The Utrecht University Honours Program review project: example based scientific publishing training aimed at bachelor medical students.

INTRODUCTION: Medical undergraduate students receive limited education on scholarly publishing. However, publishing experiences during this phase are known to influence study and career paths. The medical bachelor Honours Program (HP) at Utrecht University initiated a hands-on writing and publishing course, which resulted in nine reviews published in internationally peer reviewed academic journals. We wanted to share the project set-up, explore the academic development of the participating students and determine the impact of the reviews on the scientific community. METHODS: Thirty-one out of 50 alumni completed a digital retrospective questionnaire on for example, development of skills and benefit for their studies and career. Publication metrics of the HP review papers were retrieved from Web of Science. RESULTS: This hands-on project provides a clear teaching method on academic writing and scholarly publishing in the bachelor medical curriculum. Participants were able to obtain and improve writing and publishing skills. The output yielded well-recognized scientific papers and valuable learning experiences. 71% of the participating students published at least one additional paper following this project, and 55% of the students indicated the project influenced their academic study and/or career path. Nine manuscripts were published in journals with an average impact factor of 3.56 and cited on average 3.73 times per year. DISCUSSION: This course might inspire other medical educators to incorporate similar projects successfully into their curriculum. To this end, a number of recommendations with regard to supervision, time investment and group size are given.

Automatic measurement of short-term variability of repolarization to indicate ventricular arrhythmias in a porcine model of cardiac ischaemia.

AIMS: An automated method for determination of short-term variability (STV) of repolarization on intracardiac electrograms (STV-ARIauto) has previously been developed for arrhythmic risk monitoring by cardiac implantable devices, and has proved effective in predicting ventricular arrhythmias (VA) and guiding preventive high-rate pacing (HRP) in a canine model. Current study aimed to assess (i) STV-ARIauto in relation to VA occurrence and secondarily (ii-a) to confirm the predictive capacity of STV from the QT interval and (ii-b) explore the effect of HRP on arrhythmic outcomes in a porcine model of acute myocardial infarction (MI). METHODS AND RESULTS: Myocardial infarction was induced in 15 pigs. In 7/15 pigs, STV-QT was assessed at baseline, occlusion, 1 min before VA, and just before VA. Eight of the 15 pigs were additionally monitored with an electrogram catheter in the right ventricle, underwent echocardiography at baseline and reperfusion, and were randomized to paced or control group. Paced group received atrial pacing at 20 beats per min faster than sinus rhythm 1 min after occlusion. Short-term variability increased prior to VA in both STV modalities. The percentage change in STV from baseline to successive timepoints correlated well between STV-QT and STV-ARIauto. High-rate pacing did not improve arrhythmic outcomes and was accompanied by a stronger decrease in ejection fraction. CONCLUSION: STV-ARIauto values increase before VA onset, alike STV-QT in a porcine model of MI, indicating imminent arrhythmias. This highlights the potential of automatic monitoring of arrhythmic risk by cardiac devices through STV-ARIauto and subsequently initiates preventive strategies. Continuous HRP during onset of acute MI did not improve arrhythmic outcomes.

Pro-Arrhythmic Potential of Accumulated Uremic Toxins Is Mediated via Vulnerability of Action Potential Repolarization.

Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End-stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are among the CKD-related factors that have been linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients are cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible remain poorly understood. The current study aimed to assess the vulnerability of action potential repolarization caused by exposure to pre-identified UTs at clinically relevant concentrations. We exposed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and HEK293 chronically (48 h) to the UTs indoxyl sulfate, kynurenine, or kynurenic acid. We used optical and manual electrophysiological techniques to assess action potential duration (APD) in the hiPSC-CMs and recorded IKr currents in stably transfected HEK293 cells (HEK-hERG). Molecular analysis of KV11.1, the ion channel responsible for IKr, was performed to further understand the potential mechanism underlying the effects of the UTs. Chronic exposure to the UTs resulted in significant APD prolongation. Subsequent assessment of the repolarization current IKr, often most sensitive and responsible for APD alterations, showed decreased current densities after chronic exposure to the UTs. This outcome was supported by lowered protein levels of KV11.1. Finally, treatment with an activator of the IKr current, LUF7244, could reverse the APD prolongation, indicating the potential modulation of electrophysiological effects caused by these UTs. This study highlights the pro-arrhythmogenic potential of UTs and reveals a mode of action by which they affect cardiac repolarization.

Electrophysiology of hiPSC-Cardiomyocytes Co-Cultured with HEK Cells Expressing the Inward Rectifier Channel.

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.

LUF7244 plus Dofetilide Rescues Aberrant Kv11.1 Trafficking and Produces Functional IKv11.1.

Voltage-gated potassium 11.1 (Kv11.1) 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 because of 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 wild type (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) also restored Kv11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 µM) increased IKv 11.1 despite the presence of dofetilide (1 µM) in WT Kv11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 µM) and dofetilide (1 µM) increased IKv11.1 compared with nontreated 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. SIGNIFICANCE STATEMENT: Decreased levels of functional Kv11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of Kv11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired Kv11.1 trafficking defects, resulting in functional Kv11.1 current.

Towards the Development of AgoKirs: New Pharmacological Activators to Study Kir2.x Channel and Target Cardiac Disease.

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.

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents.

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.

Istaroxime, a positive inotropic agent devoid of proarrhythmic properties in sensitive chronic atrioventricular block dogs.

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.

Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo.

Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC-microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2±26.3nM and 144.8±35.1nM, respectively. In dog ventricular cardiomyocytes, DHE dose-dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10µM DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (cAVB) dogs. In rabbits, DHE increased the QT interval significantly by 12±10% (0.05mg/kg/5min) and 60±26% (0.5mg/kg/5min), and induced Torsade de Pointes arrhythmias (TdP, 0.5mg/kg/5min) in 2 rabbits. In cAVB dogs, 0.33mg/kg/5min DHE increased QT duration by 48±10% (P<0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), methanolic extracts of Evodia, DHE and hortiamine dose-dependently prolonged APD. At 3µM DHE and hortiamine induced EADs. hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC-CMs and dose-dependent proarrhythmic effects of DHE at micromolar plasma concentrations in cAVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR 2.1 backward trafficking.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR 2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK1 ), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR 2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR 2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 µM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 µM, 24 hrs) and late-endosomal/lysosomal KIR 2.1 accumulation. Increased KIR 2.1 expression level was also observed in the presence of Nav 1.5 co-expression. Augmented KIR 2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv 11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at -120 mV, 5 µM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at -120 mV; 2 µM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR 2.1 degradation.

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression.

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.

Short-term Variability of Repolarization Is Superior to Other Repolarization Parameters in the Evaluation of Diverse Antiarrhythmic Interventions in the Chronic Atrioventricular Block Dog.

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.

A Heart too Drunk to Drive; AV Block following Acute Alcohol Intoxication.

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.

A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs.

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIß, RIIα, and RIIß) each form a homodimer, and their dimerization domain interacts with a small helical region present in each of the more than 40 AKAPs reported so far. This allows for tight anchoring of PKA and efficient communication with other signaling proteins that interact with the AKAP scaffold in a spatial and temporal manner. The hydrophobic interaction surfaces of the PKA-R dimer and several AKAP helices have been investigated in great detail. Despite this knowledge, not every suggested AKAP has had its binding motif specified. Here we created an efficient bioinformatic tool, termed THAHIT, to accurately map the PKA binding motif and/or additional motifs of all previously reported AKAPs. Moreover, THAHIT predicts its specificity toward PKA-RIα and/or PKA-RIIα binding. To verify the validity of these newly predicted anchoring sites and their putative specificities, we used computational modeling approaches (HADDOCK), biochemical affinity studies (fluorescence anisotropy), and cellular colocalization studies. We further demonstrate the potential of THAHIT to identify novel AKAPs in cAMP-based chemical proteomics discovery data sets, and the human proteome. We retrieved numerous novel AKAP candidates, including a never reported 330 kDa AKAP observed in heart tissue, which we further characterized biochemically as a PKA-RIIα binder. Altogether, THAHIT provides a comprehensive overview of known and novel PKA-AKAP interaction domains and their PKA-R specificities.

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae).

Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 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 KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6µM) inward rectifier currents.

The network of cardiac KIR2.1: its function, cellular regulation, electrical signaling, diseases and new drug avenues.

The functioning of the human heart relies on complex electrical and communication systems that coordinate cardiac contractions and sustain rhythmicity. One of the key players contributing to this intricate system is the KIR2.1 potassium ion channel, which is encoded by the KCNJ2 gene. KIR2.1 channels exhibit abundant expression in both ventricular myocytes and Purkinje fibers, exerting an important role in maintaining the balance of intracellular potassium ion levels within the heart. And by stabilizing the resting membrane potential and contributing to action potential repolarization, these channels have an important role in cardiac excitability also. Either gain- or loss-of-function mutations, but also acquired impairments of their function, are implicated in the pathogenesis of diverse types of cardiac arrhythmias. In this review, we aim to elucidate the system functions of KIR2.1 channels related to cellular electrical signaling, communication, and their contributions to cardiovascular disease. Based on this knowledge, we will discuss existing and new pharmacological avenues to modulate their function.

Recording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions.

Background: The rapid delayed rectifier potassium current (IKr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting IKr, hERG channel, is not only permeable to K+ ions but also to Cs+ ions when present in equimolar concentrations inside and outside of the cell. Methods: In this study, IhERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs+ or K+ as the charge carrier. Equimolar Cs+ has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs+- and K+-based currents. Results: Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs+- and K+-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs+-based conductance is larger compared to the known K+-based conductance, and the Cs+ hERG conductance can be inhibited similarly to the K+-based conductance. Conclusion: Using equimolar Cs+ instead of K+ for IhERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small IKr current densities in combination with small membrane capacitances.

A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane.

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/ß 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.

Inhibiting the clathrin-mediated endocytosis pathway rescues K(IR)2.1 downregulation by pentamidine.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K(IR)2.x carried I(K1) current and that inhibiting protein degradation in the lysosome increased intracellular K(IR)2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K(IR)2.1 to further restore normal levels of functional K(IR)2.1 channels. K(IR)2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). K(IR)2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved K(IR)2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased I(K1) (at -60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute I(K1) block. Baf and CQ did not significantly enhance I(K1) densities. Pentamidine (10 µM, 48 h) reduced K(IR)2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in K(IR)2.1 degradation. Pentamidine-induced downregulation of K(IR)2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.