Changes In Action Potential Duration Research Articles

The electrophysiologic effects of KCNQ1 extend beyond expression of IKs: evidence from genetic and pharmacologic block.

While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss of function KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 loss of function allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug.In control cells, a small IKs was readily recorded but pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). By contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (μM) of moxifloxacin required to prolong APD90 100 msec was 237.4 (median, IQR 100.6-391.6, n = 7) in population cells versus 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300μM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs, and markedly prolonged APD90. Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs. Mutations in KCNQ1 - whose expression generates IKs - are the major cause of long QT syndrome. We report here that while pharmacologic IKs block in human cardiomyocytes generates minimal change in repolarization, suppressing KCNQ1 expression markedly increases both baseline repolarization duration and sensitivity to some (but not all) specific IKr blockers. Thus, beyond simply generating IKs, KCNQ1 subserves critical additional role(s) in repolarization control at baseline and in response to IKr block. Our findings imply that assessment of arrhythmic risk in individual patients and by drugs requires a framework that extends beyond a simple one gene-one ion current paradigm.

PO-01-144 ACTION POTENTIAL ALTERNANS THRIVE IN CALCIUM BUFFERED VENTRICULAR MYOCYTES

Action potential (AP) alternans (AP-ALT) are linked to increased arrhythmogenesis. It is suggested that calcium (Ca2+) transient (CaT) alternans are the primary cause of AP alternans through a bi-directional coupling feedback mechanism because CaT alternans may develop in AP alternans free conditions. Supporting this notion to date, the counterpart scenario in which AP alternans develop in the absence of CaT alternans has not yet been demonstrated. With the objective of developing a CaT free AP alternans experimental model, we revisited the idea of inducing AP alternans in the presence of the Ca2+ buffer BAPTA and investigated if Bay K 8644, an L-type calcium channel agonist and known promoter of AP alternans, was effective in Ca2+ buffered cells. We analyzed AP-ALT incidence (positive when sequences of 14 or more APs exhibited repetitive short-to-long changes in AP duration (APD)) in isolated rabbit ventricle myocytes paced at progressively shorter cycles lengths (CL) from 1000 ms to the shortest CL > 179 ms eliciting AP-ALT to determine the alternans threshold (ALT-TH). We perfused Tyrode (37 oC) alone or with 25 nM Bay k 8644 upon freely contracting myocytes or in myocytes loaded (via patch microelectrode) with 10 or 20 mM BAPTA to completely abolish CaT. AP alternans developed abundantly in Ca2+ buffered myocytes (AP-ALT incidence 65 vs 80 vs 91%, in Controls vs 10 mM BAPTA vs 20 mM BAPTA, respectively). Controls and 10 mM BAPTA had similar ALT-TH (226±37 vs 269±88 ms, p>0.05) and alternans magnitude (13±5 vs 17±9 ms, p>0.05), but both parameters were larger in the 20 mM BAPTA myocytes (273±56 ms and 21±13 ms, respectively ALT-TH and alternans magnitude, p<0.05). Concomitantly, at ALT-TH, the diastolic interval (DI) was similar in all groups (46±17 ms vs 46±18 ms vs 49±10 ms, respectively Control vs 10 mM BAPTA vs 20 mM BAPTA, p>0.05), whilst APD was similar in Control and 10 mM BAPTA (180±34 vs 223±73 ms), but significantly larger in 20 mM BAPTA (225±52 ms, p<0.02 vs Controls). Delivery of Bay K 8644 to paced alternans free myocytes loaded with BAPTA elicited APD prolongation followed by crescendo alternans (APD beat-to-beat changes of progressively increasing amplitude leading to loss of 1:1 capture) in 11/11 myocytes (CL=300 ms) and 6/6 myocytes (CL=1000 ms). In the absence of CaT AP-ALT thrive and are more sensitive to activation rate changes and/or drugs promoting alternans. Therapies aiming to reduce CaT or its dynamic response should consider this.

EN-452411-5 THE ELECTROPHYSIOLOGIC EFFECTS OF KCNQ1 EXTEND BEYOND EXPRESSION OF IKS: EVIDENCE FROM GENETIC AND PHARMACOLOGIC BLOCK

Co-expression of KCNQ1 and KCNE1 generates the repolarizing current IKs. While mutations in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in normal cardiomyocytes from human induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. A role for IKs expression as a modulator of the other major delayed rectifier in the heart, IKr, has previously been suggested. We used pharmacologic and genetic approaches to probe the role of IKs in human cardiac repolarization. We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss of function KCNQ1 variants. We compared the effects of pharmacologic IKs block with HMR-1556 to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 loss of function allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two IKr blockers in the setting of pharmacologic and genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In population cells, a small IKs was readily recorded but pharmacologic IKs block (by high-dose HMR-1556) produced no change in action potential duration at 90% repolarization (APD). By contrast, in cells with genetic ablation of KCNQ1 expression, APD was prolonged 77%. JLN cells displayed increased sensitivity to acute IKr block: the concentration of moxifloxacin required to prolong APD 100 msec (μM) was 237.4 (median, IQR 100.6-391.6, n=7) in population cells versus 22.4 (16.2-27.9, n=8) in JLN cells. In population control cells, chronic moxifloxacin exposure (300μM) mildly prolonged APD (10%), and unexpectedly increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (58%) and no increase in IKs. In cells with genetic suppression of KCNQ1 expression, moxifloxacin did not increase IKs, and markedly prolonged APD. Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.

Optogenetic termination of atrial tachyarrhythmias by brief pulsed light stimulation

AimsThe most efficient way to acutely restore sinus rhythm from atrial fibrillation (AF) is electrical cardioversion, which is painful without adequate sedation. Recent studies in various experimental models have indicated that optogenetic termination of AF using light-gated ion channels may provide a myocardium-specific and potentially painless alternative future therapy. However, its underlying mechanism(s) remain(s) incompletely understood. As brief pulsed light stimulation, even without global illumination, can achieve optogenetic AF termination, besides direct conduction block also modulation of action potential (AP) properties may be involved in the termination mechanism. We studied the relationship between optogenetic AP duration (APD) and effective refractory period (ERP) prolongation by brief pulsed light stimulation and termination of atrial tachyarrhythmia (AT). Methods and resultsHearts from transgenic mice expressing the H134R variant of channelrhodopsin-2 in atrial myocytes were explanted and perfused retrogradely. AT induced by electrical stimulation was terminated by brief pulsed blue light stimulation (470 nm, 10 ms, 16 mW/mm2) with 68% efficacy. The termination rate was dependent on pulse duration and light intensity. Optogenetically imposed APD and ERP changes were systematically examined and optically monitored. Brief pulsed light stimulation (10 ms, 6 mW/mm2) consistently prolonged APD and ERP when light was applied at different phases of the cardiac action potential. Optical tracing showed light-induced APD prolongation during the termination of AT. ConclusionOur results directly demonstrate that cationic channelrhodopsin activation by brief pulsed light stimulation prolongs the atrial refractory period suggesting that this is one of the key mechanisms of optogenetic termination of AT.

Phenomenological analysis of simple ion channel block in large populations of uncoupled cardiomyocytes.

Current understanding of arrhythmia mechanisms and design of anti-arrhythmic drug therapies hinges on the assumption that myocytes from the same region of a single heart have similar, if not identical, action potential waveforms and drug responses. On the contrary, recent experiments reveal significant heterogeneity in uncoupled healthy myocytes both from different hearts as well as from identical regions within a single heart. In this work, a methodology is developed for quantifying the individual electrophysiological properties of large numbers of uncoupled cardiomyocytes under ion channel block in terms of the parameters values of a conceptual fast-slow model of electrical excitability. The approach is applied to a population of nearly 500 rabbit ventricular myocytes for which action potential duration (APD) before and after the application of the drug nifedipine was experimentally measured (Lachaud et al., 2022, Cardiovasc. Res.). To this end, drug action is represented by a multiplicative factor to an effective ion conductance, a closed form asymptotic expression for APD is derived and inverted to determine model parameters as functions of APD and $\varDelta $APD (drug-induced change in APD) for each myocyte. Two free protocol-related quantities are calibrated to experiment using an adaptive-domain procedure based on an original assumption of optimal excitability. The explicit APD expression and the resulting set of model parameter values allow (a) direct evaluation of conditions necessary to maintain fixed APD or $\varDelta $APD, (b) predictions of the proportion of cells remaining excitable after drug application, (c) predictions of stimulus period dependency and (d) predictions of dose-response curves, the latter being in agreement with additional experimental data.

Empagliflozin and ROS-CaMKII-late sodium current signaling in heart failure with preserved ejection fraction.

Empagliflozin has been shown to improve cardiovascular outcomes in patients with heart failure and preserved ejection fraction (HFpEF), independent of diabetes status. Preclinical studies showed that empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, can directly affect heart cells. SGLT2 expression is virtually absent in cardiomyocytes, suggesting off-target beneficial effects; however, exact signaling mechanisms remain elusive. Reductions in reactive oxygen species (ROS) production, Ca/calmodulin-dependent kinase II (CaMKII) activity, and late Na+ current (INaL) have been implicated in empagliflozin's action. We tested for empagliflozin effects on INaL and consequent action potential duration (APD) changes in two complementary translational murine HFpEF models that exhibit different pathophysiology. Both HFpEF models combine metabolic and hemodynamic stresses, and exhibit diastolic dysfunction, but one has much more marked hyperglycemia. Empagliflozin pretreatment of isolated myocytes, but not its acute application, reduced the INaL, and attenuated APD prolongation and delayed afterdepolarizations in both HFpEF models. CaMKII inhibition closely mimicked these effects. Moreover, myocytes with oxidation-resistant mutations in CaMKII (MM281/282VV) prevented both H2O2-induced increase in INaL and suppression by empagliflozin. Our data suggest that empagliflozin likely regulates INaL in HFpEF by an indirect mechanism, which could include limiting ROS and its ability to activate CaMKII. In conclusion, suppression of INaL and CaMKII by empagliflozin reverses proarrhythmic APD changes and Ca handling impairments, thus may exert antiarrhythmic properties and improve diastolic function in HFpEF.

Role of AMPK pathway inactivation in high fat diet related cardiac electrophysiological remodeling.

Although impaired AMPK signaling is a hallmark of metabolic diseases (MetD), which heighten arrhythmia risk, whether and how loss of AMPK activity per se promotes adverse electrophysiological (EP) remodeling independently of confounding factors in MetD remains unknown. We investigated the EP substrate of wildtype (WT) mice on high-fat diet (HFD) relative to those on normal diet (ND) and to mice expressing the muscle-specific AMPK kinase dead isoform (AMPK-KD). After 8-weeks of HFD, mice underwent hemodynamic analysis by echocardiography and ex vivo optical action potential (AP) mapping. While HFD-fed mice did not exhibit structural or hemodynamic changes by 8-weeks, they were more prone to pacing-induced VT than their ND counterparts (57% HFD vs 11% ND). Investigation of the EP substrate revealed a marked decrease in conduction velocity (CV) in HFD vs ND-fed mice (54.9±6.7 cm/s vs 71.6±10.2 cm/s, p<0.05) with no change in AP duration (APD). Importantly, we found similar CV slowing in AMPK-KD on ND as WT on HFD. Challenging AMPK-KD mice with HFD did not reduce CV further (45.4±10.6 cm/s HFD vs 49.0±11.1 cm/s ND). In contrast, APD was decreased exclusively in AMPK-KD mice on HFD, reflecting the importance of both factors (AMPK depletion and HFD) in the modulation of myocardial repolarization. Functionally, the subset of hearts that were prone to HFD-related VT exhibited decreased CV compared to those that were protected against VT. We examined the molecular correlates of myocardial conduction, and found a paradoxical increase in Nav1.5, total and Ser368-phosphorylated Cx43 in AMPK-KD, discounting overall protein expression as a factor in CV slowing. In conclusion, impaired AMPK signaling accounts for early CV defects in HFD prior to onset of structural or mechanical remodeling. Our findings highlight AMPK as a potentially powerful anti-arrhythmic target at early stages of MetD.

Propagation of neuronal signals in intact and injured peripheral nerves: The role of nonlinear diffusion of transmembrane potential

We present results of a computational study of the influence of nonlinear diffusion on propagation of electrical excitation generated in normal and injured peripheral nerves (PN) using a one-dimensional Fitzhugh-Nagumo (FN) model. Changes in action potential duration (APD) and repolarization intervals (RI) were observed based upon modification of the FN model. Namely, the model has been altered by adding an additional power function type nonlinear diffusion term which accounted for substantial intra-extracellular charge balance changes developing during action potential formation in small peripheral nerves. It was found that changes in magnitude of nonlinear diffusion coefficient result in oscillations of APD and RI in both normal and injured PNs with partially reduced conductivity. It was also found that these oscillations in injured PNs had markedly higher amplitudes dependent on the PN's length as well as on the width of its injured part. Results of our study may be useful in improving of monitoring of peripheral nerve growth and adjusting propagation of excitation to enhance impaired connectivity in injured peripheral nerves.

Sensitivity Analysis of Cardiac Alternans and Tachyarrhythmia to Ion Channel Conductance Using Population Modeling.

Action potential duration (APD) alternans, an alternating phenomenon between action potentials in cardiomyocytes, causes heart arrhythmia when the heart rate is high. However, some of the APD alternans observed in clinical trials occurs under slow heart rate conditions of 100 to 120 bpm, increasing the likelihood of heart arrhythmias such as atrial fibrillation. Advanced studies have identified the occurrence of this type of APD alternans in terms of electrophysiological ion channel currents in cells. However, they only identified physiological phenomena, such as action potential due to random changes in a particular ion channel's conductivity through ion models specializing in specific ion channel currents. In this study, we performed parameter sensitivity analysis via population modeling using a validated human ventricular physiology model to check the sensitivity of APD alternans to ion channel conductances. Through population modeling, we expressed the changes in alternans onset cycle length (AOCL) and mean APD in AOCL (AO meanAPD) according to the variations in ion channel conductance. Finally, we identified the ion channel that maximally affected the occurrence of APD alternans. AOCL and AO meanAPD were sensitive to changes in the plateau Ca2+ current. Accordingly, it was expected that APD alternans would be vulnerable to changes in intracellular calcium concentration.

Ventricular arrhythmia and sudden cardiac death in hypertrophic cardiomyopathy: From bench to bedside.

Patients with hypertrophic cardiomyopathy (HCM) mostly experience minimal symptoms throughout their lifetime, and some individuals have an increased risk of ventricular arrhythmias and sudden cardiac death (SCD). How to identify patients with a higher risk of ventricular arrythmias and SCD is the priority in HCM research. The American College of Cardiology/American Heart Association (ACC/AHA) and the European Society of Cardiology (ESC) both recommend the use of risk algorithms to identify patients at high risk of ventricular arrhythmias, to be selected for implantation of implantable cardioverters/defibrillators (ICDs) for primary prevention of SCD, although major discrepancies exist. The present SCD risk scoring systems cannot accurately identify early-stage HCM patients with modest structural remodeling and mild disease manifestations. Unfortunately, SCD events could occur in young asymptomatic HCM patients and even as initial symptoms, prompting the determination of new risk factors for SCD. This review summarizes the studies based on patients' surgical specimens, transgenic animals, and patient-derived induced pluripotent stem cells (hiPSCs) to explore the possible molecular mechanism of ventricular arrhythmia and SCD. Ion channel remodeling, Ca2+ homeostasis abnormalities, and increased myofilament Ca2+ sensitivity may contribute to changes in action potential duration (APD), reentry circuit formation, and trigger activities, such as early aferdepolarization (EAD) or delayed afterdepolarization (DAD), leading to ventricular arrhythmia in HCM. Besides the ICD implantation, novel drugs represented by the late sodium current channel inhibitor and myosin inhibitor also shed light on the prevention of HCM-related arrhythmias. The ideal prevention strategy of SCD in early-stage HCM patients needs to be combined with gene screening, hiPSC-CM testing, machine learning, and advanced ECG studies, thus achieving individualized SCD prevention.

Validating the Arrhythmogenic Potential of High-, Intermediate-, and Low-Risk Drugs in a Human-Induced Pluripotent Stem Cell-Derived Cardiac Microphysiological System.

Evaluation of arrhythmogenic drugs is required by regulatory agencies before any new compound can obtain market approval. Despite rigorous review, cardiac disorders remain the second most common cause for safety-related market withdrawal. On the other hand, false-positive preclinical findings prohibit potentially beneficial candidates from moving forward in the development pipeline. Complex in vitro models using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) have been identified as a useful tool that allows for rapid and cost-efficient screening of proarrhythmic drug risk. Currently available hiPSC-CM models employ simple two-dimensional (2D) culture formats with limited structural and functional relevance to the human heart muscle. Here, we present the use of our 3D cardiac microphysiological system (MPS), composed of a hiPSC-derived heart micromuscle, as a platform for arrhythmia risk assessment. We employed two different hiPSC lines and tested seven drugs with known ion channel effects and known clinical risk: dofetilide and bepridil (high risk); amiodarone and terfenadine (intermediate risk); and nifedipine, mexiletine, and lidocaine (low risk). The cardiac MPS successfully predicted drug cardiotoxicity risks based on changes in action potential duration, beat waveform (i.e., shape), and occurrence of proarrhythmic events of healthy patient hiPSC lines in the absence of risk cofactors. We showcase examples where the cardiac MPS outperformed existing hiPSC-CM 2D models.

Synchronization of repolarization after cardiac resynchronization therapy: A combined clinical and modeling study.

IntroductionThe changes in ventricular repolarization after cardiac resynchronization therapy (CRT) are poorly understood. This knowledge gap is addressed using a multimodality approach including electrocardiographic and echocardiographic measurements in patients and using patient‐specific computational modeling.MethodsIn 33 patients electrocardiographic and echocardiographic measurements were performed before and at various intervals after CRT, both during CRT‐ON and temporary CRT‐OFF. T‐wave area was calculated from vectorcardiograms, and reconstructed from the 12‐lead electrocardiography (ECG). Computer simulations were performed using a patient‐specific eikonal model of cardiac activation with spatially varying action potential duration (APD) and repolarization rate, fit to a patient's ECG.ResultsDuring CRT‐ON T‐wave area diminished within a day and remained stable thereafter, whereas QT‐interval did not change significantly. During CRT‐OFF T‐wave area doubled within 5 days of CRT, while QT‐interval and peak‐to‐end T‐wave interval hardly changed. Left ventricular (LV) ejection fraction only increased significantly increased after 1 month of CRT. Computer simulations indicated that the increase in T‐wave area during CRT‐OFF can be explained by changes in APD following chronic CRT that are opposite to the change in CRT‐induced activation time. These APD changes were associated with a reduction in LV dispersion in repolarization during chronic CRT.ConclusionT‐wave area during CRT‐OFF is a sensitive marker for adaptations in ventricular repolarization during chronic CRT that may include a reduction in LV dispersion of repolarization.

Synchronization of repolarization after cardiac resynchronization therapy: time course and relation to reverse remodeling

Abstract Funding Acknowledgements Type of funding sources: None. Background The changes in ventricular repolarization after cardiac resynchronization therapy (CRT) are poorly understood. Objective Address this knowledge gap using a multimodality approach including electrocardiographic and echocardiographic measurements in patients and patient-specific computational modeling. Methods In a cohort of consecutive CRT patients, electrocardiographic and echocardiographic measurements were performed before and at various intervals after CRT, both during CRT-ON and temporary CRT-OFF. T-wave area was calculated from vectorcardiograms, reconstructed from the 12-lead ECG. Computer simulations were performed using a patient-specific eikonal model of cardiac activation with spatially varying action potential duration (APD) and repolarization rate, based on a patient’s ECG and MRI-based chest and cardiac anatomy. Results In 33 patients T-wave area during CRT-OFF doubled within 5 days of CRT (left figure) while other repolarization markers like QT-interval and peak-to-end T-wave interval hardly changed. Patients with T-wave area increase above the median increase showed a significantly larger increase in left ventricular ejection fraction than those with smaller T-wave area increase (+11±8% vs. +6±6%, respectively; p&amp;lt;0.05, n=76). Computer simulations (figure, right) show the sequence of depolarization and repolarization before CRT, obtained by the patient-specific model (BL LBBB) and following Acute CRT. Repolarization after chronic CRT-OFF was obtained by matching the measured CRT-OFF ECG with that provided by the model. The best match was obtained by changes in APD following chronic CRT that are opposite to the change in CRT-induced activation time. Such APD changes were associated with a reduction in right and left ventricular dispersion in repolarization during chronic CRT-ON compared with acute CRT-ON. Conclusions T-wave area during CRT-OFF is a sensitive marker for adaptations in ventricular repolarization after CRT that are related to CRT-induced reverse remodeling and may involve an inverse relation between activation time and APD, coinciding with lower right and left ventricular dispersion of repolarization and with larger increases in LV ejection fraction during chronic CRT.

Accurate in silico simulation of the rabbit Purkinje fiber electrophysiological assay to facilitate early pharmaceutical cardiosafety assessment: Dream or reality?

As a branch of quantitative systems toxicology, in silico simulations are of growing attractiveness to guide preclinical cardiosafety risk assessments. Traditionally, a cascade of in vitro/in vivo assays has been applied in pharmaceutical research to screen out molecules at risk for cardiac side effects and prevent subsequent risk for patients. Drug cardiosafety assessments typically employ early mechanistic, hazard-oriented in silico/in vitro assays for compound inhibition of cardiac ion channels, followed by induced pluripotent stem cells (iPSCs) or tissue-based models such as the rabbit Purkinje fiber assay, which includes the major mechanisms contributing to action potential (AP) genesis. Additionally, multiscale simulation techniques based on mathematical models have become available, which are performed in silico ‘at the heart’ of compound triage to substitute Purkinje tests and increase translatability through mechanistic interpretability. To adhere to the 3R principle and reduce animal experiments, we performed a comparative benchmark and investigated a variety of mathematical cardiac AP models, including a newly developed minimalistic model specifically tailored to the AP of rabbit Purkinje cells, for their ability to substitute experiments. The simulated changes in AP duration (dAPD90) at increasing drug concentrations were compared to experimental results from 588 internal Purkinje fiber studies covering 555 different drugs with diverse modes of action. Using our minimalistic model, 80% of the Purkinje experiments could be quantitatively reproduced. This result allows for significant saving of experimental effort in early research and justifies the embedding of electrophysiological simulations into the DMTA (Design, Make, Test, Analyze) cycle in pharmaceutical compound optimization.

Ageing Increases Cardiac Electrical Remodelling in Rats and Mice via NOX4/ROS/CaMKII-Mediated Calcium Signalling.

Objective Ageing is one of the risk factors associated with cardiovascular diseases including cardiac arrhythmias and heart failure. Ageing-related cardiac dysfunction involves a complicated pathophysiological progress. Abnormal membrane voltage and Ca2+ dynamics in aged cardiomyocytes contribute to ageing-related arrhythmias. However, its underlying mechanisms have not been well clarified. Methods Young and old rats or mice were included in this study. Cardiac electrophysiological properties and functions were assessed by ECG, echocardiography, and ex vivo heart voltage and Ca2+ optical mapping. Proteomics, phosphor-proteomics, Western blotting, Masson staining, and ROS measurement were used to investigate the underlying mechanisms. Results Ageing increased the incidence of cardiac hypertrophy and fibrosis in rats. Moreover, ageing increased the occurrence of ventricular tachycardia or ventricular fibrillation induced by rapid pacing and during isoprenaline (ISO) (1 mg/kg i.p.) challenge in mice in vivo. Optical mapping with dual dyes (membrane voltage (Vm) dye and intracellular Ca2+ dye) simultaneously recording revealed that ageing increased the action potential duration (APD) and Ca2+ transient duration (CaTD) and slowed the ventricular conduction with the Langendorff-perfused mouse heart. More importantly, ageing increased the ISO-induced (1 μM) changes of APD (ΔAPD80) and CaTD (ΔCaTD50). Ageing also delayed the decay of Ca2+ transient by extending the decay time constant from 30% to 90% (τ30−90). In addition, ageing decreased the Vm/Ca2+ latency which represented the coupling of Vm/Ca2+ including between the midpoint of AP depolarization and Ca2+ upstroke, peak transmembrane voltage and peak cytosolic calcium, and time to 50% voltage repolarization and extrusion of cytosolic calcium. Optical mapping also revealed that ageing increased the ISO-induced arrhythmia incidence and occurrence of the excitation rotor. Proteomics and phosphor-proteomics assays from rat hearts demonstrated ageing-induced protein and phosphor-protein changes, suggesting that CaMKII was involved in ageing-induced change. Ageing increased the level of ROS and the expression of NOX4, oxidative CaMKII (ox-CaMKII), phosphorated CaMKII (p-CaMKII), and periostin. Conclusion Ageing accelerates cardiac remodelling and increases the susceptibility to ventricular arrhythmias through NOX4/ROS/CaMKII pathway-mediated abnormal membrane voltage and intracellular Ca2+ handling and Vm/Ca2+ coupling.

Ventricular Dysfunction in Obese and Nonobese Rats with Metabolic Syndrome.

Obesity and dyslipidemias are both signs of metabolic syndrome, usually associated with ventricular arrhythmias. Here, we tried to identify cardiac electrical alteration and biomarkers in nonobese rats with metabolic syndrome (MetS), and these findings might lead to more lethal arrhythmias than obese animals. The MetS model was developed in Wistar rats with high-sucrose diet (20%), and after twenty-eight weeks were obtained two subgroups: obese (OMetS) and nonobese (NOMetS). The electrocardiogram was used to measure the ventricular arrhythmias and changes in the heart rate variability. Also, we measured ventricular hypertrophy and its relationship with electrical activity alterations of both ventricles, using micro-electrode and voltage clamp techniques. Also, we observed alterations in the contraction force of ventricles where a transducer was used to record mechanical and electrical papillary muscle, simultaneously. Despite both subgroups presenting long QT syndrome (0.66 ± 0.05 and 0.66 ± 0.07 ms with respect to the control 0.55 ± 0.1 ms), the changes in the heart rate variability were present only in OMetS, while the NOMetS subgroup presented changes in QT interval variability (NOMetS SD = 1.8, SD2 = 2.8; SD1/SD2 = 0.75). Also, the NOMetS revealed tachycardia (10%; p < 0.05) with changes in action potential duration (63% in the right papillary and 50% in the left papillary) in the ventricular papillary which are correlated with certain alterations in the potassium currents and the force of contraction. The OMetS showed an increase in action potential duration and the force of contraction in both ventricles, which are explained as bradycardia. Our results revealed lethal arrhythmias in both MetS subgroups, irrespectively of the presence of obesity. Consequently, the NOMetS showed mechanical-electrical alterations regarding ventricle hypertrophy that should be at the NOMetS, leading to an increase of CV mortality.

Effects of azumolene on arrhythmia substrate in a model of recurrent long-duration ventricular fibrillation

BackgroundProarrhythmic risk of conventional anti-arrhythmic agents is linked to unintended modulation of membrane voltage dynamics. We have demonstrated that the anti-fibrillatory effect of azumolene is mediated via stabilization of the hyperphosphorylated ryanodine receptor (RyR2), leading to attenuation of diastolic calcium leak. However, the concomitant effects on membrane voltage dynamics have not been evaluated yet. MethodsAfter baseline optical mapping, Langendorff-perfused rabbit hearts treated with azumolene, or vehicle, were subjected to global ischemia-reperfusion (I/R) followed by two episodes of long-duration ventricular fibrillation (LDVF). Simultaneous dual epicardial calcium transient (CaT) and voltage dynamics were studied optically. ResultsPre-treatment with azumolene was associated with higher CaT amplitude alternans ratios (0.94 ± 0.02 vs. 0.78 ± 0.03 in control hearts, at 6 Hz; p = 0.005; and action potential amplitude alternans ratio (0.95 ± 0.02 vs. 0.78 ± 0.04 at 6.0 Hz; p = 0.02), and reduction of action potential duration (APD80) dispersion (9.0 ± 4.8 msec vs. 19.3 ± 6.6 msec at 6.0 Hz p = 0.02) and optical action potential upstroke rise time (26.3 ± 2.6 msec in control vs. 13.8 ± 0.6 msec at 6.0 Hz, p = 0.02) after LDVF. No change in action potential duration (APD) was noted with azumolene treatment. ConclusionIn a model of ischemic recurrent LDVF, treatment with azumolene led to reduction of cardiac alternans, i.e., calcium and voltage alternans. Unlike conventional anti-arrhythmic agents, reduction of action potential upstroke rise time and preservation of action potential duration following azumolene treatment may reduce the proarrhythmia risk.

Electrophysiological heterogeneity in large populations of rabbit ventricular cardiomyocytes.

Cardiac electrophysiological heterogeneity includes: (i) regional differences in action potential (AP) waveform, (ii) AP waveform differences in cells isolated from a single region, (iii) variability of the contribution of individual ion currents in cells with similar AP durations (APDs). The aim of this study is to assess intra-regional AP waveform differences, to quantify the contribution of specific ion channels to the APD via drug responses and to generate a population of mathematical models to investigate the mechanisms underlying heterogeneity in rabbit ventricular cells. APD in ∼50 isolated cells from subregions of the LV free wall of rabbit hearts were measured using a voltage-sensitive dye. When stimulated at 2 Hz, average APD90 value in cells from the basal epicardial region was 254 ± 25 ms (mean ± standard deviation) in 17 hearts with a mean interquartile range (IQR) of 53 ± 17 ms. Endo-epicardial and apical-basal APD90 differences accounted for ∼10% of the IQR value. Highly variable changes in APD occurred after IK(r) or ICa(L) block that included a sub-population of cells (HR) with an exaggerated (hyper) response to IK(r) inhibition. A set of 4471 AP models matching the experimental APD90 distribution was generated from a larger population of models created by random variation of the maximum conductances (Gmax) of 8 key ion channels/exchangers/pumps. This set reproduced the pattern of cell-specific responses to ICa(L) and IK(r) block, including the HR sub-population. The models exhibited a wide range of Gmax values with constrained relationships linking ICa(L) with IK(r), ICl, INCX, and INaK. Modelling the measured range of inter-cell APDs required a larger range of key Gmax values indicating that ventricular tissue has considerable inter-cell variation in channel/pump/exchanger activity. AP morphology is retained by relationships linking specific ionic conductances. These interrelationships are necessary for stable repolarization despite large inter-cell variation of individual conductances and this explains the variable sensitivity to ion channel block.

Atrial Fibrillation in Aging and Frail Mice: Modulation by Natriuretic Peptide Receptor C.

[Figure: see text].

B-PO04-010 THE EFFECT OF CHRONIC NICOTINE EXPOSURE ON CARDIAC ELECTROPHYSIOLOGY IN THE RABBIT HEART

Tobacco smoke exposure is associated with increased risk for atrial and ventricular arrhythmias. However, the individual components of tobacco smoke that may promote arrhythmias and the underlying electrophysiological mechanisms responsible are unknown. Nicotine (NIC) is a major component of tobacco smoke and emerging tobacco products, including e-cigarettes. To determine the effects of chronic NIC exposure on autonomic and electrophysiological remodeling in the rabbit heart. Fully innervated hearts from rabbits exposed to NIC (21mg/day, transdermal patch, 28 days, n=7) or control (CT, n=4) rabbit hearts were optically mapped using voltage- (RH237) and calcium-sensitive (Rhod2-AM) indicators. Sympathetic nerve stimulation (SNS) was performed with electrical stimulation at the first thoracic vertebrae in which voltage was kept constant (7 V) and frequency was increased in 0.5 Hz increments until a 15% increase in heart rate (HR) was achieved. B-adrenergic (B-AR) responsiveness was evaluated with norepinephrine (500nM, NE). Baseline HR was higher in NIC vs. CT (215.8 BPM vs. 151 BPM, p<0.05). SNS stimulation thresholds necessary to produce an increase in HR were higher in NIC vs. CT (4.17 Hz vs. 1.38 Hz, p<0.05). Maximal HR increase after 40s of SNS trended lower in NIC vs. CT (19.38 vs. 54.30% increase, p=0.1). SNS also tended to produce a smaller change in action potential duration (APD80: -8.14 vs. -16.68% decrease, p=NS) and in calcium transient duration (CaTD80: -7.80 vs. - 20.74% decrease, p=NS) in NIC vs. CT. B-AR responsiveness was decreased in NIC vs. CT (33.38 vs. 75.77% increase in HR with NE, p=0.12). These results suggest that chronic NIC exposure may cause baseline sympathetic hyperactivity reduced B-AR sensitivity. Findings from this study will have significant implications for youth and young adults who represent the fastest growing group of nicotine users and the cardiac effects of nicotine on this potentially vulnerable population is unknown. Thus, data from this study may have implications for tobacco products and other nicotine containing products.