Prolongation Of Action Potential Duration Research Articles

Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption

Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.

Inhibition of late sodium current via PI3K/Akt signaling prevents cellular remodeling in tachypacing-induced HL-1 atrial myocytes

An aberrant late sodium current (INa,Late) caused by a mutation in the cardiac sodium channel (Nav1.5) has emerged as a contributor to electrical remodeling that causes susceptibility to atrial fibrillation (AF). Although downregulation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with AF, the molecular mechanisms underlying the negative regulation of INa,Late in AF remain unclear, and potential therapeutic approaches are needed. In this work, we constructed a tachypacing-induced cellular model of AF by exposing HL-1 myocytes to rapid electrical stimulation (1.5 V/cm, 4 ms, 10 Hz) for 6 h. Then, we gathered data using confocal Ca2+ imaging, immunofluorescence, patch-clamp recordings, and immunoblots. The tachypacing cells displayed irregular Ca2+ release, delayed afterdepolarization, prolonged action potential duration, and reduced PI3K/Akt signaling compared with controls. Those detrimental effects were related to increased INa,Late and were significantly mediated by treatment with the INa,Late blocker ranolazine. Furthermore, decreased PI3K/Akt signaling via PI3K inhibition increased INa,Late and subsequent aberrant myocyte excitability, which were abolished by INa,Late inhibition, suggesting that PI3K/Akt signaling is responsible for regulating pathogenic INa,Late. These results indicate that PI3K/Akt signaling is critical for regulating INa,Late and electrical remodeling, supporting the use of PI3K/Akt-mediated INa,Late as a therapeutic target for AF.

Empagliflozin and dapagliflozin increase Na current in human cardiomyocytes

Abstract Background Empagliflozin (EMPA) and dapagliflozin (DAPA) are sodium-glucose cotransporter 2 inhibitors (SGLT2i) used for the treatment of type 2 Diabetes Mellitus (T2DM). Both drugs reduce morbidity and mortality in heart failure (HF) patients with reduced or preserved ejection fraction, even in the absence of T2DM. Moreover, these drugs decrease ventricular arrhythmias and sudden cardiac death in HF patients. The sodium current (INa), carried by Nav1.5 channels, is responsible for cardiac action potential (AP) depolarization and determines excitability and conduction velocity. In HF patients, the expression of Nav1.5 channels is reduced, leading to a decrease of ventricular excitability that enhances the arrhythmic risk. Purpose We aimed to determine the effects of EMPA and DAPA on human cardiac INa and AP characteristics. Methods Peak INa and ventricular-like APs were recorded in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) using patch-clamp techniques. INa was also recorded in CHO cells transiently transfected with human Nav1.5+Navβ1 channels. In all cases, EMPA or DAPA (1 μM) were added to culture media and incubated for 24-h. Results APs recorded in hiPSC-CMs exhibited automatic activity and incubation with EMPA or DAPA did not modify spontaneous beating frequency (0.39±0.04 Hz; P>0.05, n≥16). In cells driven at 1 Hz, none of the drugs modified resting membrane potential (−76.7±1.4 mV; P>0.05, n≥11), but significantly increased AP amplitude from 98.6±3.6 to 105±2.2 (DAPA) and 107±2.3 mV (EMPA) (P<0.05). Interestingly, only EMPA lengthened AP duration measured at 20%, 50%, and 90% (from 605.6±31.3 to 760.5±59.0 ms, P<0.05) of repolarization. In hiPSC-CMs EMPA increased maximum INa density by 64% (from −156.0±28.0 to −256.4±28.1 pA/pF, P<0.05, n≥7) and shifted the midpoint (Vh) of the inactivation curve to more hyperpolarized potentials (from −97.3±4.5 to −108.6±4.4 mV, P<0.05, n≥7). In turn, DAPA increased maximum INa density by 24% (to −193.8±26.6 pA/pF) and shifted the Vh of the activation curve to more negative potentials (from −47.2±1.6 mV to −55.5±2.8 mV, P<0.05), an effect that would increase the INa at negative potentials coinciding with channel opening. None of the drugs modified the time course of current activation or inactivation. In CHO cells, EMPA and DAPA effects on INa were identical to those observed on hiPSC-CM. These results suggest that both SGLT2i increase INa by enhancing Nav1.5 expression into the cell membrane, by a direct gating effect on the channel, or by a combination of both. Conclusions In human cardiomyocytes, EMPA and DAPA increase INa and the AP amplitude. Moreover, EMPA, but not DAPA, prolonged AP duration. We propose that EMPA and DAPA exhibit a unique mechanism that increases cardiac excitability and conduction velocity and could contribute to the prevention of arrhythmic events in HF patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e innovaciόnInstituto de de Salud Carlos III

The interplay of sex-specific electrophysiology and repolarization heterogeneity governs ventricular arrhythmia sustainability in women

Abstract Background The role of sex-specific electrophysiology in the development of lethal ventricular arrhythmia is poorly understood. Since female ventricles have a longer action potential duration (APD) than male ventricles, we hypothesized that female hearts are more vulnerable to sustained ventricular arrhythmia in the presence of known arrhythmogenic substrates. Purpose To determine if prolonged APD in females leads to sustained ventricular arrhythmia in the presence of left ventricular (LV) hypertrophy, diffuse fibrosis, and repolarization heterogeneity. Methods Magnetic resonance imaging (MRI) data were used to develop computational biventricular models of two aortic stenosis patients (male, 54 years; female, 64 years) before (LV hypertrophy) and 3 months after (regressed LV hypertrophy) aortic valve replacement (AVR). These patients were chosen since they exhibit reduced LV mass, less fibrosis, and no ventricular arrhythmias after AVR. The models were assigned sex-specific cellular electrophysiology and an apicobasal repolarization gradient derived from human data. Simulations of apical pacing with a cycle length (CL) of 750 ms were performed while APD at 90% repolarization (APD90) and repolarization time (activation time + APD90) were calculated. Reentrant arrhythmia was induced by rapidly pacing the LV with a 2 mm wide apicobasal line electrode at CL≤265 ms. The simulations were repeated in the presence of diffuse myocardial fibrosis derived from the MRI patient data, and repeated again with an apicobasal repolarization gradient reduced by ≈35%. Ventricular mass was calculated as the product of model volume and myocardial density. Results In hypertrophic ventricles with default apicobasal repolarization gradient, rapid pacing led to reentry that lasted longer in the female (>25 s) than in the male (1.0 s) ventricles. The duration of reentry increased in the presence of fibrosis in the male ventricles (male: 6.6 s; female >25 s) and decreased in the female ventricles when the apicobasal repolarization gradient was reduced (Figure). Sustained reentry (>25 s) was not observed in the ventricles with regressed hypertrophy, regardless of the presence of fibrosis or repolarization gradient. Average APD90 was longer in the female ventricles both before (male: 267±35 ms; female: 318±34 ms) and after (male: 272±35 ms; female: 328±38 ms) AVR. The female heart had lower myocardial mass than the male heart both before (280 vs. 406 g) and after AVR (227 vs. 310 g). Conclusion Longer APD and steeper apicobasal repolarization gradients in hypertrophic female ventricles resulted in sustained arrhythmia, despite their lower mass when compared to males. The absence of sustained reentry in the post-AVR models indicates a potential electrophysiological benefit of AVR and encourages future clinical studies to quantify sex-specific differences in spatial repolarization heterogeneity. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union's Horizon 2020 research and innovation program under the ERA-NET co-fund action No. 680969 with ANR (ERA-CVD SICVALVES) and Agence Nationale de la Recherche

Cellular determinants of arrhythmic risk in hypertrophic cardiomyopathy

The most devastating consequence of HCM is sudden cardiac death (SCD) due to ventricular fibrillation. The positive correlation between the extent of late gadolinium enhancement and the arrhythmic risk in HCM suggests that ventricular arrhythmias originate from the fibrotic regions. However, recent data suggest that enhanced cellular automaticity (early- or delayed-afterdepolarizations, EADs or DADs-) may be clinically more relevant in promoting ventricular arrhythmias in patients. Aiming to better understand the cellular and molecular mechanisms of arrhythmogenesis in HCM and to establish a reliable arrhythmic risk stratification in patients, we performed a translational study in HCM patients who underwent surgical myectomy, by combining a clinical follow-up study with in vitro assessments of cellular arrhythmogenicity. We retrospectively studied 61 HCM patients who underwent surgical myectomy. At the time of surgery, fresh ventricular tissue was collected and used to isolate single ventricular cardiomyocytes (CMs), which were used for patch-clamp and Ca 2+ imaging experiments to assess the occurrence of EADs and DADs. Patients were followed up for a median time of 8 years and the occurrence of non-sustained ventricular tachycardia (NSVT) or life-threateningarrhythmic events (LAE) was monitored. EADs occurred in CMs from 36% of patients and were associated with prolonged action potential duration. DADs occurred in 24% of patients and were associated with abnormalities of intracellular Ca 2+ handling. The occurrence of NSVT/ LAE in patients strongly correlated with the presence of DADs in cardiomyocytes. The presence of pro-arrhythmic changes appears to be necessary for arrhythmia generation in HCM and seems to be related with specific alterations at ECG level, that might be used as clinical arrhythmia predictors in HCM patients. Fibrosis per se is not a major predictor of arrhythmias in HCM but may contribute to generate sustained arrhythmias in the presence of substantial cellular triggers (DADs).

Protein Kinase D1 Regulates Cardiac Hypertrophy, Potassium Channel Remodeling, and Arrhythmias in Heart Failure.

Background Structural and electrophysiological remodeling characterize heart failure (HF) enhancing arrhythmias. PKD1 (protein kinase D1) is upregulated in HF and mediates pathological hypertrophic signaling, but its role in K+ channel remodeling and arrhythmogenesis in HF is unknown. Methods and Results We performed echocardiography, electrophysiology, and expression analysis in wild-type and PKD1 cardiomyocyte-specific knockout (cKO) mice following transverse aortic constriction (TAC). PKD1-cKO mice exhibited significantly less cardiac hypertrophy post-TAC and were protected from early decline in cardiac contractile function (3 weeks post-TAC) but not the progression to HF at 7 weeks post-TAC. Wild-type mice exhibited ventricular action potential duration prolongation at 8 weeks post-TAC, which was attenuated in PKD1-cKO, consistent with larger K+ currents via the transient outward current, sustained current, inward rectifier K+ current, and rapid delayed rectifier K+ current and increased expression of corresponding K+ channels. Conversely, reduction of slowly inactivating K+ current was independent of PKD1 in HF. Acute PKD inhibition slightly increased transient outward current in TAC and sham wild-type myocytes but did not alter other K+ currents. Sham PKD1-cKO versus wild-type also exhibited larger transient outward current and faster early action potential repolarization. Tachypacing-induced action potential duration alternans in TAC animals was increased and independent of PKD1, but diastolic arrhythmogenic activities were reduced in PKD1-cKO. Conclusions Our data indicate an important role for PKD1 in the HF-related hypertrophic response and K+ channel downregulation. Therefore, PKD1 inhibition may represent a therapeutic strategy to reduce hypertrophy and arrhythmias; however, PKD1 inhibition may not prevent disease progression and reduced contractility in HF.

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Grapefruit Flavonoid Naringenin Sex-Dependently Modulates Action Potential in an In Silico Human Ventricular Cardiomyocyte Model

Recent in vitro studies showed that grapefruit (Citrus × paradisi) flavonoid naringenin alters the function of cardiac ion channels. Here, we explored the effect of naringenin on cardiomyocyte action potentials (APs) using a detailed in silico model of ventricular electrophysiology. Concentration-dependent effects of naringenin on seven major cardiac ion channels were incorporated into the Tomek–Rodriguez modification of O’Hara–Rudy (ToR-ORd) human ventricular endocardium model. To investigate the sex-dependent effect of naringenin, previously reported sex-specific ionic modifications were implemented into the model. Next, populations of 1000 models accommodating intercellular variability were generated. The results show, naringenin at various concentrations prolonged AP duration (APD) in male and female cardiomyocytes. Pacing cells at higher frequencies abbreviated APD differently in males versus females; for example, at 3 Hz, 50 μM naringenin induced AP and calcium alternans only in the female cardiomyocyte. Finally, a population modeling approach corroborated that naringenin significantly prolonged APD in a concentration-dependent manner, with a larger effect in females than in males. In conclusion, our study demonstrates that the APD-prolonging effect of naringenin was larger in females, and that pacing at faster rates induces AP alternation earlier in females, suggesting a potentially higher proarrhythmic risk of naringenin in females than in males.

Abstract P2091: Age-dependent Remodeling Of The Perinexus Modulates The Phenotype And Arrhythmic Risk Of Long Qt Syndrome Type 3

Background: The Long-QT Syndrome Type 3 (LQT3) is characterized by action potential duration (APD) prolongation due to cardiac voltage-gated sodium channel (Nav1.5) gain of function. Arrhythmia risk increases with age in LQT3 patients, but it is unknown whether this is due to progressive APD prolongation. Recent studies have shown that the LQT3 phenotype can be exacerbated by widening the perinexus, an intercalated disc nanodomain adjacent to gap junctions. However, it remains unclear if the perinexus mediates the LQT3 phenotype during aging. Objective: To investigate the role of the perinexus during aging in LQT3. Methods: Ventricular epicardial APD was obtained by optically mapping Langendorff-perfused adult (3-month) and aged (15-month) guinea pig hearts. LQT3 phenotype was induced with anemone toxin (ATXII), and the perinexus was widened with an intercalated disc adhesion inhibitor (βadp1). Perinexal width (Wp) was quantified by transmission electron microscopy. Susceptibility to arrhythmia was assessed with burst pacing in the absence and presence of βadp1 and evaluated from electrocardiograms (ECG). Results: APD was not different between adult and aged hearts at baseline, with ATXII, or ATXII+βadp1. Wp was significantly wider in adult LQT3 hearts either without or with βadp1 relative to aged hearts (without βadp1: 29.6±2.5 vs 22.9±3.8 nm; with βadp1: 38.3±1.7 vs 29.9±1.2 nm). These data suggest the perinexus narrows with aging and attenuates previously reported cellular APD prolongation such that there is no difference in whole-heart, epicardial APD between adult and aged LQT3 hearts. However, ECG Tpeak-Tend interval was not prolonged in adult, but significantly prolonged in aged hearts after treatment with ATXII+βadp1 (18.7±5.6 vs 35.5±11.5 ms), suggesting that βadp1 increases transmural dispersion of repolarization during aging in LQT3. Furthermore, ATXII+βadp1 did not induce arrhythmias in adult (0/6) but was arrhythmogenic in aged hearts (3/5). Conclusions: Perinexal narrowing associated with aging attenuates APD prolongation and reduces arrhythmic risk in aged LQT3 hearts. Loss of perinexal adhesion in aged LQT3 hearts results in significant and heterogeneous APD prolongation and increased risk of arrhythmia.

Abstract P2096: Long Qt Syndrome Type 3 Phenotype Dependence On Extracellular Sodium And The Perinexus In A Genetic Mouse Model

Background: Long QT Syndrome Type 3 (LQT3) is caused by cardiac voltage-gated sodium channel (Nav1.5) gain of function and characterized by action potential duration (APD) prolongation. A guinea pig model of drug-induced LQT3 demonstrated that elevating sodium and widening the perinexus, an intercalated disc cleft adjacent to gap junctions, individually and synergistically exacerbate APD prolongation. The synergistic effect suggests that high sodium with cardiac edema is a risk factor for LQT3 patients. However, it remains unclear if this synergistic effect occurs in a genetic model of LQT3. Purpose: To investigate individual and combined effects of sodium concentration and perinexal widening in a genetic mouse model of LQT3. Methods: Ventricular action potential duration at 30 (APD30) and 90 (APD90) percent of repolarization was measured from optically mapped, Langendorff-perfused wild type (WT) and ΔKPQ mouse hearts. Hearts were perfused with sodium concentrations of 145 or 160 mM, in the absence or presence of an intercalated disc adhesion inhibitor (βadp1) inducing perinexal widening. Results: At baseline with 145 mM sodium, APD30 and APD90 were significantly greater in ΔKPQ than WT hearts. Surprisingly, increased sodium (160 mM), significantly decreased APD90 in WT but not ΔKPQ hearts, which is inconsistent with results from the drug-induced LQT3 guinea pig model. In contrast, 160mM sodium significantly decreased APD30 in both genotypes suggesting elevated sodium increases the transient outward potassium current (Ito). Interestingly, with 145 mM sodium, βadp1 treatment did not significantly change APD30 or APD90 in either genotype. However, increasing sodium from 145 to 160 mM with βadp1 significantly increased APD90 in ΔKPQ hearts but not WT. Conclusions: Synergistic APD prolongation was modest in a genetic LQT3 mouse when elevating sodium and widening the perinexus relative to drug-induced LQT3 in guinea pig. A significant difference in action potential morphology between mice and guinea pig is the over-expression of Ito in mice and its absence in guinea pig. Future studies will investigate whether transient outward potassium channels contribute to the effect of high sodium on APD shortening during early repolarization.

Candesartan Cilexetil Attenuates Arrhythmogenicity Following Pressure Overload in Rats via the Modulation of Cardiac Electrical and Structural Remodeling and Calcium Handling Dysfunction.

BackgroundCardiac hypertrophy is associated with abnormal electrophysiology and increased arrhythmia risk. This study assessed whether candesartan cilexetil, an angiotensin II type 1 receptor blocker, could suppress arrhythmogenecity by attenuating cardiac electrical remodeling and calcium mishandling in rats with pressure‐overload hypertrophy.Methods and ResultsMale Sprague‐Dawley rats were randomly subjected to abdominal aorta banding or sham procedure and received either candesartan cilexetil (3.0 mg/kg per day) or vehicle by gavage for 5 weeks. Pressure overload was characterized by compensated left ventricular (LV) hypertrophy and fibrosis, increased LV pressure and its decay time, and prolonged corrected QT interval, all of which were attenuated by candesartan cilexetil treatment. Candesartan cilexetil–treated banded rat hearts displayed shorter QT intervals and lower vulnerability to atrial and ventricular tachyarrhythmias than vehicle‐treated banded hearts. Candesartan cilexetil prevented banding‐induced prolonged action potential duration and reduced the occurrence of triggered activity in LV papillary muscles. In addition, the prolonged time to 50% cell relengthening and calcium transient decay time were normalized in LV myocytes from candesartan cilexetil–treated banded rats, along with a normalization of decreased SERCA2a (sarco[endo]plasmic reticulum calcium‐ATPase) expression in LV tissues. Furthermore, candesartan cilexetil normalized depressed transient outward potassium current densities and protein and mRNA levels of both voltage‐gated potassium 4.2 and 4.3 channel subunits (Kv4.2 and Kv4.3) in banded rats.ConclusionsCandesartan cilexetil protects the heart from pressure overload‐induced adverse electrical remodeling by preserving potassium channel densities. In addition, calcium handling and its molecular regulation also improved after treatment. These beneficial effects may contribute to a lower susceptibility to arrhythmias in hearts from candesartan cilexetil–treated pressure‐overloaded rats.

Remdesivir (VEKLURY) for Treating COVID-19: Guinea Pig Ex Vivo and In Vivo Cardiac Electrophysiological Effects.

Bradycardia and QTc interval prolongation on the ECG have been reported with remdesivir (Veklury), an antiviral drug recently approved for treating severely ill patients with COVID-19. The objective was to evaluate the effects of remdesivir on cardiac electrophysiology ex vivo and in vivo. Ex vivo: Langendorff retroperfusion experiments were performed on isolated hearts from male Hartley guinea pigs (n = 23, total) exposed to either remdesivir 3, 10, or 30 µmol/L to assess drug-induced prolongation of the monophasic action potential duration measured at 90% repolarization (MAPD 90 ). In vivo: ECG recordings using wireless cardiac telemetry were performed in guinea pigs (n = 6) treated with daily i.p. doses of remdesivir 5 mg/kg on day 1 and 2.5 mg/kg on days 2-10. Ex vivo remdesivir (3, 10, and 30 µmol/L) had no statistically significant effect on MAPD 90 , while pacing the hearts at basic stimulation cycle lengths of 200 or 250 milliseconds, or when the hearts were not paced and beating at their intrinsic heart rate. In a second set of similar ex vivo experiments, remdesivir 10 µmol/L did not potentiate the MAPD 90 -prolonging effects of dofetilide 20 nmol/L (n = 4) hearts. In vivo remdesivir caused small but statistically significant prolongations of the RR and QTc F intervals at day 1 (5 mg/kg) and at day 10 (2.5 mg/kg). No ventricular arrhythmias were ever observed under the effect of remdesivir. Remdesivir causes bradycardia, and mild QTc prolongation, which nonetheless, could be of clinical relevance in many hospitalized patients with COVID-19 concomitantly treated with multiple drugs.

Cardiac Electrophysiological Changes and Downregulated Connexin 43 Prompts Reperfusion Arrhythmias Induced by Hypothermic Ischemia-Reperfusion Injury in Isolated Rat Hearts.

The purpose of this study was to determine the utility of the monophasic action potential (MAP) changes as an arrhythmic biomarker in hypothermic ischemia-reperfusion. The hypothermic ischemia-reperfusion model was subjected to 60min of cardioplegic arrest while the isolated rat hearts were preserved with a multidose cold K-H solution at 4°C. During the reperfusion period, the heart's arrhythmia and monophasic action potential were also monitored. The myocardial damage was assessed using HE and TTC stains. Immunohistochemistry and Western blotting were used to assess the expression and distribution of Connexin 43 (Cx43) and Akt. Collectively, prolonged action potential durations, increased dispersion of repolarization, and downregulated and lateralized Cx43 all contribute to the derangement of electrical impulse propagation that may underlie arrhythmogenesis in the cold ischemic heart following cardioplegic arrest. MAP might be used as a biomarker for arrhythmias caused by hypothermic ischemia-reperfusion.

Positive rate-dependent action potential prolongation by modulating potassium ion channels.

Pharmacological agents that prolong action potential duration (APD) to a larger extent at slow rates than at the fast excitation rates typical of ventricular tachycardia exhibit reverse rate dependence. Reverse rate dependence has been linked to the lack of efficacy of class III agents at preventing arrhythmias because the doses required to have an antiarrhythmic effect at fast rates may have pro‐arrhythmic effects at slow rates due to an excessive APD prolongation. In this report, we show that, in computer models of the ventricular action potential, APD prolongation by accelerating phase 2 repolarization (by increasing IKs) and decelerating phase 3 repolarization (by blocking IKr and IK1) results in a robust positive rate dependence (i.e., larger APD prolongation at fast rates than at slow rates). In contrast, APD prolongation by blocking a specific potassium channel type results in reverse rate dependence or a moderate positive rate dependence. Interventions that result in a strong positive rate dependence tend to decrease the repolarization reserve because they require substantial IK1 block. However, limiting IK1 block to ~50% results in a strong positive rate dependence with moderate decrease in repolarization reserve. In conclusion, the use of a combination of IKs activators and IKr and IK1 blockers could result in APD prolongation that potentially maximizes antiarrhythmic effects (by maximizing APD prolongation at fast excitation rates) and minimizes pro‐arrhythmic effects (by minimizing APD prolongation at slow excitation rates).

TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload.

Background: Cardiac hypertrophy (CH) occurs with an increase in myocardium mass as an adaptive compensation to increased stress. Prolonged CH causes decompensated heart failure (HF). Enhanced angiogenesis by vascular endothelial growth factor (VEGF) is observed in hypertrophied hearts; impaired angiogenesis by angiotensin II (AngII) is observed in failing hearts. Angiogenesis is executed by vascular endothelial cells (ECs). Abnormal Ca2+ homeostasis is a hallmark feature of hypertrophied and failing hearts. Ca2+-activated chloride channel transmembrane protein 16A (TMEM16A) is expressed in cardiomyocytes and ECs but its role in heart under stress remains unknown. Methods: Pressure-overload-induced CH and HF mouse models were established. Echocardiography was performed to evaluate cardiac parameters. Quantitative real-time PCR, traditional and simple western assays were used to quantify molecular expression. Whole-cell patch-clamp experiments were used to detect TMEM16A current (ITMEM16A) and action potential duration (APD) of cardiomyocytes. VEGF and AngII were used separately in ECs culture to simulate enhanced or impaired angiogenesis, respectively. TMEM16A low-expressed and over-expressed ECs were obtained by siRNA or lentivirus transfection. Wound healing, tube formation and ECs spheroids sprouting assays were performed to assess migration and angiogenesis. Results: Neither TMEM16A molecular expression levels nor whole-cell ITMEM16A density varied significantly during the development of CH and HF. ITMEM16A comprises transient outward current, but doesn’t account for APD prolongation in hypertrophied or failing cardiomyocytes. In cultured ECs, TMEM16A knockdown inhibited migration and angiogenesis, TMEM16A overexpression showed opposite result. Promotion of migration and angiogenesis by VEGF was decreased in TMEM16A low-expressed ECs but was increased in TMEM16A over-expressed ECs. Inhibition of migration and angiogenesis by AngII was enhanced in TMEM16A low-expressed ECs but was attenuated in TMEM16A over-expressed ECs. Conclusion: TMEM16A contributes insignificantly in myocardium remodeling during pressure-overload. TMEM16A is a positive regulator of migration and angiogenesis under normal condition or simulated stress. TMEM16A may become a new target for upregulation of angiogenesis in ischemic disorders like ischemic heart disease.

Mechanistic Insights Into Inflammation-Induced Arrhythmias: A Simulation Study.

Cardiovascular diseases are the primary cause of death of humans, and among these, ventricular arrhythmias are the most common cause of death. There is plausible evidence implicating inflammation in the etiology of ventricular fibrillation (VF). In the case of systemic inflammation caused by an overactive immune response, the induced inflammatory cytokines directly affect the function of ion channels in cardiomyocytes, leading to a prolonged action potential duration (APD). However, the mechanistic links between inflammatory cytokine-induced molecular and cellular influences and inflammation-associated ventricular arrhythmias need to be elucidated. The present study aimed to determine the potential impact of systemic inflammation on ventricular electrophysiology by means of multiscale virtual heart models. The experimental data on the ionic current of three major cytokines [i.e., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), and interleukin-6 (IL-6)] were incorporated into the cell model, and the effects of each cytokine and their combined effect on the cell action potential (AP) were evaluated. Moreover, the integral effect of these cytokines on the conduction of excitation waves was also investigated in a tissue model. The simulation results suggested that inflammatory cytokines significantly prolonged APD, enhanced the transmural and regional repolarization heterogeneities that predispose to arrhythmias, and reduced the adaptability of ventricular tissue to fast heart rates. In addition, simulated pseudo-ECGs showed a prolonged QT interval—a manifestation consistent with clinical observations. In summary, the present study provides new insights into ventricular arrhythmias associated with inflammation.

Metformin Reduces Potassium Currents and Prolongs Repolarization in Non-Diabetic Heart.

Metformin is the first choice drug for the treatment of type 2 diabetes due to positive results in reducing hyperglycaemia and insulin resistance. However, diabetic patients have higher risk of ventricular arrhythmia and sudden cardiac death, and metformin failed to reduce ventricular arrhythmia in clinical trials. In order to explore the mechanisms responsible for the lack of protective effect, we investigated in vivo the effect of metformin on cardiac electrical activity in non-diabetic rats; and in vitro in isolated ventricular myocytes, HEK293 cells expressing the hERG channel and human induced pluripotent stem cells derived cardiomyocytes (hIPS-CMs). Surface electrocardiograms showed that long-term metformin treatment (7 weeks) at therapeutic doses prolonged cardiac repolarization, reflected as QT and QTc interval duration, and increased ventricular arrhythmia during the caffeine/dobutamine challenge. Patch-clamp recordings in ventricular myocytes isolated from treated animals showed that the cellular mechanism is a reduction in the cardiac transient outward potassium current (Ito). In vitro, incubation with metformin for 24 h also reduced Ito, prolonged action potential duration, and increased spontaneous contractions in ventricular myocytes isolated from control rats. Metformin incubation also reduced IhERG in HEK293 cells. Finally, metformin incubation prolonged action potential duration at 30% and 90% of repolarization in hIPS-CMs, which is compatible with the reduction of Ito and IhERG. Our results show that metformin directly modifies the electrical behavior of the normal heart. The mechanism consists in the inhibition of repolarizing currents and the subsequent decrease in repolarization capacity, which prolongs AP and QTc duration.

Pinocembrin Decreases Atrial Fibrillation Susceptibility in a Rodent Model of Depression.

BackgroundDepression is often comorbid with cardiovascular diseases and contributes to the development and maintenance of atrial fibrillation (AF). Ample research demonstrated that pinocembrin had protective effects on the neuropsychiatric and cardiovascular systems via its pharmacological properties. However, whether pinocembrin protects from AF in depression models is not known. The present research investigated antiarrhythmic effects of pinocembrin and the underlying mechanisms in depressed rats.MethodsOne hundred and ten male Sprague Dawley rats were randomly divided into six groups: the CTL group (the normal rats administered saline), the CTP group (the normal rats administered pinocembrin), the MDD group (the depressed rats administered saline), the MDP group (the depressed rats administered pinocembrin), the MDA group (the depressed rats administered apocynin), and the MPA group (the depressed rats administered both pinocembrin and apocynin). Chronic unpredictable mild stress (CUMS) was performed for 28 days to establish the depression model. Pinocembrin was administered via gavage from Day 8 to Day 28, and apocynin was administered via intraperitoneal injection from Day 1 to Day 28. The effects were evaluated using behavioral measurements, in vitro electrophysiological studies, whole-cell patch-clamp recordings, biochemical detection, Western blot, and histological studies.ResultsPinocembrin treatment significantly attenuated the abnormality of heart rate variability (HRV), the prolongation of action potential duration (APD), the shortening of the effective refractory period (ERP), the reduction of transient outward potassium current (Ito), and the increase in L-type calcium current (ICa–L), which increase susceptibility to AF in a rat model of depression. Compared to the depressed rats, pinocembrin also increased the content of Kv4.2, Kv4.3, and atrial gap junction channel Cx40 and decreased the expression level of Cav1.2, which ameliorated oxidative stress and inhibited the ROS/p-p38MAPK pro-apoptotic pathway and the ROS/TGF-β1 pro-fibrotic pathway.ConclusionPinocembrin is a therapeutic strategy with great promise for the treatment of AF in depressed patients by reducing oxidative stress.

Determining the role of uremic toxins on cardiac electrophysiology and pro-arrhythmia during chronic kidney disease

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Young Talent Programme, CVON Introduction Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are one of the CKD-related factors already linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients is cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible still remain poorly understood. Purpose To assess the potentially increased pro-arrhythmic risk of pre-identified UTs at clinically relevant concentrations: 1) acutely in isolated adult dog ventricular cardiomyocytes (ADCMs) and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), 2) chronically in iPSC-CMs and 3) both conditions in human embryonic kidney (HEK) cells transfected with constructs that generate different ion channels, using optical and manual electrophysiological in vitro approaches. Methods and results Using the voltage sensitive membrane dye FluoVolt and manual patch clamp, action potential duration (APD) prolongation, considered as a pro-arrhythmic parameter, was assessed in ADCMs and iPSC-CMs. Acute exposure of indoxyl sulfate (IS), kynurenine (KYN) or kynurenic acid (KYNA) induced no alterations in APD in ADCMs and iPSC-CMs. iPSC-CMs, although having rather immature electrophysiological characteristics, showed significant APD prolongation when exposed chronically (48h) to those UTs. Manual patch clamp on HEK293 cells was performed to investigate individual ion currents to reveal underlying mechanisms. The repolarizing current IKr, often most sensitive and responsible for APD alterations, was not acutely affected by IS, KYN, or KYNA. However, chronic exposure (48h) to IS or KYNA significantly decreased IKr at clinically relevant concentrations. Finally, the protein level of the ion channel conducting IKr was determined in HEK293 cells, to further unveil the mechanism behind the effects of UTs on channels conducting IKr. Chronic exposure (48-72h) of the three UTs decreased total ion channel expression at pathological concentrations, which could be responsible for the decreased IKr and prolonged APD. Conclusion The results show the proof of concept to identify potentially pro-arrhythmogenic UTs and their mode of action, allowing the provision of a clinically relevant overview of (patho)physiological concentrations of UTs in both acute and chronic exposure.

Sex differences in atrial electrical properties

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss Heart Foundation Background Pronounced sex differences are known in the incidence and recurrence rates of atrial fibrillation (AF). In general, men carry a higher risk for AF - particularly at younger age - during which women are protected. Purpose In this project, we aimed to investigate electrophysiological properties of the atria that may underlie sex differences in AF development in the younger population. Methods First, we assessed sex differences in P-wave morphology (duration: PWD; area: PWA, amplitude) in 12-lead ECG in healthy volunteers (men 31.3±1.6 y, n=10; women 29.8±1.6 y, n=10) and wildtype (WT) rabbits (3-6 months, males n=12, females n=15). We investigated sex differences in atrial action potential (AP) in WT rabbits of both sexes using sharp electrode recording technique (males n=5, females n=6). To elucidate underlying mechanisms, sex differences on atrial inwardly rectifying potassium current IK1 at 37°C were evaluated using whole-cell patch-clamp technique in isolated atrial rabbit cardiomyocytes; to evaluate the long-term effect of sex hormones on IK1, we incubated the isolated cardiomyocytes for 24h with 3 or 10µM of 17β-estradiol (EST) or of dihydrotestosterone (DHT). Results Analyzing ECG parameters (figure 1), we observed a prolonged PWD and an increased PWA both in healthy men (p<0.0001) and in male rabbits (p<0.0001) as compared to healthy women or female rabbits, which may theoretically be due to a slower conduction velocity or longer AP duration in males. Intracellular AP recordings (figure 2A) showed a more negative atrial resting membrane potential (RMP, males -83.07±0.3mV vs. females -74.33±2.6mV, p<0.05) and a prolonged atrial AP duration (APD10, 18.20±3.6ms vs. 10.06±1.9ms, p=0.06; APD50, 36.60±6.6ms vs. 20.06±1.9ms, p<0.05; APD90, 91.00±10.3ms vs. 74.00±4.0ms, p=0.13) in males as compared to females. As highlighted in figure 2B, male atrial cardiomoycytes had smaller IK1 current densities, which could be due to high DHT concentration; in line with these findings (figure 2C) 24h DHT incubation of atrial cardiomyocytes from females reduced IK1, mimicking the sex differences observed. Conclusions Sex impacts on atrial electrophysiology, resulting in sex differences in P-wave morphology, AP parameters and ionic current densities. Delayed (early) repolarization in males may be causatively linked to the observed changes in P-wave morphology. The reduction of IK1 observed in males may be due to testosterone, since this reduction was observed even in female cardiomyocytes when incubated with DHT.