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Cardiac fiber direction is an important factor determining the propagation of electrical activity, as well as the development of mechanical force. In this article, we imaged the ventricles of several species with special attention to the intraventricular septum to determine the functional consequences of septal fiber organization. First, we identified a dual-layer organization of the fiber orientation in the intraventricular septum of ex vivo sheep hearts using diffusion tensor imaging at high field MRI. To expand the scope of the results, we investigated the presence of a similar fiber organization in five mammalian species (rat, canine, pig, sheep, and human) and highlighted the continuity of the layer with the moderator band in large mammalian species. We implemented the measured septal fiber fields in three-dimensional electromechanical computer models to assess the impact of the fiber orientation. The downward fibers produced a diamond activation pattern superficially in the right ventricle. Electromechanically, there was very little change in pressure volume loops although the stress distribution was altered. In conclusion, we clarified that the right ventricular septum has a downwardly directed superficial layer in larger mammalian species, which can have modest effects on stress distribution.

Modifications of the myocardial architecture can cause abnormal electrical activity of the heart. Fibro-fatty infiltrations have been implicated in various cardiac pathologies associated with arrhythmias and sudden cardiac death, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Here, we report the development of an MRI protocol to observe these modifications at 9.4 T. Two fixed ex vivo human hearts, one healthy and one ARVC, were imaged with an Iterative decomposition with echo asymmetry and least-square estimations (IDEAL) and a magnetization transfer (MT) 3D sequences. The resulting fat fraction and MT ratio (MTR) were analyzed and compared to histological analysis of the three regions (“ARVC triangle”) primarily involved in ARVC structural remodeling. In the ARVC heart, high fat content was observed in the “ARVC triangle” and the superimposition of the MTR and fat fraction allowed the identification of fibrotic regions in areas without the presence of fat. The healthy heart exhibited twice less fat than the ARVC heart (31.9%, 28.7% and 1.3% of fat in the same regions, respectively). Localization of fat and fibrosis were confirmed by means of histology. This non-destructive approach allows the investigation of structural remodeling in human pathologies where fibrosis and/or fatty tissue infiltrations are expected to occur.

Background: We report our experience of an unexpected complication of internalization of a pulmonary artery (PA) band in the vascular lumen, which occurred in a chronic porcine model of repaired tetralogy of Fallot (TOF). Methods: Twelve piglets were divided into 3 groups: (1) TOF model animals (PA band plus pulmonary valvotomy, n = 4), (2) pulmonary insufficiency (PI) animals (pulmonary valvotomy, n = 4), and (3) control animals (n = 4). A nonabsorbable, coated braided polyester tape was used to perform the main pulmonary artery banding. Echocardiography was performed 4 months postoperatively. After each animal was euthanized, PA histological analysis was performed in animals with band internalization. Results: Significant postsurgical pulmonary regurgitation and right ventricular enlargement were present in the TOF and PI, compared with control animals, whereas no significant pulmonary stenosis was observed in TOF animals when compared with PI group. Postmortem examination of all TOF animals revealed the constricting band to be intact but partially internalized into the PA lumen, allowing blood flow around the stenosis. Histological sections of the banded PA in the area of internalization showed a significant disorganization of the medial layer, with significant scarring and fibrotic reaction surrounding the outside of the band and the presence of inflammatory cells suggesting a significant inflammatory response during band internalization. Conclusions: Band internalization may occur after PA banding using a nonabsorbable, coated braided polyester tape in a chronic porcine model of repaired TOF. This unusual complication was likely due to the type of material used for banding.

Background: A large Dispersion of Repolarization (DoR) is associated with an increased arrhythmogenic risk. This can be measured clinically by calculating the Activation Recovery Interval (ARI) to estimate Action Potential Duration (APD). However, the ability of ARI to accurately predict APD dispersion in patients with repolarization abnormality has not been determined. Objective: Compare ARI calculated from patients with optical mapping of human hearts to establish the validity of ARI as a surrogate for APD. Methods: Optical mapping (OM) was performed on the left ventricles of 4 explanted human hearts. APD and repolarization times were measured endo- and epicardially on the anterior of the LV. Electroanatomic mapping was performed with CARTO over the entire endo- and epicardial surfaces of 3 patients. Activation and repolarization were calculated, dispersion of ARI was measured. Results: APD and ARI were consistent between mapping methods over most of the sub-regions studied. Epicardium ARI dispersion was consistently higher than that of the endocardium in both OM and CARTO datasets. Conclusion: APD distribution, and consequently DoR, agree between mapping methods. Measuring DoR by ARI accurately assesses the underlying repolarization abnormalities in patients.

Introduction The right ventricular outflow tract (RVOT) is the main origin of idiopathic ventricular tachycardia (IVTs). Sympathetic stimulation is involved in IVTs but mechanisms underlying this preferential origin remain unknown. Objective To determine the role of s1- and s2-adrenergic receptors (s1-AR, s2-AR) in the regional modulation of right ventricular (RV) electrophysiological properties and arrhythmias. Methods Porcine RVs were isolated and perfused via both right and left coronary arteries. Epicardial (EPI) electrical activity was optically mapped (di-4-ANEPPS 20 μM) and APD measured at 80% repolarization (APD80) in the RVOT and posterior RV free wall region (RVFW). Regional mRNA and protein expression for s1-AR and s2-AR was determined respectively by RT-PCR and Western-Blot. Results Combined s1- and s2-AR stimulation (50 nM isoprenaline, ISO) shortened APD80 EPI with a larger effect in the RVFW than the RVOT. Interestingly, isolated s2-AR stimulation (10–25 μM salbutamol, SALBU with 4 μM metoprolol, METO) only decreased APD80 significantly in the RVOT EPI (P Conlcusion These results suggest a s2-AR specific regulation of RVOT electrophysiology but its role in the triggering of arrhythmias in this region requires further studies.

Introduction The RVOT is involved in arrhythmias in several diseases such as the Brugada syndrome (BrS). In a subpopulation of patients with BrS and early repolarization (ER) syndrome, a K-ATP gain-of-function (GOF) mutation has previously been described and was suggested to contribute to arrhythmogenicity in this context. Objective To determine the role of regional K-ATP channels expression on RV repolarization heterogeneity and in arrhythmia occurrence and modulation. Methods Pig right ventricles (RV) were perfused by both right and left coronary arteries, paced at 1 or 1.5 Hz and the electrical activity optically-mapped (di-4-ANEPPS, 20 μM). The preparation was perfused with pinacidil (PINA, 20 μM), and diazoxide (DIAZ, 100 μM), and subjected to 20 min no-flow (NF) ischemia episodes followed by 20 min reperfusion (RF). Arrhythmia susceptibility was assessed using a 20 Hz burst protocol while spontaneous arrhythmias were also recorded. Protein expression was assessed by western blot. Results PINA-induced action potential duration (APD) shortening was larger in RVOT than in the free wall (FW). DIAZ induced a slight but significant reduction in APD across the RV. SUR2A expression was greater in the RVOT epicardium vs. FW but Kir6.1/2 and SUR1 expression was similar in both regions. PINA induced spontaneous ventricular fibrillation (VF) in 80% of the preparations with higher dominant frequency (DF) and regularity index in the RVOT than the FW. DF of burst-induced VF were higher in PINA but not in DIAZ compared to CTRL VF. Similarly to PINA, NF-induced APD shortening was greater in the RVOT than in the FW. No spontaneous arrhythmia occurred during NF but slow ventricular tachycardias arose in 36% of the preparations during RF. Conclusion K-ATP activation leads to an increase in RV repolarization dispersion which is likely to contribute to arrhythmogenicity in BrS or ER patients with a K-ATP channel GOF mutation and may underlie BrS phenocopy induced by RV ischemia.

International audience; The development and use of nanomaterials, especially engineered nanoparticles (NP), is expected to provide many benefits. But at the same time the development of such materials is also feared because of their potential human health risks. Indeed, NP display some characteristics similar to ultrafine environmental particles which are known to exert deleterious cardiovascular effects including pro-hypertensive ones. In this context, the effect of NP on calcium signalling, whose deregulation is often involved in hypertensive diseases, remain poorly described. We thus assessed the effect of SiO2 NP on calcium signalling by fluorescence imaging and on the proliferation response in rat pulmonary artery smooth muscle cells (PASMC). In PASMC, acute exposure to SiO2 NP, from 1 to 500μg/mL, produced an increase of the [Ca(2+)]i. In addition, when PASMC were exposed to NP at 200μg/mL, a proliferative response was observed. This calcium increase was even greater in PASMC isolated from rats suffering from pulmonary hypertension. The absence of extracellular calcium, addition of diltiazem or nicardipine (L-type voltage-operated calcium channel inhibitors both used at 10μM), and addition of capsazepine or HC067047 (TRPV1 and TRPV4 inhibitors used at 10μM and 5μM, respectively) significantly reduced this response. Moreover, this response was also inhibited by thapsigargin (SERCA inhibitor, 1μM), ryanodine (100μM) and dantrolene (ryanodine receptor antagonists, 10μM) but not by xestospongin C (IP3 receptor antagonist, 10μM). Thus, NP induce an intracellular calcium rise in rat PASMC originating from both extracellular and intracellular calcium sources. This study also provides evidence for the implication of TRPV channels in NP induced calcium rise that may highlight the role of these channels in the deleterious cardiovascular effects of NP.

Introduction We recently found the presence of a pro-arrhythmic electrophysiological substrate with increased collagen content and slow conduction in the healthy right ventricular outflow tract (RVOT). An epicardial layer of fat is commonly found in the RVOT but whether fat penetrates in the myocardium and regulates RVOT structure and electrophysiology remains unknown. Objective To assess the presence and localization of fat infiltrations (FI) in the healthy RVOT. Methods The presence of FI was assessed histologically in Masson's trichrome and Oil-Red-O stained sections from young healthy pigs (n = 4) and human donor hearts with no cardiac disease history (n = 5). High resolution MRI (9.4 T Bruker Biospin) was used to localize fat in formaldehyde-fixed human RVOTs. The expression of selected targets was assessed by qPCR and Western Blot in the subepicardial layer (EPI) of the RV free wall (RVFW) and RVOT of healthy pigs. Results Sporadic FIs could be observed in the pig RVOT but were absent in the RVFW. In human hearts, FIs were present in the RVFW, LV free wall but were larger in the RVOT. High resolution MRI showed the presence of 3 layers in the human RVOT wall with a thick epicardial fat layer, a transition layer composed of myocardial fibers and fat (up to 50% of wall thickness) and a muscular endocardial layer. Intramyocardial fat content increased with age. The mRNA expression of perilipin (PLIN1) and the adipogenic hormone adiponectin (ADIPOQ) was significantly higher in the RVOT EPI compared to the RVFW (P Conclusion FIs are in direct contact with the adjacent myocardium in the healthy porcine and human RVOT. Activin-A is likely to contribute to the higher collagen content found in the healthy RVOT as observed in the atria. Thus, these infiltrations are likely to participate to RVOT slower conduction and arrhythmogenesis.

Introduction The right ventricular outflow tract (RVOT) has a distinct embryonic development leading to specific electrophysiological properties in the healthy adult heart. These include shorter action potential duration (APD), which is likely to contribute to RVOT high susceptibility to arrhythmias in idiopathic and pathological conditions. How these differences are altered in ischemia remains unknown. Objective We sought to determine whether RVOT repolarization undergoes a specific adaptation to ischemia. Methods Pig right ventricles (RV) were perfused by both right and left anterior descending coronary arteries, paced at 1.5 Hz and the electrical activity optically-mapped on epicardial (EPI) and endocardial (ENDO) surfaces (di-4-ANEPPS, 20 μM). The preparation was perfused with pinacidil (PINA, 20 μM), diazoxide (DIAZ, 100 μM) and glibenclamide (GLIB, 10 μM), and subjected to 20 min no-flow ischemia episodes followed by 20 min reperfusion. Protein expression was assessed by western blot and localization by immunohistochemistry. Results Ischemia significantly shortened APD across the whole wedge, with a larger effect in RVOT than RVFW EPI (−50.94% ± 9.58% vs. −36.18% ± 8.38% P Conclusion RVOT repolarization seems to be more sensitive to ischemia than the RVFW. Katp channels are involved in this difference despite similar Kir6.1 and Kir6.2 expression, suggesting a role for sulphonylurea receptors. This increase in repolarization heterogeneity is likely to facilitate re-entrant arrhythmias in this context.

Introduction The right ventricular outflow tract (RVOT) is the main origin of idiopathic ventricular tachycardia (VT). Although these arrhythmias appear to be cAMP-dependent, the actual mechanisms leading to VT remain uncertain. Here we studied β-adrenergic regulation of action potential duration (APD) in the healthy pig right ventricle (RV). Objective To determine regional β-adrenergic regulation of APD and arrhythmias in the RVOT. Methods The RV of 4 pig hearts were isolated and perfused via both the coronary arteries. Epicardial electrical activity was optically mapped (di-4-ANEPPS 20 μM) and APD measured at 80% repolarization (APD80) in the RVOT and posterior RV free wall region (RVFW). Regional mRNA and protein expression of β-adrenergic receptors was determined respectively by RT-PCR and Western-Blot. Results The expression of β1-adrenergic receptor (β1-AR) was lower in the RVOT at the mRNA level (P Conlcusion β-adrenergic stimulation differentially regulates APD80 in the RVOT and RVFW. Although β1-AR expression was similar in the 2 regions, ISO preferentially shortened APD and triggered arrhythmia in the RVOT. These results suggest the presence of a high sensitivity of RVOT electrophysiology to β-adrenergic stimulation.

BackgroundTetralogy of Fallot (TOF) is associated with increasingly recognized late morbidity due to arrhythmias and right heart failure. Better understanding of the underlying mechanisms of these issues is needed to facilitate new therapeutic approaches. We aim to identify mechanisms generating arrhythmias in a swine model of repaired TOF with progressive right ventricular (RV) dysfunction.MethodsSurgery to mimic repaired TOF was done in 24 piglets (using a previously validated model); 24 animals served as control. Two, 4 and 6 months after surgery respectively, animals were sacrified for subsequent analysis. Haemodynamic parameters and ventricular remodeling were analyzed by cardiac magnetic resonance (CMR) and echocardiography before sacrifice. In isolated perfused hearts, electrical activity was measured by optical mapping. Sarcoplasmic reticular calcium handling and proteins involved in calcium management were studied in single myocytes.ResultsCompared with control animals, a right bundle branch block was present and the action potential (AP) duration was increased in the RV with a mechanical delay observed at 2 and 4 months post-operative in operated groups (p

Ventricular arrhythmias are frequent in patients with repaired tetralogy of Fallot but their underlying mechanisms remain unclear. In this study, ventricular electrical and structural remodelling was assessed in an animal model that mimics postoperative tetralogy of Fallot.Piglets underwent a tetralogy of Fallot repair-like surgery (rTOF N=6) or were sham-operated (Sham N=5). Following cardiac function assessment in vivo by MRI 3–4 months after surgery, pigs were euthanized and their hearts rapidly excised. Electrophysiological properties of right (RV) and left ventricles (LV) were obtained by optical mapping. Fibrosis was assessed histologically.RV dysfunction was evident while LV function remained unaltered in rTOF pigs. LV action potential duration (APD) was significantly longer on the epicardium (Sham 280±50ms; rTOF 390±76ms) and endocardium (Sham 301±20ms; rTOF 403±34ms) of rTOF animals (P

Background The investigation of single cardiac myocytes from healthy and diseased hearts of various species is a valuable tool to explore cardiac physio/ pathophysiology. The application of cell isolation to human donor tissue has been proofed to be difficult due to the limited amount of human tissue (mainly human right atrial appendages during cardiac surgery). Another limitation is the low viability of cardiomyocytes after isolation. In this study, we present a method to obtain single cardiac myocytes from different regions of human heart. Methods and results Human hearts rejected for transplantation were obtained from Bordeaux hospital. This protocol was approved by the Agence de la Biomedecine. Left atrial (LA) and ventricular (LV) myocytes were obtained by enzymatic dissociation. The ventricles and right atrium were removed and used for other studies (e.g. high resolution optical mapping). LA was cannulated by the circumflex artery and mounted into a Langendorff perfusion system after suture of the leaky atrial branches. LA was perfused with a Ca2+-free solution (~10 min), then collagenase and protease solution (0.08 mM Ca2+) and recirculated for ~25 min. Enzymes were washed out with a 0.2 mM Ca2+ solution. LA was separated into 4 regions: Endocardium, Epicardium, roof and pulmonary vein; LV myocytes were also obtained. Cells were re-suspended into a 1.8 mM Ca2+ solution by steps. Ca2+ transients were recorded (Fura-2, field stimulation) using an IonOptix system and cell membrane was stained with di-8 ANEPPS and visualized under confocal microscopy. Ca2+ tolerant myocytes were obtained from the 4 LA regions and LV. Human cardiac myocytes respond to electrical stimulation and Ca2+ transient can be recorded. Analysis of functional and structural data will be presented. Conclusion Isolation of single cardiac myocytes from human samples is a tedious task, but we present data showing reliable method to obtain functional and structural insights. The author hereby declares no conflict of interest

Ventricular arrhythmias are frequent in patients with repaired tetralogy of Fallot but their underlying mechanisms remain unclear. In this study, ventricular electrical and structural remodelling was assessed in an animal model that mimics postoperative tetralogy of Fallot. Piglets underwent a tetralogy of Fallot repair-like surgery (rTOF N=6) or were sham-operated (Sham N=5). Following cardiac function assessment in vivo by MRI 3-4 months after surgery, pigs were euthanized and their hearts rapidly excised. Electrophysiological properties of right (RV) and left ventricles (LV) were obtained by optical mapping. Fibrosis was assessed histologically. RV dysfunction was evident while LV function remained unaltered in rTOF pigs. LV action potential duration (APD) was significantly longer on the epicardium (Sham 280±50ms; rTOF 390±76ms) and endocardium (Sham 301±20ms; rTOF 403±34ms) of rTOF animals (P

Objective The growing adult population with surgically corrected tetralogy of Fallot (TOF) is at risk of arrhythmias and sudden cardiac death. We sought to investigate the contribution of right ventricular (RV) structural and electrophysiological remodelling to arrhythmia generation in a preclinical animal model of repaired TOF (rTOF). Methods and results Pigs mimicking rTOF underwent cardiac MRI functional characterisation and presented with pulmonary regurgitation, RV hypertrophy, dilatation and dysfunction compared with Sham-operated animals (Sham). Optical mapping of rTOF RV-perfused wedges revealed a significant prolongation of RV activation time with slower conduction velocities and regions of conduction slowing well beyond the surgical scar. A reduced protein expression and lateralisation of Connexin-43 were identified in rTOF RVs. A remodelling of extracellular matrix-related gene expression and an increase in collagen content that correlated with prolonged RV activation time were also found in these animals. RV action potential duration (APD) was prolonged in the epicardial anterior region at early and late repolarisation level, thus contributing to a greater APD heterogeneity and to altered transmural and anteroposterior APD gradients in rTOF RVs. APD remodelling involved changes in Kv4.3 and MiRP1 expression. Spontaneous arrhythmias were more frequent in rTOF wedges and more complex in the anterior than in the posterior RV. Conclusion Significant remodelling of RV conduction and repolarisation properties was found in pigs with rTOF. This remodelling generates a proarrhythmic substrate likely to facilitate re-entries and to contribute to sudden cardiac death in patients with rTOF.

The right ventricular outflow tract (RVOT) has a distinct embryological origin from the rest of the right ventricle (RV) and is a frequent origin for idiopathic and disease-related arrhythmias. We hypothesised that heterogeneous action potential duration (APD) across the right ventricle (RV) may contribute to RVOT arrhythmia generation. Pigs were anesthetized and monophasic action potentials (MAPs) recorded in sinus rhythm from the epicardium of the RV free wall and RVOT. The RV was isolated and perfused via both right and left anterior coronary arteries. The preparation was paced (1-5Hz) and the electrical activity optically mapped (di-4-ANEPPS, 10μM) on both epicardial (EPI) and endocardial (ENDO) surfaces. The expression of potassium channels was assessed by RT-PCR. In vivo, MAP durations measured at 20% and 80% repolarization were both significantly shorter in the RVOT than in the RV free wall EPI (P

The right ventricular outflow tract (RVOT) has a distinct embryological origin and is a common anatomical source of arrhythmias in the healthy and diseased myocardium. We hypothesised that specific RVOT activation and conduction properties may underlie the preferential RVOT origin of arrhythmias. Pig right ventricular (RV) wedge preparations were perfused via the left anterior descending and right coronary arteries. Electrical activation and conduction properties were obtained by optical mapping of the epicardial surface (di-4- ANEPPS 10μM) upon electrical stimulation of the preparation. Transmural needles were inserted in the RV free wall and RVOT and unipolar electrograms (EGMs) were recorded. Fiber orientation was obtained by diffusion tensor MRI. Regional mRNA expression was determined by RT-PCR and fibrosis was assessed histologically. Longitudinal and transverse conduction velocities were significantly reduced in RVOT compared to RV free wall (P