TYPE Original Research PUBLISHED 20 September 2022 DOI 10.3389/fcvm.2022.983001 Electrophysiological effects of OPEN ACCESS adipose graft transposition EDITED BY Vincenzo Lionetti, Sant’Anna School of Advanced Studies, procedure (AGTP) on the Italy REVIEWED BY post-myocardial infarction scar: Michele Miragoli, University of Parma, Italy Khatia Gabisonia, A multimodal characterization Sant’Anna School of Advanced Studies, Italy of arrhythmogenic *CORRESPONDENCE Carolina Gálvez-Montón cgalvez@igtp.cat substrate Felipe Bisbal f.bisbalvb@gmail.com Raquel Adeliño1 , Daina Martínez-Falguera1,2 , Carolina Curiel3 , † These authors share senior authorship Albert Teis1,4,5 , Roger Marsal3 , Oriol Rodríguez-Leor4,5 , SPECIALTY SECTION This article was submitted to Cristina Prat-Vidal1 , Edgar Fadeuilhe4 , Júlia Aranyó4 , Cardiovascular Biologics Elena Revuelta-López1,5 , Axel Sarrias4 , Víctor Bazan4 , and Regenerative Medicine, a section of the journal Joan F. Andrés-Cordón4 , Santiago Roura1,4,5,6 , Frontiers in Cardiovascular Medicine Roger Villuendas4,5 , Josep Lupón4,5,7 , Antoni Bayes-Genis1,4,5,7 , RECEIVED 30 June 2022 ACCEPTED 23 August 2022 Carolina Gálvez-Montón1,4,5*† and Felipe Bisbal1,4,5*† PUBLISHED 20 September 2022 1 ICREC Research Program, Germans Trias i Pujol Research Institute (IGTP), Barcelona, Spain, 2 CITATION Faculty of Medicine, University of Barcelona, Barcelona, Spain, 3 Boston Scientific Department, Adeliño R, Martínez-Falguera D, Barcelona Delegation, Barcelona, Spain, 4 Heart Institute (iCOR), Germans Trias i Pujol University Curiel C, Teis A, Marsal R, Hospital, Barcelona, Spain, 5 CIBER Cardiovascular, Instituto de Salud Carlos III, Madrid, Spain, Rodríguez-Leor O, Prat-Vidal C, 6 Faculty of Medicine, University of Vic-Central University of Catalonia (UVic-UCC), Vic, Spain, Fadeuilhe E, Aranyó J, 7 Department of Medicine, Can Ruti Campus, Autonomous University of Barcelona, Barcelona, Spain Revuelta-López E, Sarrias A, Bazan V, Andrés-Cordón JF, Roura S, Villuendas R, Lupón J, Bayes-Genis A, Gálvez-Montón C and Bisbal F (2022) Electrophysiological effects of adipose graft transposition procedure (AGTP) Objective: To assess the arrhythmic safety profile of the adipose graft on the post-myocardial infarction scar: transposition procedure (AGTP) and its electrophysiological effects on post- A multimodal characterization of arrhythmogenic substrate. myocardial infarction (MI) scar. Front. Cardiovasc. Med. 9:983001. doi: 10.3389/fcvm.2022.983001 Background: Myocardial repair is a promising treatment for patients with COPYRIGHT MI. The AGTP is a cardiac reparative therapy that reduces infarct size and © 2022 Adeliño, Martínez-Falguera, improves cardiac function. The impact of AGTP on arrhythmogenesis has Curiel, Teis, Marsal, Rodríguez-Leor, Prat-Vidal, Fadeuilhe, Aranyó, not been addressed. Revuelta-López, Sarrias, Bazan, Andrés-Cordón, Roura, Villuendas, Methods: MI was induced in 20 swine. Contrast-enhanced magnetic Lupón, Bayes-Genis, Gálvez-Montón resonance (ce-MRI), electrophysiological study (EPS), and left-ventricular and Bisbal. This is an open-access article distributed under the terms of endocardial high-density mapping were performed 15 days post-MI. Animals the Creative Commons Attribution were randomized 1:1 to AGTP or sham-surgery group and monitored License (CC BY). The use, distribution with ECG-Holter. Repeat EPS, endocardial mapping, and ce-MRI were or reproduction in other forums is permitted, provided the original performed 30 days post-intervention. Myocardial SERCA2, Connexin-43 author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cardiovascular Medicine 01 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 (Cx43), Ryanodine receptor-2 (RyR2), and cardiac troponin-I (cTnI) gene and protein expression were evaluated. Results: The AGTP group showed a significant reduction of the total infarct scar, border zone and dense scar mass by ce-MRI (p = 0.04), and a decreased total scar and border zone area in bipolar voltage mapping (p < 0.001). AGTP treatment significantly reduced the area of very-slow conduction velocity (<0.2 m/s) (p = 0.002), the number of deceleration zones (p = 0.029), and the area of fractionated electrograms (p = 0.005). No differences were detected in number of induced or spontaneous ventricular arrhythmias at EPS and Holter-monitoring. SERCA2, Cx43, and RyR2 gene expression were decreased in the infarct core of AGTP-treated animals (p = 0.021, p = 0.018, p = 0.051, respectively). Conclusion: AGTP is a safe reparative therapy in terms of arrhythmic risk and provides additional protective effect against adverse electrophysiological remodeling in ischemic heart disease. KEYWORDS myocardial infarction, adipose graft transposition procedure, myocardial repair, arrhythmic risk, ventricular tachycardia, mapping Introduction MI, with absence of arrhythmic events at 30 days post- intervention (4). In the first-in-human phase I trial, this Despite rapid progress over the past two decades, therapy proved to be safe in terms of arrhythmic events, myocardial infarction (MI) remains a leading cause of hospital admissions, and mortality (5). Currently, AGTP disability and mortality. Myocardial revascularization strategies efficacy is being tested in a multicenter, randomized, have significantly improved the natural history of MI, yet controlled phase II-III clinical trial (AGTP II Trial, post-infarct scar continues to produce ventricular dysfunction, NCT02798276) (6). heart failure, and sudden death as a result of ventricular Despite the potential beneficial effects of AGTP and arrhythmias (1, 2). Development of new reparative therapies other cardiac reparative therapies, a thorough assessment of remains a priority and continues to be a challenge in the field of their arrhythmogenic profile remains mandatory. Both pro- biomedical research. and antiarrhythmic mechanisms have been attributed to cell The adipose graft transposition procedure (AGTP) is a therapies, in most cases through ex vivo studies (7–13). reparative therapy based on placing an autologous pericardial During the pre-clinical and clinical evaluation of AGTP adipose pedicle over the infarct scar. The adipose flap therapy, no arrhythmic events have been detected, although contains mesenchymal stromal cells with immunomodulatory they were never actively sought and their true incidence could and angiogenic properties capable of migration into the have been underestimated. Accordingly, the objective of the infarcted myocardium. Since the pedicle maintains its own study was to characterize the effect of AGTP therapy on vascularization, AGTP ensures the viability of the mesenchymal tissue characteristics and the electrophysiological properties of stromal cells over time, thereby facilitating their reparative effect ischemic scar in a porcine model of chronic MI. (3–5). We and others have shown that AGTP therapy reduces the size of the scar and increases vascularization Materials and methods in the infarcted area in a porcine model of chronic This study was approved by the Animal Experimentation Unit Ethics Committee of the Germans Trias i Pujol Health Abbreviations: AGTP, adipose graft transposition procedure; BZ, Research Institute (IGTP) and by government authorities border zone; Ce-MRI, contrast-enhanced magnetic resonance; CV, conduction velocity; DZ, deceleration zones; EGM, electrograms; (Generalitat de Catalunya; Code: 10558). It complied with EPS, electrophysiological study; HDM, high-density mapping; LV, all guidelines concerning the use of animals in research and left ventricle; LVEF, left ventricular ejection fraction; MI, myocardial infarction; PVC, premature ventricular contractions; VF, ventricular teaching as defined by the Guide for the Care and Use of fibrillation; VT, ventricular tachycardia. Laboratory Animals (14). Frontiers in Cardiovascular Medicine 02 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 Study design under general anesthesia and endotracheal intubation. All images were acquired during repeated ventilator apneas The study design is depicted in Figure 1. Twenty-eight and synchronized with the ECG. Fast field echo (FFE) crossbreed Landrace X Large White pigs (50% females) cine images were acquired in 2, 3, and 4 chamber views underwent MI induction. At 15 days post-MI, evaluation and in short-axis orientation covering left ventricle from with contrast-enhanced magnetic resonance imaging (ce- base to apex. Slice thickness was 8 mm and no gap MRI), electrophysiological study (EPS), and left ventricular was used between short-axis slices. A set of conventional (LV) endocardial high-density mapping (HDM) were delayed enhancement images was obtained 10 min after bolus performed. Animals were randomly assigned to AGTP injection of gadolinium-derived contrast agent (Gadobutrol or sham surgery (Sham group). Follow-up evaluation 0.2 mmol/kg, Gadovist , Bayer, Berlin, Germany) using a R (ce-MRI, EPS, HDM) and gene/protein analysis were well-established segmented 2D inversion recovery gradient- performed 45 days post-MI (30 days post-surgery). All echo sequence. Additionally, a whole-heart high spatial procedures were carried out under general anesthesia and resolution delayed enhancement was acquired using a free- endotracheal intubation (extended details can be found in breathing, navigator-gated 3D inversion-recovery gradient-echo Supplementary material). technique. Cardiac structure and function were analyzed with Medis Medical Imaging (Leiden, Netherlands) and included end- R Myocardial infarction swine model diastolic volume (EDV), end-systolic volume (ESV), LV ejection fraction (LVEF), and LV myocardial mass (16). The model of non-reperfused MI was previously described Scar characterization with late gadolinium enhancement (15). Briefly, a left lateral MI was induced by percutaneous sequences was post-processed with the ADAS 3D software R deployment of 1–3 coils (VortX-18 Diamond 3 mm/3.3 mm (Galgo Medical, Barcelona, Spain) and included quantification coil, Boston Scientific/Target, Natick, MA, United States) at of scar subtypes (17–21). Ce-MRI provides the capability the proximal first marginal branch of the circumflex artery to assess scar 3-dimensionality by means of full-thickness or another branch as needed (4, 15). Coronary occlusion analysis. Thresholds of > 60, 40–60%, and < 40% of (TIMI flow score = 0) was confirmed by angiography in all maximum pixel intensity were used to identify dense scar, cases. To assess differences in myocardial damage between heterogeneous border zone (BZ) tissue, and normal tissue, groups, cardiac troponin-I (cTnI) serum levels were analyzed in respectively. The number and mass of MRI-detected BZ blood samples (ARCHITECT STAT High Sensitive Troponin-I; corridors were quantified. BZ corridors are automatically Architect i2000) collected at baseline and 2 h post-MI. detected as corridors of BZ tissue between two core areas or between a core area and a valve annulus and have been correlated with slow conducting channels in Adipose graft transposition procedure sinus rhythm and ventricular tachycardia (VT) isthmuses (19, 21). Two weeks after MI induction, pigs underwent left lateral thoracotomy in the fourth intercostal space to expose the heart protected by the pericardium. The pericardial adipose Electrophysiological study and tissue was detached from the fibrous layer of the pericardium, endocardial mapping maintaining its vascularization to create the adipose flap. After pericardiectomy, myocardial scar was localized in the LV LV endocardial HDM was performed via retrograde lateral wall and covered by the vascularized adipose graft in aortic access by means of the 64-pole basket catheter AGTP-treated pigs and sealed with cyanoacrylic-based surgical (Intellamap Orion, Boston Scientific, Massachusetts, glue (Glubran 2, Cardiolink, Barcelona, Spain), as previously R United States) and Rhythmia HDx 3D mapping system described (3, 4). In the Sham group, the pericardium and (Boston Scientific, Massachusetts, United States) during pericardial adipose tissue were removed. right ventricular pacing at a fixed cycle length of 400– 450 ms, depending on baseline heart rate (same cycle length was used in baseline and follow-up maps). Programmed Magnetic resonance imaging ventricular stimulation protocol was performed with S1 acquisition and post-processing trains (600–500–400 ms) and up to S5 (ventricular refractory period or 170 ms). Mapping of induced VT was attempted Ce-MRI was performed by 3T MRI scanner (Canon whenever hemodynamic conditions allowed. Extended details Vantage Galan; Ota, Tokyo, Japan), using a phased-array regarding signal filtering and acquisition can be found in four-channel surface coil and ECG gating. All animals were Supplementary material. Frontiers in Cardiovascular Medicine 03 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 FIGURE 1 Chronogram and workflow of the study. ce-MRI, contrast-enhanced magnetic resonance imaging; HD, high density; LV, left ventricular; EPS, electrophysiological study. Voltage and activation maps were analyzed off-line out of 8 isochrones comprising the entire ventricular with Rhythmia and self-customized Paraview-based activation window) (24), and areas of complex fractionated software.1 Conventional cut-offs were used for bipolar electrograms (≥ 4 deflections of the bipolar electrogram) (0.5–1.5 mV) and unipolar (6.7 mV) (22, 23) voltage. (25, 26). Conduction velocity (CV) was determined for every pair of contiguous points. Areas of CV were quantified for every 0.2 m/s steps (< 0.2–4 m/s). Lumipoint module Heart rhythm monitoring was used to determine areas of post-QRS activation and slow conduction zones, including number of deceleration Half of the animals in each group were monitored with 3- zones (DZ, defined as ≥ 3 isochrones within 1 cm lead ECG-Holter (AFT-1000-A Holter Supplies, Pairs, France) for 15 days post-surgery. Baseline rhythm, number and length of sustained and non-sustained VT episodes, and the burden of 1 www.paraview.org premature ventricular contractions (PVC) were quantified. Frontiers in Cardiovascular Medicine 04 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 Gene and protein expression analysis Myocardial infarction assessment Expression levels of Connexin 43 (Cx43), cardiac No significant differences in the increase of circulating levels Troponin-I (cTnI), sarco/endoplasmic reticulum Ca2+ - of cTnI after MI were found between AGTP and sham-surgery ATPase (SERCA2), and Ryanodine receptor-2 (RyR2) genes animals, indicating similar magnitude of myocardial ischemic were measured by quantitative real-time PCR (qRT-PCR) damage between groups (p = 0.286). (Supplementary Table 1) from infarct core, BZ, and remote myocardium in a subset of animals (Sham n = 7; AGTP n = 6). Magnetic resonance imaging For western blot analysis, 50 µg of protein from characterization of myocardial the infarct core was used to determine cTnI, Cx43, infarction SERCA2, RyR2, and α-tubulin protein expression. Immunohistochemical analyses were performed with anti- Baseline iLVEDV, iLVESV, LVEF, total scar mass, BZ mass, Cx43, SERCA2, α-SMA, and cTnI antibodies. Further dense scar mass, and number of corridors were similar between details about tissue collection, gene and protein expression, groups (Table 1). Thirty days after treatment, scar analysis and immunohistochemical analysis are included in by ce-MRI showed significant reduction in total scar mass (– Supplementary material. 2.2 ± 2.5 g vs. + 1.7 ± 3.2 g, p = 0.012), BZ mass (–1.6 ± 1.6 g vs. + 1.1 ± 2.5 g, p = 0.014), and dense scar mass (–0.6 ± 1.1 g vs. + 0.5 ± 1.1 g, p = 0.042) in the AGTP group compared to Statistical analysis the Sham group. A trend toward reduction in the number and mass of VT corridors was observed (–0.4 ± 1.3 vs. + 0.4 ± 1.2, Continuous data were reported as mean ± SD for p = 0.2 and –0.2 ± 0.5 g vs. + 0.1 ± 0.4 g, p = 0.14, respectively) normal-distributed variables or median ± IQR for non- (Figure 2). There were no differences between groups regarding normal distributed variables. Categorical data were presented LVEF or LV volumes (Table 2). Intragroup variation analysis is as percentages. Differences between groups in continuous included in Supplementary Table 2. variables were compared using Student’s t-test, ANOVA Greenhouse-Geisser correction, or Wilcoxon-rank test when the variable was not normally distributed. Chi-Square Endocardial mapping and and Fisher Exact tests were used to compare categorical electrophysiological study variables. Values of p < 0.05 were considered significant. Analyses were performed with Stata software (version 12, There was a significant reduction of total scar (– StataCorp, College Station, TX) and SPSS (19.0.1 version, SPSS, 2.2 ± 3.4 cm2 vs. + 1.1 ± 0.5 cm2 , p = 0.012) (Table 2) and BZ Inc., Chicago, IL). (–1.9 ± 2.4 cm2 vs. + 1.0 ± 1.9 cm2 , p < 0.001) (Figures 3A, 4) areas in AGTP compared to Sham group, with no significant variation of the dense scar area or the unipolar low voltage area (Table 2). The AGTP group showed an improved conduction Results properties profile compared to Sham group, with a significant reduction in the number of DZ (–0.3 ± 0.9 + 0.8 ± 0.9, p = 0.029) Study population (Figures 3B, 5), the area of very-slow (< 0.2 m/s) CV (– 3.9 ± 3.8 mm2 vs. + 1.6 ± 3.1 mm2 , p = 0.002) (Figure 3C) Initially, 28 pigs were enrolled between September 2019 and the area of highly fractionated electrograms (–1.1 ± 1.3 cm2 and January 2021. Due to COVID-19 pandemic restrictions, vs. + 0.6 ± 0.9 cm2 , p = 0.005) (Figure 3D), as well as a the experiment was temporarily suspended and 3 animals trend toward a reduction of the post-QRS activation area (– had to be excluded because of lockdown. Five animals died 0.4 ± 1.5 cm2 vs. + 0.8 ± 2.8 cm2 , p = 0.069) (Table 2). before completion of the study: 3 during MI induction [due Intragroup variation analysis is included in Supplementary to ventricular fibrillation (VF), atrioventricular block, and Table 2. coronary dissection, respectively], 1 during baseline mapping There were no significant differences in the rate of induction (due to VF), and 1 following sham surgery (due to VF). of ventricular arrhythmias in the follow-up EPS between the The remaining 20 subjects (33.9 ± 3.2 kg) were allocated AGTP and Sham groups [1 (10%) vs. 3 (30%), respectively; 1:1 to receive AGTP (n = 10; 50% female) or sham surgery p = 0.275]. None of the 4 induced ventricular arrhythmias (n = 10; 50% female). All completed the study interventions were mapped due to hemodynamic instability (3 VF and and follow-up (Figure 1). Baseline characteristics are detailed in 1 fast VT). Intragroup variation analysis is included in Table 1. Supplementary Table 2. Frontiers in Cardiovascular Medicine 05 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 TABLE 1 Baseline characteristics of AGTP and sham-surgery animals. AGTP Sham P Morphometric data Sex (female: N,%) 5, 50% 5, 50% 1.000 Weight (Kg, mean ± SD) 34.7 ± 3.1 34.1 ± 3.9 0.686 Characteristics of substrate assessed by electroanatomical mapping Total scar area (cm2 , mean ± SD) 8.4 ± 5.2 5.4 ± 4.1 0.172 BZ area (cm2 , mean ± SD) 5.6 ± 4.4 3.8 ± 2.6 0.100 Dense scar area (cm2 , median ± IQR) 1.5 ± 2.3 0.5 ± 2.6 0.596 Unipolar voltage area < 6.7 V (mm2 , median ± IQR) 80.1 ± 550.5 155.3 ± 187.8 0.677 Area of velocity < 0.2 m/s (mm2 , median ± IQR) 5.3 ± 8.6 0.3 ± 2.3 0.012 Area of velocity < 0.4 m/s (mm2 , median ± IQR) 6.4 ± 9.8 1.9 ± 7.7 0.080 Number of DZ (N, mean ± SD) 1.6 ± 1.3 0.8 ± 0.8 0.110 Post-QRS activation area (cm2 , median ± IQR) 2.0 ± 1.5 1.1 ± 1.3 0.168 Area of electrograms with ≥ 4 deflections (cm2 , mean ± SD) 4.1 ± 1.9 2.8 ± 1.2 0.120 Characteristics of substrate assessed by MRI iLVEDV (ml, mean ± SD) 124.2 ± 17.6 114.2 ± 19.2 0.271 iLVESV (ml, mean ± SD) 79.0 ± 17.6 71.9 ± 21.0 0.403 LVEF (%, mean ± SD) 38.0 ± 11.2 35.6 ± 12.0 0.674 Percentual scar size (%, mean ± SD) 10.0 ± 2.8 9.0 ± 6.1 0.700 Total scar mass (g, mean ± SD) 7.9 ± 2.9 5.8 ± 3.3 0.165 BZ mass (g, mean ± SD) 4.9 ± 1.8 4.0 ± 2.7 0.446 Dense scar mass (g, mean ± SD) 1.8 ± 1.0 3.1 ± 1.7 0.060 Corridors, number (N, median ± IQR) 1.0 ± 1.0 0.5 ± 1.0 0.241 Corridors, mass (g, mean ± SD) 0.5 ± 0.3 0.3 ± 0.3 0.074 BZ, border zone; DZ, deceleration zones; iLVEDV, indexed left ventricle end-diastolic volume; iLVESV, indexed left ventricle end-systolic volume; LVEF, left ventricular ejection fraction. Rhythm monitoring Sham (n = 6) and AGTP (n = 6) groups. However, RyR2 was higher in AGTP group compared to Sham (0.99 ± 0.60 A.U. vs. Holter-monitoring was performed in 10 animals (5 of each 2.75 ± 0.81 A.U., respectively; p = 0.002) (Figures 6B,C). No group). There were no differences between groups in number differences were detected between groups in terms of SERCA2 of PVCs, episodes of non-sustained VT, or total time in VT and Cx43 protein expression after immunohistochemical (Table 2). analysis. Positive α-SMA myofibroblasts were present in the infarct of all animals suggesting no differences between groups (Supplementary Figure 1, Upper panel). Despite viable Gene and protein expression cardiomyocytes from the border zone were positive for Cx43 (Supplementary Figure 1, Bottom panel), no differences were After MI, the loss of cTnI gene expression in the infarct core detected in infarct core with low-undetectable Cx43 expression. region of all pigs was confirmed in AGTP and Sham animals Extended data on western blot analysis is represented in (24.4 ± 10.2 vs. 620.6 ± 167.3 in AGTP, p < 0.001; 78.3 ± 55.8 Supplementary Figure 2 and Supplementary Table 3. vs. 579.4 ± 140.2 in Sham, p < 0.001; respectively), with a trend toward downregulation in AGTP animals (p = 0.064), although non-statistically significant. SERCA2, Cx43, and RyR2 genes Discussion were decreased in the infarct core of AGTP animals (p = 0.021, p = 0.018, p = 0.051, respectively). Gene expression of cTnI was The present study showed that cardiac reparative therapy partially retained in the BZ compared to remote myocardium in of chronic ischemic scar with AGTP does not increase the risk both the Sham and AGTP groups (53.4 and 56.4%, respectively), of spontaneous or induced ventricular arrhythmias and may confirming the presence of a mixture of viable myocytes and improve the arrhythmogenic milieu of the scar by reducing the fibrotic scar. In the remote myocardial samples, no significant BZ components and the areas of slow conduction, deceleration, differences between groups were detected (Figure 6A). and fractionation (Central Illustration). Regarding protein level, western blot analysis in the infarct Tissue heterogeneity of chronic ischemic scar is a hallmark core showed similar expression of cTnI and Cx43 between of VT substrate (27) and is characterized by a combination of Frontiers in Cardiovascular Medicine 06 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 FIGURE 2 Contrast-enhanced MRI-based tissue characterization. Representative examples of post-MI substrate remodeling at 30-day follow-up. Favorable remodeling was observed in the AGTP group (upper panel), with a significant reduction in border zone (BZ) and dense scar and a trend toward reduced BZ corridors. Sham group (lower panel) showed increased scar volume and a trend toward development of new BZ corridors. surviving myocardium embedded on diffuse or patchy scar (28). represent an additional mechanistic pathway for reduced The heterogeneous scar allows for slow, anisotropic conduction risk of ventricular events in individuals receiving reparative (29, 30) through viable myocytes within the scar, historically therapy with AGTP. defined as areas of intermediate bipolar voltage (BZ thresholds The mechanisms underlying the improvement of CV and 0.5 < 1.5 mV) (22). More recently, advancements in ce-MRI the reduction of total scar and BZ in both endocardial have allowed non-invasive tissue characterization (17–21). Our HDM and ce-MRI may be multifactorial. First, an increase study showed that AGTP reduces BZ areas of the scar, as in vascular supply may have enhanced cell viability by assessed by endocardial voltage mapping and by ce-MRI. This reducing myocardial tissue hypoxia. Previous studies assessing effect led to a trend in reducing the number and mass of MRI- AGTP in chronic MI porcine model showed the presence of detected BZ corridors in the AGTP group, suggesting a potential micro-vessels connecting the pericardial adipose vascular flap mechanism for reduced arrhythmic risk. with the underlying ischemic myocardium, which correlated Our study showed that AGTP may provide additional with smaller scars due to increased myocardial salvage benefits in terms of arrhythmogenicity by improving the after AGTP therapy (4). In acute MI porcine model, cell electrophysiological properties of the scar. Treated individuals migration and trafficking from adipose pedicle to the infarcted showed a reduction in the number of DZ and in the size myocardium was also demonstrated (3). Cardiac adipose of areas with very-slow CV (<0.2 m/s) and those with a tissue contains progenitor cells with inherent cardiac-like high degree of fractionation. Previous data support that all phenotype that can differentiate into endothelial cell types these conduction abnormalities are commonly present at the (34, 35). Recent data from our group has demonstrated critical sites of the VT circuit (26, 31–33). Improvement that adipose cardiac tissue-resident progenitor cells may of overall conduction properties of the ischemic scar could promote local vascularization by secretion of proangiogenic Frontiers in Cardiovascular Medicine 07 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 TABLE 2 Results of MRI, LV endocardial HDM and rhythm monitoring of AGTP and sham-surgery animals at 30-day follow-up from the surgery. AGTP Sham P MRI LV and scar analysis iLVEDV (ml, mean ± SD) –3.2 ± 18.5 + 2.4 ± 18.9 0.532 iLVESV (ml, mean ± SD) –5.7 ± 18 –4.2 ± 16 0.938 LVEF (%, mean ± SD) + 2.6 ± 9.4 +4.4 ± 9.6 0.695 Total scar mass (g, mean ± SD) –2.2 ± 2.5 + 1.7 ± 3.2 0.012 BZ mass (g, mean ± SD) –1.6 ± 1.6 + 1.1 ± 2.5 0.014 Dense scar mass (g, mean ± SD) –0.6 ± 1.1 + 0.5 ± 1.1 0.042 Corridors, number (g, mean ± SD) –0.4 ± 1.3 + 0.4 ± 1.2 0.203 Corridors, mass (g, mean ± SD) –0.2 ± 0.5 + 0.1 ± 0.4 0.136 HD electroanatomical mapping Total scar area (cm2 , mean ± SD) –2.2 ± 3.4 1.1 ± 0.5 0.012 BZ area (cm2 , median ± IQR) –1.9 ± 2.4 + 1.0 ± 1.9 < 0.001 Dense scar area (cm2 , mean ± SD) 0.3 ± 1.3 –0.2 ± 0.9 0.353 Unipolar low voltage area (mm2 , median ± IQR) + 0.1 ± 2.8 –0.2 ± 7.9 0.449 Area of velocity < 0.2 m/s (mm2 , mean ± SD) –3.9 ± 3.8 + 1.6 ± 3.1 0.002 Area of velocity < 0.4 m/s (mm2 , mean ± SD) –3.1 ± 10.3 1.0 ± 6.7 0.087 DZ (N, mean ± SD) –0.3 ± 0.9 + 0.8 ± 0.9 0.029 Post-QRS activation area (cm2 , median ± IQR) –0.4 ± 1.5 + 0.8 ± 2.8 0.069 Area of electrograms with ≥ 4 deflections (cm2 , mean ± SD) –1.1 ± 1.3 + 0.6 ± 0.9 0.005 Rhythm monitoring Number of PVC (mean ± SD) 500 ± 472 600 ± 442 0.687 Number of non-sustained VT episodes (mean ± SD) 6.0 ± 8.6 4.5 ± 4.9 0.572 Total time in VT (seconds, mean ± SD) 13.0 ± 25.3 8.2 ± 9.6 0.345 BZ, border zone; DZ, deceleration zones; iLVEDV, indexed left ventricle end-diastolic volume; iLVESV, indexed left ventricle end-systolic volume; LVEF, left ventricular ejection fraction; PVC, premature ventricular contractions; VT, ventricular tachycardia. FIGURE 3 Baseline and follow-up mapping data. Boxplots shows significant differences in the area of border zone tissue (A), number of deceleration zones (B), very-slow conduction velocity (C), and highly fractionated electrogram area (D). BZ, border zone; CV, conduction velocity; DZ, deceleration zones. Frontiers in Cardiovascular Medicine 08 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 FIGURE 4 Scar detection by endocardial mapping. High density endocardial mapping of the left ventricle showing post-MI low voltage area. After 30 days, animals receiving AGTP showed a reduction in border zone area (0.5–1 mV) (upper panel) whereas those in the Sham group showed overall increase in total scar area (bottom panel). FIGURE 5 Isochronal mapping and deceleration zones. Representative isochronal activation maps of AGTP (top) and sham-surgery (bottom) animals; the AGTP group showed significant reduction in the number of deceleration zones over time, whereas an increase was observed in the sham subjects (A). (B) Illustrates an example of a follow-up high density map of a sham subject exhibiting a late and fractionated electrogram (yellow box) at the latest activation area (infero-basal left ventricular wall). Frontiers in Cardiovascular Medicine 09 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 FIGURE 6 Gene and protein expression profiles. (A) cTnI, Cx43, SERCA2, and RyR2 gene expression in sham (n = 7) and AGTP-treated (n = 6) animals determined by normalizing the expression for each gene to GUSB following the 2−1 Ct method. (B) Western Blot images of RyR2 presence and the respective Ponceau S staining to depict protein loading (top) and the corresponding histogram of RyR2 protein expression of sham and AGTP groups (bottom). (C) cTnI, Cx43, and RyR2 protein expression determined by western blot in sham and AGTP-treated animals represented in boxplots. CENTRAL ILLUSTRATION Experimental model and main findings of the study. BZ, border zone; DZ, deceleration zones; EGM, electrogram; EPS, electrophysiological study; HD, high density; MRI, magnetic resonance imaging. Frontiers in Cardiovascular Medicine 10 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 factors (e.g., extracellular vesicles, cytokines) as a paracrine tolerated fast monomorphic VTs; thus, no correlation effect (35). Additionally, adipose-derived hormones can exert between critical isthmus of the VT and the voltage and cardio-protective effects against ischemia by cardiomyocyte activation map could be established. The scar following apoptosis attenuation, infarct size reduction, and modulation first marginal occlusion may have eventually affected of collagen I/III ratio into the myocardial scar, as we myocardium beneath the posteromedial papillary muscle; previously reported (3). Consistently with previous data this would likely lead to underestimation of infarct size by (4), our study showed no benefit in LV contractility in endocardial mapping but not by MRI. Epicardial adhesions chronic phase of MI. Scar heterogeneity has been shown following prior AGTP/sham surgery preclude epicardial to be better correlated with arrhythmogenicity than with mapping; thus, epicardial arrhythmogenic substrate was LV function (36). The lack of improvement in LVEF in not assessed. The study did not include a non-infarcted AGTP-treated animals is not necessarily related to the sham group, according to the 3R principle as it was not electrophysiological remodeling of the scar; thus, AGTP may expected to add significant information to the study. LV impact the arrhythmogenic milieu without effect on LV dyssynchrony or regional wall motion abnormalities were systolic function. Notably, relatively small scars resulted in not analyzed. Previous data on functional impact of AGTP a remarkable impairment of the LVEF. Unpublished data therapy can be found in previous publications from our group of another, unrelated set of animals showed the impact of (4). Heart failure related biomarkers (i.e., BNP) were not scar location on global LV systolic function. Non-significant analyzed. Finally, despite a high SD value of cTnI values, differences in LVEF decline were observed at 30-day following there were no statistically significant differences between anterior (mid LAD, 36.4 ± 7.5%) vs. lateral infarction groups in post-MI circulant cTnI or in the total scar mass (first marginal branch of the circumflex artery, 42.5 ± 5.3) assessed by ce-MRI, therefore ensuring similar baseline MI (p = 0.055), despite significant differences in infarct size extension in both groups. (21.1 ± 5 vs. 9.4 ± 6.1% LGE Mass 5SD, respectively; p = 0.001). These results suggest that smaller lateral scars may lead to similar impact on global LVEF than more Clinical perspectives extensive anterior scars. In addition to the reduced size of scar in AGTP- The AGTP reduces the scar size in chronic MI porcine treated animals, a trend toward a lower cTnI expression model (4). In the first-in-human phase I trial (adiFLAP might suggest higher homogeneity, although not reaching Trial; NCT01473433), no adverse or arrhythmic event statistical significance. A lower gene expression of SERCA2, related to AGTP were detected (5). In the present study, a Cx43, and RyR2 in the AGTP group may lead to a thorough assessment of the electrophysiological remodeling more homogeneous electrical propagation and contribute of the scar following AGTP was carried out. Our findings to a more unexcitable tissue, which may ultimately suggest that this reparative therapy is safe and may provide reduce arrhythmic events as previously described (37). protective antiarrhythmic effects, adding a potential benefit Specifically, SERCA2 and RyR2 are responsible for against arrhythmic events on top of reducing scar size and Ca2 + intracellular transport (38, 39), and Cx43 for heterogeneity and improving LV function. These findings intercellular communication and electric conductance. provide additional safety information that will require The downregulation of these proteins in the AGTP group further confirmation from the ongoing AGTP II randomized may parallel the reduction of tissue border zone, suggesting clinical trial (phase II-III; NCT02798276) (6). The AGTP a reduction of viable cells intermingled with scar tissue. is a simple technique that does not require exogenous Cx43 is a component of the gap junction and is involved cell therapy or cardiac tissue engineering products and in the inter-myocyte communication; a lower presence of avoids histocompatibility issues, making the procedure Cx43 at the myocardial scar and viable cardiomyocytes readily available to any patient with chronic MI undergoing may contribute to decreased pro-arrhythmic milieu in cardiac surgery. AGTP-treated animals. Conclusion Study limitations Treatment with AGTP of non-reperfused chronic MI is a The number of induced ventricular arrhythmias was safe reparative therapy in terms of arrhythmic risk and provides low, with no differences between groups. Limited infarct additional protective effect against adverse electrophysiological size derived from first marginal artery embolization may remodeling by homogenizing the scar, reducing border zone have contributed to the low VT inducibility rate and to components, and improving areas of slow conduction and the high proportion of fast polymorphic VT/VF or poorly fractionation. Frontiers in Cardiovascular Medicine 11 frontiersin.org Adeliño et al. 10.3389/fcvm.2022.983001 Data availability statement Acknowledgments The original contributions presented in this study are We are grateful to the Center of Comparative Medicine and included in the article/Supplementary material, further Bioimaging of Catalonia (CMCiB) for their contribution to the inquiries can be directed to the corresponding author/s. successful execution of the animal model. Ethics statement Conflict of interest The animal study was reviewed and approved by the Animal Experimentation Unit Ethics Committee of the Germans Trias FB served as a consultant for Biosense Webster and Abbott i Pujol Health Research Institute (IGTP) and the Government and has received speaker honoraria from Boston Scientific, Authorities (Generalitat de Catalunya; Code: 10558). Biosense Webster, Biotronik, and Abbott. The remaining authors declare that the research was conducted in the absence of any commercial or financial Author contributions relationships that could be construed as a potential conflict of interest. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. Publisher’s note All claims expressed in this article are solely those of the Funding authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the This work was supported by the Instituto de Salud Carlos reviewers. Any product that may be evaluated in this article, or III (ISCIII PI18/01227, PI18/00256, INT20/00052, PIC18/00014, claim that may be made by its manufacturer, is not guaranteed and PI21/01700), Sociedad Española de Cardiología, Generalitat or endorsed by the publisher. de Catalunya (2017-SGR-483), Red RICORS (RD21/0017/0022), and CIBER Cardiovascular (CB16/11/00403) as part of the Plan Nacional de I + D + I, and co-funded by ISCIII- Subdirección General de Evaluación and the Fondo Europeo Supplementary material de Desarrollo Regional (FEDER). RA has been sponsored by a grant (2021FI_B1 00202) from the Agència de Gestió The Supplementary Material for this article can be d’Ajuts Universitaris i de Recerca (AGAUR) and Fons found online at: https://www.frontiersin.org/articles/10.3389/ Social Europeu (FSE). fcvm.2022.983001/full#supplementary-material References 1. Timmis A, Townsend N, Gale CP, Torbica A, Lettino M, Petersen SE, et al. 6. 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