High arrhythmic risk in antero-septal acute myocardial ischemia is explained by increased transmural reentry occurrence

Acute myocardial ischemia is a precursor of sudden arrhythmic death. Variability in its manifestation hampers understanding of arrhythmia mechanisms and challenges risk stratification. Our aim is to unravel the mechanisms underlying how size, transmural extent and location of ischemia determine arrhythmia vulnerability and ECG alterations. High performance computing simulations using a human torso/biventricular biophysically-detailed model were conducted to quantify the impact of varying ischemic region properties, including location (LAD/LCX occlusion), transmural/subendocardial ischemia, size, and normal/slow myocardial propagation. ECG biomarkers and vulnerability window for reentry were computed in over 400 simulations for 18 cases evaluated. Two distinct mechanisms explained larger vulnerability to reentry in transmural versus subendocardial ischemia. Macro-reentry around the ischemic region was the primary mechanism increasing arrhythmic risk in transmural versus subendocardial ischemia, for both LAD and LCX occlusion. Transmural micro-reentry at the ischemic border zone explained arrhythmic vulnerability in subendocardial ischemia, especially in LAD occlusion, as reentries were favoured by the ischemic region intersecting the septo-apical region. ST elevation reflected ischemic extent in transmural ischemia for LCX and LAD occlusion but not in subendocardial ischemia (associated with mild ST depression). The technology and results presented can inform safety and efficacy evaluation of anti-arrhythmic therapy in acute myocardial ischemia.

human torso/ventricular models, which are very costly and require high performance computing. In this study, we exploit high resolution datasets obtained from high performance computing simulations using a human torso/ biventricular electrophysiology model, constructed and evaluated using extensive experimental and clinical data from ionic dynamics to the ECG. Electrophysiological changes in phase 1A ischemia are different from those observed in other stages post-occlusion, such as phase 1B ischemia or infarction, which have been the focus of previous investigations 12,13 .

Methods
Human torso/biventricular electrophysiological model in acute ischemia. A human biventricular model embedded in a torso was used to simulate electrophysiological activity from ionic dynamics to body surface potentials (Fig. 1A, left) 14 . Electrophysiological alterations in the ischemic region were modelled as in previous studies 15,16 , including ischemic core zone (ICZ), lateral border zone (BZ) and endocardial BZ. Membrane Stimulation protocols. Sinus rhythm was simulated using a realistic activation sequence by applying endocardial stimulation 14 for 3 beats (S1) with a cycle length of 600 ms. To evaluate reentry vulnerability, ectopic stimulation (S2) was applied transmurally at the BZ. This was based on the experimental evidence from 22 reporting that the earliest activity of premature beats in acute myocardial ischemia was found to occur in the normal myocardium adjacent to the ischemic region, and that no important time differences were found between endo-and epicardium. S2 was applied at varying coupling intervals (CI, i.e. time difference between the last S1 and S2) and for each CI, simulated electrical activity was analysed to identify reentry occurrence. The vulnerability window (VW) was quantified as the range of CIs for which S2 resulted in at least two reentrant cycles. As the relative location of the ectopic stimulation in the ischemic BZ with respect to ventricular anatomy and ischemic region may influence the VW, we considered six S2 locations equally spaced around the BZ. Simulations were conducted using the numerical solver CHASTE 23 . By combining multiple scenarios, CIs and ectopic locations, we conducted a computationally-expensive study of more than 400 simulations of 3-11 hours on 720 CPUs.

Results
credibility of human torso-ventricular model for ecG and reentry vulnerability investigations in control and acute ischemia. Simulation results with the human torso-biventricular model in acute ischemia were first evaluated through comparison with experimental and clinical recordings. Firstly, as demonstrated in our previous study 17 and in Supplementary Tables ST1 and ST2, the electrophysiological consequences of acute myocardial ischemia at ionic, cellular and tissue level are reproduced in the simulations with the human ventricular tissue model, in agreement with experimental recordings 3,4,17,24,25 . Specifically, in the simulations, ischemic tissue exhibits human ventricular action potential duration shortening, elevation of resting potential, prolonged post-repolarization refractoriness and decreased conduction velocity (see Supplementary Tables ST1  and ST2) as shown in previous experimental, clinical and computational recordings 3,4,17,[24][25][26][27][28] . These are the key electrophysiological properties relevant for evaluation of reentrant dynamics and ECG changes in acute ischemia.
Furthermore, as described in our previous study 14 , the endocardial activation model imposed to simulate sinus rhythm yields activation sequence and QRS complex in the 12-lead ECG, consistent with experimental and clinical recordings [29][30][31] . This is further illustrated in Supplementary Fig. SF2, which shows the agreement of simulated ECG in healthy conditions compared to a clinical recording from a healthy volunteer from the PTB database 32 , both in terms of QRS complex and T wave morphology.
Introduction of acute regional ischemia in the human torso/biventricular model causes QRS alterations and ST deviations in agreement with clinical recordings for both transmural and subendocardial ischemia ( Thus, the consistency of simulation results with experimental and clinical recordings for ionic, cellular, tissue, whole-ventricular and ECG properties yields credibility to the findings presented below. impact of location, transmural extent and size of acute regional ischemia on ecG biomarkers.   Differences in wavelength for reentry explain the effect of ischemic region size and myocardial conduction velocity on arrhythmic risk. Figure 4 displays the results of the VWs obtained for the 16 different ischemia scenarios with premature stimulus S2 in the ischemic BZ in the LV mid-cavity for both LAD and LCX occlusion. No reentry was induced without ischemia with the same protocol. Consistent with the theory on reentrant circuits, slow myocardial propagation (as imposed in the grey-coloured cases in Figs 2 and 4) facilitates reentry by reducing the wavelength of cardiac impulse (i.e. conduction velocity multiplied by refractory period). This is demonstrated in Fig. 4 by the wider VWs reported in each grey-coloured scenario with slow conduction velocity versus their correspondent case with normal conduction velocity in the remote myocardium. Additionally, larger ICZ were always more arrhythmogenic than smaller ones (Fig. 4).
The mechanisms are illustrated in Fig. 5, for large versus small ischemic regions (Fig. 5A,B, respectively), and normal versus slow myocardial propagation for small ischemic regions (Fig. 5B,C, respectively). In agreement with previous studies 5, 15 , propagation following the ectopic stimulus is blocked in the still refractory ischemic region (red cross, 382 ms), but circles around it towards the RV through base and apex (white arrows, 382 ms). The larger pathway through the large ischemic region (Fig. 5A) allows enough time, firstly, for ischemic tissue to recover and to allow retrograde propagation through the large ischemic region (Fig. 5A, 424 ms), and secondly, for the normal tissue to recover once propagation has traversed the ischemic region, ensuring the continuation of the reentry (Fig. 5A, 544 ms).
In contrast, the small ischemic region (Fig. 5B) does not allow enough time for the recovery of ischemic tissue, preventing retrograde propagation and leading to bidirectional conduction block (Fig. 5B, 424 ms), and no reentry established (Fig. 5B, 544 ms). Reducing conduction velocity promotes the formation of reentrant circuits by delaying propagation around the ischemic region (Fig. 5C, 382 ms), allowing the recovery of the ischemic tissue and ensuring retrograde propagation and reentry (Fig. 5C, 424, 544 ms), even with small ischemic region.
Arrhythmia vulnerability in subendorcardial LAD occlusion is explained by septo-apical transmural microreentrant pathways. We then investigated the mechanisms explaining how the effect of location and transmural extent of the ischemic region may modulate arrhythmic risk. We hypothesized that the intersection of the ischemic border zone with the septum, as in LAD occlusion, promotes the establishment of transmural micro-reentrant pathways. In the investigation of differences between LAD and LCX occlusion, the location of the ectopic stimulus with respect to the LV anatomy is likely to modulate arrhythmic risk. We therefore computed the VWs for six different S2 locations around the BZ, for large transmural and subendocardial ischemic areas in LAD versus LCX occlusion (Fig. 6). Overall, more reentries are induced in LAD versus LCX occlusion (54 versus 33 reentries for CIs = 225-300 ms), considering both transmural and subendocardial ischemia, for the conditions tested. Furthermore, more reentries are established in transmural versus subendocardial ischemia: 27 versus 6 reentries, respectively, in LCX occlusion, and 35 versus 19 reentries, respectively, in LAD occlusion (Fig. 6).
Macro-reentry around the ischemic region, as described in Fig. 5 and in previous studies 5,15 , was established in transmural ischemia, for both LCX and LAD occlusions and most S2 locations (Fig. 6, light grey boxes).
As reported by Janse et al. 5 , an additional mechanism consisting of transmural micro-reentry was identified (Fig. 6, dark grey boxes), for both LAD and LCX occlusion. Even though transmural micro-reentries occurred both in transmural and subendocardial ischemia, they were the key mechanism explaining vulnerability to reentry in subendocardial ischemia (Fig. 6). Figure 7 demonstrates the differences in reentrant patterns in transmural versus subendocardial ischemia. As in Figs 5A,C, 7A illustrates the different stages of macro-reentry, with unidirectional conduction block in the ICZ, followed by propagation around the BZ (400 ms) proceeding retrogradely into the ICZ (480 ms) and finally reentering into the NZ (620 ms). The reentrant wave continues (710 ms), sustaining the macro-reentry around the ICZ.
However, in subendocardial ischemia (Fig. 7B), the epicardial view shows that the ectopic stimulus propagates through the ventricles (260 to 480 ms), with subsequent recovery (620 ms) until a breakthrough occurs (710 ms). In contrast, intramural dynamics are very different as shown in the transmural view through the septum in Fig. 7C. Following the ectopic stimulus (260 ms), propagation is blocked unidirectionally (red cross) in the subendocardial ischemic region (400 ms) but proceeds towards the base and also surrounds the ischemic region through the LV wall towards the apex (white arrows). Propagation continues to surround the BZ (480 ms, 550 ms), blocked in the ischemic region due to refractoriness (red cross), but reentering through normal tissue towards the apex (white arrows, 620 ms). It is then that a spiral wave is established, anchored in the BZ in the septo-apical www.nature.com/scientificreports www.nature.com/scientificreports/ region (Fig. 7C, 620 ms-802 ms). Figure 7D demonstrates the existence of a stable I-type filament in the septo-apical region. Simulated electrocardiographic signal in lead V3 of both reentries analysed in Fig. 7A,B/C/D are shown in 7E, associating macro reentries with larger wave amplitude than transmural micro-reentries (~1.3 versus ~0.5 mV).
The mechanisms underlying the smaller number of reentries in subendocardial versus transmural ischemia post-LCX occlusion are illustrated in Fig. 8A,B. Macro-reentry around the transmural ischemic region is established in Fig. 8A, similarly to the patterns shown in Fig. 7A. However, the subendocardial ischemic region is not able to sustain the conditions for reentry establishment, as shown in Fig. 8B. Figure 8C shows the electrocardiogram signal  www.nature.com/scientificreports www.nature.com/scientificreports/ simulated in lead V6 for the cases displayed in Fig. 8A,B. More details about the formation of reentries can be found in the Supplementary Videos (SV1-SV4).

Discussion
In this simulation study, the human ventricles affected by acute regional ischemia are shown to sustain two key types of reentrant patterns that determine differences in arrhythmic risk with varying ischemic region location, transmurality and size using high performance simulations. Macro-reentry around the ischemic region occurs primarily for large fully-transmural ischemic regions leading to ST elevation, for both LAD and LCX occlusion. Large ischemic regions and slow myocardial propagation favoured the establishment of macro-reentries. Furthermore, transmural micro-reentry at the ischemic border zone was identified as critical to explain arrhythmic risk, particularly for subendocardial ischemic regions, associated with mild ST depression. www.nature.com/scientificreports www.nature.com/scientificreports/ Our simulation results also show a higher arrhythmic risk in LAD versus LCX post-occlusion, for the conditions tested, in agreement with clinical reports 34 . Simulations reveal that this is due to a higher propensity for the establishment of transmural reentrant circuits at the intersection of ischemic region and septo-apical region in LAD occlusion, rather than in the LV for LCX occlusion 8,11 . Furthermore, simulations show that QRS downslopes may provide useful information on ischemic extent for subendocardial ischemia, whereas ST elevation is an indicator of size for fully-transmural ischemic regions 19,35 .
The credibility of the findings is supported by the consistency of the simulation results with experimental and clinical recordings at the different scales involved, as proposed by Carusi et al. 36 . Firstly, ischemic tissue displays key electrophysiological alterations known to determine ECG biomarkers and reentrant patterns post-occlusion, and specifically prolonged post-repolarization refractoriness and slow conduction velocity 3,4,16,24,25 . This also includes electrophysiological gradients in the ischemic border zone, as in our previous studies 15,26,28 . Furthermore, the activation sequence used to simulate sinus rhythm has also been shown to be consistent with experimental recordings 29 . Finally, ECG patterns in control and in acute ischemia are in agreement with clinical 12-lead ECGs recordings 21,32,33 . This is both in terms of QRS complex progression between precordial leads ( Supplementary  Fig. SF2) and ischemia-induced alterations in QRS slopes and ST elevation (Fig. 2), shown to be in the range with values reported in clinical studies 35,37 .
The simulations with the human ventricular acutely-ischemic model also reproduce the figure-of-eight reentrant patterns around the ischemic region, as reported experimentally by 5,15 . These macro-reentries are favoured by large transmural ischemic regions, and their likelihood is explained by the wavelength for reentry. Simulations also reveal micro-reentries, also reported experimentally 5 , which are facilitated by slow intramural conduction close to the BZ. Whereas they also occur in transmural ischemia, micro-reentries are the only observed mechanism of reentry in subendocardial ischemia. Post-LAD occlusion, the intersection of the BZ and the septo-apical region favours the establishment of micro-reentry by providing larger intramural pathways than in the LV free wall. This can explain the higher arrhythmic risk in LAD versus LCX occlusion 8,11,38 . The existence of two pro-arrhythmic mechanisms of reentry in acute ischemia determined by the transmurality and location of the ischemic region may offer opportunities for targeted anti-arrhythmic treatments.
Slow propagation in the remote myocardium had a substantial effect on arrhythmic risk in our simulations, even though with negligible ECG effects (Fig. 4). Fibrosis 39 , diabetes 40 , or hypertrophy 41 are main risk factors potentially affecting cell coupling and consequently decreasing myocardial conduction, albeit with unclear ECG signature 42 . In our simulations, slow conduction promoted reentry in acute regional ischemia. This is in close correspondence with ex-vivo studies linking the occurrence of transmural reentries to conduction delays produced by fibrotic tissue 43 . It could also explain that patients suffering from fibrosis as a consequence of pathologies like hypertrophic cardiomyopathy 44 or diabetes mellitus 45 are at higher risk under ischemic conditions. Moreover, subendocardial ischemia was associated with slight ST depression in our simulations, as in Wilhelms et al. 46 , but also with high arrhythmia vulnerability, for LAD occlusion. The significance of potential risk predictors in silent myocardial ischemia remains an open discussion 47,48 . Significant ST elevation as main clinical marker of acute ischemia severity 48 was only correlated in our results with fully-transmural scenarios supporting macro-reentry (Fig. 3). Conversely, changes in QRS downslope 19 accurately represented the proportion of affected tissue and arrhythmic risk for LAD occlusion.

Limitations of the Study
The consequences of acute myocardial ischemia are complex and dynamic. In this study we focus on simulating the electrophysiological consequences of the first 10 minutes post-occlusion on action potential, refractoriness and conduction velocity to investigate ECG alterations and reentry vulnerability in one human ventricular anatomical model of phase 1A ischemia. In addition to these factors, variability in anatomy, perfusion areas, ischemic region shape and severity, and baseline electrophysiological properties could also modulate arrhythmic risk [49][50][51][52] . Additional factors to consider are that simulations are undertaken in a stationary (non-contracting) ventricular model, and anatomical features such as endocardial trabeculations, papillary muscles and autonomic balance could play a role in reentrant pathways during ischemia [53][54][55][56] . Our study did not consider the role the Purkinje system may have during reentrant activity in acute ischemia. Building on the new insights provided by our study, further studies can investigate those additional factors.

conclusion
In this study, we present a human torso/biventricular modelling and simulation study into the mechanisms of variability in arrhythmic risk and ECG biomarkers in acute regional ischemia (phase 1A). The credibility of the simulation findings is supported by the consistency of simulated electrophysiological properties obtained with the human multiscale model with experimental and clinical recordings for ionic, cellular, tissue, and ECG properties. We identified transmural micro-reentries, in addition to the established macro-reentry around the ischemic region, as key pro-arrhythmic mechanisms in acute regional ischemia. Transmural acute ischemia (causing ST elevation) led to both macro-and micro-reentries, whereas arrhythmic risk in subendocardial ischemia (associated with mild ST depression) was predominantly explained by transmural micro-reentry. The modelling and simulation technology and results presented can inform the safety and efficacy evaluation of anti-arrhythmic therapy in acute myocardial ischemia.