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Aims Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR–Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. Methods and results The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1–S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. Conclusion Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs. [ABSTRACT FROM AUTHOR]

Aims Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). Methods and results Optical mapping of action potentials (APs) and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K+/Mg++ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ −40 mV and had upstroke dF/dtmax and Vm-Ca2+ dynamics compatible with I CaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of I CaL. In experiments and the model, sub-maximal L-type Ca2+ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. Conclusion These data demonstrate that I CaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS. [ABSTRACT FROM AUTHOR]

Aims Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. Methods and results We employed a phenotype-driven N -ethyl- N -nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2 p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2 p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs—leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2 p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. Conclusions Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common arrhythmia worldwide. It is associated with significant increases in morbidity in the form of stroke and heart failure, and a doubling in all-cause mortality. The pathophysiology of AF is incompletely understood, and this has contributed to a lack of effective treatments and disease-modifying therapies. An important cellular process that may explain how risk factors give rise to AF includes post-translational modification of proteins. As the most commonly occurring post-translational modification, protein phosphorylation is especially relevant. Although many methods exist for studying protein phosphorylation, a common and highly resolute technique is mass spectrometry (MS). This review will discuss recent evidence surrounding the role of protein phosphorylation in the pathogenesis of AF. MS-based technology to study phosphorylation and uses of MS in other areas of medicine such as oncology will also be presented. Based on these data, future goals and experiments will be outlined that utilize MS technology to better understand the role of phosphorylation in AF and elucidate its role in AF pathophysiology. This may ultimately allow for the development of more effective AF therapies. [ABSTRACT FROM AUTHOR]

Aims We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. Methods and results Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. Conclusions We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects. [ABSTRACT FROM AUTHOR]

Despite significant advances in its detection, understanding and management, atrial fibrillation (AF) remains a highly prevalent cardiac arrhythmia with a major impact on morbidity and mortality of millions of patients. AF results from complex, dynamic interactions between risk factors and comorbidities that induce diverse atrial remodelling processes. Atrial remodelling increases AF vulnerability and persistence, while promoting disease progression. The variability in presentation and wide range of mechanisms involved in initiation, maintenance and progression of AF, as well as its associated adverse outcomes, make the early identification of causal factors modifiable with therapeutic interventions challenging, likely contributing to suboptimal efficacy of current AF management. Computational modelling facilitates the multilevel integration of multiple datasets and offers new opportunities for mechanistic understanding, risk prediction and personalized therapy. Mathematical simulations of cardiac electrophysiology have been around for 60 years and are being increasingly used to improve our understanding of AF mechanisms and guide AF therapy. This narrative review focuses on the emerging and future applications of computational modelling in AF management. We summarize clinical challenges that may benefit from computational modelling, provide an overview of the different in silico approaches that are available together with their notable achievements, and discuss the major limitations that hinder the routine clinical application of these approaches. Finally, future perspectives are addressed. With the rapid progress in electronic technologies including computing, clinical applications of computational modelling are advancing rapidly. We expect that their application will progressively increase in prominence, especially if their added value can be demonstrated in clinical trials. [ABSTRACT FROM AUTHOR]

Aims Inflammation plays a role in atrial fibrillation (AF), but classical anti-inflammatory molecules are ineffective. Recent evidence suggests that failure of inflammation-resolution causes persistent inflammatory signalling and that a novel drug-family called resolvins promotes inflammation-resolution. Right heart disease (RHD) is associated with AF; experimental RHD shows signs of atrial inflammatory-pathway activation. Here, we evaluated resolvin-therapy effects on atrial arrhythmogenic remodelling in experimental RHD. Methods and results Pulmonary hypertension and RHD were induced in rats with an intraperitoneal injection of 60 mg/kg monocrotaline (MCT). An intervention group received daily resolvin-D1 (RvD1), starting 1 day before MCT administration. Right atrial (RA) conduction and gene-expression were analysed respectively by optical mapping and qPCR/gene-microarray. RvD1 had no or minimal effects on MCT-induced pulmonary artery or right ventricular remodelling. Nevertheless, in vivo transoesophageal pacing induced atrial tachyarrhythmias in no CTRL rats vs. 100% MCT-only rats, and only 33% RvD1-treated MCT rats (P  < 0.001 vs. MCT-only). Conduction velocity was significantly decreased by MCT, an effect prevented by RvD1. RHD caused RA dilation and fibrosis. RvD1 strongly attenuated RA fibrosis but had no effect on RA dilation. MCT increased RA expression of inflammation- and fibrosis-related gene-expression pathways on gene-microarray transcriptomic analysis, effects significantly attenuated by RvD1 (334 pathways enriched in MCT-rats vs. control; only 177 dysregulated by MCT with RvD1 treatment). MCT significantly increased RA content of type 1 (proinflammatory) CD68-positive M1 macrophages without affecting type 2 (anti-inflammatory) M2 macrophages. RvD1-treated MCT-rat RA showed significant reductions in proinflammatory M1 macrophages and increases in anti-inflammatory M2 macrophages vs. MCT-only. MCT caused statistically significant increases in protein-expression (western blot) of COL3A1, ASC, CASP1, CASP8, IL1β, TGFβ3, CXCL1, and CXCL2, and decreases in MMP2, vs. control. RvD1-treatment suppressed all these MCT-induced protein-expression changes. Conclusion The inflammation-resolution enhancing molecule RvD1 prevents AF-promoting RA remodelling, while suppressing inflammatory changes and fibrotic/electrical remodelling, in RHD. Resolvins show potential promise in combating atrial arrhythmogenic remodelling by suppressing ongoing inflammatory signalling. [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient's derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). Methods and results Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the I f and I CaL currents. No differences were observed in the repolarizing current I Kr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. Conclusions Our results demonstrate that the common genetic background of the patients induces functional alterations of I f and I CaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first report that, using patient-derived CMs differentiated from iPSC, suggests a plausible cellular mechanism underlying this complex familial form of AF. [ABSTRACT FROM AUTHOR]

Aims Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. Methods and results We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. Conclusion Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo. [ABSTRACT FROM AUTHOR]

Aims Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. Methods and results We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. Conclusion This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III CIBERCV Introduction Cardiac arrhythmias can originate as a consequence of the proarrhythmic effects of some therapeutical regenerative products. In vitro studies have demonstrated different levels of electrophysiological complexity but these models usually lack of direct translation into the clinical practice. Objective To develop an arrhythmia complexity evaluation method that enables to characterize electrophysiological complexity in translational scenarios. Methodology: To build a new evaluation method, frequency and rotor biomarkers from in vitro cell cultures presenting fibrillatory activity (N=15) were evaluated using optical mapping recordings (Rhod-2AM Ca2+ transient dye, 30-minute incubation, excited with green light). The patterns observed at in vitro level were analyzed and characterized by means of frequency and rotor biomarkers. Next, we performed a forward translational study to evaluate these electrophysiological biomarkers at a clinical level. In 206 patients with atrial fibrillation (AF) with non-invasive body surface recordings using the Acorys system from Corify (67 electrodes, 4 second segments) (STRATIFY-AF study) were evaluated to analyze the same frequency and rotor biomarkers previously explored in vitro. Moreover, we build a complexity score that described from 0 to 1 the complexity at electrophysiological level, where 0 corresponded to simple scenarios and 1 to complex arrhythmic patterns. Patients were followed for 12 months to evaluate long term evolution. Results Complexity evaluation method identified the highest dominant frequency, median frequency and rotor time as the most important predictors for arrhythmia complexity definition. In vitro cultures results showed that samples presenting higher dominant frequency were also associated with small dominant frequency area extension and lower rotor time maintenance, i.e. with a high value of the complexity biomarker (Figure 1B). At clinical level, patients with high values of the complexity score were identified with Persistent AF patients showing complex patterns in whom AF recurred after ablation at 12 months (Figure 1D). Conclusions We developed a translational approach that enables to characterize arrhythmia complexity in different frameworks, which is crucial to evaluate potential undesirable effects of therapeutical regenerative products during the complete development and validation of the product. Open in new tab Download slide In vitro and patient complexity examples [ABSTRACT FROM AUTHOR]

Background A recent genome-wide association study identified a susceptibility locus for atrial fibrillation at the KCNN3 gene. Since the KCNN3 gene encodes for a small conductance calcium-activated potassium channel, we hypothesized that overexpression of the SK3 channel increases susceptibility to cardiac arrhythmias. Methods and results We characterized the cardiac electrophysiological phenotype of a mouse line with overexpression of the SK3 channel. We generated homozygote (SK3T/T) and heterozygote (SK3+/T) mice with overexpression of the channel and compared them with wild-type (WT) controls. We observed a high incidence of sudden death among SK3T/T mice (7 of 19 SK3T/T mice). Ambulatory monitoring demonstrated that sudden death was due to heart block and bradyarrhythmias. SK3T/T mice displayed normal body weight, temperature, and cardiac function on echocardiography; however, histological analysis demonstrated that these mice have abnormal atrioventricular node morphology. Optical mapping demonstrated that SK3T/T mice have slower ventricular conduction compared with WT controls (SK3T/T vs. WT; 0.45 ± 0.04 vs. 0.60 ± 0.09 mm/ms, P = 0.001). Programmed stimulation in 1-month-old SK3T/T mice demonstrated inducible atrial arrhythmias (50% of SK3T/T vs. 0% of WT mice) and also a shorter atrioventricular nodal refractory period (SK3T/T vs. WT; 43 ± 6 vs. 52 ± 9 ms, P = 0.02). Three-month-old SK3T/T mice on the other hand displayed a trend towards a more prolonged atrioventricular nodal refractory period (SK3T/T vs. WT; 61 ± 1 vs. 52 ± 6 ms, P = 0.06). Conclusion Overexpression of the SK3 channel causes an increased risk of sudden death associated with bradyarrhythmias and heart block, possibly due to atrioventricular nodal dysfunction. [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K+ Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. Methods and results Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. Conclusion Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility. [ABSTRACT FROM AUTHOR]

Aims Cardiac hypertrophy is accompanied by reprogramming of gene expression, where the altered expression of ion channels decreases electrical stability and increases the risk of life-threatening arrhythmias. However, the underlying mechanisms are not fully understood. Here, we analysed the role of the depolarizing current If which has been hypothesized to contribute to arrhythmogenesis in the hypertrophied ventricle. Methods and results We used transverse aortic constriction in mice to induce ventricular hypertrophy. This resulted in an increased number of If positive ventricular myocytes as well as a strongly enhanced and accelerated If when compared with controls. Of the four HCN (hyperpolarization-activated cyclic nucleotide-gated channels) isoforms mediating If, HCN2 and HCN4 were the predominantly expressed subunits in healthy as well as hypertrophied hearts. Unexpectedly, only the HCN1 transcript was significantly upregulated in response to hypertrophy. However, the combined deletion of HCN2 and HCN4 disrupted ventricular If completely. The lack of If in hypertrophic double-knockouts resulted in a strong attenuation of pro-arrhythmogenic parameters characteristically observed in hypertrophic hearts. In particular, prolongation of the action potential was significantly decreased and lengthening of the QT interval was reduced. Conclusions We suggest that the strongly increased HCN channel activity in hypertrophied myocytes prolongs the repolarization of the ventricular action potential and thereby may increase the arrhythmogenic potential. Our results provide for the first time a direct link between an upregulation of ventricular If and a diminished repolarization reserve in cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Aims Cardiac hypertrophy and heart failure are associated with QT prolongation and lethal ventricular arrhythmias resulting from decreased K+ current densities and impaired repolarization. Recent studies in mouse models of physiological cardiac hypertrophy revealed that increased phosphoinositide-3-kinase-α (PI3Kα) signalling results in the up-regulation of K+ channels and the normalization of ventricular repolarization. The experiments here were undertaken to test the hypothesis that increased PI3Kα signalling will counteract the adverse electrophysiological remodelling associated with pathological hypertrophy and heart failure. Methods and results In contrast to wild-type mice, left ventricular (LV) hypertrophy, induced by transverse aortic constriction (TAC), did not result in prolongation of ventricular action potentials or QT intervals in mice with cardiac-specific expression of constitutively active PI3Kα (caPI3Kα). Indeed, repolarizing K+ currents and K+ channel subunit transcripts were increased in caPI3Kα + TAC LV myocytes in proportion to the TAC-induced cellular hypertrophy. Congestive heart failure in a transgenic model of dilated cardiomyopathy model is accompanied by prolonged QT intervals and ventricular action potentials, reduced K+ currents and K+ channel transcripts. Increased PI3Kα signalling, but not renin–angiotensin system blockade, in this model also results in increased K+ currents and improved ventricular repolarization. Conclusion In the setting of pathological hypertrophy or heart failure, enhanced PI3Kα signalling results in the up-regulation of K+ channel subunits, normalization of K+ current densities and preserved ventricular function. Augmentation of PI3Kα signalling, therefore, may be a useful and unique strategy to protect against the increased risk of ventricular arrhythmias and sudden death associated with cardiomyopathy. [ABSTRACT FROM PUBLISHER]

Aims Fibrosis is known to promote cardiac arrhythmias by disrupting myocardial structure. Given recent evidence that myofibroblasts form gap junctions with myocytes at least in co-cultures, we investigated whether myofibroblast–myocyte coupling can promote arrhythmia triggers, such as early afterdepolarizations (EADs), by directly influencing myocyte electrophysiology. Methods and results Using the dynamic voltage clamp technique, patch-clamped adult rabbit ventricular myocytes were electrotonically coupled to one or multiple virtual fibroblasts or myofibroblasts programmed with eight combinations of capacitance, membrane resistance, resting membrane potential, and gap junction coupling resistance, spanning physiologically realistic ranges. Myocytes were exposed to oxidative (1 mmol/L H2O2) or ionic (2.7 mmol/L hypokalaemia) stress to induce bradycardia-dependent EADs. In the absence of myofibroblast–myocyte coupling, EADs developed during slow pacing (6 s), but were completely suppressed by faster pacing (1 s). However, in the presence of myofibroblast–myocyte coupling, EADs could no longer be suppressed by rapid pacing, especially when myofibroblast resting membrane potential was depolarized (−25 mV). Analysis of the myofibroblast–myocyte virtual gap junction currents revealed two components: an early transient-outward Ito-like current and a late sustained current. Selective elimination of the Ito-like component prevented EADs, whereas selective elimination of the late component did not. Conclusion Coupling of myocytes to myofibroblasts promotes EAD formation as a result of a mismatch in early vs. late repolarization reserve caused by the Ito-like component of the gap junction current. These cellular and ionic mechanisms may contribute to the pro-arrhythmic risk in fibrotic hearts. [ABSTRACT FROM PUBLISHER]

Aims Increasing evidence indicates that congenital long QT syndromes (LQTSs) promote atrial fibrillation. The atrial action potential (AP) has a short plateau, and whether LQTS atrial cardiomyocytes generate triggered activity via early afterdepolarizations (EADs) is unclear. Atrial cellular arrhythmia mechanisms have not been defined in congenital LQTS. Therefore, we studied atrial cardiomyocyte electrophysiology in mice with an LQTS3 SCN5A inactivation-impairing mutation (ΔKPQ heterozygotes). Methods and results Peak and late Na+ current (INaP and INaL) were measured with whole-cell patch clamp in left atrial (LA) cardiomyocytes. APs were recorded in multicellular LA preparations with floating microelectrodes. INaL was increased by 110% in LA cardiomyocytes of ΔKPQ mice, whereas INaP was unchanged. AP duration (APD) was prolonged over all frequencies in ΔKPQ mice, but particularly at lower frequencies [e.g. APD90 at 0.5 Hz: 197 ± 8 ms vs. wild-type (WT) 82 ± 2 ms, P< 0.001]. EADs occurred at 0.5 Hz in 10/18 ΔKPQ (56%) vs. 1/10 WT (10%) atria (P< 0.05). EADs immediately preceded premature APs in other LA regions, suggesting triggered activity. Ranolazine preferentially inhibited INaL (50% inhibitory concentration: 12.5 vs. 151.8 µM for INaP) in ΔKPQ myocytes. At 10 µM, ranolazine shortened APD (e.g. APD90 at 0.5 Hz to 122 ± 4 ms, P= 0.01) without changing APD in WT and suppressed EAD occurrence and triggered activity (from 10/18 to 1/9 preparations, 11%, P< 0.05). Conclusion This study implicates increased INaL in excessive atrial APD prolongation and arrhythmic EAD occurrence in a congenital LQTS3 mouse model. Our observations provide the first direct demonstration of atrial EADs and triggered activity in a genetically defined animal model of human LQTS and have potential clinically-relevant mechanistic and therapeutic implications. [ABSTRACT FROM PUBLISHER]

Aims Heart failure is a common antecedent to atrial fibrillation; both heart failure and atrial fibrillation are associated with increased myocardial oxidative stress. Chronic canine heart failure reduces atrial action potential duration and atrial refractoriness. We hypothesized that inducible nitric oxide synthase 2 (NOS2) contributes to atrial oxidative stress and electrophysiologic alterations. Methods and results A 16-week canine tachypacing model of heart failure was used (n= 21). At 10 weeks, dogs were randomized to either placebo (n = 12) or active treatment (n = 9) with NOS cofactor, tetrahydrobiopterin (BH4, 50 mg), and NOS substrate (l-arginine, 3 g) twice daily for 6 weeks. A group of matched controls (n = 7) was used for comparison. Heart failure increased atrial NOS2 and reduced atrial BH4, while l-arginine was unchanged. Treatment reduced inducible atrial fibrillation and normalized the heart failure-induced shortening of the left atrial myocyte action potential duration. Treatment increased atrial [BH4] while [l-arginine] was unchanged. Treatment did not improve left ventricular function or dimensions. Heart failure-induced reductions in atrial [BH4] resulted in NOS uncoupling, as measured by NO and superoxide anion (O2·−) production, while BH4 and l-arginine treatment normalized NO and O2·−. Heart failure resulted in left atrial oxidative stress, which was attenuated by BH4 and l-arginine treatment. Conclusion Chronic non-ischaemic heart failure results in atrial oxidative stress and electrophysiologic abnormalities by depletion of BH4 and uncoupling of NOS2. Modulation of NOS2 activity by repletion of BH4 may be a safe and effective approach to reduce the frequency of atrial arrhythmias during heart failure. [ABSTRACT FROM PUBLISHER]

Aims: Treatment with the anticancer drug taxol (TXL), which polymerizes the cytoskeleton protein tubulin, may evoke cardiac arrhythmias based on reduced human cardiac sodium channel (Nav1.5) function. Therefore, we investigated whether enhanced tubulin polymerization by TXL affects Nav1.5 function and expression and whether these effects are β1-subunit-mediated. [ABSTRACT FROM PUBLISHER]

Aims: Left atrial (LA) stretch-associated electrophysiological changes in patients with mitral stenosis (MS) predispose to atrial fibrillation. We hypothesized that the normalization of the pressure gradient by percutaneous transvenous mitral balloon valvotomy (PTMV) affects LA but not right atrial (RA) conduction, depending on the site of stimulation. Because direction-dependent (asymmetric) changes of conduction may contribute to arrhythmogenesis, we assessed conduction symmetry in MS patients and tested whether it is restored by PTMV. [ABSTRACT FROM PUBLISHER]

Aims: The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. [ABSTRACT FROM PUBLISHER]

Gap junctions form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart: connexin43 (Cx43), Cx40 and Cx45 are found in distinctive combinations and relative quantities in different, functionally-specialized subsets of cardiac myocyte. Mutations in genes that encode connexins have only rarely been identified as being a cause of human cardiac disease, but remodelling of connexin expression and gap junction organization are well documented in acquired adult heart disease, notably ischaemic heart disease and heart failure. Remodelling may take the form of alterations in (i) the distribution of gap junctions and (ii) the amount and type of connexins expressed. Heterogeneous reduction in Cx43 expression and disordering in gap junction distribution feature in human ventricular disease and correlate with electrophysiologically identified arrhythmic changes and contractile dysfunction in animal models. Disease-related alterations in Cx45 and Cx40 expression have also been reported, and some of the functional implications of these are beginning to emerge. Apart from ventricular disease, various features of gap junction organization and connexin expression have been implicated in the initiation and persistence of the most common form of atrial arrhythmia, atrial fibrillation, though the disparate findings in this area remain to be clarified. Other major tasks ahead focus on the Purkinje/working ventricular myocyte interface and its role in normal and abnormal impulse propagation, connexin-interacting proteins and their regulatory functions, and on defining the precise functional properties conferred by the distinctive connexin co-expression patterns of different myocyte types in health and disease. [ABSTRACT FROM PUBLISHER]

Abstract: Objectives: The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown. Methods: The effect of K201 on excitation–contraction (E–C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca2+ measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes. Results: Acute application of <1 μmol/L K201 had no significant effect on E–C coupling. K201 at 1 μmol/L decreased Ca2+ transient amplitude (to 83±7%) without affecting I Ca,L or the SR Ca2+ content. At 3 μmol/L K201 caused a larger reduction of Ca2+ transient amplitude (to 60±7%) with accompanying reductions in I Ca,L amplitude (to 66±8%) and SR Ca2+ content (74±9%). Spontaneous SR Ca2+ release during diastole was induced by increasing intracellular [Ca2+]. At 1 μmol/L K201 reduced the frequency of spontaneous Ca2+ release. The effect of K201 on SR-mediated Ca2+ waves and Ca2+ sparks was examined in β-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 μmol/L) reduced the frequency and velocity of SR Ca2+ waves despite no change in SR Ca2+ content. At 3 μmol/L K201 completely abolished Ca2+ waves and reduced the SR Ca2+ content (to ∼73%). K201 at 1 μmol/L reduced Ca2+ spark amplitude and frequency. Assays specific to SR Ca2+-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca2+ uptake and release. Conclusions: K201 modifies E–C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca2+ release during Ca2+ overload without significantly affecting Ca2+ transient amplitude. [Copyright &y& Elsevier]

Abstract: Objective: The aims of this study were to: i) investigate the role of angiotensin in mediating changes to myocardial electromechanical properties during the development and regression of left ventricular hypertrophy (LVH) generated by constriction of the thoracic aorta; ii) identify any role of angiotensin-1 receptor blockade on ameliorating changes to these electromechanical properties. Methods: LVH was induced in guinea-pigs by constricting the ascending aorta (AC groups). After 42±3 days, the constriction was either removed or left in place. Following the second operation animals were fed losartan (10 mg·kg−1·day−1) or saline for 42±3 days. Sham-operated animals served as controls. In other groups, LVH was generated by subcutaneous angiotensin II (200 ng·kg−1·min−1) infusion for 42±3 days with or without losartan administration (AT groups), and compared to animals undergoing aortic constriction for a similar period. Electromechanical changes were recorded in isolated left ventricular myocardial preparations. Results: Wet and dry heart-to-body weight ratios (HBR) increased significantly in the AC and AT models compared to control. Losartan prevented the increase of HBR in the AT group. Removal of the constriction allowed LVH to regress to control. The force–frequency relationship was reduced in both models and recovered fully on regression. However, the two models generated different electrophysiological changes: in the AC group, longitudinal conduction velocity was reduced and transverse conduction increased, with a consequent reduction of the anisotropic conduction ratio. On regression recovery was only partial; action potential duration was prolonged and did not recover. In the AT group, electrophysiological changes were limited: only an increase of transverse conduction and a reduction of the anisotropic conduction ratio were observed. Losartan had no effect on HBR or electromechanical variables in the aortic constricted animals, nor did it affect the extent of recovery in animals with regression of LVH. Conclusions: The electromechanical changes to hypertrophied myocardium are different in these two models of LVH. Moreover, losartan was ineffective in modulating the consequences of hypertrophy induced by constriction of the thoracic aorta. [Copyright &y& Elsevier]

Abstract: Increased consumption of fish rich in omega-3 polyunsaturated fatty acids (ω3-PUFAs) is associated with decreased incidence of sudden cardiac death in post-myocardial infarction patients, but is also related to increased incidence of sudden death and arrhythmias in patients with acute myocardial ischemia. This review discusses the possible pro- and antiarrhythmic mechanisms of ω3-PUFAs in relation to various cardiac pathologies. Differences between circulating and incorporated ω3-PUFAs with respect to electrophysiology are emphasized. We conclude that ω3-PUFAs alter cardiac electrophysiology and thereby may be pro- or antiarrhythmic, dependent on the mechanism of arrhythmia. As ω3-PUFAs may be antiarrhythmic under conditions that favour triggered activity, they may facilitate re-entrant arrhythmias. This may explain the contradictory outcomes of increased intake of fish oil on sudden death and arrhythmias in clinical trials. Advice to increase intake of ω3-PUFA supplements or fatty fish should be tailored to individual patients with respect to the arrhythmogenic mechanisms associated with the underlying pathology. [Copyright &y& Elsevier]

Objective: The restitution mechanism has been the focus of attention as the possible mechanism behind ventricular fibrillation (VF). However, its contribution in chronic ischemic heart has not been established. Methods: We investigated chronic ischemic dogs with occlusion of left anterior descending artery. Sixty unipolar electrograms were simultaneously recorded from an entire cardiac surface. Activation-recovery intervals (ARIs) and QRST deflection area (AQRST) were measured during constant atrial pacing. The ischemic dogs were divided into two groups, five dogs in VF(+) group or seven dogs in VF(-) group, according to VF occurrence by programmed electrical stimulation. Results: When investigating ARI dispersions on an epicardium, there was no difference between VF(+) and VF(-) groups. The relationship between ARIs and diastolic intervals was quantified as an electrical restitution curve. The slopes of the ARI restitution curve for the anterior left ventricle in VF(+) dogs were significantly steeper than those of VF(-) dogs. The amplitude of AQRST alternans were significantly greater in VF(+) dogs than VF(-) dogs. Conclusions: Combined observation of steep restitution slopes and increased electrical alternans supported the restitution mechanism as being involved in the arrhythmia. Dynamic restitution properties and not static single-beat ARI dispersion may play an important role in the VF arrhythmia in the chronic ischemic heart. [Copyright &y& Elsevier]

Objectives: This study evaluated the in vivo electrophysiological effects of a highly selective slow delayed-rectifier K+-current blocker, HMR 1556, to gain insights into the consequences of selectively inhibiting the slow delayed-rectifier current in vivo. Methods: Atrial and ventricular effective refractory periods, sinus node recovery time, Wenckebach cycle-length, atrial fibrillation duration and electrocardiographic intervals were measured before and after intravenous HMR 1556. Results: HMR 1556 increased atrial and ventricular refractory periods (e.g. by 6±4% and 27±6% at cycle lengths of 360 and 400 ms, respectively), QT intervals and sinus-node recovery times. Beta-adrenoceptor blockade with nadolol abolished all effects except those on ventricular refractoriness and changed positive use-dependent effects on refractoriness to reverse use-dependent ones. In the presence of dofetilide to block rapid delayed-rectifier current, HMR 1556 effects were potentiated (e.g. atrial and ventricular refractory periods increased by 26±3% and 34±3% at cycle lengths of 360 and 400 ms, respectively). HMR 1556 reduced vagal atrial fibrillation duration from 1077±81 to 471±38 s, an effect abolished by nadolol and greatly potentiated by dofetilide (duration 77±30 s). HMR 1556 increased Wenckebach cycle length only in the presence of dofetilide. Conclusions: Slowed delayed-rectifier current inhibition affects atrial repolarization, sinus node function and atrial fibrillation in vivo, but only in the presence of intact beta-adrenergic tone, and delays ventricular repolarization even when beta-adrenoceptors are blocked. The slow delayed-rectifier current is particularly important when rapid delayed-rectifier current is suppressed, illustrating the importance of repolarization reserve. [Copyright &y& Elsevier]

Objective: The aim of this study was to investigate changes following regression of left ventricular hypertrophy (LVH). Methods: Electrophysiolological changes were recorded in isolated guinea-pig myocardial preparations. LVH was induced by constriction of the thoracic aorta and regression was followed after removal of the constriction. Sham-operated animals served as controls. Results: During 42 days constriction, heart/body weight ratio increased (3.19±0.49 vs. 3.85±0.83 g kg−1) and was accompanied by an increase of cell size. Forty-two days after clip removal, values had returned to control values. LVH increased action potential (AP) duration (mean 112% of control) and decreased conduction velocity (60.4±3.3 vs. 45.9±4.6 cm−1). These changes did not return to control after regression of LVH. The changes to condition velocity were attributed solely to increases of intracellular resistivity. The positive staircase response also decreased with LVH, but did recover upon regression. In isolated whole hearts, no changes to subepicardial action potential duration, QRS complex duration or AP refractory period were observed in LVH or its regression. During low-flow ischaemia AP duration shortened reversibly, the rate of shortening was more rapid in hypertrophied hearts but similar to control in regressed hearts. The incidence of ventricular tachyarrhythmias of fibrillation during low-flow ischaemia was similar in control, hypertrophied and regressed hearts. Conclusion: Morphological regression of LVH is not accompanied by reversal of electrophysiological changes measured in isolated preparations, whereas some aspects of contractile function to recover. [Copyright &y& Elsevier]

Background: Cellular electrical coupling is essential for normal propagation of the cardiac action potential, whereas reduced electrical coupling is associated with arrhythmias. Known cellular uncoupling agents have severe side effects on membrane ionic currents. We investigated the effect of carbenoxolone on cellular electrical coupling, membrane ionic currents, and atrial and ventricular conduction. Methods and Results: In isolated rabbit left ventricular and right atrial myocytes, carbenoxolone (50 μmol/l) had no effect on action potential characteristics. Calcium, potassium, and sodium currents remained unchanged. Dual current clamp experiments on poorly coupled cell pairs revealed a 21±3% decrease in coupling conductance by carbenoxolone (mean±S.E.M., n=4, p<0.05). High-density activation mapping was performed in intact rabbit atrium and ventricle during Langendorff perfusion of the heart. The amplitude of the Laplacian of the electrograms, a measure of coupling current in intact hearts, decreased from 1.45±0.66 to 0.75±0.51 μA/mm3 (mean±SD, n=32, p<0.05) after 15 min of carbenoxolone. Carbenoxolone reversibly decreased longitudinal and transversal conduction velocity from 66±15 to 49±16 cm/s and from 50±14 to 35±15 cm/s in ventricle, respectively (mean±SD, n=5, both p<0.05). In atrium, longitudinal and transversal conduction velocity decreased from 80±29 to 60±16 cm/s and from 49±10 to 38±10 cm/s (mean±SD, n=8, both p<0.05). Conclusions: Carbenoxolone-induced uncoupling causes atrial and ventricular conduction slowing without affecting cardiac membrane currents. Activation delay is larger in poorly coupled cells. [Copyright &y& Elsevier]

Objective: The Andersen’s syndrome is a hereditary disease, which is characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Recently, mutations of the KCNJ2 gene, which encodes the inward rectifying potassium channel subunit Kir2.1, have been identified in affected individuals. However, the functional effects of these mutations have not yet been fully elucidated. Methods and Results: To clarify this situation we generated known Andersen disease mutants of KCNJ2 which did not yield any measurable K+ currents in CHO cells indicating that the Andersen mutants failed to form functional homomultimeric complexes. EGFP-tagged KCNJ2 wild-type and mutant channels distributed in a similar homogeneous pattern in the cell membrane suggesting that protein trafficking was not altered by the Andersen mutations but rather implicating that the mutations rendered the KCNJ2 channel non-functional. In heterologous coexpression experiments the Andersen mutants exerted a dominant-negative effect on wild-type KCNJ2. However, the extent of suppression varied between the different KCNJ2 mutants. Given our results in CHO cells, we expressed the disease mutant KCNJ2-S136F in neonate rat cardiomyocytes using adenoviral gene transfer to test the effect of Andersen mutants on native IK1. IK1 density was indeed significantly reduced in KCNJ2-S136F-infected cells (n=9) compared to control cells (n=9) over a voltage range from −70 to −150 mV (P<0.05). Conclusion: These results support that Kir2.x channels are a critical component of native IK1 in neonate rat cardiomyocytes and that a dominant-negative suppression of IK1 in native cells is the pathophysiological correlate of the Andersen’s syndrome. [Copyright &y& Elsevier]

Objective: The interval from the peak to the end of the T wave (TpTe) has been proposed to reflect the heterogeneity of action potential durations within the ventricular wall. Several studies have previously described TpTe to be independent of heart rate, which contradicts the in vitro observation of marked changes in transmural repolarisation heterogeneity due to cycle length changes. Because of this inconsistency, we investigated heart rate related changes of TpTe interval. Methods: During 24-h recordings (SEER MC, Marquette GE) in healthy young women (n=25, 26±7 years) and men (n=25, 27±8 years), a 10-s 12-lead ECG was obtained every 30 s. Recordings were repeated after 1 day, 1 week, and 1 month and results in each subject were pooled together and grouped for women and men. The QT and QTpeak intervals were obtained automatically using QT Guard software (Marquette) and TpTe was computed as the difference between QT and QTpeak. In each subject TpTe values were averaged over 10-ms RR interval bands from 550 to 1150 ms. Results: In both sexes, TpTe interval showed marked rate dependence with prolongation at long RR intervals. TpTe intervals in men were significantly longer over the entire range of investigated RR intervals (P=1.4×10−25). However, whereas the difference between sexes was marked at short cycle length (RR interval bin 540–550 ms: women 87±5 vs. men 95±9, P=5.1×10−4) it decreased at long cycle lengths (RR interval bin 1140–1150 ms: women 99±5 vs. men 106±6, P=9.3×10−4). Conclusion: There is a marked rate dependence of TpTe interval, which differs between women and men. The finding is consistent with the TpTe interval being an approximate surrogate of the intraventricular repolarisation gradient. The rate dependent increase in transmural repolarisation heterogeneity might be one of the reasons for the increased propensity of torsades de pointes in women. [Copyright &y& Elsevier]

Objective: Our goal was to study rate adaptation of atrial action potentials in non-steady and steady states to further our understanding of mechanisms determining inducibility and stability of atrial fibrillation. Methods: We used standard microelectrode techniques to examine the characteristics of steady-state action potentials paced at regular cycle lengths (CL) and of nonsteady-state action potentials observed after an abrupt change of CL in atria from canine hearts that had been rapidly paced. Results: We compared action potential characteristics among normal atria, atria in which chronic atrial fibrillation (cAF, lasting more than 3 days) had been induced and atria in which only nonsustained atrial fibrillation (nAF, lasting less than 12 h) had been induced. In steady-state, the rate adaptation of maximum diastolic potential (MDP) and action potential duration (APD) was markedly reduced in both cAF and nAF. Action potential characteristics did not differ between cAF and nAF atria, suggesting that factors other than electrophysiological properties determine the chronicity of AF. The time course of change in APD after an abrupt change of CL was altered in nAF/cAF atria; i.e., when CL was prolonged, APD also prolonged at the first beat, and then shortened during several subsequent beats (initial phase). Thereafter, APD slowly prolonged to a new steady-state (slow phase). In nAF/cAF atria, the initial phase was enhanced (greater shortening of APD) and the slow phase was reduced (less prolongation of APD). This latter phase was modified by ryanodine. Conclusions: Thus the reduced rate adaptation of steady-state APD is explained mainly by the loss of a slow phase of APD adaptation in nAF/cAF which is reversed in the presence of ryanodine. Therefore, in both nAF and cAF atria, rate adaptation of MDP as well as APD are reduced, nonsteady state as well as steady state, AP characteristics are markedly altered and these changes are partially explicable by Cai-dependent processes. [ABSTRACT FROM PUBLISHER]

The aim was to determine whether dofetilide, a new class III antiarrhythmic agent, could reduce the heterogeneity of repolarisation produced by rapid cardiac pacing and to compare it with quinidine. Increased heterogeneity of repolarisation times may be causally linked to malignant cardiac re-entrant arrhythmias. Methods: Studies were performed in open chest, artificially ventilated, pentobarbitone anaesthetised beagle dogs of 13 to 15 kg body weight. Myocardial electrocardiograms were simultaneously recorded from 31 electrodes located on the left and right ventricular surfaces of the in situ canine heart. Recordings were obtained over a single cardiac cycle during rapid ventricular pacing at the maximum following frequency. Computer assisted measurements of activation time, activation-repolarisation interval, and repolarisation time were performed and the heterogeneity of these measurements calculated. Measurements were performed before treatment and after each incremental intravenous dose of dofetilide (3, 10, 30, and 100 μg·kg−1), quinidine (1, 3, and 10 mg·kg−1), or saline vehicle. Results: Dofetilide increased repolarisation time via a selective prolongation of activation-repolarisation interval, activation time being unchanged. It reduced the heterogeneity of repolarisation time and activation-repolarisation interval, heterogeneity of activation time being unchanged. Quinidine increased repolarisation time by a non-selective increase of both activation-repolarisation interval and activation time. It increased the heterogeneity of activation times which offset a decrease in heterogeneity of activation-repolarisation intervals to produce no change in heterogeneity of repolarisation times. Conclusions: Dofetilide, but not quinidine, reduces pacing induced asynchrony of repolarisation in the canine heart and this effect may contribute to its antiarrhythmic activity.Cardiovascular Research 1992;26:1102-1108 [ABSTRACT FROM PUBLISHER]

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1-2% in young adults but increasing to more that 10% in 80+ years patients. Albeit AF is a frequent electrophysiological disorder, to date, the genetic bases of AF remain rather elusive. Point mutations in a large variety of ion channels have been described in familial cases of AF, yet explaining a minority of cases. Recently, genome wide association studies (GWAS) have unraveled risk SNPs highly associated with AF, among which the most significant are located in 4q25 locus. Surprisingly these risk alleles are located in a gene desert, being the closest gene PITX2. Experimental evidences of Pitx2 loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factors plays a pivotal role in atrial electrophysiology. Therefore these data, underscore PITX2 as a candidate gene for AF. In this context, we have recruited 31 AF patients from the Regional Hospital for genetic analyses of both the risk alleles and PITX2 ORF re-sequencing. Among those patients, we have found two point mutations in the HD of PITX2 and three other mutations in the 5'untranslated region. A 65 years male patient with recurrent AF displayed two distinct HD-mutations, G947A (Q103H) and G1008A (E124Q), which both resulted in a change within a highly conserved amino acid position. Curiously, no 4q25 risk variants were present in this subject. Both PITX2 HD mutations were further followed for functional studies. We generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2 MD4 and PITX2 MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, in a similar range as PITX2 DN (50%). Co-transactivation with other cardiac-enriched transcription factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2 function. We are currently evaluating the functional consequence of these mutations in a cardiomyocyte context. Preliminary data suggest that distinct AF-related genes are similarly impaired. In summary, we have identified novel PITX2 mutations in an AF patient, which functionally impairs the transactivating capacity of PITX2. [ABSTRACT FROM AUTHOR]

Arrhythmias arise from a complex interaction between structural changes in the myocardium and changes in cellular electrophysiology. Electrophysiological balance requires precise control of sarcolemmal ion channels and exchangers, many of which are regulated by phospholipid, phosphatidylinositol(4,5)bisphosphate. Phosphatidylinositol(4,5)bisphosphate is the immediate precursor of inositol(1,4,5)trisphosphate, a regulator of intracellular Ca2+ signalling and, therefore, a potential contributor to arrhythmogenesis by altering Ca2+ homeostasis. The aim of the present review is to outline current evidence that this signalling pathway can be a player in the initiation or maintenance of arrhythmias. [ABSTRACT FROM AUTHOR]