1.5 is essential for cardiac impulse propagation and its dysfunction has been linked to conduction disease. Methods Rats were immunized with a peptide sequence derived from the third extracellular loop of the first domain of NaV1.5. After 28 days, we evaluated in vivo both the electrical and mechanical parameters of cardiac function. Histopathology, myocardial gene and protein expression were assessed. Whole-cell patch-clamp was used to measure sodium current (INa) density in isolated cardiomyocytes. Results NaV1.5-immunized rats had high titers of autoantibodies against NaV1.5. On ECG recording, NaV1.5-immunized animals showed significantly prolonged PR-intervals. During Holter ECG-monitoring we observed repeated prolonged episodes of third-degree atrioventricular and sinoatrial block in every NaV1.5-immunized animal, but not in controls. Immunization had no effect on cardiac function. In comparison to controls, myocardial NaV1.5 mRNA and protein levels were decreased in immunized rats. INa density was reduced in cardiomyocytes incubated with sera from NaV1.5-immunized rats and from patients with idiopathic atrioventricular block (AVB) in comparison to sera from respective controls. In patients with idiopathic AVB, we observed autoantibodies against NaV1.5 that were absent in sera from healthy controls. Conclusions Provocation of an autoimmune response against NaV1.5 induces conductance defects probably caused by a reduced expression level and an inhibition of NaV1.5 by autoantibodies, resulting in decreased INa.

The caged xanthone gambogic acid (GA) is a novel anti-cancer agent which exhibits anti-proliferative, anti-inflammatory and cytotoxic effects in many types of cancer tissues. In a recent phase IIa study, GA exhibits a favourable safety profile. However, limited data are available concerning its interaction with cardiac ion channels. Heteromeric assembly of Kir2.x channels underlies the cardiac inwardly rectifying I

Class IA antiarrhythmic drug quinidine was one of the first clinically used compounds to terminate atrial fibrillation and acts as multichannel inhibitor with well-documented inhibitory effects on several cardiac potassium channels. In the mammalian heart, heteromeric assembly of Kir2.1-2.3 channels underlies I

The cardiac I

Kir2.x channels form the molecular basis of cardiac I(K1) current and play a major role in cardiac electrophysiology. However, there is a substantial lack of selective Kir2 antagonists. We found the β(3)-adrenoceptor antagonist SR59230A to be an inhibitor of Kir2.x channels. Therefore, we characterized the effects of SR59230A on Kir2.x and other relevant cardiac potassium channels. Cloned channels were expressed in the Xenopus oocyte expression system and measured with the double-microelectrode voltage clamp technique. SR59230A inhibited homomeric Kir2.1 channels with an IC(50) of 33μM. Homomeric Kir2.2 and Kir2.3 channels and Kir2.x heteromers were also inhibited by SR59230A with similar potency. In contrast, no relevant inhibitory effects of SR59230A were found in cardiac Kv1.5, Kv4.3 and KvLQT1/minK channels. In hERG channels, SR59230A only induced a weak inhibition at a high concentration. These findings establish SR59230A as a novel inhibitor of Kir2.1-2.3 channels with a favorable profile with respect to additional effects on other cardiac repolarizing potassium channels.

There is genetic and epidemiological evidence demonstrating the association between Lp(a) levels and cardiovascular disease. While evolocumab reduces Lp(a) by 25-30%, the impact of PCSK9 inhibition on plaque at different Lp(a) levels is unknown. GLAGOV compared the effects of the PCSK9 inhibitor

Background and purpose: Atomoxetine is a selective noradrenaline reuptake inhibitor, recently approved for the treatment of attention-deficit/hyperactivity disorder. So far, atomoxetine has been shown to be well tolerated, and cardiovascular effects were found to be negligible. However, two independent cases of QT interval prolongation, associated with atomoxetine overdose, have been reported recently. We therefore analysed acute and subacute effects of atomoxetine on cloned human Ether-a-Go-Go-Related Gene (hERG) channels. Experimental approach: hERG channels were heterologously expressed in Xenopus oocytes and in a human embryonic kidney cell line and hERG currents were measured using voltage clamp and patch clamp techniques. Action potential recordings were made in isolated guinea-pig cardiomyocytes. Gene expression and channel surface expression were analysed using quantitative reverse transcriptase polymerase chain reaction, Western blot and the patch clamp techniques. Key results: In human embryonic kidney cells, atomoxetine inhibited hERG current with an IC50 of 6.3 µmol·L−1. Development of block and washout were fast. Channel activation and inactivation were not affected. Inhibition was state-dependent, suggesting an open channel block. No use-dependence was observed. Inhibitory effects of atomoxetine were attenuated in the pore mutants Y652A and F656A. In guinea-pig cardiomyocytes, atomoxetine lengthened action potential duration without inducing action potential triangulation. Overnight incubation with high atomoxetine concentrations resulted in a decrease of channel surface expression. Conclusions and implications: Whereas subacute effects of atomoxetine seem negligible under therapeutically relevant concentrations, hERG channel block should be considered in cases of atomoxetine overdose and when administering atomoxetine to patients at increased risk for the development of acquired long-QT syndrome.

Dronedarone is a novel class III antiarrhythmic drug that is widely used in atrial fibrillation. It has been shown in native cardiomyocytes that dronedarone inhibits cardiac inwardly rectifying current IK1 at high concentrations, which may contribute both its antifibrillatory efficacy and its potential proarrhythmic side effects. However, the underlying mechanism has not been studied in further detail to date. In the mammalian heart, heterotetrameric assembly of Kir2.x channels is the molecular basis of IK1 current. Therefore, we studied the effects of dronedarone on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Dronedarone inhibited Kir2.1 currents but had no effect on Kir2.2 or Kir2.3 currents. Onset of block was slow but completely reversible upon washout. Blockade of Kir2.1 channels did not exhibit strong voltage dependence or frequency dependence. In a screening with different Kir2.1 mutants lacking specific binding sites within the cytoplasmic pore region, we found that residue E224 is essential for binding of dronedarone to Kir2.1 channels. In conclusion, direct block of Kir2.1 channel subunits by dronedarone through binding at E224 may underlie its inhibitory effects on cardiac IK1 current.

Ajmaline is a class Ia anti-arrhythmic compound that is widely used for the diagnosis of Brugada syndrome and the acute treatment of atrial or ventricular tachycardia. For ajmaline, inhibitory effects on a variety of cardiac K+ channels have been observed, including cardiac Kv1 and Kv4 channels. However, the exact pharmacological properties of channel blockade have not yet been addressed adequately. Using two different expression systems, we analysed pharmacological effects of ajmaline on the potassium channels Kv1.5 and Kv4.3 underlying cardiac I Kur and I to current, respectively. When expressed in a mammalian cell line, we find that ajmaline inhibits Kv1.5 and Kv4.3 with an IC50 of 1.70 and 2.66 μM, respectively. Pharmacological properties were further analysed using the Xenopus expression system. We find that ajmaline is an open channel inhibitor of cardiac Kv1.5 and Kv4.3 channels. Whereas ajmaline results in a mild leftward shift of Kv1.5 activation curve, no significant effect on Kv4.3 channel activation could be observed. Ajmaline did not significantly affect channel inactivation kinetics. Onset of block was fast. For Kv4.3 channels, no significant effect on recovery from inactivation or channel deactivation could be observed. Furthermore, there was no use-dependence of block. Taken together, we show that ajmaline inhibits cardiac Kv1.5 and Kv4.3 channels at therapeutic concentrations. These data add to the current understanding of the electrophysiological basis of anti-arrhythmic action of ajmaline.

In order to prevent human error, it is essential to understand the nature of the user’s behaviour. This chapter proposes a combined approach to increase knowledge of user behaviour by instantiating a programmable user model with data gathered from a user profile. Together, the user profile and user model represent, respectively, the static and dynamic characteristics of user behaviour. Typically, user models have been employed by system designers to explore the user decision-making process and its implications, since user profiles do not account for the dynamic aspects of a user interaction. In this chapter, the user profile and model are employed to study human errors—supporting an investigation of the relationship between user errors and user characteristics. The chapter reviews the literature on user profiles and models and presents the proposed user profile and model. It concludes by discussing the application of the proposed approach in the context of electrical systems’ operation.

article i nfo The transient outward current Ito is an important determinant of the early repolarization phase. Ito and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and Ito by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Go6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCβ, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCβ siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent Ito reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native Ito. These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.

Documenting accidents and incidents in a clear, precise and unambiguous way is essential to ground the error study and to propose preventive measures. Research in the field of Human Error has yielded many taxonomies and categorizations of errors which are used by companies when analyzing and reporting on accidents and incidents. However, there are difficulties regarding the appropriateness of the terms as applied to a company's specific context, since taxonomies tend to be specific. Moreover, the lack of a clear organization and the adoption of concepts which are not universal among users with different background render their taxonomy's use rather difficult. This paper proposes a taxonomy and an associated supporting tool for describing accidents and incidents in the operation of electrical systems. The proposed taxonomy addresses the concepts and the terms related to the causes of error as well as concepts related to the user cognitive processes that led to the error.

International audience; Accident and incident analysis is essential to the study of human error and the development of error prevention measures. Human Error research deals essentially with the classification of error and the identification of the causal relation between the error detected and the level of human performance at which it occurred. As a result the literature proposes many error categorization methods and taxonomies. These are not, in themselves, sufficient, however, to analyze (and understand) the circumstances surrounding the error occurrence. For a more complete understanding of human error, it is necessary to associate each error with the sequence of steps taken by the human operator during the task that led to it. This paper proposes an extension of the existing error categorization found in the literature and applies it to the analysis of human error reports originating in the electricity industry.

The antidepressant mianserin exhibits a tetracyclic structure that is different from typical tricyclic antidepressants (TCA) and that of selective serotonin reuptake inhibitors. In comparison to the older TCA, mianserin has been shown to have a superior risk profile regarding proarrhythmic effects, both in vitro and in vivo. However, the underlying molecular electrophysiological basis has not been elucidated to date. Therefore, we studied the effects of mianserin on cardiac hERG potassium channels, the predominant target of drug-induced proarrhythmia. HERG channels were expressed in the Xenopus oocyte expression system and in human embryonic kidney (HEK) cells and currents were measured with two-microelectrode voltage-clamp and whole-cell patch-clamp, respectively. Mianserin inhibited hERG currents in a dose-dependent manner with an IC(50) of 3.2 micromol/l in HEK cells. Onset of blockade was slow and the inhibitory effect was not reversible upon wash-out of the drug. In hERG channel mutants, Y652A and F656A, lacking aromatic residues in the S6 domain, the effect of mianserin was significantly reduced in comparison to the wild type. Mianserin inhibited hERG currents in the open and inactivated state, but not in the closed states. HERG inactivation kinetics were significantly altered by mianserin without marked effects on channel activation kinetics. The inhibitory effect was not frequency dependent. In conclusion, mianserin is a low-affinity hERG-blocking agent. However, taken together with the lack of APD-prolongation shown in other studies, mianserin seems to have a good safety profile. Lack of consistent QT prolonging effects of mianserin in previous studies may therefore be linked to additional effects such as inhibition of other cardiac ion channels. However, as demonstrated by clinical case reports, mianserin can induce proarrhythmic effects in susceptible patients. Therefore, in patients with complex co-medication (i.e., additional hERG-blocking agents) and in patients with risk factors for acquired long QT syndrome as well as in cases of overdose, adequate monitoring should be recommended.

Inhibition of I(K1) currents by adrenergic alpha(1) receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native I(K1) current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved. Adrenergic alpha(1A) receptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native I(K1) currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes. Activation of co-expressed adrenergic alpha(1A) receptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of I(K1) current by adrenergic alpha(1) receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of alpha(1A) receptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein. In summary, on the molecular level the inhibitory regulation of I(K1) currents by adrenergic alpha(1A) receptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native I(K1) current by adrenergic alpha(1) receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation.

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr(157) and Thr(239) (termed AQP1 DeltaPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr(157) or Thr(239) caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 DeltaPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both Thr(157) and Thr(239) are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 DeltaPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.

Nadine Vonderlin,1 Fathima Fischer,1 Edgar Zitron,1,2 Claudia Seyler,1 Daniel Scherer,1 Dierk Thomas,1,2 Hugo A Katus,1,2 Eberhard P Scholz1 1Department of Internal MedicineIII, University Hospital Heidelberg, 2German Centre for Cardiovascular Research, Partner Site Heidelberg/Mannheim, Heidelberg, Germany Abstract: Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 µM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 µM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam. Keywords: midazolam, anesthetics, human ether-à-go-go-related gene, potassium channels

beta(3)-adrenoceptors have recently been shown to induce a complex modulation of intracellular signaling pathways including cyclic guanine monophosphate, cyclic adenosine monophosphate, nitric oxide, and protein kinases A and C. They are expressed in a broad variety of tissues including the myocardium, vascular smooth muscle, and endothelium. In those tissues, resting membrane potential is controlled mainly by inwardly rectifying potassium channels of the Kir2 family namely, Kir2.1 in the vascular smooth muscle, Kir2.1-2.3 in the myocardium, and Kir2.1-2.2 in the endothelium. In the present study, we investigated the possible modulation of Kir2 channel function by beta(3)-adrenoceptors in an expression system. Human-cloned beta(3)-adrenoceptors and Kir2.1 (KCNJ2), Kir2.2 (KCNJ12), and Kir2.3 (KCNJ4) channels were coexpressed in Xenopus oocytes, and currents were measured with double-microelectrode voltage clamp. Activation of beta(3)-adrenoceptors with isoproterenol resulted in markedly increased currents in Kir2.1 and in Kir2.2 potassium channels with EC50 values of 27 and 18 nM, respectively. In contrast, Kir2.3 currents were not modulated. Coapplication of specific inhibitors of protein kinase A (KT-5720) and calmodulin kinase II (KN-93) had no effects on the observed regulation in Kir2.1. However, coapplication of protein kinase C (PKC) inhibitors staurosporine and chelerythrine suppressed the observed effect. In Kir2.2, coapplication of KT-5720 reduced the effect of beta(3)-adrenoceptor activation. No differences in current increase after application of isoproterenol were observed between mutant Kir2.2 potassium channels lacking all functional PKC phosphorylation sites and Kir2.2 wild-type channels. In heteromeric Kir2.x channels, all types of heteromers were activated. The effect was most pronounced in Kir2.1/Kir2.2 and in Kir2.2/Kir2.3 channels. In summary, homomeric and heteromeric Kir2.x channels are activated by beta(3)-adrenoceptors via different protein kinase-dependent pathways: Kir2.1 subunits are modulated by PKC, whereas Kir2.2 is modulated by protein kinase A. In heteromeric composition, a marked activation of currents can be observed particularly with involvement of Kir2.2 subunits. This regulation may contribute to the hyperpolarizing effects of beta(3)-adrenoceptors in tissues that exhibit modulation by Kir2 channel function.

To elucidate the ionic mechanism of endothelin-1 (ET-1)-induced focal ventricular tachyarrhythmias, the regulation of I(K1) and its main molecular correlates, Kir2.1, Kir2.2 and Kir2.3 channels, by ET-1 was investigated. Native I(K1) in human atrial cardiomyocytes was studied with whole-cell patch clamp. Human endothelin receptors were coexpressed with human Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes. Currents were measured with a two-microelectrode voltage clamp. In human cardiomyocytes, ET-1 induced a marked inhibition of I(K1) that could be suppressed by the protein kinase C (PKC) inhibitor staurosporine. To investigate the molecular mechanisms underlying this regulation, we studied the coupling of ET(A) receptors to homomeric and heteromeric Kir2.1, Kir2.2 and Kir2.3 channels in the Xenopus oocyte expression system. ET(A) receptors coupled functionally to Kir2.2 and Kir2.3 channels but not to Kir2.1 channels. In Kir2.2 channels lacking functional PKC phosphorylation sites, the inhibitory effect was abolished. The inhibition of Kir2.3 currents could be suppressed by the PKC inhibitors staurosporine and chelerythrine. The coupling of ET(A) receptors to heteromeric Kir2.1/Kir2.2 and Kir2.2/Kir2.3 channels resulted in a strong inhibition of currents comparable with the effect observed in Kir2.2 homomers. Surprisingly, in heteromeric Kir2.1/Kir2.3 channels, no effect was observed. ET-1 inhibits human cardiac I(K1) current via a PKC-mediated phosphorylation of Kir2.2 channel subunits and additional regulatory effects on Kir2.3 channels. This mechanism may contribute to the intrinsic arrhythmogenic potential of ET-1.

Objective: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, IKr. The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and IKr currents was investigated in this study. Methods: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native IKr currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. Results: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC50 values of 1.0 and 13.2 AM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of 20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 AM BIM I reduced native IKr currents by 69.2% and lead to action potential prolongation. Conclusion: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and IKr currents.

Nadine Vonderlin,1 Fathima Fischer,1 Edgar Zitron,1,2 Claudia Seyler,1 Daniel Scherer,1 Dierk Thomas,1,2 Hugo A Katus,1,2 Eberhard P Scholz1 1Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany; 2German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim, Heidelberg, GermanyAbstract: Midazolam is a short-acting benzodiazepine that is widely used in anesthesia. Despite its widespread clinical use, detailed information about cardiac side effects of midazolam is largely lacking. Using the double-electrode voltage clamp technique, we studied pharmacological effects of midazolam on heterologously expressed Kv1.5 channels underlying atrial repolarizing current IKur. Midazolam dose-dependently inhibited Kv1.5 current, yielding an IC50 of 17 µM in an HEK cell line and an IC50 of 104 µM in Xenopus oocytes. We further showed that midazolam did not affect the half-maximal activation voltage of Kv1.5 channels. However, a small negative shift of the inactivation curve could be observed. Midazolam acted as a typical open-channel inhibitor with rapid onset of block and without frequency dependence of block. Taken together, midazolam is an open channel inhibitor of cardiac Kv1.5 channels. These data add to the current understanding of the pharmacological profile of midazolam.Keywords: anesthetics, potassium channels, pharmacology