Monophasic Action Potential Recordings Research Articles

Loss of protein phosphatase 2A regulatory subunit PPP2R5A is associated with increased incidence of stress-induced proarrhythmia.

Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear. The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca2+ transients under increased pacing frequencies and determination of total K+ channel currents (I K). ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT. These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca2+ tansients observed.

Acute afterload leads to increased electrophysiological heterogeneity after myocardial infarction

Abstract Background Myocardial infarction (MI) results in altered mechanical loading and changes in the cardiac electrical properties. The infarct border zone is pro-arrhythmic but the exact role of mechano-electrical coupling remains unclear. Objective We studied spatial electrical heterogeneity in MI animals during acute afterload increase using a novel E-field methodology for high resolution mapping of local activation-repolarization intervals (ARI) in vivo. Methods Anterior-septal MI was induced in five domestic pigs by 120-minute occlusion of the left anterior descending artery followed by reperfusion. This led to an infarct size of 17.7±2.1% of the left ventricle. After 1 month, electro-anatomical mapping was performed before and during an acute afterload challenge induced by partially inflating a balloon in the descending aorta. A non-contact recording of a 64-electrode array was translated to 2048 non-contact electrograms distributed over the left ventricle. The non-contact electrograms were processed to determine the ARIs using a custom-made algorithm, previously validated against monophasic action potential recordings. Based on the contact map we defined border zone (BZ, voltage 0.5 to 1.5 mV) and remote (>1.5mV) regions. Heterogeneity was defined as the interquartile range (IQR) of ARIs in fixed neighborhoods of 1cm radius (figure 1A) and analyzed in 10 segments (5 BZ and 5 remote) of a modified version of the AHA model (49 segments by dividing the 16 non-apical segments). Other segments were discarded due to artefacts mainly caused by the array touching the septal and apical wall. Results Acute afterload challenge resulted in an increase of the systolic left ventricular pressure of 41.7±5.4% and increased left ventricular repolarization heterogeneity (IQR 4.03±1.23ms baseline to 4.85±1.38ms during inflation, p=0.004). There was a significant increase in heterogeneity in both BZ (4.78±1.60ms to 5.64±1.66ms, p=0.020) and remote (2.24±0.17ms to 3.00±0.86ms, p=0.034) regions (figure 1B). The IQR in the infarct BZ was higher compared to the remote zone at rest (4.78±1.60ms vs 2.24±0.17ms, p=0.010) as well as during inflation (5.64±1.66ms vs 3.00±0.86ms, p=0.008) (figure 1B). Both BZ and remote regions responded equally to acute afterload (p for interaction = 0.803). Conclusion Increased afterload leads to increased repolarization heterogeneity. This heterogeneity is higher in the infarct BZ. These alterations could provide a functional substrate for reentry. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): KU Leuven - C1 funding

Feasibility of simultaneous multichannel magnetocardiographic mapping, multiple monophasic action potential recording and laser photocoagulation of isolated and perfused rabbit ventricular myocardium

Abstract Background Multichannel magnetocardiographic mapping (MCG) has been validated as an accurate non-invasive method for 3D localization of arrhythmogenic substrates suitable for ablation treatment and of intracardiac electrophysiology catheters (1,2). High resolution (HR) MCG-guided multiple simultaneous monophasic action potential (multi-MAP) recording and pacing with a single patented amagnetic catheter (AC) detects local arrhythmogenic mechanisms (3). Laser photocoagulation (LP) providing more controlled and homogeneous myocardial lesions (4), may be an alternative to radiofrequency (RF) ablation, without unwanted electromagnetic noise effect during real-time MCG. Purpose This study aimed to experimentally test the feasibility of simultaneous multi-MAP recording and LP guided by real-time MCG. Methods An unshielded 36 channels SQUID system (sensitivity of 30 fT/Hz1/2, at 1 Hz) (CMI, USA) was used to study Langendorff-perfused isolated rabbit heart with MCG (Figure 1A). Four epicardial MAPs were simultaneously recorded (inter MAP distance: 1 mm) with a single multi-MAP AC (bandpass: DC-500 Hz; sampling frequency: 2 kHz) (GE-Prucka Cardiolab). LP was performed with a NdYAG laser (Dornier Medilas 5100) and a catheter system for safe myocardium LP (5). The study was carried out according to NIH guidelines for animal care and use. Results MCG imaging of rabbit ventricular magnetic field distribution and inverse source localization with the magnetic dipole model are shown in Figure 1B. The difference in morphology of local HR ventricular multi-MAP signals was compatible with myocardial anisotropy (Figure 1C). With appropriate cooling of the optic fiber-tissue interface, no epicardial damage was induced by LP, whereas deeper well defined intramural myocardial lesions were appreciable, of different size as a function of the energy delivered (Figure 1D). Multi-MAP recording was not significantly disturbed by LP. Keeping the Medilas unit at least 5 meters away from the MCG sensors, no electromagnetic artifacts were observed during MCG. Conclusion MCG, single catheter multi-MAP recording and well-defined LP lesions of ventricular myocardium were simultaneously feasible on Langendorff-perfused isolated rabbit heart, without major electromagnetic interferences, in an unshielded catheterization laboratory. With appropriate technological developments, safe MCG-guided single-catheter laser ablation of focal arrhythmogenic substrates is foreseen as a possible minimally invasive alternative to current interventional approaches. Funding Acknowledgement Type of funding sources: None.

Dapagliflozin but not empagliflozin prevents atrial fibrillation in an experimental whole-heart model

Abstract Background The SGLT2 inhibitors dapagliflozin and empagliflozin have an evolving role in heart failure therapy. Observational data suggest a lower risk of atrial fibrillation (AF) for patients on SGLT2 inhibitors. So far, it is unclear whether this is related to a direct antiarrhythmic potential of SGLT2 inhibitors or just a marker of improved heart function. Experimental data on the impact of SGTL2 inhibitors on atrial electrophysiology is scarce. Purpose This study aimed at evaluating the effects of the two SGLT2 inhibitors dapagliflozin and empagliflozin on atrial electrophysiology. Methods 25 rabbit hearts were explanted and retrogradely perfused employing the Langendorff-setup. Two catheters were placed on both atria to record monophasic action potentials. Hearts were paced at three different cycle lengths (350ms, 250ms, 150ms), thereby obtaining cycle-length dependent atrial action potential durations (aAPD90) and effective refractory periods (aERP). Atrial post-repolarization (aPRR) refractoriness was determined by the difference between aERP and aAPD90. AF was induced in the presence of acetylcholine (ACh, 1μM) and isoproterenol (Iso, 1μM). Thereafter, hearts were treated with dapagliflozin (n=13, 3 μM) or empagliflozin (n=12, 3 μM). Results Ach/Iso abbreviated aAPD90, aERP and prolonged aPRR in both groups (dapagliflozin group: aAPD90: −49 ms, p<0.01; aERP: −33 ms, p<0.01; aPRR: +18 ms, p=0.06; empagliflozin group: aAPD90: −40 ms, p<0.01; aERP: −25 ms, p<0.05; aPRR: +18 ms, p=0.05). Further treatment with dapagliflozin increased aAPD90 (+13 ms, p<0.01), aERP (+34 ms, p<0.05) and aPRR (+6 ms, p=ns). Additional administration of empagliflozin had no significant effect on aAPD90 (−8 ms, p=ns), aERP (−1 ms, p=ns) or aPRR (+9 ms, p=ns). Vulnerability to AF was assessed by a standardized protocol consisting of several trains of burst pacing. Ach/Iso increased the vulnerability to AF episodes in both groups (dapagliflozin group: 71 episodes vs. 1 under baseline conditions, p<0.01; empagliflozin group: 27 episodes vs. 2 under baseline conditions, p=0.08). Additional perfusion of dapagliflozin significantly reduced the occurrence of AF (to 30 episodes, p<0.05) while additional empagliflozin treatment had no significant antiarrhythmic effect (32 episodes, p=ns). Conclusion Dapagliflozin and empagliflozin had opposing effects on atrial electrophysiology. Dapagliflozin prolonged aAPD90 and aERP, thereby preventing AF. In contrast, empagliflozin did not change aAPD90 or aERP, resulting in no antiarrhythmic effect. Our findings hint at a potential antiarrhythmic benefit of dapagliflozin. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Hans and Gertie Fischer-Foundation (to C.E.)

Adrenergic stimulation amplifies the difference in beat-to-beat variability between the scar border zone and remote region

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): KU Leuven BOF-C1 “Blood pressure induced premature ventricular beats as triggers for ventricular arrhythmia in ischemic cardiomyopathy” Background Myocardial infarction (MI) results in a dense scar region surrounded by a heterogeneous region of fibrosis and remodeled myocytes called the border zone (BZ). Beta-adrenergic stimulation results in increased beat-to-beat variability of repolarization (BVR) which could increase spatial heterogeneity and arrhythmia vulnerability. Objective To examine the effect of adrenergic stimulation on the beat-to-beat variability in the BZ, compared to the remote region, using novel methodology for determining spatially dense activation-repolarization intervals. Methods Anterior-septal myocardial infarction (MI) was induced in 10 domestic pigs by 120-minute occlusion of the left anterior descending artery followed by reperfusion. Electro-anatomical mapping was performed after one month. The BZ was defined using contact mapping as the region with bipolar voltage between 0.5 and1.5mV. A non-contact recording of a 64-electrode array was translated to 2048 non-contact electrograms distributed over the LV (EnSite PrecisionTM, St. Jude/Abbott Medical). Electrophysiological recordings were made during baseline and during an isoproterenol (ISO) infusion (incremental doses of 0.01µg/kg until 0.04µg/kg). In each of the 2048 points non-contact electrograms over 25 consecutive beats were processed to determine the BVR using a custom-made algorithm, validated against monophasic action potential recordings. Results During baseline conditions the maximal BVR was increased in the BZ compared to the remote region (BZ: 3.28±0.90 ms vs remote: 2.61±0.67 ms, P=0.002). During ISO infusion the maximal BVR was also increased in the BZ (BZ: 3.55±0.74 ms vs remote: 2.21±0.60 ms, P<0.001). During baseline the BZ exhibited a larger spatial variance of BVR than the remote region (BZ: 0.20±0.11 ms2 vs remote: 0.087±0.055 ms2, P=0.002). During ISO infusion the spatial variance of BVR was larger in the BZ (BZ: 0.23±0.12 ms2 vs remote: 0.083±0.056 ms2, P=0.001). The maximal BVR was not significantly different during baseline and ISO in the BZ, nor the remote region (P>0.05). However, the difference of the maximal BVR between BZ and remote regions was significantly increased during ISO (baseline: 0.67±0.48 ms vs ISO: 1.34±0.49ms, P=0.001). Conclusion The MI BZ showed increased temporal heterogeneity in repolarization that could serve as functional substrate for re-entry. Adrenergic stimulation amplified this vulnerability by increasing the difference in maximal BVR between BZ and remote regions.

Discrete sites of frequent premature ventricular complexes cluster within the infarct border zone and coincide with high frequency of delayed afterdepolarizations under adrenergic stimulation.

Sympathetic activation in ischemic heart disease can cause lethal arrhythmias. These often are preceded by premature ventricular complexes (PVCs), which at the cellular level could result from delayed afterdepolarizations. The purpose of this study was to identify and map vulnerable areas for arrhythmia initiation after myocardial infarction (MI) and to explore the link between PVCs and cellular events. Anterior-septal wall MI was induced by 120 minutes of coronary occlusion followed by reperfusion (27 MI and 16 sham pigs). After 4 weeks, EnSite™ electroanatomic mapping combined with imaging was performed to precisely locate PVC sites of origin and subsequently record monophasic action potentials. Cardiomyocytes were isolated from different regions to study regional cellular remodeling. Isoproterenol was used as a surrogate for adrenergic stimulation both invivo and in cardiomyocytes. PVCs originated from the MI border zone (BZ) and occurred at discrete areas with clusters of PVCs within the BZ. At these sites, frequent delayed afterdepolarizations and occasional associated spontaneous action potentials translating to a PVC were present. Cardiomyocytes isolated from the MI BZ exhibited more spontaneous action potentials than cardiomyocytes from remote regions. Sensitivity to adrenergic stimulation was increased in MI, invivo and in cardiomyocytes. In awake, freely moving MI animals, frequent PVCs, ventricular arrhythmia, and sudden cardiac death occurred spontaneously at moderately elevated heart rates. Post-MI, arrhythmias initiate from discrete vulnerable areas within the BZ, where delayed afterdepolarizations, related to increased adrenergic response of BZ cardiomyocytes, can generate PVCs.

Repolarization heterogeneity within the myocardial infarction border zone correlates with variability of myocyte remodeling

Abstract Background Myocardial infarction (MI) results in a regional scar, with a border zone (BZ) of surviving myocytes interspersed with fibrosis providing an anatomical substrate for re-entry. Heterogeneous repolarization within the BZ may add a functional component aggravating re-entrant arrhythmias. Purpose We studied BZ heterogeneity and developed novel methodology for high resolution mapping of local in vivo activation-repolarization intervals (ARI) within the BZ and for studying the relation to cellular action potential (AP) profiles of cells isolated from the BZ. Methods Anterior-septal myocardial infarction was induced in 5 domestic pigs by 120-minute occlusion of the left anterior descending artery followed by reperfusion (18.9±4.7% of the left ventricle). After 1-month, electro-anatomical mapping was performed. Contact mapping was used to define the BZ (bipolar voltage 0.5–1.5mV). A non-contact recording of a 64-electrode array was translated to 2048 non-contact electrograms distributed over the LV. The non-contact electrograms were processed to determine the ARIs using a custom-made algorithm, validated against monophasic action potential recordings. After 2–4 days recovery, single cardiomyocytes were enzymatically isolated from the anterior-septal BZs and remote regions. Cardiomyocytes were field stimulated at 1Hz at 37°C and cellular AP duration (APD) was optically recorded (fluorescent voltage-sensitive dye Di-8-Annepps). Results In vivo, regional ARIs tended to be longer in the BZs than remote. ARI heterogeneity, quantified as the standard deviation of ARIs in a neighborhood of 1cm radius, was increased in the BZ (anterior BZ: 3.4±1.0 ms, P=0.052, septal BZ: 3.6±1.7 ms, P=0.027 vs remote: 2.0±0.5 ms). Cellular APD was measured in large population samples (>100 cells per region in each pig) and was longer in BZ myocytes compared to the remote region. Cellular APD heterogeneity, measured as the standard deviation within cell population samples pooled by region per animal, was increased in the BZ (anterior BZ: 105.9±17.0 ms, P=0.0010; septal BZ: 98.1±20.8 ms, P=0.0127 vs remote: 73.9±8.6 ms). Cell APD correlated to in vivo ARI (R2=0.34, P=0.021) and cellular heterogeneity correlated strongly with in vivo heterogeneity (R2=0.67, P=0.002). Conclusion In the BZ of MI, in vivo regional heterogeneity adds a functional substrate for re-entry that may result from heterogeneous cellular remodeling and increased cell-cell APD variability. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): KU Leuven BOF-C1 “Blood pressure induced premature ventricular beats as triggers for ventricular arrhythmia in ischemic cardiomyopathy”

Insights into sinus arrhythmia of the dog: Acetylcholine perfusion of canine right atrium results in beat-to-beat patterns that mimic sinus arrhythmia supporting exit block in the sinoatrial conduction pathways

Sinus arrhythmia of the dog is unique because of the pronounced alternating beat-to-beat intervals. The clustering of these short (faster rates) and long (slower rates) intervals is not just influenced by autonomic input from breathing; sinus arrhythmia can persist in the panting or apneic dog. The multiplicity of central and peripheral influences on the sinus node complicates the unraveling of the mechanisms of sinus arrhythmia. Studies of the sinus node suggest that acetylcholine can slow cellular depolarization and block sinoatrial conduction. Electrocardiographic monitoring of the dog supports this notion in that abrupt bifurcation into short and long intervals develop at lower heart rates. We sought to determine whether this phenomenon could be recapitulated in canine atrial preparations perfused with acetylcholine and whether selective pharmacologic blockade of the voltage and calcium clocks could provide insight into its mechanism. Spontaneous beat to beat (A–A) intervals were obtained from monophasic action potential recordings of perfused canine right atrial preparations before and during perfusion with acetylcholine (2−5 μM). The calcium clock was blocked with ryanodine (2−3 μM). The membrane clock was blocked with diltiazem hydrochloride (ICa,L blocker; 0.25 μM) and ZD7288 (If blocker; 3 μM). Hyperpolarization was hindered by blockade of IK,Ado/IK,Ach with tertiapin Q (100 nM) before and during acetylcholine perfusion.Acetylcholine resulted in beat clusters similar to those seen in sinus arrhythmia of the dog. Beat clusters were consistent with intermittent 2:1 and 3:1 sinoatrial conduction block. Tertiapin Q abolished this patterning suggesting a role of IK,Ado/IK,ACh in the mechanism of these acetylcholine-induced beat-to-beat patterns.

Antiarrhythmic effect of ranolazine in combination with selective NCX-inhibition in an experimental model of atrial fibrillation

Abstract Background Previous studies showed an antiarrhythmic effect of ranolazine in different clinical and experimental studies. The aim of this study was to investigate the effects of a combination of ranolazine with different selective NCX-inhibitors in an established experimental model of atrial fibrillation. Methods and results 18 hearts of New Zealand white rabbits were retrogradely perfused. Left and right atrial catheters were used to record monophasic action potentials. Hearts were paced at three different cycle lengths (350ms, 250ms, 150ms). Thereby, cycle-length dependent atrial action potential durations (aAPD90), atrial effective refractory periods (aERP) and atrial post-repolarization refractoriness (aPRR=aERP-aAPD90) were obtained. Vulnerability to AF was tested by a standardized protocol employing several trains of burst pacing. After generating baseline data, the hearts were perfused with a combination of acetylcholine (ACh, 1μM) and isoproterenol (Iso, 1μM) to increase occurrence of AF. Afterwards, the hearts were assigned to two groups and additionally perfused with a combination of 10 μM ranolazine and 1 μM of the selective NCX-inhibitor ORM-10103 (group 1: Rano-ORM) or 10 μM ranolazine and 1 μM of another NCX-inhibitor, SEA0400 (group 2: Rano-SEA). Infusion of ACh/Iso led to a shortening of aAPD90 (group 1/2: −27.2ms/−24.5ms p<0.05), aERP (−29.2ms/−35.6ms p<0.05), aPRR (−12.2ms/−13.7ms p=ns) and the occurrence of AF-Episodes was significantly increased (group 1: baseline 6 episodes to 34 episodes under ACh/Iso p<0.05, group 2: baseline 7 episodes to 26 episodes under ACh/Iso p<0.05). Additional perfusion with ranolazine and ORM-10103 did not alter aAPD90 (−0,47ms) while effective refractory periods (+21.4ms, p<0.05) and aPRR (+43ms, p<0.05) were significantly prolonged and AF episodes were effectively reduced to 15 episodes (p<0.05). In group 2, Rano-SEA led to a slight decrease in aAPD90 (−9.5ms) while aERP (+13.3ms, p<0.05) and aPRR (+19.8ms, p<0.05) were prolonged. The occurrence of AF episodes was reduced to 15 episodes (p=0.1). Conclusion To our knowledge this is the first study investigating the effect of ranolazine combined with different selective NCX-inhibitors in an isolated whole-heart model of AF. Atrial repolarization was not significantly changed with either of the combinations. However, both combinations prolonged aERP and aPRR and thereby suppressed induction of AF. Combining novel cellular targets may therefore lead to new potentially interesting options for antiarrhythmic AF therapy that have to be tested in clinical studies. Funding Acknowledgement Type of funding source: None

Antiarrhythmic Effect of Ranolazine in Combination with Selective NCX-Inhibition in an Experimental Model of Atrial Fibrillation

The aim of this study was to investigate the effects of a combination of ranolazine with different selective inhibitors of the Na+/Ca2+-exchanger (NCX) in an established experimental model of atrial fibrillation (AF). Eighteen hearts of New Zealand white rabbits were retrogradely perfused. Atrial catheters were used to record monophasic action potentials (aPRR). Hearts were paced at three different cycle lengths. Thereby, atrial action potential durations (aAPD90), atrial effective refractory periods (aERP) and atrial post-repolarization refractoriness were obtained. Isoproterenol and acetylcholine were employed to increase the occurrence of AF. Thereafter, the hearts were assigned to two groups (n = 9 each group) and additionally perfused with a combination of 10 µM ranolazine and 1 µM of the selective NCX-inhibitor ORM-10103 (group A: Rano-ORM) or 10 µM ranolazine and 1 µM of another NCX-inhibitor, SEA0400 (group B: Rano-SEA). The infusion of Iso/ACh led to a shortening of aAPD90, aERP, aPRR and the occurrence of AF episodes was significantly increased. Additional perfusion with ranolazine and ORM-10103 (group A) significantly prolonged the refractory periods and aPRR and AF episodes were effectively reduced. In group B, Rano-SEA led to a slight decrease in aAPD90 while aERP and aPRR were prolonged. The occurrence of AF episodes was consecutively reduced. To our knowledge, this is the first study investigating the effect of ranolazine combined with different selective NCX-inhibitors in an isolated whole-heart model of AF. Both combinations prolonged aERP and aPRR and thereby suppressed the induction of AF.

Eleutheroside B, a selective late sodium current inhibitor, suppresses atrial fibrillation induced by sea anemone toxin II in rabbit hearts.

Eleutheroside B (EB) is the main active constituent derived from the Chinese herb Acanthopanax senticosus (AS) that has been reported to possess cardioprotective effects. In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation (AF). Whole-cell recording was conducted in isolated rabbit atrial myocytes. The intracellular calcium ([Ca2+]i) concentration was measured using calcium indicator Fura-2/AM fluorescence. Monophasic action potential (MAP) and electrocardiogram (ECG) synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system. We showed that EB dose-dependently inhibited late sodium current (INaL), transient sodium current (INaT), and sea anemone toxin II (ATX II)-increased INaL with IC50 values of 167, 1582, and 181 μM, respectively. On the other hand, EB (800 μM) did not affect L-type calcium current (ICaL), inward rectifier potassium channel current (IK), and action potential duration (APD). Furthermore, EB (300 μM) markedly decreased ATX II-prolonged the APD at 90% repolarization (APD90) and eliminated ATX II-induced early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and triggered activities (TAs). Moreover, EB (200 μM) significantly suppressed ATX II-induced Na+-dependent [Ca2+]i overload in atrial myocytes. In the Langendorff-perfused rabbit hearts, application of EB (200 μM) or TTX (2 μM) substantially decreased ATX II-induced incidences of atrial fibrillation (AF), ventricular fibrillation (VF), and heart death. These results suggest that augmented INaL alone is sufficient to induce AF, and EB exerts anti-AF actions mainly via blocking INaL, which put forward the basis of pharmacology for new clinical application of EB.

The AMP-activated protein kinase modulates hypothermia-induced J wave.

The mechanism underlying the occurrence of the J wave in low temperature remains unclear. However, low temperature is associated with metabolic disorder and 5' AMP-activated protein kinase (AMPK), which modulates ionic currents and cardiac metabolism. This study investigated whether AMPK regulation can modulate the occurrence of the J wave at low temperature. Unipolar and bipolar leads were used to record monophasic action potential (the endocardium and epicardium) and pseudo-electrocardiograms (inferior leads) to study the cardiac electrical activity. Measurements were taken in isolated Langendorff rabbit hearts at both 30℃ and 37℃ before and after administration of 4-aminopyridine (an ultrarapid delayed rectifier potassium current inhibitor, IKur , 50µmol L-1 ), PF06409577 (an AMPK activator, 1µmol L-1 ), compound C (an AMPK inhibitor, 10µmol L-1 ) and glibenclamide (an ATP-sensitive inward rectifier potassium channel inhibitor, IKATP , 20µmol L-1 ). The amplitude of the J wave (2.46±0.34mV vs. 1.11±0.23mV, P<.01) at 30℃ (n=15) was larger than that at 37℃ (n=15). PF06409577 (1µmol L-1 ) increased the J waves at both 30℃ and 37℃. In contrast, compound C (10µmol L-1 ) reduced J wave at both 37℃ and 30℃. Low-temperature-induced J waves were individually suppressed by 4-AP (50µmol L-1 ) and glibenclamide (20µmol L-1 ). AMPK inhibition reduces low-temperature-induced J waves and possible ventricular arrhythmogenesis by modulating IKATP and IKur channels.

Upregulation of MMP-9 and CaMKII prompts cardiac electrophysiological changes that predispose denervated transplanted hearts to arrhythmogenesis after prolonged cold ischemic storage

BackgroundAlthough the importance of the potential mechanisms of arrhythmogenesis, including cardiac electrophysiology dysfunction and gap junction electrical uncoupling by Cx43 degradation under pathological conditions, has been widely and thoroughly established, the correlations between these mechanisms and biomarkers of cold ischemic insult have not yet been confirmed. Limited evidence suggests that these biomarkers are significantly associated with cardiac dysrhythmia. Thus, in this study, we analyzed such correlations in denervated transplanted hearts. Methods and resultsRat hearts were divided into two groups according to a random number table. The hearts in the first group were stored in histidine-tryptophan-ketoglutarate (HTK) solution for 6 h to produce cold ischemic insult (insult group, n = 8), while the hearts in the second group were left untreated (control group, n = 8). Ventricular arrhythmias (VAs) were successively observed in 8/8 hearts stored in HTK solution and 0/8 untreated hearts (P < 0.05). The analysis of the electrophysiological parameters by programmed electrical stimulation (PES) and monophasic action potential (MAP) recording demonstrated that compared with the control hearts, the insult hearts had increased transmural dispersion of repolarization (TDR) and effective refractory periods (ERPs) (P < 0.001), decreased conduction velocities (CVs) and excitation wavelengths (CV × ERP, λ) (P < 0.05), and longer times between peak MAP values and 90% repolarization (MAPD90) in the epicardium (P < 0.05). However, no significant difference in endocardial MAPD90 was observed between the two groups of hearts. The investigation of biomarkers and Cx43 proteins by a western blot analysis showed that the matrix metalloproteinase-9 (MMP-9) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) levels were strongly elevated and that the Cx43 protein levels were markedly reduced in the insult hearts. ConclusionMMP-9 overexpression mediates gap junction (GJ) electrical uncoupling induced by Cx43 decreases, while CaMKII upregulation induces transmural dispersion of repolarization (TDR) increase, both of which increase the risk of ventricular arrhythmias (VAs) in denervated transplanted hearts after prolonged cold ischemic storage.

Contact Forces Required to Record Monophasic Action Potentials: A Complement to Catheter Contact Force Measurement.

The ability to monitor catheter contact force (CF) plays a major role in assessing radiofrequency ablation, impacting lesion size and arrhythmia recurrence, and dictating ablation duration and/or overall patient safety. Our study sought to determine the relative CFs required to elicit reproducible monophasic action potential (MAP) recordings. The study utilized four swine in which: first, median sternotomies were performed and MAPs were collected from seven ventricular locations on the epicardial surface of each heart; and second, a subset of endocardial signals was recorded from a reanimated heart. In these studies, the initial elicitation and then loss of stable MAP waveforms were recorded, as were their associated catheter CFs (n = 371). Mean CF at the onset of stable MAP recordings was 14.2 ± 2.9 g for epicardial and 16.6 ± 2.5g for endocardial locations. Across epicardial locations, no significant differences in CF were required to elicit MAPs. Additionally, endocardial and epicardial CFs for MAPs did not significantly differ for respective locations, i.e., right ventricular septum endocardial versus epicardial. In our study, the catheter CFs required to elicit MAPs were within optimal ranges previously reported for eliciting clinically viable radiofrequency ablations. We believe that MAP recordings could complement CF measurements with electrical data, providing additional clinical feedback for physicians performing cardiac ablation. If applied clinically, MAP recordings could potentially improve ablation outcomes in patients with cardiac arrhythmias.

Antiarrhythmic effect of sevoflurane as an additive to HTK solution on reperfusion arrhythmias induced by hypothermia and ischaemia is associated with the phosphorylation of connexin 43 at serine 368

BackgroundReperfusion ventricular arrhythmia (RA) associated with hypothermic ischaemic storage is increasingly recognized as a substantial contributor to adverse consequences after heart transplantation. Ischemia- or hypothermia-induced gap junction (GJ) remodelling is closely linked to RA. Reducing GJ remodelling contributes to RA attenuation and is important in heart transplantation. However, sevoflurane has an antiarrhythmic effect associated with the connexin 43 (Cx43) protein that has not yet been fully established.MethodsHearts were divided into two groups according to a random number table: all hearts were arrested by an infusion of histidine-tryptophan-ketoglutarate (HTK) solution (4 °C) followed by (1) storage in HTK solution (4 °C) alone for 6 h (n = 8, Control group) or (2) storage in HTK solution supplemented with sevoflurane (2.5%) (4 °C) for 6 h (n = 8, Sevo-HTK group). First, the total Cx43 level and the phosphorylation of Cx43 at Ser368 (Cx43-pS368) were assessed by Western blotting, and the distribution of Cx43 was assessed by immunohistochemistry. Second, programmed electrical stimulation (PES) and monophasic action potential (MAP) recording were used to analyse the MAP duration (MAPD), conduction velocity (CV) and transmural repolarization dispersion (TDR). In addition, haematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) staining were individually used to investigate the degree of myocardial pathological damage and cell apoptosis. Finally, bipolar electrograms were used to record the graft re-beating time and monitor RA during reperfusion for 15 to 30 min.ResultsSevo-HTK solution relatively increased the total Cx43 (P < 0.01) and Cx43-pS368 (P < 0.01) levels and prevented Cx43 redistribution (P < 0.05) and CV slowing (P < 0.001) but did not change TDR (P > 0.05). Additionally, the Cx43-pS368/total Cx43 ratio (P>0.05) was similar in the two groups. However, with Sevo-HTK solution, the graft re-beating times were shortened, myocardial pathological damage was ameliorated, and the number of apoptotic cells was markedly decreased.ConclusionThe reduction in hypothermia and ischaemia-induced reperfusion arrhythmias by the addition of sevoflurane to HTK solution may be related to the phosphorylation of Cx43 at serine 368.

Determinants of slowed conduction in premature ventricular beats induced during programmed stimulations in perfused guinea-pig heart.

What is the central question of this study? Is the slowed conduction upon premature ventricular activations during clinical electrophysiological testing attributable to the prolonged activation latency, or increased impulse propagation time, or both? What is the main finding and its importance? Prolonged activation latency at the stimulation site is the critical determinant of conduction slowing and associated changes in the ventricular response intervals in premature beats initiated during phase 3 repolarization in perfused guinea-pig heart. These relations are likely to have an effect on arrhythmia induction and termination independently of the presence of ventricular conduction defects or the proximity of the stimulation site to the re-entrant circuit. During cardiac electrophysiological testing, slowed conduction upon premature ventricular activation can limit the delivery of the closely coupled impulses from the stimulation site to the region of tachycardia origin. In order to examine the contributing factors, in this study, cardiac conduction intervals and refractory periods were determined from left ventricular (LV) and the right ventricular (RV) monophasic action potential recordings obtained in perfused guinea-pig hearts. A premature activation induced immediately after the termination of the refractory period was associated with conduction slowing. The latter was primarily accounted for by the markedly increased (+54%) activation latency at the LV stimulation site, with only negligible changes (+12%) noted in the LV-to-RV delay. The prolonged activation latency was acting to limit the shortest interval at which two successive action potentials can be induced in the LV and RV chambers. The prolongation of the activation latency in premature beats was accentuated upon an increase in the stimulating current intensity, or during hypokalaemia. This change was related to the reduced ratio of the refractory period to the action potential duration, which allowed extrastimulus capture to occur earlier during phase 3 repolarization. Flecainide, a Na+ channel blocker, prolonged both the activation latency and the LV-to-RV delay, without changing their relative contributions to conduction slowing. In summary, these findings suggest that the activation latency is the critical determinant of conduction slowing and associated changes in the ventricular response intervals upon extrastimulus application during phase 3 of the action potential.

Unanesthetized Rodents Demonstrate Insensitivity of QT Interval and Ventricular Refractory Period to Pacing Cycle Length.

Aim: The cardiac electrophysiology of mice and rats has been analyzed extensively, often in the context of pathological manipulations. However, the effects of beating rate on the basic electrical properties of the rodent heart remain unclear. Due to technical challenges, reported electrophysiological studies in rodents are mainly from ex vivo preparations or under deep anesthesia, conditions that might be quite far from the normal physiological state. The aim of the current study was to characterize the ventricular rate-adaptation properties of unanesthetized rats and mice.Methods: An implanted device was chronically implanted in rodents for atrial or ventricular pacing studies. Following recovery from surgery, QT interval was evaluated in rodents exposed to atrial pacing at various frequencies. In addition, the frequency dependence of ventricular refractoriness was tested by conventional ventricular programmed stimulation protocols.Results: Our findings indicate total absence of conventional rate-adaptation properties for both QT interval and ventricular refractoriness. Using monophasic action potential recordings in isolated mice hearts we could confirm the previously reported shortening of the action potential duration at fast pacing rates. However, we found that this mild shortening did not result in similar decrease of ventricular refractory period.Conclusion: Our findings indicate that unanesthetized rodents exhibit flat QT interval and ventricular refractory period rate-dependence. This data argue against empirical use of QT interval correction methods in rodent studies. Our new methodology allowing atrial and ventricular pacing of unanesthetized freely moving rodents may facilitate more appropriate utility of these important animal models in the context of cardiac electrophysiology studies.

Medium-term Electrophysiologic Effects of a Cellularized Scaffold Implanted in Rats After Myocardial Infarction.

BackgroundCardiac repair strategies are being evaluated for myocardial infarctions, but the safety issues regarding their arrhythmogenic potential remain unresolved. By utilizing the in-vivo rat model, we have examined the medium-term electrophysiologic effects of a biomaterial scaffold that has been cellularized with spheroids of human adipose tissue, derived from mesenchymal stem cells and umbilical vein endothelial cells.MethodsMesenchymal stem cells, which exhibit adequate differentiation capacity, were co-cultured with umbilical vein endothelial cells and were seeded on an alginate based scaffold. After in-vitro characterization, the cellularized scaffold was implanted in (n=15) adult Wistar rats 15 min post ligation of the left coronary artery, with an equal number of animals serving as controls. Two weeks thereafter, monophasic action potentials were recorded and activation-mapping was performed with a multi-electrode array. An arrhythmia score for inducible ventricular tachyarrhythmias was calculated after programmed electrical stimulation.ResultsThe arrhythmia score was comparable between the treated animals and controls. No differences were detected in the local conduction at the infarct border and in the voltage rise in monophasic action potential recordings. Treatment did not affect the duration of local repolarization, but tended to enhance its dispersion.ConclusionsThe fabricated bi-culture cellularized scaffold displayed favorable properties after in-vitro characterization. Medium-term electrophysiologic assessment after implantation in the infarcted rat myocardium revealed low arrhythmogenic potential, but the long-term effects on repolarization dispersion will require further investigation.

Kvβ2 Subunit Regulates Cardiac Levels of SLC41a3 a Sodium‐Magnesium Transporter.

BackgroundClinical evidence has demonstrated that administration of magnesium can provide cardioprotective benefits during acute MI as well as suppression of cardiac arrhythmias. Further, meta‐analysis data has uncovered an inverse relation to circulatory levels of magnesium and risk for cardiac injury. Magnesium influx/efflux behaviors have been recorded in isolated cardiomyocytes; further the addition of magnesium during and prior to cardiac injury such as ischemia reperfusion was demonstrated to significantly reduce ischemic injury. While clinical evidence highlights magnesium's cardiovascular benefits, identification and subsequent regulation remain largely unknown of the magnesium channels responsible. Recent research has identified one of the key magnesium/sodium exchange channels as SLC41a1‐3 family. Murine cardiac expression levels demonstrate significant mRNA levels of SLC41a3 in particular.Methods and ResultsA recent microarray study performed in hearts from Kvβ2 knockout mice demonstrated a significant decrease in SLC41a3 mRNA expression compared with control. Protein expression validated the decrease in expression levels of SLC41a3. These findings may indicate the Kvβ2 subunit acts as a novel regulator to SLC41a3, previous research identified Kvβ2 having protein‐protein interactions with SLC39a1 another member of the solute carrier family (SLC). Cardiac function measured by monophasic action potential (AP) recordings presented with a significant reduction in AP duration values in control hearts perfused with magnesium (MgSO4) while Kvβ2 KO hearts demonstrate little to no alterations in AP duration post magnesium exposure. Further functionality will be assessed in isolated cardiomyocytes testing the influx and efflux of magnesium to understand Kvβ2's role in channel activity.HypothesisBased upon our preliminary data we hypothesis that the Kvβ2 subunit plays significant roles in SLC41a3 expression as well as function within the heart.ConclusionThese novel roles of Kvβ2 may shed new light on the molecular understanding of how administration of magnesium can benefit both cardiac injury and arrhythmias.Support or Funding InformationResearch Supported: This Research work was funded by National Institutes of Health (NIH) Grant R01‐HL‐102171 (S. M. Tipparaju).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autonomic conflict exacerbates long QT associated ventricular arrhythmias

This study tested the hypothesis that concomitant sympathetic and parasympathetic stimulation (“autonomic conflict”) may act as a trigger for arrhythmias in long QT syndrome (LQTS). Studies were performed in isolated innervated rabbit hearts treated with clofilium (100 nmol/L); a potassium channel blocker. The influence of vagus nerve stimulation (VNS) on spontaneous ventricular arrhythmias was assessed in the absence/presence of sustained noradrenaline perfusion (100 nmol/L) and with sudden adrenergic stress (injections of noradrenaline into the perfusion line). Hearts were instrumented for a pseudo-electrocardiogram and monophasic action potential recordings. VNS, which slows heart rate, was associated with a stimulation frequency-dependent incidence of spontaneous early after-depolarisations (EADs) and ventricular tachycardia (VT), best predicted by the duration of the electrocardiographic T-wave and by triangulation of the ventricular action potential. In the presence of sustained (steady-state) noradrenaline perfusion, the incidence of EADs and VT with VNS was decreased from 73/55% to 45/27%, respectively. However, sudden adrenergic stress, imposed during periods of sustained VNS, was associated with a transient increase in the incidence of severity of observed arrhythmias, as indicated by an increase in the average arrhythmias score (1.6 ± 0.4 vs. 2.1 ± 0.7, p = .01). Analysis of electrophysiological parameters suggests that sudden adrenergic stress is associated with a transient prolongation, and increased triangulation, of the ventricular action potential, which may predispose to triggered activity. This study demonstrates that autonomic conflict is a pro-arrhythmic stimulus in LQTS. However, combined adrenergic and parasympathetic stimulation has a complex relationship with arrhythmogenicity, with differences in the effects of steady-state adrenergic activation vs. sudden adrenergic stress.