Mapping data of human ventricular fibrillation (VF) are limited. We performed detailed mapping of the activities underlying the onset of VF and targeted ablation in patients with structural cardiac abnormalities. We evaluated 54 patients (50 ± 16 years) with VF in the setting of ischaemic (n = 15), hypertrophic (n = 8) or dilated cardiomyopathy (n = 12), or Brugada syndrome (n = 19). Ventricular fibrillation was mapped using body-surface mapping to identify driver (reentrant and focal) areas and invasive Purkinje mapping. Purkinje drivers were defined as Purkinje activities faster than the local ventricular rate. Structural substrate was delineated by electrogram criteria and by imaging. Catheter ablation was performed in 41 patients with recurrent VF. Sixty-one episodes of spontaneous (n = 10) or induced (n = 51) VF were mapped. Ventricular fibrillation was organized for the initial 5.0 ± 3.4 s, exhibiting large wavefronts with similar cycle lengths (CLs) across both ventricles (197 ± 23 vs. 196 ± 22 ms, P = 0.9). Most drivers (81%) originated from areas associated with the structural substrate. The Purkinje system was implicated as a trigger or driver in 43% of patients with cardiomyopathy. The transition to disorganized VF was associated with the acceleration of initial reentrant activities (CL shortening from 187 ± 17 to 175 ± 20 ms, P < 0.001), then spatial dissemination of drivers. Purkinje and substrate ablation resulted in the reduction of VF recurrences from a pre-procedural median of seven episodes [interquartile range (IQR) 4–16] to 0 episode (IQR 0–2) (P < 0.001) at 56 ± 30 months. The onset of human VF is sustained by activities originating from Purkinje and structural substrate, before spreading throughout the ventricles to establish disorganized VF. Targeted ablation results in effective reduction of VF burden. The initial phase of human ventricular fibrillation (VF) is critical as it involves the primary activities leading to sustained VF and arrhythmic sudden death. The origin of such activities is unknown. Body-surface mapping shows that most drivers (≈80%) during the initial VF phase originate from electrophysiologically defined structural substrates. Repetitive Purkinje activities can be elicited by programmed stimulation and are implicated as drivers in 37% of cardiomyopathy patients. The onset of human VF is mostly associated with activities from the Purkinje network and structural substrate, before spreading throughout the ventricles to establish sustained VF. Targeted ablation reduces or eliminates VF recurrence. Ventricular fibrillation (VF) onset in humans—Purkinje and structural substrate govern the transition from trigger to disorganized VF. Schematic view of initial VF activities in patients with cardiac structural abnormalities. The upper panel shows an electrocardiogram of spontaneous VF onset in a patient with a prior history of myocardial infarction. The three illustrations show the sequence of trigger, initial organized VF, and disorganized VF. The trigger is shown as a red star close to the structural substrate (mottled white area). Initial VF activities are represented as localized waves generated from the ventricular or Purkinje substrate. Then the acceleration of activities in parallel with previously described changes (reduction in action potential duration, Ca handling…) leads to dissemination of activities and VF disorganization.

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.

Cardiovascular magnetic resonance (CMR) can through the two methods 3D FLASH and diffusion tensor imaging (DTI) give complementary information on the local orientations of cardiomyocytes and their laminar arrays. Eight explanted rat hearts were perfused with Gd-DTPA contrast agent and fixative and imaged in a 9.4T magnet by two types of acquisition: 3D fast low angle shot (FLASH) imaging, voxels 50 × 50 × 50 μm, and 3D spin echo DTI with monopolar diffusion gradients of 3.6 ms duration at 11.5 ms separation, voxels 200 × 200 × 200 μm. The sensitivity of each approach to imaging parameters was explored. The FLASH data showed laminar alignments of voxels with high signal, in keeping with the presumed predominance of contrast in the interstices between sheetlets. It was analysed, using structure-tensor (ST) analysis, to determine the most (v 1 ST ), intermediate (v 2 ST ) and least (v 3 ST ) extended orthogonal directions of signal continuity. The DTI data was analysed to determine the most (e 1 DTI ), intermediate (e 2 DTI ) and least (e 3 DTI ) orthogonal eigenvectors of extent of diffusion. The correspondence between the FLASH and DTI methods was measured and appraised. The most extended direction of FLASH signal (v 1 ST ) agreed well with that of diffusion (e 1 DTI ) throughout the left ventricle (representative discrepancy in the septum of 13.3 ± 6.7°: median ± absolute deviation) and both were in keeping with the expected local orientations of the long-axis of cardiomyocytes. However, the orientation of the least directions of FLASH signal continuity (v 3 ST ) and diffusion (e 3 ST ) showed greater discrepancies of up to 27.9 ± 17.4°. Both FLASH (v 3 ST ) and DTI (e 3 DTI ) where compared to directly measured laminar arrays in the FLASH images. For FLASH the discrepancy between the structure-tensor calculated v 3 ST and the directly measured FLASH laminar array normal was of 9 ± 7° for the lateral wall and 7 ± 9° for the septum (median ± inter quartile range), and for DTI the discrepancy between the calculated v 3 DTI and the directly measured FLASH laminar array normal was 22 ± 14° and 61 ± 53.4°. DTI was relatively insensitive to the number of diffusion directions and to time up to 72 hours post fixation, but was moderately affected by b-value (which was scaled by modifying diffusion gradient pulse strength with fixed gradient pulse separation). Optimal DTI parameters were b = 1000 mm/s2 and 12 diffusion directions. FLASH acquisitions were relatively insensitive to the image processing parameters explored. We show that ST analysis of FLASH is a useful and accurate tool in the measurement of cardiac microstructure. While both FLASH and the DTI approaches appear promising for mapping of the alignments of myocytes throughout myocardium, marked discrepancies between the cross myocyte anisotropies deduced from each method call for consideration of their respective limitations.

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.

Alteration of action potential duration (APD) heterogeneity contributes to arrhythmogenesis. Purkinje-muscle junctions (PMJs) present differential electrophysiological properties including longer APD. The goal of this study was to determine if Purkinje-related or myocardial focal activation modulates ventricular repolarization differentially in healthy and ischemic myocardium. Simultaneous epicardial (EPI) and endocardial (ENDO) optical mapping was performed on sheep left ventricular (LV) wedges with intact free-running Purkinje network (N = 7). Preparations were paced on either ENDO or EPI surfaces, or the free-running Purkinje fibers (PFs), mimicking normal activation. EPI and ENDO APDs were assessed for each pacing configuration, before and after (7 min) of the onset of no-flow ischemia. Experiments were supported by simulations. In control conditions, maximal APD was found at endocardial PMJ sites. We observed a significant transmural APD gradient for PF pacing with PMJ APD = 347 ± 41 ms and EPI APD = 273 ± 36 ms (p < 0.001). A similar transmural gradient was observed when pacing ENDO (49 ± 31 ms; p = 0.005). However, the gradient was reduced when pacing EPI (37 ± 20 ms; p = 0.005). Global dispersion of repolarization was the most pronounced for EPI pacing. In ischemia, both ENDO and EPI APD were reduced (p = 0.005) and the transmural APD gradient (109 ± 55 ms) was increased when pacing ENDO compared to control condition or when pacing EPI (p < 0.05). APD maxima remained localized at functional PMJs during ischemia. Local repolarization dispersion was significantly higher at the PMJ than at other sites. The results were consistent with simulations. We found that the activation sequence modulates repolarization heterogeneity in the ischemic sheep LV. PMJs remain active following ischemia and exert significant influence on local repolarization patterns.

The pig is commonly used as an experimental model of human heart disease, including for the study of mechanisms of arrhythmia. However, there exist differences between human and porcine cellular electrophysiology: The pig action potential (AP) has a deeper phase-1 notch, a longer duration at 50% repolarization, and higher plateau potentials than human. Ionic differences underlying the AP include larger rapid delayed-rectifier and smaller inward-rectifier K⁺-currents (I_Kr and I_K1 respectively) in humans. AP steady-state rate-dependence and restitution is steeper in pigs. Porcine Ca²⁺ transients can have two components, unlike human. Although a reliable computational model for human ventricular myocytes exists, one for pigs is lacking. This hampers translation from results obtained in pigs to human myocardium. Here, we developed a computational model of the pig ventricular cardiomyocyte AP using experimental datasets of the relevant ionic currents, Ca²⁺-handling, AP shape, AP duration restitution, and inducibility of triggered activity and alternans. To properly capture porcine Ca²⁺ transients, we introduced a two-step process with a faster release in the t-tubular region, followed by a slower diffusion-induced release from a non t-tubular subcellular region. The pig model behavior was compared with that of a human ventricular cardiomyocyte (O’Hara-Rudy) model. The pig, but not the human model, developed early afterdepolarizations (EADs) under block of I_K1, while I_Kr block led to EADs in the human but not in the pig model. At fast rates (pacing cycle length = 400 ms), the human cell model was more susceptible to spontaneous Ca²⁺ release-mediated delayed afterdepolarizations (DADs) and triggered activity than pig. Fast pacing led to alternans in human but not pig. Developing species-specific models incorporating electrophysiology and Ca^2+-handling provides a tool to aid translating antiarrhythmic and arrhythmogenic assessment from the bench to the clinic.