No entanto, estudos têm demonstrado o valor dos biomarcadores de fibrose miocárdica na progressão da patogênese da DCh.6A inflamação miocárdica persistente e a fibrose representam as principais características patológicas da DCh que estariam relacionadas com a sua progressão.7 apresentam dados que contribuem para o nosso entendimento relacionado à carga da fibrose miocárdica em diferentes estágios da DCh. Para tanto, os autores avaliaram a presença (ou não) da Galectina-3 , um biomarcador de fibrose miocárdica, em diferentes estágios da doença comparados à um grupo controle e se ela está associada a mortalidade ou necessidade de transplante de coração no estágio mais avançado da doença. Para esse propósito, foram realizados 2 estudos com desenhos distintos. Inicialmente, a fim de estratificar os grupos pelo status de cardiomiopatia chagásica (CC) utilizando um desenho de estudo transversal, 330 pacientes soropositivos para T. cruzi  foram incluídos, além de 153 controles soronegativos (pareados por idade e sexo). Desses pacientes soropositivos, 97 com formas cardíacas mais graves de CC fizeram parte do estudo longitudinal prospectivo censurado até o evento .Nesta edição, Fernandes et al.,s = -0,16, p = 0,001). Foram observados eventos em 28 pacientes (29%). Em pacientes com cardiopatia e FEVE <50%, o modelo de regressão linear ajustado mostrou uma associação significativa entre os níveis de Gal-3 e morte ou transplante cardíaco durante um seguimento de cinco anos .Os resultados foram resumidos e analisados nos diferentes grupos. A mediana de idade foi de 49 ± 9,2 anos, com mediana de seguimento de 58 meses. Pacientes com doença de Chagas sem cardiomiopatia (n = 187) e aqueles com cardiomiopatia e FEVE >50% (n = 46) tinham níveis de Gal-3 semelhantes aos de controles saudáveis (n = 153), mas aqueles com cardiomiopatia e FEVE <50% (n = 97) apresentaram níveis de Gal-3 significativamente maiores do que os controles saudáveis . Uma correlação significativa foi observada entre os níveis de Gal-3 e FEVE ; assim, os resultados das análises multivariadas, que incluíram cinco variáveis independentes, devem ser considerados com cautela devido à provável supersaturação do modelo.A interpretação desses resultados deve considerar o pequeno número de pacientes com seguimento de cinco anos e experiência em centro único já citados pelos autores. No grupo de pacientes com cardiomiopatia e FEVE <50%, possivelmente foram utilizados diferentes graus de comprometimento miocárdico, já que havia um amplo espectro de FEVE (variando de 20% a 40%) levando a diferentes perfis de pacientes incluídos nos grupos.T. cruzi .10 Isso significaria um avanço no manejo da DCh.Um dos maiores desafios da DCh é a dificuldade da identificação, em um estágio inicial, os indivíduos infectados que farão parte dos 20% a 30% que podem desenvolver cardiomiopatia. Infelizmente, no momento, não há como prever essa possível progressão. Assim, encontrar biomarcadores confiáveis de progressão da doença significaria o maior salto na história da DCh desde sua descoberta em 1909 pelo Dr. Carlos Chagas. Por esse motivo, existem inúmeros grupos de pesquisa que se dedicam à busca de biomarcadores derivados do hospedeiro e do  1 In Brazil, to date, it is the neglected disease with the highest burden, of which ~30% of individuals will progress to the symptomatic tissue disruptive stage within 20 – 30 years after being infected2 .3In recent decades, the epidemiology of Chagas disease (Chd) has changed significantly as a consequence of urbanization and migration.5 The use of myocardial fibrosis biomarkers to predict progression in patients with normal or near normal LV function is certainly important in Chd, where traditional risk factors, such as LVEF, may not be as useful. Nevertheless, studies have shown the value of myocardial fibrosis biomarkers in the progression of Chd pathogenesis.6Persistent myocardial inflammation and fibrosis represent the main pathological characteristics of Chd that would be correlated with its progression.7 present data that contributes to our understanding related to the burden of myocardial fibrosis in different stages of Chd. To this end, the authors assessed the presence (or not) of Galectin-3 , a myocardial fibrosis biomarker, in different stages of the disease compared to a control group and whether it is associated with mortality or need for a heart transplant in the most advanced stage of the disease. For this purpose, 2 studies with different designs were carried out. Initially, in order to stratify the groups by the Chagas cardiomyopathy (CC) status using a cross-sectional study design, 330 patients seropositive for T. cruzi  were included, in addition to 153 seronegative controls (matched by age and gender). Of these seropositive patients, 97 with more severe cardiac forms of CC were part of the prospective longitudinal study censored until the event .In this edition, Fernandes et al.s=-0.16, p=0.001). Events were observed in 28 patients (29%). In patients with cardiopathy and LVEF< 50%, the adjusted linear regression model showed a significantly association between Gal-3 levels and death or heart transplantation during a five-year follow-up . The authors concluded that in patients with the cardiac form, higher Gal-3 levels were significantly associated with severe forms of the disease and a higher long-term mortality rate, which means that they can be effectively used to identify high-risk patients.The results were summarized and analysed in the different groups. The median age was 49 ± 9.2 years, with a median follow-up of 58 months. Chagas disease patients without cardiomyopathy (n = 187) and those with cardiomyopathy and LVEF >50% (n = 46) had Gal-3 levels similar to those of healthy controls (n = 153), but those with cardiomyopathy and LVEF <50% (n = 97) had significantly higher Gal-3 levels than the healthy controls (p = 0.0001). A significant correlation was observed between Gal-3 levels and LVEF ; thus, the results from the multivariate analyses, which included 5 independent variables, should be taken with caution due to likely model oversaturation.The interpretation of these results should consider the small number of patients who had a five-year follow-up and single center experience already quoted by the authors. In the group of patients with cardiomyopathy and LVEF <50%, different degrees of myocardial involvement were possibly used, since there was a wide spectrum of LVEF (ranging from 20% to 40%) leading to different patient profiles included in the groups.10 This would mean a breakthrough in the management of ChdOne of the mayor challenges in Chd is the difficulty in identifying at an early stage the infected subjects who will be part of those 20% to 30% who might develop cardiomyopathy. Unfortunately, at present there is no way to predict this possible progression. Thus, finding reliable biomarkers of disease progression would mean the greatest leap forward in the history of Chd since its discovery in 1909 by Dr. Carlos Chagas. For this reason, there are numerous research groups devoted to the search of both host-derived and T. cruzi-derived biomarkers.


Ventricular arrhythmias arise from complex electroanatomical substrates in patients with structural heart disease. There have been significant advancements in technologies and techniques for ventricular tachycardia catheter ablation. A systematic approach to mapping and ablation is paramount, and catheter ablation has shifted to be a potential first-line therapy for patients needing early intervention, particularly for those with post-infarction arrhythmias. Furthermore, imaging integration, coupled with a systematic, detailed substrate characterization, has shown promise and provides a safe and effective approach for long-term arrhythmia control. The majority of VT cases in patients with structural heart disease are due to scar-based reentry. In those individuals with prior myocardial infarctions, VT commonly originates from the subendocardial region of the infarcted myocardium. These areas of myocardial scar alter the impulse propagation, thereby producing slow and anisotropic conduction.13 The reentrant circuits associated with different VT morphologies are rarely from disparate scar locations and are commonly within the same infarcted region.14 Furthermore, a “shared” isthmus is present in approximately 43% of patients with multiple VTs.15 In addition to scar-based reentry, the subendocardial Purkinje fibers also play an important role in ventricular arrhythmia in these patients. Purkinje fibers arborize along the border zone of the subendocardial scar, and may serve as a localized reentry substrate for monomorphic VT16 and also as triggers for polymorphic ventricular arrhythmias .17Multiple morphologies of spontaneous or inducible VTs were observed in a majority (60%–80%) of post-infarction patients who underwent catheter ablation.18 In contrast with patients with postinfarction ventricular arrhythmias, the majority of patients with NICM have only limited areas of endocardial scar. The scar location does not conform to the distribution of the coronary arteries, and is often located near the perivalvular region .19 Delayed enhancement magnetic resonance imaging (MRI) and electroanatomic mapping has demonstrated the presence of mid-myocardial and epicardial low-voltage areas compared with the endocardial surface, located near the valve annulus.20Nonischemic cardiomyopathy (NICM) constitutes a group of heterogeneous diseases resulting in myocardial dysfunction that is in the absence of significant coronary artery disease. In addition to Purkinje fascicular-related reentry and focal automaticity, the predominant mechanism (62%–89%) of VT in NICM remains as myocardial scar-based reentry.21 In the absence of sustained VT, the morphology of premature ventricular contractions can also be useful to locate the regions of potential arrhythmia substrate or to ascertain the possibility of Purkinje involvement. Furthermore, various QRS morphologic criteria have been used to distinguish endocardial from epicardial or non-endocardial sites of origins, especially for VTs associated with NICMs (see later discussion below).24To maximize the success of VT ablation in patients with structural heart disease, a thorough preoperative assessment is essential. Careful attention should be paid to the 12-lead electrocardiograms (ECGs). The presence of Q-waves in sinus rhythm on the ECG provides a noninvasive clue for localization of myocardial scarring, and identifies a potential arrhythmia substrate in patients with ischemic cardiomyopathy (ICM). Careful analysis of the 12-lead ECG allows for localization of the “exit site” for ischemic VTs with a reasonable predictive accuracy (> 70%) to guide the initial mapping efforts.Conventional mapping techniques, such as activation and/or entrainment mapping, identify the functional components and the critical isthmus of a reentrant VT circuit. Such mapping strategies, however, are dependent on the inducibility and hemodynamic tolerance of the arrhythmia. In clinical practice, the majority of induced VTs are poorly tolerated, and therefore activation and entrainment mapping have a limited role. A paradigm shift focusing more on a substrate-based mapping strategy is generally required for successful VT ablation.25 These predefined voltage criteria have been validated in animal models and human histopathology, and help to identify the location end and extent of endocardial scar.27The basis of electroanatomical characterization of the myocardial substrate relies on high-density endocardial bipolar voltage mapping during sinus or paced rhythm. Areas of the endocardium that are relatively healthy or “normal” have endocardial bipolar voltages greater than 1.5 mV, while areas with low voltage (< 0.5 mV) are arbitrarily classified as “dense scar.” Border zones are regions between healthy and scarred myocardium, and typically have bipolar voltages ranging from 0.5 mV to 1.5 mV..28 Once the low-voltage “scar” areas have been identified, pace mapping along the scar border zone (0.5–1.5 mV) can then be performed to approximate the “exit” site of the VT circuit. At the putative “exit,” the paced QRS morphology should be similar to the spontaneous arrhythmia, with a short stimulus-QRS interval (≤ 40 ms). Once identified, further mapping efforts can be made into the scar region and away from the border zone, where a critical isthmus is often located.Based on detailed entrainment mapping of hemodynamically tolerated arrhythmias, most VT isthmus or entrance sites (84%) are identified within the dense scar (< 0.5 mV), and most of the exits are located in the border zone (0.5– 1.5 mV) 29Electrically unexcitable scar (EUS) is defined by areas of high-output unipolar pacing (< 10 mA at 2-ms pulse width) in regions of low amplitude electrograms (< 0.5 mV) that fail to capture myocardium. EUS has been correlated with areas of dense scar with local bipolar voltages of < 0.25 mV. Since areas of EUS are commonly located in proximity to VT isthmuses and potential conduction channels, a potential VT isthmus is often located in between areas of EUS.9 These potential conduction channels can be identified in the majority of patients by making careful adjustments to the voltage color gradient of the bipolar voltage maps of lowvoltage areas (< 1.5 mV) during baseline sinus or paced rhythm. A “conducting channel” is defined by the presence of a “corridor” of consecutive electrograms differentiated by higher bipolar voltage amplitudes than the surrounding tissue.30 However, a single cutoff threshold value is often not sufficient due to the heterogeneous nature of the scar tissue, and frequent adjustments of voltage color thresholds are often required to visualize potential channels.The histology of an endocardial resection specimen from post-infarction patients with sustained VT revealed isolated bundles of viable myocardial fibers within the infarct tissue, often associated with fractionation of electrograms and slow conduction..30 These channels have been correlated with the critical VT isthmuses by entrainment mapping,28 and can be identified non-invasively by contrast-enhanced MRI.31 The presence of late or fractionated potentials within the channel significantly improves its specificity for identifying VTrelated isthmus.32 Further mapping efforts are needed to establish the functional significance of these VT-related channels, such as activation mapping, pace mapping, and entrainment mapping.A majority of VT-related conducting channels can be identified in areas of scar when the bipolar voltage threshold is set to between 0.2 mV and 0.3 mV 34 Most LAVAs reside in scar or border zones, and may appear as late potentials or electrograms with isolated delayed components recorded after the ventricular activation/surface QRS.36LAVA is a general term that incorporates all abnormal ventricular electrograms that represent near-field signals recorded from non-uniform anisotropic slow conduction of surviving viable myocardial fibers within the potential VT isthmus.37 Integration of contrast-enhanced CT imaging with three-dimensional electroanatomic mapping helps to guide substrate-based ablation in post-infarction VTs.In patients with post-infarction VTs, the endocardial low voltage scar (< 1.5 mV) and the distribution of LAVA recordings have been located in regions of myocardial wall thinning (< 5 mm) on contrast-enhanced computed tomography (CT). Very late LAVA (> 100 ms after QRS complex) were almost exclusively (93%) detected within the thinnest scar area (< 3 mm thickness).38 Ventricular pacing or pacing at different rates provides different wavefront propagations that may unmask areas of slow conduction/LAVA .39 Delivery of ventricular extrastimuli also can reveal decremental impulse propagation in areas of slow conduction demonstrating increased fractionation of LAVA/late potential signals . In patients with left ventricular (LV) cardiomyopathies/VT substrates, LV stimulation may be more useful than right ventricular (RV) pacing in unmasking LAVA. This may be achieved by either inserting a catheter into a LV venous branch via the coronary sinus or by having simultaneous retrograde and trans-septal LV access for LV stimulation and recording.However, the latency and the ability to detect LAVA/late potentials are dependent on the activation wavefronts with variable fusion of near-field versus far-field signals, and thus are affected to a large extent by the anatomical locations of scar. LAVAs were more frequently detected after the QRS complex in the epicardium (91%) than in the endocardium , and only 43% of septal LAVA were detectable and separated from far-field ventricular electrograms.40 Topographically, when pacing at the exit side of the isthmus of the VT circuit, the activation wavefront propagates more rapidly to the exit site, resulting in a good ECG match as that of the clinical VT. In contrast, when pacing at the entrance side of the VT isthmus, the activation propagates preferentially out of the entrance site, resulting in a very different paced QRS morphology and a poor QRS match compared with the clinical VT .Pacemapping is also useful in localizing the critical isthmus of post-infarction VTs..High-density pacemapping (bipolar or unipolar) is performed within abnormal low-voltage regions during sinus rhythm. Conceptually, stepwise pacemapping from the exit to the isthmus should lead to a gradual prolongation of the S-QRS interval while maintaining a nearperfect QRS matching to the VT. Importantly, an abrupt transition from an area of perfect pacemapping to an area of poor matching (such as the entrance side of the isthmus) corresponds with the VT isthmus. By quantifying the degree of paced QRS match at each location using a color gradient, this identifies the virtual transition zone (from the best to the worst matching sites), and thus the course of the VT circuit can be characterized ® ; the Relexion™ HD spiral catheter and the DuoDeca Livewire™ catheter ; and the IntellaMap Orion™ catheter and/or Rhythmia™ mapping system .44 The local electrogram voltages and morphologies are dependent on the recording electrode size, interelectrode spacing, and direction of wavefront propagation.45 When compared with the standard ablation catheter with the 3.5 mm to 4.0 mm distal electrode, tissue characterization using a catheter with small (1.0 mm), closely spaced electrodes  resulted in a 22% smaller low-voltage area (< 1.5 mV) and a 47% reduction in dense scar size (< 0.5 mV) in an animal model of infarction.42 High-density, multielectrode mapping minimizes the effects of farfield signals and helps to identify heterogeneity within the low-voltage scar, allowing for better localization of channels of surviving myocardial bundles and fractionated electrograms or LAVA detected within these channels.46High-density multielectrode mapping VT substrate characterization has evolved with the emergence of highdensity multielectrode mapping catheters, including the PentaRay47The use of multipolar catheter mapping is associated with a higher mapping density with better endocardial LAVA identification in patients with post–myocardial infarction VT. Overall, the impact of multipolar catheter use on patient outcomes seems to be mediated by a more comprehensive characterization of the VT substrate, which translated to a lower VT recurrence rate following catheter ablation.48 Importantly, the size of the endocardial unipolar lowvoltage area surrounding the bipolar scar (penumbra) reflects the intramural/epicardial scar and diffuse myocyte loss. This is associated with an increased mortality and arrhythmia recurrence following ablation.49In patients with post-infarction VT, endocardial scar extension and density predict scar transmurality and the endoepicardial presence of LAVA. The presence of LAVA predicts a favorable VT ablation outcome. However, endocardial bipolar voltage may underestimate the scar size as compared with MRI, possibly due to the presence of hibernating myocardium, early reperfusion, collateral circulation, or a concomitant cardiomyopathy.. In these cases, a late potential/LAVA-guided ablation strategy is associated with less favorable outcomes than post-infarction patients.50In comparison with post-infarction VT, patients with NICM have no predilection for endocardial scar substrate, with a nearly equal extent of scar occurring on the endocardium and epicardium, respectively. Importantly, less dense scar (< 0.5 mV) was observed in patients with NICM, and late or fractionated potentials refl ecting conduction delay are significantly less prevalent 51 However, the distribution and extent of endocardial versus epicardial scars may vary in individual patients, and the utility of endocardial voltage mapping in identifying non- endocardial scar is limited.52 Unipolar recordings provide a larger field of view than bipolar recordings, allowing for the detection of epicardial or intramural scars during endocardial mapping. Based on clinical and MRI data, a cutoff value of unipolar endocardial voltage of ≤ 6.78 mV and < 8.27 mV best differentiated the presence of underlying intramural and epicardial scars, respectively.54The majority of NICM patients who failed prior endocardial ablations often have more extensive areas of abnormal voltage on the epicardium rather than on the endocardium, and an epicardial ablation may be needed in up to 50% of these individuals.56 In the subset of patients with inferolateral scars, epicardial VT substrate with late potentials were frequently (63%–81%) identified. In contrast, the arrhythmia substrate in patients with the anteroseptal scar subtype were mainly identified in the periaortic and intramural septal locations, and were associated with more extensive endocardial unipolar scar and a higher VT recurrence rate. These .ndings provide a better understanding of the arrhythmogenic substrate in patients with NICM and the prognostic significance. Epicardial mapping may be considered in selected patients when (1) the 12-lead ECG of the VT suggests an epicardial origin ; (2) there is evidence of epicardial substrate on imaging studies ; (3) unipolar electrogram voltage abnormality in the presence of no or minimal bipolar (< 1.5 mV) endocardial scar; and (4) failure of prior endocardial ablation .Recent studies of electroanatomical mapping and MRI use in patients with NICM demonstrated two predominant scar patterns, specifically (1) basal anteroseptal and (2) inferolateral locations of nearly equal distribuions.The primary goal of catheter ablation of scar-related VT is the interruption of critical areas of slow conduction responsible for the development and maintenance of the reentrant circuit. Most patients with scar-related VT present with unstable arrhythmias that are not amenable to conventional mapping techniques based on point-by-point activation mapping or overdrive entrainment. In these scenarios, a “substrate-based” ablation strategy facilitates workflow, optimizes lesion sets, and improves patient safety and procedural outcomes., has been shown to be an effective strategy of VT control in patients with structural heart disease, particularly in those with ICM.57 With high-density mapping and extensive ablations across the endocardial ± epicardial surfaces, the elimination of all LAVA may be achieved in ~70% of patients.58 Complete LAVA elimination is associated with a reduction in recurrent VT or death (p = 0.035) compared with the persistence of LAVA , and represents a useful and effective procedural endpoint for substrate-based VT ablation. Although non-inducibility predicts a lower risk of VT occurrence, a combined endpoint of LAVA elimination of VT non-inducibility is associated with the lowest VT recurrence rate (16%), in comparison with non-inducibility alone.58Various substrate modification techniques have been described for unmappable or hemodynamically intolerable VT. Linear radiofrequency lesions are usually created to transect the potential isthmuses or channels, or to connect the VT exit sites to anatomic barriers such as the EUS or a valvular annulus. An ablation strategy guided by abnormal potentials, coupled with local voltage profile analysis 59 In some individuals, LAVA could not be identifi ed or eliminated due to intramural or epicedial scar; septal (or early activated) substrate locations; inadequate sampling; or difficult anatomical characteristics, such as the papillary muscles.However, the success of such potential guided ablation strategies may depend on the substrate location and the patient’s anatomy.60 Other novel ablation strategies include “core isolation” and “scar dechanneling.” Core isolation involves identifying an area of interest within dense scar that includes areas of voltage channels, late potentials, and isthmus sites. Contiguous ablation lesions are created to either completely surround a particular region or to anchor it to nearby anatomic structures, with the endpoint of exit block or inexcitability within this core area.61 Scar dechanneling represents an alternative method to LAVA abolition with focal ablation performed at sites at which the earliest fractionated potentials are recorded near the entrance of the conducting channels, aiming toward the endpoint of eliminating a consecutive series of LAVA.62 These substrate-based ablation approaches may be particularly useful in patients who are non-inducible at baseline or in those who have multiple, unmappable arrhythmias. Importantly, these techniques are achievable in most patients and represent definable procedural endpoints. Overall, an extensive substrate-based ablation strategy is associated with a 38% relative risk reduction in VT recurrence as compared with limited substrate modification .63The presence of a complex arrhythmia substrate with multiple potential conducting channels and possible midwall or epicardial VT exit sites provided the rationale for an extensive endocardial  ablation strategy in those with structural heart disease. Such “scar homogenization” increases the freedom from arrhythmia at long-term follow-up in patients with post-infarction VT storm.64 The structural abnormality may be segmented as areas of delayed gadolinium enhancement on MRI, and/or as areas of wall thinning on multidetector CT scan, and are correlated with local conduction delay defined by late potentials or LAVA.65 Real-time image integration for substrate localization/characterization for VT ablation will soon become an accepted standard.Real-time image integration to guide catheter ablation is feasible in large series of patients with scar-related VTs, facilitating workflow with significant impacts on procedural management with improved outcomes. Imaging integration provides important complementary information to electroanatomical mapping, particularly in patients with NICM or in those undergoing epicardial ablations.47 Complete LAVA elimination, the use of real-time image integration, and high-density mapping with multipolar catheters were independent predictors of VT free survival after catheter ablation for post-myocardial infarction VT . These findings strongly suggest that complete LAVA elimination is a predictive procedural endpoint associated with a twofold reduction in VT recurrence.The impact of technical innovations and substrate-based ablation approaches on VT ablation has recently been evaluated.. A substrate-based ablation approach is less applicable, and a greater proportion of patients require epicardial ablation. The ablation procedures are associated with significantly longer operation time and fluoroscopic exposure, with a lower long-term cumulative VT-free survival than post-infarction VTs.66 Serial electroanatomic mapping also demonstrated new voltage abnormalities in approximately 50% of NICM patients with VT, often accompanied by progressive ventricular remodeling.68 Despite these limitations, an improvement in overall VT burden with a reduced need for antiarrhythmic drugs can be achieved with aggressive endocardial and adjuvant epicardial ablation. Catheter ablation remains a safe and effective approach for longterm arrhythmia control in most NICM patients.69However, catheter ablation of ventricular arrhythmias in the setting of non-ischemic dilated cardiomyopathy remains a major challenge 70 The recently published Ventricular Tachycardia Ablation versus Escalated Antiarrhythmic Drug Therapy in Ischemic Heart Disease (VANISH) trial prospectively compared the use of antiarrhythmic drugs containing amiodarone and/ or mexiletine with catheter ablation. A significantly lower rate of composite primary outcomes of death, VT storm, or appropriate ICD shock were observed in patients who underwent catheter ablation versus those who received an escalating dosage of amiodarone or the addition of mexiletine.71 However, no significant difference was observed among patients who were not treated with amiodarone at baseline. Nonetheless, these findings support the preemptive use of early catheter ablation in the management of ventricular arrhythmias in patients with structural heart disease refractory to antiarrhythmic drugs.The International VT Ablation Center Collaborative Group represents the largest retrospective multicenter study that demonstrated that freedom from VT recurrence after catheter ablation was associated with an improved transplant-free survival, independent of ejection fraction, heart failure, and type of cardiomyopathy  in patients with structural heart disease.74However, catheter ablation of VT in patients with NICM remains a challenge compared with that of post-infarction patients. Imaging integration coupled with a systemic, detailed substrate characterization has shown promise in providing a safe and effective approach for long-term arrhythmia control in most NICM patients. Despite a shift towards a more substrate-based ablation strategy, activation and entrainment mapping helps to identify the critical isthmus and to avoid potential unnecessary ablations. However, hemodynamic instability limits such conventional mapping techniques in the majority of patients, and is associated with an increased mortality.75Percutaneous left ventricular assist devices (pLVADs) commonly used in VT ablation include the TandemHeart™ left atrial-to-femoral artery bypass device  and the Impellas left ventricle-toascending aorta pump . The use of pLVADs during unstable VT in patients with structural heart disease appears to be feasible and safe. pLVADs provide adequate end-organ perfusion during VTs, facilitate extensive mapping, and require fewer rescue shocks during the procedure. However, improvement in long-term procedural outcome has not been demonstrated. The use of hemodynamic support during VT ablation therefore needs to be weighed against the risk of complications, longer procedure time, and the incremental cost.Ventricular arrhythmias arise from complex electroanatomical substrates and are a significant cause of morbidity and mortality in patients with structural heart disease. Catheter ablation is historically implemented as a treatment of last-resort. There have been recent advancements in mapping technologies and techniques allowing for better definition of potential surrogate targets of the VT substrate. Various substrate-based mapping and ablation strategies such as targeting LAVA, along with homogenization or dechanneling strategies improve the procedural outcomes and minimize complications. A systematic approach to mapping and ablation of VT is paramount, and catheter ablation has shifted to a potential first-line therapy for early intervention purposes, particularly in patients with post-infarction VTs.


OM was adapted to monitor cardiac electrical behaviour in an open-chest pig heart model with physiological or artificial blood circulation. We detail the methods and display the OM data obtained using di-4-ANBDQBS and Cardiogreen. Activation time, action potential duration, repolarization time and conduction velocity maps were constructed. The technique was applied to track cardiac electrical activity during regional ischaemia and arrhythmia. Our study is the first to apply high-spatiotemporal-resolution OM in the pig heart in situ to record cardiac electrical activity qualitatively under artificial blood perfusion. The use of an FDA-approved voltage-sensitive dye and artificial blood perfusion in a swine model, which is generally accepted as a valuable pre-clinical model, demonstrates the promise of OM for clinical application.The emergence of optical imaging has revolutionized the investigation of cardiac electrical activity and associated disorders in various cardiac pathologies. The electrical signals of the heart and the propagation pathways are crucial for elucidating the mechanisms of various cardiac pathological conditions, including arrhythmia. The synthesis of near-infrared voltage-sensitive dyes and the voltage sensitivity of the FDA-approved dye Cardiogreen have increased the importance of optical mapping (OM) as a prospective tool in clinical practice. We aimed to develop a method for the high-spatiotemporal-resolution OM of the large animal hearts  The electrical signals in the heart and the associated propagation pathways are crucial for elucidating the mechanisms of various cardiac pathological conditions, including arrhythmia. The first heart activation maps were constructed based on electrical signals obtained by arrays of electrodes4 or optical fibres5 from hundreds of recording sites separated by a few millimetres. Because of the limited number of points, such techniques have low resolution. Meanwhile, the main characteristic of current optical mapping (OM) methods is high spatiotemporal resolution6. Because of this property, OM with voltage-sensitive dyes is widely used in the investigation of cardiac arrhythmogenesis at the cellular, tissue and organ levels. Isolation of the heart from an organism has been demonstrated to influence the mechanisms of arrhythmia formation7. Therefore, in situ investigations are of particular importance for identifying mechanisms similar to those that occur in nature during pathophysiological conditions. The synthesis of near-infrared (NIR) voltage-sensitive dyes that can be used in blood-perfused hearts8 and the demonstration of the voltage sensitivity of the FDA-approved dye Cardiogreen11 have increased the potential of OM as a prospective useful tool in clinical practice.The emergence of optical imaging has undoubtedly revolutionized the investigation of cardiac electrical activityin situ was reported by Lee et al.12, who developed a cardiopulmonary bypass circuit in rats, recorded voltage and calcium signals, and demonstrated the feasibility of the multiparametric imaging of a mammalian heart in vivo. However, due to the challenging procedures and problems associated with motion artefacts in live organisms, the use of high-spatiotemporal-resolution OM for large animal hearts in situ has not been reported for a long time since the OM technique was developed. Moreover, the emergence of studies investigating the impact of autonomic nervous regulation on arrhythmia formation in the intact heart14 have encouraged the development of OM systems adapted for large animals/humans in situ. The swine model is generally accepted as a valuable pre-clinical model because of its large heart and body weight and the similarity of its cardiovascular system to that of humans16. Recently, OM was performed in freely contracting pig hearts17. The authors used an optical fibre array and recorded optical signals (OSs) from 16 locations on the pig heart and also performed a ratiometric analysis in the whole heart. Using excitation ratiometry, the electrical activity of the heart was recorded during the application of a high stimulation frequency or during fibrillation, i.e., the conditions when the impact of contraction on the OS is at least partially suppressed. Despite this, the maps of various action potential (AP) parameters were not provided, suggesting that their technique lacks one of the most important OM features, i.e., registration at high spatial resolution. Evidently, excitation ratiometry helps to minimize contraction artefacts. However, comprehensive studies have demonstrated that additional mechanical heart immobilization or mathematical ascertainment of the pixel and heart surface are required to use ratiometric OM in freely contracting hearts18.The first attempt to apply OM in situ. In this paper, we present a detailed description of the methods used and high-spatiotemporal-resolution OM data obtained from pig hearts in situ under normal and pathophysiological conditions. The use of artificial blood perfusion and an FDA-approved voltage-sensitive dye demonstrates the promise of the registration of cardiac electrical activity for clinical application, such as in open-heart surgery.Our aim was to develop an OM system adapted for large animals and suitable for the high-spatiotemporal-resolution recording of cardiac electrical activity under artificial blood perfusion, a condition that is widely used in cardiac surgery. Additional experiments were performed in freely contracting heart using glued metal frame for the immobilization of a part of anterior surface of the heart under physiological blood circulation. We used a swine model and performed open-chest heart surgery. Using the fluorescent, voltage-sensitive dyes di-4-ANBDQBS and Cardiogreen, we recorded the electrical activity of the heart First, we attempted to register the electrical activity of the pig heart under physiological blood circulation using different heart immobilization methods. We performed experiments using a hand-made frame Fig.  or a cliOnline Supplementary Material, Movie vs. RV2). The coronary arteries also acted as a morphological obstacle that significantly reduced the intensity of the OSs from this area. The maximal fractional change in the fluorescence signal (ΔF/F) was 6.1 ± 1.8% (n = 5), on average.The results of the OM of the pig heart immobilized by the frame are presented in Fig. Similar optical data were obtained using an Octopus tissue stabilizer (data not shown).in situ under physiological blood circulation.Due to the low quality of the recordings, we did not continue investigations of OM In experiments with artificial blood circulation using di-4-ANBDQBS, the ΔF/F was 16.17 ± 2.08% (n = 4). Representative recordings of the optical signals obtained during ventricular pacing with a 500-ms period under the control are presented in Fig. in situ under artificial blood perfusion can be used to evaluate the activation time, the direction of excitation propagation and the conduction velocity. The average conduction velocity independent of stimulation location was higher in the RV than in the left ventricle (LV), i.e., 1.21 ± 0.13 m/s vs. 0.91 ± 0.1 m/s .Figure Online Supplementary Material . Under different excitation wavelengths, OAPs originate from different myocardial depths22. In our presented example, the OAP alternans were recorded in the epicardial layer of the left myocardium. Meanwhile, the results show that the ratio barely reflects the alternans. Thus, fluorescence ratiometry should be used with caution in arrhythmia investigations performed to detect the differences between myocardial layers that may underlie the mechanisms of various arrhythmias.In addition to stimulation from different heart locations, various pacing cycle periods were applied. Normalized OAPs obtained under different stimulation periods (200–500 ms) and excitation wavelengths , as well as OAPs calculated as the ratio of the OS under green and red light excitation, are presented in the ial Fig. . Movies l Movies –S6. OAP ght Fig. vs. b. UOnline Supplementary Material , that is generally accepted to stop cardiac contractions, sometimes did not sufficiently suppress the motion. Moreover, a while after blebbistatin injection, contractions were partly restored, which was possibly related to metabolism of the drug; therefore, the same dose of uncoupler was injected again. In some experiments, diltiazem was also applied, which fully stopped the motion and prevented ventricular arrhythmia.in situ, we used the dye Cardiogreen. Previously, we showed that the OS obtained by Cardiogreen is composed of two components, fast and slow, and that the characteristics are dependent on the excitation/emission wavelengths (λex and λem)11. To demonstrate that Cardiogreen can be used for the OM of the heart in situ, we applied three optimal excitation/emission conditions suitable for recording cardiac electrical activity, i.e., λex = 660 nm/λem = 775 nm, λex = 660 nm/λem = 720 nm, and λex = 780 nm/λem = 808 nm. Representative results showing the activation time, OAPD50 and repolarization time maps and the OAPs from the RV, artery (Ar), and LV, indicated by asterisks in the activation maps obtained at various λex and λem values, are presented in Fig. ex = 660 nm and λem = 775 nm, positive OSs were obtained11. At such λex/λem values, the OS of Cardiogreen consisted mainly of the fast component, and the OAP exhibited a characteristic steep upstroke, which allowed for the construction of an activation time map . The repolarization phase of the OS was clearly distorted; therefore, construction of the OAPD map was irrelevant. The OS recorded at λex = 780 nm and λem = 808 nm included two components, which were both negative . The presence of the slow component makes the upstroke of the OS less steep than that of the OS obtained at λex = 660 nm, and the signal has a longer duration than the AP of a single cell. Notwithstanding, the signals were sufficient to construct activation, OAPD50 and repolarization time maps . OSs recorded at λex = 660 nm/λem = 720 nm were used only for the calculation of the ratio and are not presented.In the next set of experiments on recording the electrical activity of the pig heart in situ. Recordings of cardiac electrical activity composed of OS relations obtained by Cardiogreen in the pig heart are presented in Movie Online Supplementary Material).Thus, the results show that the FDA-approved dye Cardiogreen, which is clinically used for vessel visualization, can be used for the registration of cardiac electrical activity in the pig heart ex = 660 nm/λem = 720 nm and λex = 780 nm/λem = 808 nm.To compare the OSs obtained by the fluorescent dyes di-4-ANBDQBS and Cardiogreen, parameters, such as the maximal ΔF/F, maximal signal-to-noise ratio (SNR) and SNR decay rate, were evaluated. The parameters were calculated from the OSs obtained ~15 min after dye injection. The average values are presented in Table The data show that the maximal ΔF/F and SNR were significantly lower and the SNR decay rate was higher for Cardiogreen than for di-4-ANBDQBS.in situ. Furthermore, we attempted to evaluate the applicability of this method in situ during various pathological conditions.The data above clearly show the capability of using OM for the registration of myocardial electrical activity Online Supplementary Material in Fig. Online Supplementary Material, Fig. e) vs. ischaemia (f)).In some experiments, acute regional ischaemia was induced by occlusion of the left anterior descending coronary artery by encircling it with surgical sutures. The experiments were performed under artificial blood circulation using di-4-ANBDQBS. The activation and OAPD maps, which are presented in the in situ, ventricular tachycardia (VT) or ventricular fibrillation (VF) was induced by applying burst pacing with a frequency higher than 10 Hz. During VT/VF, reentrant-type disorders generally dominate. Usually, these disorders are related to the formation of two types of mother rotors, i.e., functional mother rotors, which appear to form because of the distribution of APDs and occur at different sites in the heart but are more common in the RV, and anatomical obstacles, which usually develop at sites where cardiac tissue, usually in the LV, is damaged by various pathological conditions27.To evaluate the possibility of OM for recording arrhythmias in situ allowed us to register different types of wavefront circularities during VT/VF. An example of the multidirectional spread of excitation waves with different directions of propagation in every excitation cycle was obtained under physiological blood circulation using the hand-made frame for heart immobilization , multiple wave breakups occur, and the regularity of the signals is destroyed. The OSs were normalized to the background fluorescence. Therefore, if the whole ventricular wall was electrically excited, then the OSs from the same locations should be of the same amplitude. The smaller amplitudes of certain OAPs .In our experiments, we did not aim to investigate the mechanisms underlying reentrant arrhythmia development; however, OM of the pig heart ion Fig. . The action Fig.  show thein situ were obtained under artificial blood circulation using di-4-ANBDQBS. The formation of VF and reentrant arrhythmias around stable potential anatomical obstacles are shown in Fig. see also Online Supplementary Material, Fig. Online Supplementary Material). In the movie, the main excitation waves can be observed to circulate around the damaged tissue. The main circulation wave draws in the excitation waves from the left side of the LV. Both waves are shown by white arrows at the bottom of the activation map . We can also see the secondary propagation waves of the excitation from the main sources propagating to the RV. These secondary waves generate multiple excitations throughout the heart. The OAPD50 maps  show the notable differences in the OAPD that coincide with the sites of severe conduction impairment (see locations in the activation maps with the greatest crowding of isochrones).The impairment of excitation wave propagation and an OAPD distribution that may indicate the formation of an anatomical obstacle in the LV of the pig heart by OM in situ. The registration of cardiac OAPs using di-4-ANBDQBS or Cardiogreen was performed under physiological or artificial blood circulation. The low quality of electrical activity recordings under physiological blood circulation encouraged us to develop an OM system for the pig heart in situ under artificial blood perfusion, a condition that is widely used in cardiac surgery. Overall, the results confirm that cardiac electrical activity could be optically mapped in situ under normal or arrhythmic conditions in a large animal model.Our study was designed to adapt the high-spatiotemporal-resolution OM technique widely used in isolated hearts to the cardiac imaging of pig hearts in situ, including in humans, was performed using multiple arrays of electrodes13. However, OM using voltage-sensitive dyes has a great advantage over electrical mapping in that the spatiotemporal resolution is much higher. The dual OM of voltage and calcium in small mammalian hearts in situ has been reported12. Recently, a paper in which OM was used in a pig model in vivo was published. The OS from di-4-ANBDQBS was recorded in the contracting heart. At physiological contraction rates, the OAPs were recorded using a 2D fibre array from 16 locations of the heart and ratiometry17. At first glance, our work may seem to present similar data as the recent publication by Lee et al.17. However, in principle, Lee and co-authors present two methods for recording cardiac electrical activity in an in vivo pig model: excitation ratiometry in a freely contracting heart and optical fibre recordings from 16 points. In our study, we present two different modes for OM in an in situ pig model: mechanical immobilization and the chemical arrest of heart contractions. In our opinion, cardiac OM is as interesting as it allows the recording of electrical activity at both a high spatial and temporal resolution. The strength of OM is that it allows the construction of various maps, such as maps of the activation time, AP duration and other parameters, across the entire heart surface. Lee et al.17 presented activation maps recorded in both modes . However, the OAPD maps were constructed using only optical fibre recordings from 16 points in an area of 12 × 12 mm of the heart, and no OAPD maps from mapping of the whole heart using excitation ratiometry were presented. Considering that the anterior surface of the pig heart is at least 50 × 50 mm in size, obviously, recordings from an area of 12 × 12 mm cannot provide detailed information about the propagation of electrical signals in the heart and cannot be considered to provide high-spatial-resolution mapping. Our presented data include activation time, OAPD, repolarization time, and conduction velocity maps constructed from OSs obtained from a large mapping area, i.e., 55 × 55 mm, under artificial blood circulation. Additionally, other disadvantages of the ratiometric analysis used in the study by Lee et al.17 could be emphasized, such as its limited potential for application in freely contracting hearts. Excitation ratiometry can reduce the influence of heart contraction movements, but it cannot eliminate the effects of the heart twisting without any immobilization, which is always present in freely contracting hearts. It has been demonstrated that to eliminate the effects of twisting, heart immobilization or mathematical data processing should be used18.Previously, electrical mapping of the heart The open-chest pig heart model can be used in two ways: with and without an artificial blood circulation system. Under physiological blood circulation, heart contractions are obligatory, while under artificial blood circulation, the heart can be fully stopped.19. Chemical and mechanical immobilization and online/offline computational methods are used to create suitable conditions for OM to reduce the influence of heart motion on the OSs .To use voltage-sensitive dyes for the registration of electrical activity, the pixel registered by the camera and the exact site of tissue registration should be alignedin situ was successful because the obtained signals were sufficient to register electrical behaviour throughout almost the entire mapping area. However, the OS was not ideal, since contraction artefacts still occurred. This phenomenon may restrict the available voltage-sensitive dye options because under such conditions, only dyes (preferably NIR dyes) with high fractional fluorescence could be used. Notwithstanding, the use of a frame or cardiac surgical tissue stabilizer enables the registration of optical cardiac electrical activity in situ under normal conditions or VT/VF. The main disadvantage of mechanical motion arrest using a frame or tissue stabilizer is that the field of view is quite limited; therefore, the technique may not be suitable for the investigation of certain patho/physiological conditions.Heart contractions are essential for supplying the organism with oxygen and removing metabolites, and the chemical blocking of contractions is generally unusable. Therefore, during experiments performed under physiological blood circulation, we mechanically immobilized part of the heart. Our experiments show that the optical registration of cardiac electrical activity in situ. Under artificial blood circulation, heart contractions are not necessary for blood circulation; therefore, activity of the heart can be fully stopped. OM under artificial blood circulation approximates the conditions under Langendorff perfusion of the isolated heart because under both conditions, the heart is perfused in a retrograde manner through the aorta. Artificial blood circulation enables the use of chemical uncouplers and the performance of experiments in situ without excision of the heart from the organism, which may change the activity of the heart7. Moreover, OM in situ allows evaluation of the influence of the autonomic nervous system as well as humoural regulation on the electrical activity of the heart. The chemical arrest of contractions allowed the use of the fluorescent dye Cardiogreen, whose ΔF/F is markedly lower than that of di-4-ANBDQBS. The results supplemented those of our previous report on the voltage sensitivity of Cardiogreen10. However, the data on the effect of blebbistatin on cardiac electrical activity are controversial25. Our results show that under artificial blood circulation, the one-time bolus containing blebbistatin did not have a significant effect on the electrogram of the pig heart in situ. Our data are in agreement with those of recent studies, which did not observe obvious electrophysiological changes following blebbistatin perfusion in the intact guinea pig heart14 or pig heart under Langendorff perfusion6. Notwithstanding, the lack of comprehensive investigations of the cardiotoxicity of this uncoupler is still a limiting factor for the application of OM in pre-clinical/clinical studies.Our results show that standard artificial blood circulation provides controlled conditions and that under such conditions, OM can be applied to record the electrical activity of the heart in large animals in situ means that the dyes enter the blood environment; therefore, only NIR dyes can be used effectively to match the haemoglobin optical window8. As these dyes are excited by long wavelengths of light, the emitted fluorescence originates from a considerable tissue depth. This behaviour is of particular value in the OM of large mammalian hearts, which have thick ventricular walls and massive coronary vessels. Although the voltage-sensitive dyes used in this study require long excitation wavelengths, our results show that the anatomical site of the main subepicardial coronary vessels located in the anterior interventricular sulcus distorts the OAPs. This result is consistent with data from a simulation approach demonstrating that the OAP upstroke is prolonged near large subepicardial vessels or shows a distinct “humped” morphology30.The use of voltage-sensitive dyes in OM in situ to record cardiac electrical activity qualitatively under artificial blood perfusion. The use of an FDA-approved, voltage-sensitive dye and artificial blood perfusion in a swine model, which is generally accepted as a valuable pre-clinical model, demonstrates the promise of OM for clinical application.Thus, our study is the first to apply high-spatiotemporal-resolution OM in the pig heart Experiments were performed in an open-chest pig heart model under physiological or artificial blood circulation. All experiments were performed according to the European Community guiding principles and approved by the State Food and Veterinary Service of the Republic of Lithuania .Twenty pigs weighing 35–40 kg were premedicated intramuscularly with atropine sulphate , xylazine hydrochloride  and ketamine hydrochloride . An Edan IM70 data monitor  was then connected to the tail to monitor the status of the anaesthetised animal. Under sterile conditions, an 18-G catheter  was inserted into the lateral auricular vein. A three-way connector  and infusion system  were connected, and the administration of 0.9% sodium chloride saline  was started. The pigs received 25,000 IU of heparin sodium  intravenously to prevent blood clotting.Online Supplementary Material. Animals were subjected to long-term, deep anaesthesia by the intravenous injection of propofol  by a Harvard Apparatus infusion pump . Opioids were used for analgesia, and butorphanol tartrate  was administered every hour. Myorelaxation was provided intravenously using pipecuronium bromide , which was repeated every hour during surgery. Before the thoracic cavity incisions, local subcutaneous anaesthesia was established with lidocaine  solution (20 mg/mL).Thiopental sodium  was injected intravenously to stop breathing. Then, a ventilation apparatus  for artificial lung ventilation was connected. A detailed description of the intubation procedure is provided in the Electrocardiography, pulse, and arterial blood pressure monitoring was performed by an Edan IM70 device  throughout the experiment. The arterial blood gas and electrolyte levels were measured as well. All parameters were maintained within normal ranges throughout the study.After the experiments, the pigs were euthanized intravenously with pentobarbital sodium .The animals were subjected to the cardiopulmonary bypass system, to achieve full haemodinamic stability and conditions similar to open heart surgery (on pump approach). Cardiopulmonary bypass was established by cannulating the distal ascending aorta. An aortic root cannula with a vent line was introduced into the ascending aorta below the aortic cannula for the delivery of dye solutions into the coronary circulation. After establishment of the cardiopulmonary bypass system, artificial lung ventilation was stopped.Online Supplementary Material.All surgical techniques and artificial blood circulation connections are presented in detail in the During open-heart surgery under physiological blood circulation and lung ventilation, two techniques were applied. First, a hand-made metal frame was used as an alternative for the mechanical contraction arrest of the heart to immobilize an area of the heart surface Fig. . The metIn some experiments, an Octopus tissue stabilizer was used  Fig. . This deIn experiments with artificial blood circulation, which allows the use of chemical uncouplers, heart motion was eliminated by the injection of a 100-mL bolus containing 600 µmol/L blebbistatin together with fluorescent dye directly into a cannula inserted into the aorta. The final concentration of blebbistatin in the animal blood (~4000 mL) was 15 µmol/L.11. The dyes were injected through the aortic root cannula, and the vent line was inserted into the aorta. To obtain maximal staining of the heart tissue, during the dye injection the aorta was clamped below the arterial return cannula. The dye injection lasted no more than 2 min. Other details of the staining procedure differed depending on the blood circulation model.Two fluorescent dyes were used: the NIR fluorescent, voltage-sensitive dye di-4-ANBDQBS and indocyanine green (Cardiogreen), which also has voltage-sensitive propertiesOnline Supplementary Material) bolus containing 20 µmol/L di-4-ANBDQBS was injected using a peristaltic pump, and the perfusion velocity was gradually increased to obtain the appropriate pressure. The pressure was monitored by a monometer attached to the tube connected to the aorta catheter. The injection was performed under pressure starting at 80 mmHg and gradually increasing to a maximum of ~140 mmHg until the staining became apparent by monitoring of the heart with an EMCCD camera. The visualization of the coronary arteries with the NIR fluorescent, voltage-sensitive dye is described and presented in the Online Supplementary Material , staining was performed under pressure control to obtain sufficient delivery of the dye into the coronary circulation to avoid dye leakage in the left ventricular chamber. A 300-mL Ringer acetate Fresenius infusion saline .Under artificial blood circulation (n = 13), a 100-mL Ringer acetate Fresenius infusion saline bolus containing 50 µmol/L di-4-ANBDQBS (n = 8) or 120 µmol/L Cardiogreen (n = 5) and blebbistatin was injected directly through the infusion system. The di-4-ANBDQBS and Cardiogreen loading process is presented in Movies Under both experimental conditions, the final concentration of di-4-ANBDQBS in the circulating blood (~4000 mL) was ~1.5 µmol/L, and the final concentration of Cardiogreen was ~3 µmol/L.Physiological saline was always heated to +38 °C before use. Five millilitres of an aqueous stock solution of Cardiogreen (2 mg/mL) was freshly prepared before every experiment.in situ during open-heart surgery  at a wavelength of either 660 nm or 780 nm . Altogether, eight excitation LEDs were placed in a stable wheel Fig. . Cardioget al.31, Navitar lenses with such parameters are one of the best choices available for OM of the heart of big animal. The anterior surface of the heart, including the RV, anterior interventricular sulcus, and LV, was imaged. The overall mapping area was 55 × 55 mm. When the frame was used, the effective mapping area was 40 × 40 mm. Using imaging software (Andor SOLIS x-3467), optical movies were acquired at a frame rate of 500 Hz with a resolution of 128 × 128 pixels. One pixel corresponds to 0.43 mm square on the heart surface. For optical recordings, the heart was paced via bipolar electrodes with 2-ms stimuli at twice the diastolic threshold. Bipolar hook electrodes were embedded into the atrium, ventricle or septum.Optical movies were obtained with a cooled (−100 °C), fast, 14-bit EMCCD camera  equipped with a 50-mm focal length with F/0.95 high speed objective MVL50HS . Based on study of Bien The optical movies were processed using ImageJ software. The OAPs presented in the figures were taken from an area of 5 × 5 pixels. The background fluorescence (F) was subtracted from every frame of the recording. The OS was normalized with respect to the background fluorescence to obtain the fractional change in the fluorescence signal (ΔF/F). The OAP maps were constructed using custom Scroll 1.16 software developed by Dr. S. Mironov (University of Michigan). Before creating the maps, the OSs were additionally filtered using three-point triangular time and 5 × 5 pyramidal kernel space filters. The activation time maps show the time interval from stimulus to 50% depolarization of the OAP. The OAPD50 and OAPD80 maps show the duration of the OAP at the level of 50% and 80% repolarization from activation time, respectively. The repolarization time maps show the OAPD50 along with the activation time.The VT/VF dominant frequency (DF) was obtained from the frequency spectrum of the OSs using a Fourier transform.The significance of the difference between various parameters of OSs recorded from different dyes under normal or artificial blood circulation was evaluated using one-way ANOVA. A value of p < 0.05 was considered statistically significant.Online Supplementary Material.Movie S1.Movie S2.Movie S3.Movie S4.Movie S5.Movie S6.Movie S7.Movie S8.Movie S9.Movie S10.Movie S11.


Acute cellular rejection (ACR) is a major complication after heart transplantation. Endomyocardial biopsy (EMB) remains the gold standard for its diagnosis, but it has concerning complications. We evaluated the usefulness of speckle tracking echocardiography (STE) and biomarkers for detecting ACR after heart transplantation.We prospectively studied 60 transplant patients with normal left and right ventricular systolic function who underwent EMB for surveillance 6 months after transplantation. Sixty age- and sex-matched healthy individuals constituted the control group. Conventional echocardiographic parameters, left ventricular global longitudinal, radial and circumferential strain , left ventricular systolic twist (LV-twist) and right ventricular free wall longitudinal strain (RV-FWLS) were analyzed just before the procedure. We also measured biomarkers at the same moment.p=0.002; 13.9° ± 4.8° vs 17.1° ± 3.2°, p=0.048; 16.6% ± 2.9% vs 21.4%± 3.2%, p < 0.001; respectively), while no differences were observed between the LV-GRS or LV-GCS. All of these parameters were lower in the transplant group without ACR than in the nontransplant control group, except for the LV-twist. Cardiac troponin I levels were significantly higher in patients with significant ACR than in patients without significant ACR . The combination of troponin with LV-GLS, RV-FWLS and LV-Twist had an area under curve for the detection of ACR of 0.80 (0.68–0.92), 0.89 (0.81–0.93) and 0.79 (0.66–0.92), respectively.Among the 60 studied patients, 17 (28%) had severe ACR (grade ≥ 2R), and 43 (72%) had no significant ACR (grade 0 – 1R). The absolute values of LV-GLS, LV-twist and RV-FWLS were lower in transplant patients with ACR degree ≥ 2 R than in those without ACR (12.5% ± 2.9% vs 14.8% ± 2.3%, Heart transplant patients have altered left ventricular dynamics compared with control individuals. The combination of troponin with strain parameters had higher accuracy for the detection of ACR than the isolated variables and this association might select patients with a higher risk for ACR who will benefit from an EMB procedure in the first year after heart transplantation. Heart transplantation is the treatment of choice for selected patients with end-stage heart failure , 2. AlthEndomyocardial biopsy (EMB) is the widely accepted gold standard for the diagnosis of acute cellular rejection (ACR) . HoweverTherefore, there has been considerable effort exerted to develop noninvasive and accurate methods that can reduce the need for EMB, including biomarker detection, imaging techniques and genetic tests , 14. TheIn the present study, we aimed to assess the value of STE-derived strain measurements and biomarkers for the noninvasive detection of ACR after heart transplantation. We also evaluated the ventricular dynamics of heart transplant patients in comparison with control individuals using STE.From January 2014 to November 2018, we prospectively studied heart transplant patients who underwent EMB in the 6th month after orthotopic heart transplantation for the diagnosis of ACR during routine surveillance. All of the included patients were asymptomatic and did not present with any hemodynamic compromise at the time of enrollment.The exclusion criteria were as follows: age < 18 years, arrhythmia, left ventricular ejection fraction < 0.55, right ventricular dysfunction of any degree , vascular graft disease, humoral rejection, two or more previous cellular rejection episodes, chronic kidney disease, Chagas disease reactivation, limited echocardiographic window for STE analysis and an inconclusive EMB analysis.All patients underwent the same protocol according to the predefined steps. First, enrolled patients underwent a complete echocardiographic analysis for the assessment of left and right ventricular function. For those with a normal systolic function, images were acquired for mechanical analysis by STE, and blood was taken for biomarker tests. The patients then underwent EMB and, according to the results, were divided into the following groups: 1) without significant ACR (grades 0 and 1R) and 2) with significant ACR (grades 2R and 3R).Individuals without a clinical history of any disease known to interfere with myocardial physiology or structure and who were matched by sex and age with the transplant patients constituted the control group. This group underwent STE for the analysis of ventricular mechanics and was compared to the groups of transplant patients with and without significant ACR. The study protocol was approved by our ethical committee, and all patients gave written informed consent to participate.EMB was performed through the internal jugular or femoral vein under radioscopy. A minimum of 3 distinct ventricular myocardial fragments were collected (each consisting of at least 50% myocardium) and sent for anatomopathological analysis. A sample was considered sufficient when at least 3 myocardial fragments were obtained for analysis by optical microscopy after fixation in 10% formalin and staining of the laminae with hematoxylin and eosin. Two experienced cardiac pathologists blinded to the echocardiographic results analyzed all biopsies. The grade of rejection was based upon the recommendations of the International Society for Heart and Lung Transplantation . The resOn the day of EMB, just before the procedure, patients underwent echocardiographic examination on a commercially available machine equipped with an MS5 probe . Image acquisition and assessment were performed according to the recommendations of the American Society of Echocardiography . Left veTo assess the ventricular mechanics, 3 consecutive cardiac cycles were recorded. Left ventricular short-axis and apical views were acquired using two-dimensional grayscale second-harmonic imaging at a frame rate of 50–80 frames per second. Left ventricular short-axis views at the basal, mid (papillary muscles) and apex levels were acquired to analyze the circumferential (LV-GCS) and radial strain (LV-GRS), while the left ventricular apical 4-, 2- and 3-chamber views were used to assess the left ventricular global longitudinal strain (LV-GLS) and the apical 4-chamber view focused on the right ventricle was used to analyze the right ventricular free wall longitudinal strain (RV-FWLS).STE analysis was performed offline using dedicated software . All echocardiographic measurements were performed by one specialist blinded to the clinical data. End-systole was determined by pulsed-wave Doppler at the time of aortic valve closure. After the ventricular endocardial border was manually traced, the epicardial borders were automatically defined to create regions of interest according to the ventricular segmentation; if necessary, adjustments were made by the operator. In particular, care was taken to not include the myocardial trabeculae and the pericardium. Following this step, the myocardial speckles were automatically tracked by the dedicated software, and, in the case of suboptimal tracking, further manual adjustments were allowed, resulting in strain curves that were exported to a spreadsheet. Global longitudinal strain, circumferential strain, and radial strain were calculated as averages of peak systolic strain values obtained from all segments in the respective views. Accordingly, the LV-GLS was obtained from the mean of 18 segments acquired in the apical 4-, 2-, and 3-chamber views, while circumferential and radial strain were obtained from 12 segments in LV short-axis views at the basal, papillary muscle levels and apex. Left ventricular twist (LV-Twist) is the wringing motion of the heart around its long axis. It was calculated as the net absolute difference between the apical and basal rotations . By widely assumed convention, apical rotation has positive values and basal had negative values . The RV-The intraobserver reproducibility of the strain measurements was assessed in a subsample of 30 randomly selected patients 3 months after the initial evaluation; the observer was blinded to the previous results. Interobserver variability was assessed in the same subsample by a second blinded experienced echocardiographer.Biomarker analysis was performed before EMB. For this, a 20 mL blood sample was collected from a peripheral veinto determine the plasma levels of cardiac troponin I and BNP.Cardiac troponin I levels were quantified with a high sensitivity 3-step sandwich immunoassay using direct chemiluminescent technology and consistent amounts of 2 monoclonal antibodies. An auxiliary reagent was included to reduce nonspecific binding using an Advia Centaur TnI-Ultra commercial kit . The level of detection was 0.006 ng/mL (levels < 0.006 were reported as 0.005 ng/mL). The normal range of cardiac troponin I was considered < 0.04 ng/mL.Plasma concentrations of BNP were determined with a 2-step sandwich immunoassay using direct chemiluminescent technology and consistent amounts of 2 monoclonal antibodies using an Advia Centaur commercial kit . The level of detection was 2 pg/mL. Levels < 2 were reported as 1 pg/mL.Categorical variables were compared using Pearson chi-square tests, Fisher exact tests, or likelihood ratio tests. Continuous variables were compared usingthe analysis of variance and Tukey’s test  or the Kruskal-Wallis and Dunn’s tests. The results were expressed as the means with standard deviations or as the medians with interquartile ranges. Linear correlations were tested using the Spearman rank method.Receiver operating characteristic curves were constructed for variables significantly associated with the presence of ACR degree 2R, and the optimal cutoff values were calculated as the point with the highest sums of sensitivity and specificity. Receiver operating characteristic curves were also constructed for troponin combined with strain parameters significantly associated with the presence of ACR degree 2R.The interobserver and intraobserver reproducibility of LV-GLS, LV-Twist and RV-FWLSwere assessed using intraclass correlation coefficients and 95% confidence intervals (CIs) in one-way random and two-way mixed models.p-value < 0.05 was considered statistically significant.All analyses were performed using SPSS version 17 . A Ninety-five patients were initially enrolled in the study. Among these patients, 35 were excluded because of the following characteristics: 2 due to cardiac arrhythmia, 4 due to left ventricular systolic dysfunction, 4 due to right ventricular systolic dysfunction, 14 due to two or more previous ACR episodes, 4 due to a limited echocardiographic acoustic window for STE analysis, 3 due to inconclusive results of EMB, 2 due to humoral rejection and 2 due to Chagas disease recurrence. A total of 60 heart transplant patients and 60 control individuals constituted the final study population  did not show significant ACR (grade 0 or 1R), while 17(28%) showed significant ACR (grade 2R or 3R).The baseline characteristics of the heart transplant patients included in this study are described in Table p=0.007]. No significant difference in BNP levels was found between patients with significant ACR and patients without significant ACR .Cardiac troponin I levels as assessed by the ultrasensitive assay were significantly higher in patients with significant ACR than in patients without significant ACR [0.19 ng/mL (0.09–1.31) vs 0.05 ng/mL (0.01–0.18), The variables obtained by conventional echocardiography are shown in Table p = 0.002;13.9° ± 4.8° vs 17.1° ± 3.0°, p = 0.048;16.6% ± 2.9%vs21.4%± 3.2%, p< 0.001; respectively), as shown in Fig. The LV-GLS, LV-GCS, LV-GRS, RV-FWLS and LV-twist values are shown in Table When we combined the strain parameters assessed as significant to detect ACR with troponin, we found that it increased the accuracy compared with the variables used individually. We report it in the model described below, based on the area under the curve (AUC) Fig. .Fig. 3aThe intraclass correlation coefficient of LV-GLS was 0.92 (95% CI = 0.85–0.96) for interobserver variability. For intraobserver variability, the intraclass correlation coefficient of the LV-GLS was 0.93 (95% CI = 0.86–0.97). The intraclass correlation coefficients of the RV-FWLS were 0.90 (95% CI = 0.80–0.95) and 0.92 (95% CI = 0.84–0.96) for inter- and intraobserver variability, respectively. For the LV-Twist, the inter and intraobserver variability were 0.79 (95% CI = 0.35–0.94) and 0.78 (95% CI = 0.33–0.94), respectively.The main results of our work are that patients with ACR had significantly lower values of LV-GLS, RV-FWLS and LV-Twist and higher level of troponin I than patients without significant ACR. In addition, the combination of troponin and these parameters may improve the diagnostic value of strain in heart transplant patients with preserved left and right ventricular systolic functions. The combination of RV-FWLS and troponin presented a higher accuracy for the detection of ACR degree ≥2R. The AUC of the combination of troponin (> 0.05 ng/mL) with RV-FWLS  was 0.89(95%CI = 0.81–0.93). Thus, it may be considered for use as a screening tool for the detection of ACR and could reduce the number of EMBs after heart transplantation.ACR is a significant and frequent complication of heart transplantation. In the first year, it is the most common cause of mortality. Currently, EMB is the clinical gold standard in screening for graft rejection after heart transplantation and is actually the only tool for the diagnosis and classification of allograft rejection . ConsideTo the best of our knowledge, this is the first study to analyze all strain parameters using STE  in the same population of heart transplanted patients with normal right and left ventricular systolic function, and its combination with biomarkers, to diagnosis clinically significant ACR. Extra care was given in our study to restrict patient selection to a fixed period of time (6 months post-heart transplantation) to minimize the possible influence of the time since heart transplantation on ventricular strain, as recommended by the current guidelines . AdditioPrevious studies have shown that myocardial strain has a higher sensitivity than conventional echocardiography, and therefore, may be an important tool to detect early subclinical cardiac dysfunction . AlthougRight ventricular strain analysis has been poorly described to date in this scenario. Mingo-Santos et al. demonstrated a predictive role of STE parameters in the diagnosis of ACR  . That stp < 0.048). In parallel to our results, a unique previous study that applied STE derived LV-Twist measurements to detect rejection in heart transplanted patients demonstrated that the LV-Twist decreased more in the group with ACR than in the group without ACR (9.6°±2.7° vs 12.2° ±2.3°), p < 0.0001, [As we showed in our results, the LV-Twist values were significantly lower in the group with significant ACR than in the group without significant ACR , loading conditions and donor age –45.The limitations of this study should be addressed. First, this was a single-center study with a small number of patients and a limited number of rejection episodes graded equal to or above 2R (17 out of 60 samples). Despite its extensive validation, STE is still an evolving technique, and improvements such as better tracking accuracy are still needed. Additionally, STE accuracy is highly dependent on image quality. Suboptimal resolution can produce a negative impact on the final results. Nevertheless, despite these limitations, we were able to successfully perform speckle-tracking analysis of both left and right ventricular longitudinal strain in 95% of the patients. The reproducibility of the parameters was good and was similar to that reported in other studies. Finally, our results must be independently validated in a prospective external cohort, preferably in multicenter studies, before they can be used in clinical practice.Heart transplant patients have altered left ventricular dynamics compared with control individuals. The association of strain parameters derived from STE, particularly RV-FWLS, with troponin seems to be able to detect ACR with higher accuracy than these variables used individually. These parameters might be considered a screening method that can be added to the management of heart transplant patients to safely reduce the number of unnecessary EMB.With the increase in heart transplantation, new tools for detecting acute cellular rejection are highly desirable. Our study findings show that the analysis of strain parameters with troponin levels might be useful to noninvasively detect patients who are at high risk of cellular rejection.


Pulmonary valve (PV) endocarditis is a frequent complication during follow-up in patients with repaired right ventricular outflow tract (RVOT) obstruction and poses relevant diagnostic and treatment challenges. We aimed to describe in details the possible different clinical presentations of this rare condition and to highlight the role of both transthoracic and transoesophageal echocardiography which, in experienced hands, may provide comprehensive useful information for the clinicians.We below describe the clinical presentation and the echo findings of three cases of pulmonary valve endocarditis complicating disease course after different repair modalities of congenital right ventricular outflow tract obstruction.The present case series outlines the diagnostic challenges of this increasingly frequent complication during follow-up of patients with congenital RVOT dysfunction after both surgical and percutaneous repair. Despite the diffusion of multimodality imaging, echocardiography with PV-dedicated views play a pivotal role in diagnosing such condition and guiding clinical management. Furthermore, this case series highlight that the suspicion of infective endocarditis should be raised whenever a sudden increase in transvalvular gradient is found during follow-up. In adults with congenital heart disease, infective endocarditis (IE) may involve both native and prosthetic pulmonary valve (PV) after surgical or transcatheter repair.In the era of multimodality imaging, transthoracic and transoesophageal echocardiography with PV-dedicated views play a pivotal role in diagnosing such condition and guiding clinical management.The predominant echo feature of PV endocarditis is right ventricular outflow tract obstruction. Infective endocarditis should be suspected whenever a sudden increase in transvalvular gradient is found during follow-up.,In the general population, infective endocarditis (IE) involving pulmonary valve (PV) is an extremely rare event, accounting for 1.5–2% of all cases.Here, we present three cases of PV endocarditis complicating three different repair modalities of congenital RVOT obstruction.TM valve  was successfully performed with symptoms relief. Three years later, the patient was referred to our Adult Congenital Heart Disease (ACHD) Unit for fever following a dental abscess and persisting despite appropriate antibiotic treatment. On physical examination at admission, the patient was febrile, the heart rate (HR) was 110 b.p.m., blood pressure was 100/60 mmHg, and the oxygen saturation was 99% on room air. Inflammatory markers were markedly raised and transthoracic echocardiogram (TTE) showed normal biventricular systolic function  and a well-seated and normally functioning bioprosthetic PV with thin and mobile leaflets. However, from high parasternal short-axis view, a mobile filamentous echogenic mass was visualized attached to the struts of the proximal segment of the stent in RVOT . No signs of pulmonary embolization were demonstrated at the positron emission tomography (PET)/computed tomography (CT) imaging. Serial blood cultures identified Streptococcus anginosus as the causative microorganism and, accordingly, a treatment with teicoplanine was commenced. Six weeks later, the patient was afebrile and in good clinical status and the inflammatory markers returned to normal values. However, TTE at 2-month follow-up showed persistent PV mass, albeit significantly reduced in size, hyperechogenic, and less mobile . Considering that the patient was haemodynamically stable with no signs of active infection, and relying on the echocardiographic features suggestive of low risk of embolization, a conservative watchful management was followed. The 6-month follow-up TTE showed complete resolution of the vegetation.A 24-year-old man with double outlet right ventricle and pulmonary stenosis (PS), with previous surgical palliation with Blalock–Taussig (BT) shunt in early infancy and subsequent biventricular repair with right ventricle (RV) to pulmonary artery (PA) conduit placement, developed a progressive conduit stenosis during follow-up. A percutaneous pulmonary prosthesis implant with a MelodyFigure  and C). Normal biventricular systolic function  was demonstrated. Doppler analysis revealed severe PS with peak gradient measured at 93 mmHg with no significant pulmonary regurgitation (PR) detectable . Moreover, a large and mobile vegetation was visualized attached to the ventricular side of the PV . No signs of pulmonary embolization were demonstrated at the PET/CT imaging. Streptococcus sanguinis was isolated from blood cultures and adequate antibiotic regimen with gentamicin and ceftriaxone was administered for 6 weeks with clinical improvement and echo evidence of progressive reduction in size of the vegetation. At 6-month follow-up, TTE showed complete resolution of the PV vegetation , and the patient was managed conservatively.A 19-year-old man with complex congenital heart disease was referred to our division for persisting fever 1 month after a percutaneous procedure. The cardiac diagnosis was mesocardia, congenitally corrected transposition of the great arteries with non-restrictive ventricular septal defect (VSD), pulmonary atresia with hypoplastic pulmonary arteries supplied by numerous major aortopulmonary collateral arteries. During infancy, he underwent bilateral BT shunts followed by transcatheter perforation of the PV. As significant PS with clinical signs of severe pulmonary hypoperfusion persisted, 1 month before attending our ACHD Unit, a percutaneous PV balloon dilatation was successfully performed at other institution with subsequent improvement in effort tolerance. On admission at our unit, the patient reported severe shortness of breath. On physical examination, the patient was febrile, HR was 105 b.p.m., room air oxygen saturation was 90%, and a harsh 4/6 Levine systolic murmur was appreciable at the cardiac base. At TTE, a narrow RVOT with a dysplastic PV overriding a large, unrestricted VSD were visualized from subcostal and modified apical views  conduit placement, presented to our ACHD Unit after a syncopal episode occurred during a mild effort. He reported, 1 month earlier, an episode of intermittent fever associated with gastrointestinal symptoms. On admission, he was afebrile and well perfused with normal vital signs. A harsh 4/6 Levine systolic and a 3/6 diastolic murmur were heard at the cardiac base. Routine blood tests demonstrated elevation of the inflammatory markers. TTE showed dilated and mildly hypokinetic RV  with significantly raised RV systolic pressure. Although the conduit was non-visible on 2D echocardiography, Doppler signal at RVOT level revealed severe PS with peak gradient of 171 mmHg . LV systolic function was preserved (LVEF 60%). The transoesophageal echocardiogram (TOE) with upper oesophageal (UE) views allowed clear visualization of the severely stenotic conduit with degenerated and thickened PV leaflets determining moderate PR. Moreover, multiple small, mobile masses arising from the ventricular side of the PV were visualized . The CT imaging demonstrated severely reduced inner conduit lumen size with a minimum diameter of 3 mm, while no signs of embolization were found. Staphylococcus aureus was isolated from serial blood cultures. Based on the TOE results, the patient underwent a successful surgical PV replacement with a homograft.A 27-year-old man with tetralogy of Fallot (TOF) repaired during childhood and reoperated at the age of 14 with an RV to PA ContegraThe findings from our series show that, in ACHD patients, PV IE may involve both native and bioprosthetic PV after surgical or transcatheter repair leading to relevant diagnostic and clinical difficulties during follow-up.Nowadays, PV is the cardiac valve less frequently affected by IE, with the majority of cases confined to patients with congenital RVOT dysfunction. In the last decades, surgical advances allowed to a growing proportion of patients with successfully repaired congenital heart disease to reach adulthood with residual lesions and sequelae, often requiring further treatment during their lifetimes. Accordingly, the increase in prevalence of patients who had undergone either surgical or percutaneous PV procedures results in a parallel increase in the population at risk for IE. In fact, both surgical PV replacementEven though echocardiography remains the mainstay imaging modality in the diagnostic flowchart of IE, difficulties in images acquisition resulting from the variable position of the conduits or shadowing artefacts from stented valves may prevent accurate PV leaflets visualization. Notwithstanding, our case series demonstrates that, in tertiary centres with expertise in imaging in ACHD, echocardiography provides fundamental and reliable morphological and functional information for a correct diagnosis, for guiding the management strategy and for monitoring treatment response. Multiple and off-axis unconventional echo views may be helpful to clearly visualize all the structures in the RVOT.Transoesophageal echocardiogram is often underused to image RVOT because PV visualization may be limited by its anterior position in chest, thus appearing in the far field. However, TOE role in suspected PV endocarditis should not be neglected, especially when TTE is insufficient to accurately rule out the involvement of other cardiac structures, such as other valves and subvalvular apparatus, patches or intra-cardiac shunts. Furthermore, using PV-dedicated views, TOE may in some cases allows a comprehensive visualization of the anteriorly seated valved conduits as shown in Case 3.Moreover, it should be noted that, unlike IE involving other cardiac valves, PV endocarditis more often presents with a stenotic lesion, as previous studies have demonstrated.Pulmonary valve endocarditis is a frequent complication during follow-up of patients with congenital RVOT dysfunction after both surgical and percutaneous repair. Echo images may be challenging, nevertheless, using unconventional PV-dedicated views, echocardiography provides fundamental info not just for diagnosis but also for treatment guidance.Dr Giancarlo Scognamiglio, MD, PhD, is a Consultant Cardiologist and Lead of the Imaging Programme in Adult Congenital Heart Disease at the GUCH Unit of Monaldi Hospital in Naples, Italy. During his training, he specialized in transthoracic and transoesophageal echocardiography and congenital heart disease. His fields of interest include congenital heart disease, valvular heart disease, pulmonary hypertension, ICU, transthoracic and transoesophageal echocardiography, and 3D echo. He is author of chapters in 4 scientific books and more than 40 scientific papers on Peer-Reviewed Journals. Dr Scognamiglio is Professor of Congenital Heart Disease in the Specialty Training Programme in Cardiology at the University of Campania Luigi Vanvitelli.European Heart Journal - Case Reports online.Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Consent: The author/s confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patients in line with COPE guidance.Conflict of interest: none declared.ytaa195_Supplementary_DataClick here for additional data file.