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Background: Irreversible electroporation is an energy form utilizing high-voltage pulsed electric field, leading to cellular homeostasis disruption and cell death. Recently, irreversible electroporation has shown promising results for the treatment of cardiac arrhythmias. However, reversible and irreversible effects of pulsed electric field on cardiac myocytes remain poorly understood. Here, we evaluated the influence of a monophasic single electric pulse (EP) on the contractility, Ca 2+ homeostasis and recovery of cardiac myocytes., Methods: Isolated rat left ventricular myocytes were electroporated using single monophasic EP of different durations and voltages. Sarcomere length and intracellular Ca 2+ were simultaneously monitored for up to 20 minutes after EP application in Fura-2 loaded left ventricular myocytes. Lethal voltage thresholds were determined using 100 µs and 10 ms pulses and by discriminating cell orientation with respect to the electric field., Results: Electroporation led to an immediate increase in intracellular Ca 2+ which was dependent upon the voltage delivered to the cell. Intermediate-voltage EP (140 V, 100 µs) increased sarcomere shortening, Ca 2+ transient amplitude, and diastolic Ca 2+ level measured 1 minute post-EP. Although sarcomere shortening returned to pre-EP level within 5 minutes, Ca 2+ transient amplitude decreased further below pre-EP level and diastolic Ca 2+ level remained elevated within 20 minutes post-EP. Spontaneous contractions were observed after sublethal EP application but their frequency decreased progressively within 20 minutes. Lethal EP voltage threshold was lower in myocytes oriented perpendicular than parallel to the electric field using 100 µs pulses while an opposite effect was found using 10 ms pulses., Conclusions: Sublethal EP affected rat left ventricular myocytes contractility and disrupted Ca 2+ homeostasis as a function of the EP voltage. Moreover, EP-induced lethality was preceded by a large increase in intracellular Ca 2+ and was dependent upon the EP duration, amplitude and left ventricular myocytes orientation with respect to the electric field. These findings provide new insights into the effect of pulsed electric field on cardiac myocytes.

Background: Right ventricular outflow tract (RVOT) is a common source of ventricular tachycardia, which often requires ablation. However, the mechanisms underlying the RVOT's unique arrhythmia susceptibility remain poorly understood due to lack of detailed electrophysiological and molecular studies of the human RVOT., Methods: We conducted optical mapping studies in 16 nondiseased donor human RVOT preparations subjected to pharmacologically induced adrenergic and cholinergic stimulation to evaluate susceptibility to arrhythmias and characterize arrhythmia dynamics., Results: We found that under control conditions, RVOT has shorter action potential duration at 80% repolarization relative to the right ventricular apical region. Treatment with isoproterenol (100 nM) shortened action potential duration at 80% repolarization and increased incidence of premature ventricular contractions ( P =0.003), whereas acetylcholine (100 μM) stimulation alone had no effect on action potential duration at 80% repolarization or premature ventricular contractions. However, acetylcholine treatment after isoproterenol stimulation reduced the incidence of premature ventricular contractions ( P =0.034) and partially reversed action potential duration at 80% repolarization shortening ( P =0.029). Immunolabeling of RVOT (n=4) confirmed the presence of cholinergic marker VAChT (vesicular acetylcholine transporter) in the region. Rapid pacing revealed RVOT susceptibility to both concordant and discordant alternans. Investigation into transmural arrhythmia dynamics showed that arrhythmia wave fronts and phase singularities (rotors) were relatively more organized in the endocardium than in the epicardium ( P =0.006). Moreover, there was a weak but positive spatiotemporal autocorrelation between epicardial and endocardial arrhythmic wave fronts and rotors. Transcriptome analysis (n=10 hearts) suggests a trend that MAPK (mitogen-activated protein kinase) signaling, calcium signaling, and cGMP-PKG (protein kinase G) signaling are among the pathways that may be enriched in the male RVOT, whereas pathways of neurodegeneration may be enriched in the female RVOT., Conclusions: Human RVOT electrophysiology is characterized by shorter action potential duration relative to the right ventricular apical region. Cholinergic right ventricular stimulation attenuates the arrhythmogenic effects of adrenergic stimulation, including increase in frequency of premature ventricular contractions and shortening of wavelength. Right ventricular arrhythmia is characterized by positive spatial-temporal autocorrelation between epicardial-endocardial arrhythmic wave fronts and rotors that are relatively more organized in the endocardium.

Background: Studies of ventricular fibrillation (VF) in humans are limited because of the short available duration. We sought to study surface ECG waveforms and effect of ablation in long-lasting VF in patients with left assist devices., Methods: Continuous 12-lead ECG of 5 episodes of long-lasting VF occurring in 3 patients with left ventricular assist device were analyzed. Spectral analysis (dominant frequency) and quantification of waveform amplitude, regularity (Unbiased Regularity Index), and complexity (Nondipolar Index) were performed over a median of 24 minutes of VF. Radiofrequency ablation was performed during VF in 2 patients., Results: There was a significant increase in dominant frequency between VF onset and termination but none of the other parameters significantly changed. Some VF parameters varied from patient to patient and from lead to lead. Dominant frequency decreased after radiofrequency ablation in both cases and VF terminated spontaneously shortly after ablation in one case. The previously incessant VFs in these 2 patients did not recur afterward., Conclusions: VF rate increases over time in patients with left ventricular assist devices and is lowered by ablation. Long-lasting VF may be modified or even terminated by ablation.

Background: Ventricular arrhythmias are frequent in patients with repaired tetralogy of Fallot (rTOF), but their origin and underlying mechanisms remain unclear. In this study, the involvement of left ventricular (LV) electrical and structural remodeling was assessed in an animal model mimicking rTOF sequelae., Methods: Piglets underwent a tetralogy of Fallot repair-like surgery (n=6) or were sham operated (Sham, n=5). Twenty-three weeks post-surgery, cardiac function was assessed in vivo by magnetic resonance imaging. Electrophysiological properties were characterized by optical mapping. LV fibrosis and connexin-43 localization were assessed on histological sections and protein expression assessed by Western Blot., Results: Right ventricular dysfunction was evident, whereas LV function remained unaltered in rTOF pigs. Optical mapping showed longer action potential duration on the rTOF LV epicardium and endocardium. Epicardial conduction velocity was significantly reduced in the longitudinal direction in rTOF LVs but not in the transverse direction compared with Sham. An elevated collagen content was found in LV basal and apical sections from rTOF pigs. Moreover, a trend for connexin-43 lateralization with no change in protein expression was found in the LV of rTOFs. Finally, rTOF LVs had a lower threshold for arrhythmia induction using incremental pacing protocols., Conclusions: We found an arrhythmogenic substrate with prolonged heterogeneous action potential duration and reduced conduction velocity in the LV of rTOF pigs. This remodeling precedes LV dysfunction and is likely to contribute to ventricular arrhythmias and sudden cardiac death in patients with rTOF.

Background Papillary muscles are an important source of ventricular tachycardia (VT). Yet little is known about the role of the right ventricular (RV) endocavity structure, the moderator band (MB). The aim of this study was to determine the characteristics of the MB that may predispose to arrhythmia substrates. Methods Ventricular wedge preparations with intact MBs were studied from humans (n=2) and sheep (n=15; 40-50 kg). RV endocardium was optically mapped, and electrical recordings were measured along the MB and septum. S1S2 pacing of the RV free wall, MB, or combined S1-RV S2-MB sites were assessed. Human (n=2) and sheep (n=4) MB tissue constituents were assessed histologically. Results The MB structure was remarkably organized as 2 excitable, yet uncoupled compartments of myocardium and Purkinje. In humans, action potential duration heterogeneity between MB and RV myocardium was found (324.6±12.0 versus 364.0±8.4 ms; P<0.0001). S1S2-MB pacing induced unidirectional propagation via MB myocardium, permitting sustained macroreentrant VT. In sheep, the incidence of VT for RV, MB, and S1-RV S2-MB pacing was 1.3%, 5.1%, and 10.3%. Severing the MB led to VT termination, confirming a primary arrhythmic role. Inducible preparations had shorter action potential duration in the MB than RV (259.3±45.2 versus 300.7±38.5 ms; P<0.05), whereas noninducible preparations showed no difference (312.0±30.3 versus 310.0±24.6 ms, respectively). Conclusions The MB presents anatomic and electrical compartmentalization between myocardium and Purkinje fibers, providing a substrate for macroreentry. The vulnerability to sustain VT via this mechanism is dependent on MB structure and action potential duration gradients between the RV free wall and MB.

Background: Sudden cardiac death because of ventricular fibrillation (VF) is commonly unexplained in younger victims. Detailed electrophysiological mapping in such patients has not been reported., Methods: We evaluated 24 patients (29±13 years) who survived idiopathic VF. First, we used multielectrode body surface recordings to identify the drivers maintaining VF. Then, we analyzed electrograms in the driver regions using endocardial and epicardial catheter mapping during sinus rhythm. Established electrogram criteria were used to identify the presence of structural alterations., Results: VF occurred spontaneously in 3 patients and was induced in 16, whereas VF was noninducible in 5. VF mapping demonstrated reentrant and focal activities (87% versus 13%, respectively) in all. The activities were dominant in one ventricle in 9 patients, whereas they had biventricular distribution in others. During sinus rhythm areas of abnormal electrograms were identified in 15/24 patients (62.5%) revealing localized structural alterations: in the right ventricle in 11, the left ventricle in 1, and both in 3. They covered a limited surface (13±6 cm 2 ) representing 5±3% of the total surface and were recorded predominantly on the epicardium. Seventy-six percent of these areas were colocated with VF drivers ( P <0.001). In the 9 patients without structural alteration, we observed a high incidence of Purkinje triggers (7/9 versus 4/15, P =0.033). Catheter ablation resulted in arrhythmia-free outcome in 15/18 patients at 17±11 months follow-up., Conclusions: This study shows that localized structural alterations underlie a significant subset of previously unexplained sudden cardiac death. In the other subset, Purkinje electrical pathology seems as a dominant mechanism., (© 2018 The Authors.)

Background: Ventricular tachycardia recurrence can occur after ventricular tachycardia ablation because of incomplete and nontransmural ventricular lesion formation. We sought to compare the lesions made by a novel irrigated needle catheter to conventional radiofrequency lesions., Methods and Results: Thirteen female sheep (4.6±0.7 years, 54±8 kg) were studied. In 7 sheep, 60-s radiofrequency applications were performed using an irrigated needle catheter. In 6 sheep, conventional lesions were made using a 4-mm irrigated catheter. 1.5T in vivo and high-density magnetic resonance imaging (9.4T) were performed on explanted hearts from animals receiving needle radiofrequency. Conventional lesion volume was calculated as (1/6)×π×(A×B(2)+C×D(2)/2). Needle lesion volume was measured as Σ(π×r(2))/2 with a slice thickness of 1 mm. The dimensions of all lesions were also measured on gross pathology. Additional histological analysis of the needle lesions was performed. One hundred twenty endocardial left ventricular ablation lesions (conventional, n=60; needle, n=60) were created. At necropsy, more lesions were found using needle versus conventional radiofrequency (90% versus 75%; P<0.05). Comparing needle versus conventional radiofrequency: lesion volume was larger (1030±362 versus 488±384 mm(3); P<0.001), lesion depth was increased (9.9±2.7 versus 5±2.4 mm; P<0.001), and more transmural lesions were created (62.5% versus 17%; P<0.01). Pericardial contrast injection was observed in 4 apical attempts using needle radiofrequency, however, with no adverse effects. Steam pops occurred in 3 attempts using conventional radiofrequency., Conclusions: Irrigated needle ablation is associated with more frequent, larger, deeper, and more often transmural lesions compared with conventional irrigated ablation. This technology might be of value to treat intramural or epicardial ventricular tachycardia substrates resistant to conventional ablation., (© 2015 American Heart Association, Inc.)

Background: Therapy strategies for atrial fibrillation based on electric characterization are becoming viable personalized medicine approaches to treat a notoriously difficult disease. In light of these approaches that rely on high-density surface mapping, this study aims to evaluate the presence of 3-dimensional electric substrate variations within the transmural wall during acute episodes of atrial fibrillation., Methods and Results: Optical signals were simultaneously acquired from the epicardial and endocardial tissue during acute fibrillation in ovine isolated left atria. Dominant frequency, regularity index, propagation angles, and phase dynamics were assessed and correlated across imaging planes to gauge the synchrony of the activation patterns compared with paced rhythms. Static frequency parameters were well correlated spatially between the endocardium and the epicardium (dominant frequency, 0.79 ± 0.06 and regularity index, 0.93 ± 0.009). However, dynamic tracking of propagation vectors and phase singularity trajectories revealed discordant activity across the transmural wall. The absolute value of the difference in the number, spatial stability, and temporal stability of phase singularities between the epicardial and the endocardial planes was significantly >0 with a median difference of 1.0, 9.27%, and 19.75%, respectively. The number of wavefronts with respect to time was significantly less correlated and the difference in propagation angle was significantly larger in fibrillation compared with paced rhythms., Conclusions: Atrial fibrillation substrates are dynamic 3-dimensional structures with a range of discordance between the epicardial and the endocardial tissue. The results of this study suggest that transmural propagation may play a role in atrial fibrillation maintenance mechanisms., (© 2015 American Heart Association, Inc.)

Background: Electric excitability in the ventricular wall is influenced by cellular electrophysiology and passive electric properties of the myocardium. Action potential (AP) rise time, an indicator of myocardial excitability, is influenced by conduction pattern and distance from the epicardial surface. This study examined AP rise times and conduction velocity as the depolarizing wavefront approaches the epicardial surface., Methods and Results: Two-photon excitation of di-4-aminonaphthenyl-pyridinum-propylsulfonate was used to measure electric activity at discrete epicardial layers of isolated Langendorff-perfused rabbit hearts to a depth of 500 μm. Endo-to-epicardial wavefronts were studied during right atrial or ventricular endocardial pacing. Similar measurements were made with epi-to-endocardial, transverse, and longitudinal pacing protocols. Results were compared with data from a bidomain model of 3-dimensional (3D) electric propagation within ventricular myocardium. During right atrial and endocardial pacing, AP rise time (10%-90% of upstroke) decreased by ≈50% between 500 and 50 μm from the epicardial surface, whereas conduction velocity increased and AP duration was only slightly shorter (≈4%). These differences were not observed with other conduction patterns. The depth-dependent changes in rise time were larger at higher pacing rates. Modeling data qualitatively reproduced the behavior seen experimentally and demonstrated a parallel reduction in peak I(Na) and electrotonic load as the wavefront approaches the epicardial surface., Conclusions: Decreased electrotonic load at the epicardial surface results in more rapid AP upstrokes and higher conduction velocities compared with the bulk myocardium. Combined effects of tissue depth and pacing rate on AP rise time reduce conduction safety and myocardial excitability within the ventricular wall.