Opto-electronic feedback control of membrane potential for real-time control of action potentials.

To unlock new research possibilities by acquiring control of action potential (AP) morphologies in excitable cells, we developed an opto-electronic feedback loop-based system integrating cellular electrophysiology, real-time computing, and optogenetic approaches and applied it to monolayers of heart muscle cells. This allowed accurate restoration and preservation of cardiac AP morphologies in the presence of electrical perturbations of different origin in an unsupervised, self-regulatory manner, without any prior knowledge of the disturbance. Moreover, arbitrary AP waveforms could be enforced onto these cells. Collectively, these results set the stage for the refinement and application of opto-electronic control systems to enable in-depth investigation into the regulatory role of membrane potential in health and disease.

Light transmittance in human atrial tissue and transthoracic illumination in rats support translatability of optogenetic cardioversion of atrial fibrillation.

BACKGROUND: Optogenetics could offer a solution to the current lack of an ambulatory method for the rapid automated cardioversion of atrial fibrillation (AF), but key translational aspects remain to be studied. OBJECTIVE: To investigate whether optogenetic cardioversion of AF is effective in the aged heart and whether sufficient light penetrates the human atrial wall. METHODS: Atria of adult and aged rats were optogenetically modified to express light-gated ion channels (i.e., red-activatable channelrhodopsin), followed by AF induction and atrial illumination to determine the effectivity of optogenetic cardioversion. The irradiance level was determined by light transmittance measurements on human atrial tissue. RESULTS: AF could be effectively terminated in the remodeled atria of aged rats (97%, n = 6). Subsequently, ex vivo experiments using human atrial auricles demonstrated that 565-nm light pulses at an intensity of 25 mW/mm2 achieved the complete penetration of the atrial wall. Applying such irradiation onto the chest of adult rats resulted in transthoracic atrial illumination as evidenced by the optogenetic cardioversion of AF (90%, n = 4). CONCLUSION: Transthoracic optogenetic cardioversion of AF is effective in the aged rat heart using irradiation levels compatible with human atrial transmural light penetration.

A Possible Explanation for the Low Penetrance of Pathogenic KCNE1 Variants in Long QT Syndrome Type 5.

Long QT syndrome (LQTS) is an inherited cardiac rhythm disorder associated with increased incidence of cardiac arrhythmias and sudden death. LQTS type 5 (LQT5) is caused by dominant mutant variants of KCNE1, a regulatory subunit of the voltage-gated ion channels generating the cardiac potassium current IKs. While mutant LQT5 KCNE1 variants are known to inhibit IKs amplitudes in heterologous expression systems, cardiomyocytes from a transgenic rabbit LQT5 model displayed unchanged IKs amplitudes, pointing towards the critical role of additional factors in the development of the LQT5 phenotype in vivo. In this study, we demonstrate that KCNE3, a candidate regulatory subunit of IKs channels minimizes the inhibitory effects of LQT5 KCNE1 variants on IKs amplitudes, while current deactivation is accelerated. Such changes recapitulate IKs properties observed in LQT5 transgenic rabbits. We show that KCNE3 accomplishes this by displacing the KCNE1 subunit within the IKs ion channel complex, as evidenced by a dedicated biophysical assay. These findings depict KCNE3 as an integral part of the IKs channel complex that regulates IKs function in cardiomyocytes and modifies the development of the LQT5 phenotype.

In vivo and cellular antiarrhythmic and cardiac electrophysiological effects of desethylamiodarone in dog cardiac preparations.

BACKGROUND AND PURPOSE: The aim of the present study was to study the antiarrhythmic effects and cellular mechanisms of desethylamiodarone (DEA), the main metabolite of amiodarone (AMIO), following acute and chronic 4-week oral treatments (25-50 mg·kg-1 ·day-1 ). EXPERIMENTAL APPROACH: The antiarrhythmic effects of acute iv. (10 mg·kg-1 ) and chronic oral (4 weeks, 25 mg·kg-1 ·day-1 ) administration of DEA were assessed in carbachol and tachypacing-induced dog atrial fibrillation models. Action potentials were recorded from atrial and right ventricular tissue following acute (10 µM) and chronic (p.o. 4 weeks, 50 mg·kg-1 ·day-1 ) DEA application using the conventional microelectrode technique. Ionic currents were measured by the whole cell configuration of the patch clamp technique in isolated left ventricular myocytes. Pharmacokinetic studies were performed following a single intravenous dose (25 mg·kg-1 ) of AMIO and DEA intravenously and orally. In chronic (91-day) toxicological investigations, DEA and AMIO were administered in the oral dose of 25 mg·kg-1 ·day-1 ). KEY RESULTS: DEA exerted marked antiarrhythmic effects in both canine atrial fibrillation models. Both acute and chronic DEA administration prolonged action potential duration in atrial and ventricular muscle without any changes detected in Purkinje fibres. DEA decreased the amplitude of several outward potassium currents such as IKr , IKs , IK1 , Ito , and IKACh , while the ICaL and late INa inward currents were also significantly depressed. Better drug bioavailability and higher volume of distribution for DEA were observed compared to AMIO. No neutropenia and less severe pulmonary fibrosis was found following DEA compared to that of AMIO administration. CONCLUSION AND IMPLICATIONS: Chronic DEA treatment in animal experiments has marked antiarrhythmic and electrophysiological effects with better pharmacokinetics and lower toxicity than its parent compound. These results suggest that the active metabolite, DEA, should be considered for clinical trials as a possible new, more favourable option for the treatment of cardiac arrhythmias including atrial fibrillation.

Optical ventricular cardioversion by local optogenetic targeting and LED implantation in a cardiomyopathic rat model.

AIMS: Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs (i) in the pathologically remodelled heart using an (ii) implantable multi-LED device for (iii) in vivo closed-chest, local illumination. METHODS AND RESULTS: In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place 3 weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin or Citrine for control experiments. At 8-10 weeks after TAC, VTs were induced ex vivo and in vivo, followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination. CONCLUSION: Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion.

The Effects of Repetitive Use and Pathological Remodeling on Channelrhodopsin Function in Cardiomyocytes.

Aim: Channelrhodopsins (ChRs) are a large family of light-gated ion channels with distinct properties, which is of great importance in the selection of a ChR variant for a given application. However, data to guide such selection for cardiac optogenetic applications are lacking. Therefore, we investigated the functioning of different ChR variants in normal and pathological hypertrophic cardiomyocytes subjected to various illumination protocols. Methods and Results: Isolated neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with lentiviral vectors to express one of the following ChR variants: H134R, CatCh, ReaChR, or GtACR1. NRVMs were treated with phenylephrine (PE) to induce pathological hypertrophy (PE group) or left untreated [control (CTL) group]. In these groups, ChR currents displayed unique and significantly different properties for each ChR variant on activation by a single 1-s light pulse (1 mW/mm2: 470, 565, or 617 nm). The concomitant membrane potential (V m) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average V m during illumination (V plateau: -38 mV) as compared with a V plateau > -20 mV for the other ChR variants. On repetitive activation at increasing frequencies (10-ms pulses at 1-10 Hz for 30 s), peak currents, which are important for cardiac pacing, decreased with increasing activation frequencies by 17-78% (p < 0.05), while plateau currents, which are critical for arrhythmia termination, decreased by 10-75% (p < 0.05), both in a variant-specific manner. In contrast, the corresponding V plateau remained largely stable. Importantly, current properties and V m responses were not statistically different between the PE and CTL groups, irrespective of the variant used (p > 0.05). Conclusion: Our data show that ChR variants function equally well in cell culture models of healthy and pathologically hypertrophic myocardium but show strong, variant-specific use-dependence. This use-dependent nature of ChR function should be taken into account during the design of cardiac optogenetic studies and the interpretation of the experimental findings thereof.

Long-Term Endurance Exercise Training Alters Repolarization in a New Rabbit Athlete's Heart Model.

In the present study, the effect of long-term exercise training was investigated on myocardial morphological and functional remodeling and on proarrhythmic sensitivity in a rabbit athlete's heart model. New-Zealand white rabbits were trained during a 12-week long treadmill running protocol and compared with their sedentary controls. At the end of the training protocol, echocardiography, in vivo and in vitro ECG recordings, proarrhythmic sensitivity with dofetilide (nM) were performed in isolated hearts, and action potential duration (APD) measurements at different potassium concentrations (4.5 and 2 mM) were made in the isolated papillary muscles. Expression levels of the slow component of delayed rectifier potassium current and fibrosis synthesis and degradation biomarkers were quantified. Echocardiography showed a significantly dilated left ventricle in the running rabbits. ECG PQ and RR intervals were significantly longer in the exercised group (79 ± 2 vs. 69 ± 2 ms and 325 ± 11 vs. 265 ± 6 ms, p < 0.05, respectively). The in vivo heart rate variability (HRV) (SD of root mean square: 5.2 ± 1.4 ms vs. 1.4 ± 0.2 ms, p < 0.05) and Tpeak-Tend variability were higher in the running rabbits. Bradycardia disappeared in the exercised group in vitro. Dofetilide tended to increase the QTc interval in a greater extent, and significantly increased the number of arrhythmic beats in the trained animals in vitro. APD was longer in the exercised group at a low potassium level. Real-time quantitative PCR (RT-qPCR) showed significantly greater messenger RNA expression of fibrotic biomarkers in the exercised group. Increased repolarization variability and higher arrhythmia incidences, lengthened APD at a low potassium level, increased fibrotic biomarker gene expressions may indicate higher sensitivity of the rabbit "athlete's heart" to life-threatening arrhythmias.

Impaired cytoplasmic domain interactions cause co-assembly defect and loss of function in the p.Glu293Lys KNCJ2 variant isolated from an Andersen-Tawil syndrome patient.

AIMS: Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1. METHODS AND RESULTS: The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels. CONCLUSIONS: Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.

Increased Ca2+ content of the sarcoplasmic reticulum provides arrhythmogenic trigger source in swimming-induced rat athlete's heart model.

Sudden cardiac death among top athletes is very rare, however, it is 2-4 times more frequent than in the age-matched control population. In the present study, the electrophysiological consequences of long-term exercise training were investigated on Ca2+ homeostasis and ventricular repolarization, together with the underlying alterations of ion channel expression, in a rat athlete's heart model. 12-week swimming exercise-trained and control Wistar rats were used. Electrophysiological data were obtained by using ECG, patch clamp and fluorescent optical measurements. Protein and mRNA levels were determined by the Western immunoblot and qRT-PCR techniques. Animals in the trained group exhibited significantly lower resting heart rate, higher incidence of extrasystoles and spontaneous Ca2+ release events. The Ca2+ content of the sarcoplasmic reticulum (SR) and the Ca2+ transient amplitude were significantly larger in the trained group. Intensive physical training is associated with elevated SR Ca2+ content, which could be an important part of physiological cardiac adaptation mechanism to training. However, it may also sensitize the heart for the development of spontaneous Ca2+ release and extrasystoles. Training-associated remodeling may promote elevated incidence of life threatening arrhythmias in top athletes.

Self-restoration of cardiac excitation rhythm by anti-arrhythmic ion channel gating.

Homeostatic regulation protects organisms against hazardous physiological changes. However, such regulation is limited in certain organs and associated biological processes. For example, the heart fails to self-restore its normal electrical activity once disturbed, as with sustained arrhythmias. Here we present proof-of-concept of a biological self-restoring system that allows automatic detection and correction of such abnormal excitation rhythms. For the heart, its realization involves the integration of ion channels with newly designed gating properties into cardiomyocytes. This allows cardiac tissue to i) discriminate between normal rhythm and arrhythmia based on frequency-dependent gating and ii) generate an ionic current for termination of the detected arrhythmia. We show in silico, that for both human atrial and ventricular arrhythmias, activation of these channels leads to rapid and repeated restoration of normal excitation rhythm. Experimental validation is provided by injecting the designed channel current for arrhythmia termination in human atrial myocytes using dynamic clamp.

Age-related regulation and region-specific distribution of ion channel subunits promoting atrial fibrillation in human left and right atria.

AIMS: Age-induced changes and electrical remodelling are important components of the atrial fibrillation (AF) substrate. To study regional distribution and age-dependent changes in gene expression that may promote AF in human atria. METHODS AND RESULTS: Human left atrial (LA) and right atrial (RA) tissue samples were obtained from donor hearts unsuitable for transplantation and from patients undergoing mitral valve repair. Atrial fibrillation was mimicked in vitro by tachypacing of human atrial tissue slices. Ionic currents were studied by the whole-cell patch-clamp technique; gene expression was analysed by real-time qPCR and immunoblotting. Both healthy RA and RA from older patients showed greater CACNA1c mRNA and CaV1.2 protein expression than LA. No age-dependent changes of Kir2.1 expression in both atria were seen. Remodelling occurred in a qualitatively similar manner in RA and LA. IK1 and Kir2.1 protein expression increased with AF. MiR-1, miR-26a, and miR-26b were down-regulated with AF in both atria. ICa,L was decreased. CACNA1c and CACNA2b expression decreased and miR-328 increased in RA and LA during AF. Ex vivo tachypacing of human atrial slices replicated these findings. There were age-dependent increases in miR-1 and miR-328, while miR-26a decreased with age in atrial tissues from healthy human donor hearts. CONCLUSION: Features of electrical remodelling in man occur in a qualitatively similar manner in both human atria. Age-related miR-328 dysregulation and reduced ICa,L may contribute to increased AF susceptibility with age.

Beat-to-beat variability of cardiac action potential duration: underlying mechanism and clinical implications.

Beat-to-beat variability of cardiac action potential duration (short-term variability, SV) is a common feature of various cardiac preparations, including the human heart. Although it is believed to be one of the best arrhythmia predictors, the underlying mechanisms are not fully understood at present. The magnitude of SV is basically determined by the intensity of cell-to-cell coupling in multicellular preparations and by the duration of the action potential (APD). To compensate for the APD-dependent nature of SV, the concept of relative SV (RSV) has been introduced by normalizing the changes of SV to the concomitant changes in APD. RSV is reduced by ICa, IKr, and IKs while increased by INa, suggesting that ion currents involved in the negative feedback regulation of APD tend to keep RSV at a low level. RSV is also influenced by intracellular calcium concentration and tissue redox potential. The clinical implications of APD variability is discussed in detail.

A novel 'splice site' HCN4 Gene mutation, c.1737+1 G>T, causes familial bradycardia, reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability.

BACKGROUND: The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome. METHODS AND RESULTS: A novel c.1737+1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated family members [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rate<60/min on resting ECG). Minimum [36 (SD 7) vs. 47 (SD 5) bpm, p=0.0087) and average heart rates [62 (SD 8) vs. 73 (SD 8) bpm, p=0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings. CONCLUSIONS: The identified novel 'splice site' HCN4 gene mutation, c.1737+1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers.

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments.

BACKGROUND: In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined. METHODS: Ion currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 µg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts. RESULTS: ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 µM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 µM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts. CONCLUSIONS: The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain.

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene.

BACKGROUND AND PURPOSE: The reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel-in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve. EXPERIMENTAL APPROACH: We have developed a model for the long QT syndrome type-5 in rabbits (LQT5 ) with cardiac-specific overexpression of a mutant (G52R) KCNE1 ß-subunit of the channel that carries the slow delayed-rectifier K(+) -current (IKs ). ECG parameters, including short-term variability of the QT interval (STVQT ), a biomarker for proarrhythmic risk, and arrhythmia development were recorded. In vivo, arrhythmia susceptibility was evaluated by i.v. administration of the IKr blocker dofetilide. K(+) currents were measured with the patch-clamp technique. KEY RESULTS: Patch-clamp studies in ventricular myocytes isolated from LQT5 rabbits revealed accelerated IKs and IKr deactivation kinetics. At baseline, LQT5 animals exhibited slightly but significantly prolonged heart-rate corrected QT index (QTi) and increased STVQT . Dofetilide provoked Torsade-de-Pointes arrhythmia in a greater proportion of LQT5 rabbits, paralleled by a further increase in STVQT . CONCLUSION AND IMPLICATIONS: We have created a novel transgenic LQT5 rabbit model with increased susceptibility to drug-induced arrhythmias that may represent a useful model for testing proarrhythmic potential and for investigations of the mechanisms underlying arrhythmias and sudden cardiac death due to repolarization disturbances.

Role of the dysfunctional ryanodine receptor - Na(+)-Ca(2+)exchanger axis in progression of cardiovascular diseases: What we can learn from pharmacological studies?

Abnormal Ca(2+)homeostasis is often associated with chronic cardiovascular diseases, such as hypertension, heart failure or cardiac arrhythmias, and typically contributes to the basic ethiology of the disease. Pharmacological targeting of cardiac Ca(2+)handling has great therapeutic potential offering invaluable options for the prevention, slowing down the progression or suppression of the harmful outcomes like life threatening cardiac arrhythmias. In this review we outline the existing knowledge on the involvement of malfunction of the ryanodine receptor and the Na(+)-Ca(2+)exchanger in disturbances of Ca(2+)homeostasis and discuss important proof of concept pharmacological studies targeting these mechanisms in context of hypertension, heart failure, atrial fibrillation and ventricular arrhythmias. We emphasize the promising results of preclinical studies underpinning the potential benefits of the therapeutic strategies based on ryanodine receptor or Na(+)-Ca(2+)exchanger inhibition.

A Common CCK-B Receptor Intronic Variant in Pancreatic Adenocarcinoma in a Hungarian Cohort.

OBJECTIVES: Variant c.811+32C>A in intron 4 of the cholecystokinin-B receptor gene (CCKBR) was reported to correlate with higher pancreatic cancer risk and poorer survival. The variant was suggested to induce retention of intron 4, resulting in a new splice form with enhanced receptor activity. Our objective was to validate the c.811+32C>A variant as an emerging biomarker for pancreatic cancer risk and prognosis. METHODS: We genotyped variant c.811+32C>A in 122 pancreatic adenocarcinoma case patients and 106 control subjects by sequencing and examined its association with cancer risk and patient survival. We tested the functional effect of variant c.811+32C>A on pre-messenger RNA splicing in human embryonic kidney 293T and Capan-1 cells transfected with CCKBR minigenes. RESULTS: The allele frequency of the variant was similar between patients and control subjects (18.4% and 17.9%, respectively). Survival analysis showed no significant difference between median survival of patients with the C/C genotype (266 days) and patients with the A/C or A/A genotypes (257 days). CCKBR minigenes with or without variant c.811+32C>A exhibited no difference in expression of the intron-retaining splice variant. CONCLUSION: These data indicate that variant c.811+32C>A in CCKBR does not have a significant impact on pancreatic cancer risk or survival in a Hungarian cohort.

Identification and functional characterisation of a novel KCNJ2 mutation, Val302del, causing Andersen-Tawil syndrome.

Loss-of-function mutations of the KCNJ2 gene encoding for the inward rectifier potassium channel subunit Kir2.1 cause Andersen-Tawil Syndrome (ATS), a rare genetic disorder characterised by periodic paralysis, ventricular arrhythmias, and dysmorphic features. Clinical manifestations of the disease appear to vary greatly with the nature of mutation, therefore, functional characterisation of ATS-causing mutations is of clinical importance. In this study, we describe the identification and functional analysis of a novel KCNJ2 mutation, Val302del, identified in a patient with ATS. Heterologously expressed wild type (WT) and Val302del mutant alleles showed similar subcellular distribution of the Kir2.1 protein with high intensity labelling from the membrane region, demonstrating normal membrane trafficking of the Val302del Kir2.1 variant. Cells transfected with the WT allele displayed a robust current with strong inward rectification, while no current above background was detected in cells expressing the Val302del Kir2.1 subunit. Co-transfection of CHO cells with the WT and the Val302del Kir2.1 revealed a dose-dependent inhibitory effect of the Val302del Kir2.1 mutant subunit on WT Kir2.1 currents. These observations indicate that the WT and the Val302del mutant subunits co-assemble in the cell membrane and that the mutation affects potassium conductivity and (or) gating of the WT/Val302del heteromeric Kir2.1 channels.

Fibroblast inward-rectifier potassium current upregulation in profibrillatory atrial remodeling.

RATIONALE: Fibroblasts are involved in cardiac arrhythmogenesis and contribute to the atrial fibrillation substrate in congestive heart failure (CHF) by generating tissue fibrosis. Fibroblasts display robust ion currents, but their functional importance is poorly understood. OBJECTIVE: To characterize atrial fibroblast inward-rectifier K(+) current (IK1) remodeling in CHF and its effects on fibroblast properties. METHODS AND RESULTS: Freshly isolated left atrial fibroblasts were obtained from controls and dogs with CHF (ventricular tachypacing). Patch clamp was used to record resting membrane potential (RMP) and IK1. RMP was significantly increased by CHF (from -43.2±0.8 mV, control, to -55.5±0.9 mV). CHF upregulated IK1 (eg, at -90 mV from -1.1±0.2 to -2.7±0.5 pA/pF) and increased the expression of KCNJ2 mRNA (by 52%) and protein (by 80%). Ba(2+) (300 µmol/L) decreased the RMP and suppressed the RMP difference between controls and dogs with CHF. Store-operated Ca(2+) entry (Fura-2-acetoxymethyl ester) and fibroblast proliferation (flow cytometry) were enhanced by CHF. Lentivirus-mediated overexpression of KCNJ2 enhanced IK1 and hyperpolarized fibroblasts. Functional KCNJ2 suppression by lentivirus-mediated expression of a dominant negative KCNJ2 construct suppressed IK1 and depolarized RMP. Overexpression of KCNJ2 increased Ca(2+) entry and fibroblast proliferation, whereas the dominant negative KCNJ2 construct had opposite effects. Fibroblast hyperpolarization to mimic CHF effects on RMP enhanced the Ca(2+) entry. MicroRNA-26a, which targets KCNJ2, was downregulated in CHF fibroblasts. Knockdown of endogenous microRNA-26 to mimic CHF effects unregulated IK1. CONCLUSIONS: CHF upregulates fibroblast KCNJ2 expression and currents, thereby hyperpolarizing RMP, increasing Ca(2+) entry, and enhancing atrial fibroblast proliferation. These effects are likely mediated by microRNA-26a downregulation. Remodeling-induced fibroblast KCNJ2 expression changes may play a role in atrial fibrillation promoting fibroblast remodeling and structural/arrhythmic consequences.