Differences In Action Potential Duration Research Articles

Maturing hiPSC-cardiomyocytes in cardiac microtissues for accurate preclinical in vitro modelling of long-QT syndrome type 1

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): reNEW - Novo Nordisk Foundation Center for Stem Cell Medicine Background Long-QT syndrome type 1 (LQT1), a cardiac arrhythmia often leading to sudden cardiac death, is due to mutations in KCNQ1 gene. Human iPSC-derived cardiomyocytes (hiPSC-CMs) have been widely used to model LQT1, promoting the concept of their use in preclinical studies. However, KCNQ1 locus is subjected to developmentally regulated genomic imprinting in the heart: in fetal cardiomyocytes the paternal allele is repressed, while both alleles become expressed after birth. Since hiPSC-CMs are typically immature fetal-like cells, residual imprinting may affect evaluation of KCNQ1 mutations in this system. Purpose This study wants to address the imprinting status of KCNQ1 in hiPSC-CMs and propose maturing hiPSC-CMs as a solution for reliable LQT1 modelling. Methods We derived hiPSC-CMs from a LQT1 patient carrying a heterozygous KCNQ1 mutation on the paternal allele and compared their electrical properties with the isogenic corrected hiPSC-CMs by patch clamp. We analysed KCNQ1 expression by ddPCR and DNA methylation. We used three-dimensional cardiac microtissues (MTs) composed by hiPSC-CMs, hiPSC-derived cardiac fibroblasts and endothelial cells to induce cardiomyocte maturation. Results LQT1 and isogenic corrected hiPSC-CMs showed no difference in action potential duration (APD). We hypothesized that residual KCNQ1 imprinting in hiPSC-CMs due to their immaturity was masking the phenotype. When we analyzed different hiPSC-CM lines, we found in all an unbalanced KCNQ1 allelic expression, with paternal allele accounting for 5-15% of the total. We then investigated whether maturing hiPSC-CMs could remove KCNQ1 imprinting. We included hiPSC-CMs in MTs, as this system previously showed to promote functional maturation and upregulation of KCNQ1. Here, we observed a substantial increased expression of the paternal allele and a reduction in the expression of KCNQ1OT1, the long non-coding RNA inducing paternal allele repression. LQT1 hiPSC-CMs dissociated from MTs showed prolonged APD compared to the corrected line. We also demonstrated that this was due to the reduction of the ionic current mediated by KCNQ1 (IKs) in LQT1 hiPSC-CMs compared to the corrected controls, thus revealing the mutation causative effect. Conclusions Our findings show that residual KCNQ1 imprinting in immature hiPSC-CMs can lead to underestimate (or overestimate) functional effects of pathological variants based on the parental allele that carries the mutation. This is overcome by maturation of hiPSC-CMs in tri-cellular cardiac microtissue which promotes KCNQ1 bi-allelic expression and allow dissecting mutation mechanisms. This study brings attention to the epigenetic regulation of hiPSC models and demonstrates the utility of using matured hiPSC-CMs as in vitro preclinical model for cardiac arrhythmias.

Combining pharmacokinetic and electrophysiological models for early prediction of drug-induced arrhythmogenicity

Background and ObjectiveIn silico methods are gaining attention for predicting drug-induced Torsade de Pointes (TdP) in different stages of drug development. However, many computational models tended not to account for inter-individual response variability due to demographic covariates, such as sex, or physiologic covariates, such as renal function, which may be crucial when predicting TdP. This study aims to compare the effects of drugs in male and female populations with normal and impaired renal function using in silico methods. MethodsPharmacokinetic models considering sex and renal function as covariates were implemented from data published in pharmacokinetic studies. Drug effects were simulated using an electrophysiologically calibrated population of cellular models of 300 males and 300 females. The population of models was built by modifying the endocardial action potential model published by O'Hara et al. (2011) according to the experimentally measured gene expression levels of 12 ion channels. ResultsFifteen pharmacokinetic models for CiPA drugs were implemented and validated in this study. Eight pharmacokinetic models included the effect of renal function and four the effect of sex. The mean difference in action potential duration (APD) between male and female populations was 24.9 ms (p<0.05). Our simulations indicated that women with impaired renal function were particularly susceptible to drug-induced arrhythmias, whereas healthy men were less prone to TdP. Differences between patient groups were more pronounced for high TdP-risk drugs. The proposed in silico tool also revealed that individuals with impaired renal function, electrophysiologically simulated with hyperkalemia (extracellular potassium concentration [K+]o = 7 mM) exhibited less pronounced APD prolongation than individuals with normal potassium levels. The pharmacokinetic/electrophysiological framework was used to determine the maximum safe dose of dofetilide in different patient groups. As a proof of concept, 3D simulations were also run for dofetilide obtaining QT prolongation in accordance with previously reported clinical values. ConclusionsThis study presents a novel methodology that combines pharmacokinetic and electrophysiological models to incorporate the effects of sex and renal function into in silico drug simulations and highlights their impact on TdP-risk assessment. Furthermore, it may also help inform maximum dose regimens that ensure TdP-related safety in a specific sub-population of patients.

Long-term changes in heart rate and electrical remodeling contribute to alternans formation in heart failure: a patient-specific in silico study.

Individuals with chronic heart failure (CHF) have an increased risk of ventricular arrhythmias, which has been linked to pathological cellular remodeling and may also be mediated by changes in heart rate. Heart rate typically fluctuates on a timescale ranging from seconds to hours, termed heart rate variability (HRV). This variability is reduced in CHF, and this HRV reduction is associated with a greater risk for arrhythmias. Furthermore, variations in heart rate influence the formation of proarrhythmic alternans, a beat-to-beat alternation in the action potential duration (APD), or intracellular calcium (Ca). In this study, we investigate how long-term changes in heart rate and electrical remodeling associated with CHF influence alternans formation. We measure key statistical properties of the RR-interval sequences from ECGs of individuals with normal sinus rhythm (NSR) and CHF. Patient-specific RR-interval sequences and synthetic sequences (randomly generated to mimicking these statistical properties) are used as the pacing protocol for a discrete time-coupled map model that governs APD and intracellular Ca handling of a single cardiac myocyte, modified to account for pathological electrical remodeling in CHF. Patient-specific simulations show that beat-to-beat differences in APD vary temporally in both populations, with alternans formation more prevalent in CHF. Parameter studies using synthetic sequences demonstrate that increasing the autocorrelation time or mean RR-interval reduces APD alternations, whereas increasing the RR-interval standard deviation leads to higher alternans magnitudes. Importantly, we find that although both the CHF-associated changes in heart rate and electrical remodeling influence alternans formation, variations in heart rate may be more influential.NEW & NOTEWORTHY Using patient-specific data, we show that both the changes in heart rate and electrical remodeling associated with chronic heart failure influence the formation of proarrhythmic alternans in the heart.

The effect of sex and age on ex vivo cardiac electrophysiology: insight from a guinea pig model.

Highlighting the importance of sex as a biological variable, we recently reported sex differences in guinea pig in vivo electrocardiogram (ECG) measurements. However, substantial inconsistencies exist in this animal model, with conflicting reports of sex-specific differences in cardiac electrophysiology observed in vivo and in vitro. Herein, we evaluated whether sexual dimorphism persists in ex vivo preparations, using an isolated intact heart preparation. Pseudo-ECG recordings were collected in conjunction with dual optical mapping of transmembrane voltage and intracellular calcium from Langendorff-perfused hearts. In contrast to our in vivo results, we did not observe sex-specific differences in ECG parameters collected from isolated hearts. Furthermore, we observed significant age-specific differences in action potential duration (APD) and Ca2+ transient duration (CaD) during both normal sinus rhythm (NSR) and in response to dynamic pacing but only a modest sex-specific difference in CaD30. Similarly, the alternans fluctuation coefficient, conduction velocity during sinus rhythm or in response to pacing, and electrophysiology parameters (atrioventricular nodal effective refractory period, Wenckebach cycle length) were comparable between males and females. Results of our study suggest that the observed sex-specific differences in in vivo ECG parameters from guinea pigs are diminished in ex vivo isolated heart preparations, although age-specific patterns are prevalent. To assess sex as a biological variable in cardiac electrophysiology, a comprehensive approach may be necessary using both in vitro measurements from cardiomyocyte or intact heart preparations with secondary follow-up in vivo studies.NEW & NOTEWORTHY We evaluated whether the guinea pig heart has intrinsic sex-specific differences in cardiac electrophysiology. Although we observed sex-specific differences in in vivo ECGs, these differences did not persist ex vivo. Using a whole heart model, we observed similar APD, CaD, conduction velocity, and alternans susceptibility in males and females. We conclude that sex-specific differences in guinea pig cardiac electrophysiology are likely influenced by the in vivo environment and less dependent on the intrinsic electrical properties of the heart.

Abstract 12847: Atrial Fibrillation and Atrial Flutter: What Are the Substrate Differences Seen in 3D Panoramic Optical Mapping?

Background: Panoramic optical mapping (POM) is an effective tool for 3D visualization and analysis of action potential duration (APD) heterogeneity, a key driver for initiation and persistence of reentry. POM may elucidate cardiac electrophysiological predisposition to develop chaotic arrhythmia such as atrial fibrillation (AF) or organized arrhythmia such as atrial flutter (AFL). Objective: To determine the APD differences in AF and AFL in a transgenic (TG) murine model. Methods: We performed high resolution POM with 4 simultaneous CMOS cameras on 14 Langendorff perfused TG murine hearts (SCN5A mutation at lidocaine binding site F1759A) with spontaneous AF and AFL. We generated serial 2D activation and APD maps in normal sinus rhythm (NSR), while phase movies were created in AF/AFL. All optical maps were then reconstructed in 3D. We averaged the APD80 and APD dispersion values over each atrial chamber and compared the mean differences: i) within AF and AFL across right atrium (RA) and left atrium (LA); and ii) within RA and LA across AF and AFL. Results: AF (n=7) and AFL (n=7) were present at baseline (8/14) or induced by 10Hz burst pacing (6/14). Mean APD80 in RA/LA was: 57.03 ± 4.66 / 50.93± 3.38ms (p&lt;0.05) in AF vs 77.18 ± 4.30 / 58.25 ± 3.88ms (p&lt; 0.01) in AFL, and mean dispersion: 6.23 ± 1.04/ 7.25± 1.71ms in AF vs 5.49 ± 0.80/ 8.42 ± 1.15ms (p&lt;0.01) in AFL. High mean APD80 in the RA with low intra-atrial dispersion in the RA lead to AFL rotational reentry cores, while lower overall APD80 values with high intra-atrial dispersion in the LA resulted in the presence of AF phase singularity points. Conclusion: Development of AF was associated with high intra-atrial APD dispersion in NSR, whereas AFL was associated with high inter-atrial APD dispersion in NSR (1.4-fold higher mean APD80 in the RA). Certain atrial APD dispersion patterns observed in NSR are associated with development of chaotic vs. organized atrial arrhythmias.

Remodelling of potassium currents underlies arrhythmic action potential prolongation under beta-adrenergic stimulation in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) patients often present an enhanced arrhythmogenicity that can lead to lethal arrhythmias, especially during exercise. Recent studies have indicated an abnormal response of HCM cardiomyocytes to β-adrenergic receptor stimulation (β-ARS), with prolongation of their action potential rather than shortening. The mechanisms underlying this aberrant response to sympathetic stimulation and its possible proarrhythmic role remain unknown. The aims of this study are to investigate the key ionic mechanisms underlying the HCM abnormal response to β-ARS and the resultant repolarisation abnormalities using human-based experimental and computational methodologies. We integrated and calibrated the latest models of human ventricular electrophysiology and β-ARS using experimental measurements of human adult cardiomyocytes from control and HCM patients. Our major findings include: (1) the developed in silico models of β-ARS capture the behaviour observed in the experimental data, including the aberrant response of HCM cardiomyocytes to β-ARS; (2) the reduced increase of potassium currents under β-ARS was identified as the main mechanism of action potential prolongation in HCM, rather than a more sustained inward calcium current; (3) action potential duration differences between healthy and HCM cardiomyocytes were increased upon β-ARS, while endocardial to epicardial differences in HCM cardiomyocytes were reduced; (4) models presenting repolarisation abnormalities were characterised by downregulation of the rapid delayed rectifier potassium current and the sodium‑potassium pump, while inward currents were upregulated. In conclusion, our results identify causal relationships between the HCM phenotype and its arrhythmogenic response to β-ARS through the downregulation of potassium currents.

Spatial uniformity of action potentials indicates base-to-apex depolarization and repolarization of rainbow trout (Oncorhynchus mykiss) ventricle.

The spatial pattern of electrical activation is crucial for a full understanding of fish heart function. However, it remains unclear whether there is regional variation in action potential (AP) morphologies and underlying ion currents. Because the direction of depolarization and spatial differences in the durations of ventricular APs set limits to potential patterns of ventricular repolarization, we determined AP morphologies, underlying ion currents and ion channel expression in four different ventricular regions (spongy myocardium; and apex, base and middle of the compact myocardium), and correlated them with in vivo electrocardiograms (ECGs) in rainbow trout (Oncorhynchus mykiss). ECGs recorded from three leads indicated that the depolarization and repolarization of APs propagate from base to apex, and the main depolarization axis of the ventricle is between +90 and +120deg. AP shape was uniform across the whole ventricle, and little regional differences were found in the density of repolarizing K+ currents or depolarizing Ca2+ and Na+ currents and the underlying transcripts of ion channels, providing compelling evidence for the suggested excitation pattern. The spatial uniformity of AP durations and base-to-apex propagation of activation with a relatively slow velocity of propagation indicates no special ventricular conduction pathway in the trout ventricle such as the His-Purkinje system of mammalian hearts. The sequence of repolarization is solely determined by activation time without being affected by regional differences in AP duration.

BS-400-08 HETEROGENOUS CAMP SIGNALING IN THE INTACT HEART IS SEX DEPENDENT

cAMP is key for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. Yet, if cAMP signaling occurs heterogeneously throughout the intact heart, and how this translates into functional responses, has not been explored. To determine the spatiotemporal kinetics of cAMP signaling in the intact heart and the underlying mechanisms responsible. Male and female cardiac-specific CAMPER reporter mice that report cAMP binding by changes in FRET were used at 12 weeks. Hearts were excised and Langendorff-perfused for simultaneous cAMP and Vm imaging on a novel integrated whole heart optical imaging system. Following perfusion, tissue samples from base and apex were assessed for phosphodiesterase (PDE) activity. In male hearts, cAMP was uniformly activated in response to β-AR stimulation with bolus norepinephrine (NE, 1.5 μM). Conversely, in female hearts NE led to a greater change in cAMP activity in basal regions vs. the apex (n=7, p<0.05). Moreover, cAMP deactivation was slower in the base vs. apex, in female (n=6, p<0.01) but not male hearts (n=6). Apex-base differences were also evident following PDE inhibition with IBMX (100 μM), with a greater change in cAMP activity in the apex vs. base in both female (n=7, p<0.001) and male hearts (n=5, p<0.05). Likewise, PDE activity assays showed higher total PDE activity in apical regions (n=10, p<0.01), with more apical PDE activity in female vs. male hearts (n=5, p<0.05). In female hearts, faster apical cAMP deactivation following bolus NE was associated with a significant difference in action potential duration (APD80) between apex and base (n=3, p<0.05), but APD80 was not significantly different between regions in male hearts. Using novel whole heart imaging, we have shown female hearts display lower maximal cAMP activity and faster deactivation in the apex, in part, due to elevated PDE activity in this region. This heterogeneity was not observed in male hearts. These findings may have important implications for electrophysiological responses regulated by the cAMP pathway, particularly in heart failure, where PDE activity is altered.

Abstract 10418: Complex Action Potential Regulation in Cardiomyocytes from Induced Pluripotent Stem Cells (iPSC-CMs) Reveals a Novel Arrhythmia Susceptibility Mechanism

Background: While the cardiac sodium channel ( SCN5A ) variants S1103Y and R1193Q have been associated with LQT3 and drug-induced arrhythmia, they are too common to be highly arrhythmogenic: S1103Y allele frequency is 8.1% in Africans and R1193Q 6.1% in East Asians. We have previously reported that S1103Y had no effect on QT in a large biobank or on action potential duration (APD) in iPSC-CMs. Here, we identify a mechanism underlying control of normal baseline repolarization with these variants and study whether the mechanism is generalizable. Methods: Late sodium current (I Na-L ), I Kr , and sensitivity to the blockers dofetilide (I Kr ) and GS967 (I Na-L ) were measured in natural or genome-edited iPSC-CMs carrying S1103Y, R1193Q, or R518X, a common KCNQ1 nonsense variant in the Jervell Lange-Nielsen (JLN) syndrome and unaffected family members. Results: Although APD was unaffected by S1103Y, I Na-L was increased 2~16-fold in SY/YY compared to SS cells (p&lt;0.01) and GS967 shortened APDs only in SY/YY but not in SS cells (p&lt;0.001). Further, I Kr was increased in SY/YY ([SY] 1.2 pA/pF, n=17, p&lt;0.001, [YY] 1.2 pA/pF, n=11, p&lt;0.01) compared to SS cells (0.67 pA/pF, n=20) and challenge with dofetilide prolonged APD by 100 msec at much lower concentrations in SY/YY (median 4.1 nM [SY], 4.2 [YY], n=5-11) compared to SS cells (249 nM, n=14, p&lt;0.001); afterdepolarizations were seen more often in SY/YY (8/16) than in SS cells (1/14). R1193Q also displayed no difference in baseline APD but increased I Kr and I Na-L and increased dofetilide sensitivity. In R518X JLN iPSC-CMs, I Ks was absent as expected but I Kr was increased (1.9 pA/pF, n=5), and editing R518X to wild-type increased I Ks (1.3 pA/pF, n=10) and dramatically decreased I Kr (0.2 pA/pF, n=4). Conclusions: S1103Y and R1193Q increase I Na-L but unexpectedly do not affect baseline repolarization. We hypothesize that increased I Kr through as yet undetermined mechanisms maintains normal repolarization but at the cost of increased sensitivity to I Kr block. The findings with R518X suggest this may be a general mechanism. Our data show that preposition to genetically- or drug-mediated arrhythmias is determined by a complex interaction among multiple repolarizing currents that is not fully captured by heterologous expression systems.

L-type channel inactivation balances the increased peak calcium current due to absence of Rad in cardiomyocytes.

The L-type Ca2+ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (ICa,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates ICa,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of ICa,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak ICa,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in ICa,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell ICa,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of ICa,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β1,β2-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β1,β2-AR still yielded modulated ICa,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca2+ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β1,β2-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

AimsGp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.Methods and resultsNOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype.ConclusionTogether, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

Measures of repolarization variability predict ventricular arrhythmogenesis in heptanol-treated Langendorff-perfused mouse hearts.

BackgroundTime-domain and non-linear methods can be used to quantify beat-to-beat repolarization variability but whether measures of repolarization variability can predict ventricular arrhythmogenesis in mice have never been explored.MethodsLeft ventricular monophasic action potentials (MAPs) were recorded during constant right ventricular 8 ​Hz pacing in Langendorff-perfused mouse hearts, in the presence or absence of the gap junction and sodium channel inhibitor heptanol (0.1, 0.5, 1 or 2 ​mM).ResultsUnder control conditions, mean action potential duration (APD) was 39.4 ​± ​8.1 ​ms. Standard deviation (SD) of APDs was 0.3 ​± ​0.2 ​ms, coefficient of variation was 0.9 ​± ​0.8% and the root mean square (RMS) of successive differences in APDs was 0.15 ​± ​0.14 ​ms. Poincaré plots of APDn+1 against APDn revealed ellipsoid morphologies with a SD along the line-of-identity (SD2) to SD perpendicular to the line-of-identity (SD1) ratio of 4.6 ​± ​2.1. Approximate and sample entropy were 0.61 ​± ​0.12 and 0.76 ​± ​0.26, respectively. Detrended fluctuation analysis revealed short- and long-term fluctuation slopes of 1.49 ​± ​0.27 and 0.81 ​± ​0.36, respectively. Heptanol at 2 ​mM induced ventricular tachycardia in five out of six hearts. None of the above parameters were altered by heptanol during which reproducible electrical activity was observed (KW-ANOVA, P ​> ​0.05). Contrastingly, SD2/SD1 decreased to 2.03 ​± ​0.41, approximate and sample entropy increased to 0.82 ​± ​0.12 and 1.45 ​± ​0.34, and short-term fluctuation slope decreased to 0.82 ​± ​0.19 during the 20-s period preceding spontaneous ventricular tachy-arrhythmias (n ​= ​6, KW-ANOVA, P ​< ​0.05).ConclusionMeasures of repolarization variability, such as SD2/SD1, entropy, and fluctuation slope are altered preceding the occurrence of ventricular arrhythmogenesis in mouse hearts. Changes in these variables may allow detection of impending arrhythmias for early intervention.

Exposure to Secondhand Smoke and Arrhythmogenic Cardiac Alternans in a Mouse Model.

Background:Epidemiological evidence suggests that a majority of deaths attributed to secondhand smoke (SHS) exposure are cardiovascular related. However, to our knowledge, the impact of SHS on cardiac electrophysiology, handling, and arrhythmia risk has not been studied.Objectives:The purpose of this study was to investigate the impact of an environmentally relevant concentration of SHS on cardiac electrophysiology and indicators of arrhythmia.Methods:Male C57BL/6 mice were exposed to SHS [total suspended particles (THS): , nicotine: , carbon monoxide: , or filtered air (FA) for 4, 8, or 12 wk (]. Hearts were excised and Langendorff perfused for dual optical mapping with voltage- and -sensitive dyes.Results:At slow pacing rates, SHS exposure did not alter baseline electrophysiological parameters. With increasing pacing frequency, action potential duration (APD), and intracellular alternans magnitude progressively increased in all groups. At 4 and 8 wk, there were no statistical differences in APD or alternans magnitude between SHS and FA groups. At 12 wk, both APD and alternans magnitude were significantly increased in the SHS compared to FA group (). SHS exposure did not impact the time constant of transient decay () at any exposure time point. At 12 wk exposure, the recovery of transient amplitude with premature stimuli was slightly (but nonsignificantly) delayed in SHS compared to FA hearts, suggesting that release via ryanodine receptors may be impaired.Conclusions:In male mice, chronic exposure to SHS at levels relevant to social situations in humans increased their susceptibility to cardiac alternans, a known precursor to ventricular arrhythmia. https://doi.org/10.1289/EHP3664

Quantification of Beat-To-Beat Variability of Action Potential Durations in Langendorff-Perfused Mouse Hearts.

Background: Beat-to-beat variability in action potential duration (APD) is an intrinsic property of cardiac tissue and is altered in pro-arrhythmic states. However, it has never been examined in mice.Methods: Left atrial or ventricular monophasic action potentials (MAPs) were recorded from Langendorff-perfused mouse hearts during regular 8 Hz pacing. Time-domain, frequency-domain and non-linear analyses were used to quantify APD variability.Results: Mean atrial APD (90% repolarization) was 23.5 ± 6.3 ms and standard deviation (SD) was 0.9 ± 0.5 ms (n = 6 hearts). Coefficient of variation (CoV) was 4.0 ± 1.9% and root mean square (RMS) of successive differences in APDs was 0.3 ± 0.2 ms. The peaks for low- and high-frequency were 0.7 ± 0.5 and 2.7 ± 0.9 Hz, respectively, with percentage powers of 39.0 ± 20.5 and 59.3 ± 22.9%. Poincaré plots of APDn+1 against APDn revealed ellipsoid shapes. The ratio of the SD along the line-of-identity (SD2) to the SD perpendicular to the line-of-identity (SD1) was 8.28 ± 4.78. Approximate and sample entropy were 0.57 ± 0.12 and 0.57 ± 0.15, respectively. Detrended fluctuation analysis revealed short- and long-term fluctuation slopes of 1.80 ± 0.15 and 0.85 ± 0.29, respectively. When compared to atrial APDs, ventricular APDs were longer (ANOVA, P < 0.05), showed lower mean SD and CoV but similar RMS of successive differences in APDs and showed lower SD2 (P < 0.05). No difference in the remaining parameters was observed.Conclusion: Beat-to-beat variability in APD is observed in mouse hearts during regular pacing. Atrial MAPs showed greater degree of variability than ventricular MAPs. Non-linear techniques offer further insights on short-term and long-term variability and signal complexity.

Age‐related changes in sympathetic responsiveness and cardiac electrophysiology

IntroductionAging is associated with chronic increases in sympathetic activity, yet neurodegeneration also occurs, leading to a loss of functional sympathetic fibers in the heart. How these age‐related changes in sympathetic structure and function directly impact cardiac electrophysiology and intracellular Ca2+ handling have not been systematically characterized.ObjectiveTo determine how aging affects electrophysiological responses to sympathetic stimulation in intact mouse hearts.MethodsYoung (3 – 4 months, YWT, n=10) and aged (20 – 24 months, AGED, n=11) C57Bl6 female mice were studied. Optical mapping was performed on fully innervated hearts loaded with voltage‐ (Vm, Rh237) and calcium‐sensitive (Ca2+, Rhod2‐AM) indicators. Two stimulation electrodes were used – one on the apex of the heart for ventricular pacing and one inserted into the spinal canal (T1‐T3) for sympathetic nerve stimulation (SNS). SNS thresholds were determined as the lowest stimulation frequency (SNSL) necessary to elicit a 5% increase in heart rate (HR); the high SNS stimulation frequency (SNSH) was set at SNSL + 5 Hz. After SNS, hearts were perfused with isoproterenol (1 μM, ISO) to assess β‐adrenergic responsiveness. Hearts were then preserved for immunohistochemistry for tyrosine hydroxylase (TH: sympathetic fibers) and neurotransmitter content with HPLC analysis.ResultsBaseline HRs trended higher in AGED vs. YWT hearts (225.14±26.24 bpm vs. 166.50±13.11 bpm; p=NS). The SNSL threshold was increased in AGED vs. YWT hearts (5.36±0.34 Hz vs. 3.81±0.44 Hz, p&lt;0.05). A time course of HR increase and return to baseline in response to SNSL showed significant increases at 5 and 10s of SNS in YWT compared baseline (5 sec: 73±23% increase, p=&lt;0.0001 vs. time 0; 10 sec: 53±19% increase, p&lt;0.05 vs. time 0). There were no statistically significant changes in HR at any time point in AGED compared to baseline. However, both AGED and YWT hearts had similarly increased HRs in response to ISO. Despite an increased susceptibility to pacing‐induced arrhythmias in AGED, there were no significant differences in action potential duration compared to YWT. Ca2+ transient decay constants (tau) were decreased in YWT with SNS, but AGED tau were unaffected by SNS. AGED had significantly lower nerve fiber density than YWT (0.99±0.14% vs. 2.50±0.09% TH+, p&lt;0.01) and trended toward lower norepinephrine (NE) content (2.61±0.214 vs. 3.61±0.184 pmol/mg, p=NS).ConclusionsInterestingly, baseline HRs tended to be higher in AGED vs. YWT hearts, suggesting an increase in basal sympathetic tone, even in a de‐centralized innervated heart preparation. Stimulation thresholds for SNS‐induced changes in HR were increased in AGED and stimulation with supra‐threshold frequencies resulted in minimal changes in HR, action potential, or Ca2+ handling properties compared to YWT responses. With ISO, however, HR and action potential responses were similar in AGED and YWT hearts, suggesting that altered nerve density and function, and not necessarily β‐adrenergic responsiveness, are responsible for these differences.Support or Funding InformationThis project was funded by the NIH (NIH R01 HL111600; to C.M. Ripplinger), the Training Program in Pharmacology at UC Davis (T32GM099608; to S.D. Francis Stuart) and an Achievement Rewards for College Scientists scholarship (to S.D. Francis Stuart).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metformin prevents ischaemic ventricular fibrillation in metabolically normal pigs.

Metformin is the drug most often used to treat type 2 diabetes. Evidence suggests that metformin may reduce mortality of individuals with type 2 diabetes, but the mechanism of such an effect is unknown and outcomes of metformin treatment in people without diabetes have not been determined. If metformin favourably affected mortality of non-diabetic individuals, it might have even broader therapeutic utility. We evaluated the effect of metformin on myocardial energetics and ischaemic ventricular fibrillation (VF) in metabolically normal pigs. Domestic farm pigs were treated with metformin (30mgkg-1day-1 orally for 2-3weeks; n=36) or received no treatment (n=37). Under anaesthesia, pigs underwent up to 90min low-flow regional myocardial ischaemia followed by 45min of reperfusion. Pigs were monitored for arrhythmia, monophasic action potential morphology, haemodynamics and myocardial substrate utilisation, AMP-activated protein kinase (AMPK) phosphorylation activity and ATP concentration. Death due to VF occurred in 12% of pigs treated with metformin compared with 50% of untreated controls (p=0.03). The anti-fibrillatory effect of metformin was associated with attenuation of action potential shortening in ischaemic myocardium (p=0.02) and attenuation of the difference in action potential duration between ischaemic and non-ischaemic regions (p<0.001) compared with untreated controls. Metformin had no effect on myocardial contractile function, oxygen consumption, or glucose or lactate utilisation. During ischaemia, however, metformin treatment amplified the activation of AMPK and preserved ATP concentration in myocardium compared with untreated controls (each p<0.05). Chronic treatment of metabolically normal pigs with metformin at a clinically relevant dose reduces mortality from ischaemic VF. This protection is associated with preservation of myocardial energetics during ischaemia. Maintenance of myocardial ATP concentration during ischaemia is likely to prevent action potential shortening, heterogeneity of repolarisation, and propensity for lethal arrhythmia. The findings suggest that metformin might be protective in non-diabetic individuals with coronary heart disease.

Atrial Fibrillation Dynamics and Ionic Block Effects in Six Heterogeneous Human 3D Virtual Atria with Distinct Repolarization Dynamics.

Atrial fibrillation (AF) usually manifests as reentrant circuits propagating through the whole atria creating chaotic activation patterns. Little is yet known about how differences in electrophysiological and ionic properties between patients modulate reentrant patterns in AF. The goal of this study is to quantify how variability in action potential duration (APD) at different stages of repolarization determines AF dynamics and their modulation by ionic block using a set of virtual whole-atria human models. Six human whole-atria models are constructed based on the same anatomical structure and fiber orientation, but with different electrophysiological phenotypes. Membrane kinetics for each whole-atria model are selected with distinct APD characteristics at 20, 50, and 90% repolarization, from an experimentally calibrated population of human atrial action potential models, including AF remodeling and acetylcholine parasympathetic effects. Our simulations show that in all whole-atria models, reentrant circuits tend to organize around the pulmonary veins and the right atrial appendage, thus leading to higher dominant frequency (DF) and more organized activation in the left atrium than in the right atrium. Differences in APD in all phases of repolarization (not only APD90) yielded quantitative differences in fibrillation patterns with long APD associated with slower and more regular dynamics. Long APD50 and APD20 were associated with increased interatrial conduction block and interatrial differences in DF and organization index, creating reentry instability and self-termination in some cases. Specific inhibitions of IK1, INaK, or INa reduce DF and organization of the arrhythmia by enlarging wave meandering, reducing the number of secondary wavelets, and promoting interatrial block in all six virtual patients, especially for the phenotypes with short APD at 20, 50, and/or 90% repolarization. This suggests that therapies aiming at prolonging the early phase of repolarization might constitute effective antiarrhythmic strategies for the pharmacological management of AF. In summary, simulations report significant differences in atrial fibrillatory dynamics resulting from differences in APD at all phases of repolarization.

Intravenous anti-influenza drug oseltamivir will not induce torsade de pointes: Evidences from proarrhythmia model and action-potential assay

We evaluated proarrhythmic risk of intravenous oseltamivir with chronic atrioventricular block canine model (n = 4) and action-potential assay on guinea-pig right ventricle (n = 5). Oseltamivir in doses of 3–30 mg/kg, i.v. did not induce torsade de pointes in the canine model, whereas that in concentrations of 30–300 μM decreased maximum rate of phase 0 depolarization, shortened action potential duration at 30%, 60% and 90% repolarization levels, but prolonged difference in action-potential duration between 30% and 90% repolarization levels in a concentration-related manner. These results indicate that oseltamivir will not induce torsade de pointes clinically, since it inhibits both inward and outward currents.

Computational Representations of Myocardial Infarct Scars and Implications for Arrhythmogenesis.

Image-based computational modeling is becoming an increasingly used clinical tool to provide insight into the mechanisms of reentrant arrhythmias. In the context of ischemic heart disease, faithful representation of the electrophysiological properties of the infarct region within models is essential, due to the scars known for arrhythmic properties. Here, we review the different computational representations of the infarcted region, summarizing the experimental measurements upon which they are based. We then focus on the two most common representations of the scar core (complete insulator or electrically passive tissue) and perform simulations of electrical propagation around idealized infarct geometries. Our simulations highlight significant differences in action potential duration and focal effective refractory period (ERP) around the scar, driven by differences in electrotonic loading, depending on the choice of scar representation. Finally, a novel mechanism for arrhythmia induction, following a focal ectopic beat, is demonstrated, which relies on localized gradients in ERP directly caused by the electrotonic sink effects of the neighboring passive scar.

Transmural APD gradient synchronizes repolarization in the human left ventricular wall.

The duration and morphology of the T wave predict risk for ventricular fibrillation. A transmural gradient in action potential duration (APD) in the ventricular wall has been suggested to underlie the T wave in humans. We hypothesize that the transmural gradient in APD compensates for the normal endocardium-to-epicardium activation sequence and synchronizes repolarization in the human ventricular wall. We made left ventricular wedge preparations from 10 human donor hearts and measured transmural activation and repolarization patterns by optical mapping, while simultaneously recording a pseudo-ECG. We also studied the relation between local timings of repolarization with the T wave in silico. During endocardial pacing (1 Hz), APD was longer at the subendocardium than at the subepicardium (360 ± 17 vs. 317 ± 20 ms, P < 0.05). The transmural activation time was 32 ± 4 ms and resulted in final repolarization of the subepicardium at 349 ± 18 ms. The overall transmural dispersion in repolarization time was smaller than that of APD. During epicardial pacing, the dispersion in repolarization time increased, whereas that of APD remained similar. The morphology of the T wave did not differ between endocardial and epicardial stimulation. Simulations explained the constant T wave morphology without transmural APD gradients. The intrinsic transmural difference in APD compensates for the normal cardiac activation sequence, resulting in more homogeneous repolarization of the left ventricular wall. Our data suggest that the transmural repolarization differences do not fully explain the genesis of the T wave.