Optical Mapping Experiments Research Articles

Cholinergic regulation of heart rate: Functional importance of l-type cav1.3 channels

The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying IKACh), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of several ion channels involved in pacemaking, including “funny” f-(HCN4) and L-type Cav1.3 calcium channels. To determine the importance of Cav1.3, GIRK1/4 and HCN4 in cholinergic regulation of HR. We recorded the HR of isolated Langendorff perfused hearts and the pacemaker leading site in ex vivo sinus nodes of mice in control or during ACh perfusion. We used control wild-type (WT) mice, and five genetically modified mouse models: Cav1.3KO (no Cav1.3-mediated L-type current), Girk4KO (no IKACh), HCN4-CNBD (no cAMP-dependent regulation of HCN4), Girk4KO/HCN4-CNBD and Girk4KO/Cav1.3KO. Perfusion of 1 μM ACh significantly reduced HR in hearts from control (18%), Girk4KO (12%) and HCN4-CNBD (23%) animals, but not in hearts from in Cav1.3KO, Girk4KO/HCN4-CNBD and Girk4KO/Cav1.3 mice. 3 μM ACh significantly reduced HR in all the strains tested except for Girk4KO/Cav1.3KO animals. Preliminary results obtained from optical mapping experiments show ACh-induced shift of pacemaker leading site in WT and HCN4-CNBD mice. Girk4KO/Cav1.3KO mice show a larger shift of the leading pacemaker site than WT and HCN4-CNBD mice, to regions outside the sinus node area. Our data indicate that Cav1.3 channels play an important role in the cholinergic regulation of heart rate. Indeed, concomitant ablation of GIRK1/4 and Cav1.3 channels nearly abolished regulation in isolated murine hearts. Preliminary optical mapping results may indicate that this concomitant ablation increases pacemaker shift in cholinergic stimulation.

Regional repolarization shortening is more arrhythmogenic than regional conduction slowing in an ex vivo porcine experimental model

Ventricular fibrillation (VF) is a major cause of sudden cardiac death. In patients without apparent structural heart disease, J-point elevation and J waves are often associated with an increased risk to VF. The mechanisms underlying these ECG patterns and the associated arrhythmogenesis are still debated. To investigate the respective role of conduction and repolarization heterogeneities in the emergence of J-waves and in the vulnerability to VF. Epi- and endocardial optical mapping experiments were performed in porcine right ventricular (RV) wedge preparations ( n = 26). Both right coronary and left anterior descending (LAD) arteries were perfused separately with Tyrode, followed by addition of either pinacidil (PINA; 20 μM, n = 14) or flecainide (FLECA; 2 μM, n = 12) in the LAD only. RV action potentials (AP) properties were assessed at 1 to 4 Hz stimulation frequencies, and a bath ECG was recorded to evaluate ST segment changes. Spontaneous or induced arrhythmias were quantified and analyzed to calculate dominant frequencies (DF) and regularity index (RI). Local perfusion of PINA induced strong AP duration and repolarization time (RT) heterogeneity on epi- and endocardial surfaces while regional FLECA perfusion significantly prolonged total activation time and increased RT heterogeneity. Both models were associated with changes in ST segment, but those of conduction heterogeneity were more subtle. They both decreased RI of arrhythmias compared to the baseline (0.53 vs. 0.84 P = 0.02 for PINA; 0.47 vs. 0.76 P = 0.06 for FLECA), and DF were significantly higher for PINA compared to FLECA (9.41 vs. 7.74 P = 0.03). Spontaneous VFs are more frequent with a local perfusion of PINA compared to FLECA (86 vs. 8% P = 0.0001). Regional repolarization shortening and conduction slowing result in specific ECG phenotypes and distinct VF dynamics. In our porcine model, repolarization heterogeneity is associated with a higher risk to VF.

Abstract MP114: Cardiotropic Adenovirus Increases Arrhythmia Susceptibility During Acute Infection

Myocarditis is responsible for up to 42% of sudden cardiac death in young adults yet mechanisms underlying virally-induced arrhythmia remain elusive. Adenovirus is a leading etiological agent of myocarditis but due to species-specificity, in vivo models are limited. Altered cardiac gap junction function underlies sudden cardiac death and given that gap junctions also propagate antiviral immune responses, we hypothesized adenovirus would target intercellular coupling, leading to arrhythmogenesis. Employing a recently described strain of mouse adenovirus, MAdV-3, we investigated cardiotropism, arrhythmia susceptibility, and molecular mechanisms of a virally-induced arrhythmogenic substrate. Adult C57BL/6 mice were inoculated with MAdV-3 and infection allowed to progress for one week. Viral genomes were detected by qPCR to measure tissue tropism together with whole animal histopathology. Echo- and electrocardiography were used to measure cardiac function and electrical activity. Viral effects on transcription and junctional protein/ion channel expression and localization were quantified using RT-qPCR, confocal microscopy, and western blotting. Finally, ex vivo optical mapping was employed to test conduction defects and susceptibility to arrhythmia. We find MAdV-3 viral genomes specifically enriched in heart tissue, confirming cardiotropism. Immune cell infiltration was not detected in the heart and no cardiomyopathy was apparent by echocardiography. RR interval widening, however, does occur in MAdV-3 infected mice with reductions in cardiac ion channel and gap junction mRNA transcripts. Optical mapping experiments show that conduction velocity is altered in acutely infected hearts and norepinephrine challenge uncovered electrical impairment presenting as atrioventricular dissociation with increased premature ventricular contraction burden. Our data demonstrate that MAdV-3 is cardiotropic and acute infection result in changes in cardiac electrical activity prior to development of cardiomyopathy or a detectable immune response. MAdV-3 infection is a novel model for adenoviral myocarditis, providing insights into viral subversion of cardiac intercellular coupling and mechanisms of arrhythmogenesis.

Variation in RARG increases susceptibility to doxorubicin-induced cardiotoxicity in patient specific induced pluripotent stem cell-derived cardiomyocytes

Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DNA breaks, reactive oxygen species production, and cell death. Conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in DIC.

Spatiotemporal correlation uncovers characteristic lengths in cardiac tissue.

Complex spatiotemporal patterns of action potential duration have been shown to occur in many mammalian hearts due to period-doubling bifurcations that develop with increasing frequency of stimulation. Here, through high-resolution optical mapping experiments and mathematical modeling, we introduce a characteristic spatial length of cardiac activity in canine ventricular wedges via a spatiotemporal correlation analysis, at different stimulation frequencies and during fibrillation. We show that the characteristic length ranges from 40 to 20cm during one-to-one responses and it decreases to a specific value of about 3cm at the transition from period-doubling bifurcation to fibrillation. We further show that during fibrillation, the characteristic length is about 1cm. Another significant outcome of our analysis is the finding of a constitutive phenomenological law obtained from a nonlinear fitting of experimental data which relates the conduction velocity restitution curve with the characteristic length of the system. The fractional exponent of 3/2 in our phenomenological law is in agreement with the domain size remapping required to reproduce experimental fibrillation dynamics within a realistic cardiac domain via accurate mathematical models.

Development of new voltage-sensitive fluorescent dyes for cardiac optical mapping

Cardiac arrhythmias are an important cause of morbidity and mortality. Optical mapping using voltage-sensitive dyes (VSDs) allows to study these disorders in isolated perfused tissues. Until recently, this technique remained limited to the myocardial surface due to the short excitation wavelengths of common VSDs. Using red excitation light allows to by-pass this problem, but availability of such dyes remains limited. To develop new families of red-shifted VSDs and to characterize both their spectral and potentiometric properties. Three families of VSDs were designed. The spectral properties of each VSD was assessed by spectrofluorometry in Langendorff perfused rat hearts. The potentiometric properties were assessed through optical mapping in Langendorff perfused rat hearts. For spectrofluorometry, hearts were freely beating and systolic and diastolic pressures were continuously monitored. In optical mapping experiments hearts were mechanically arrested by supplementation of blebbistatin (10 μM). Out of the three families, one showed a strong shift in excitation and emission wavelengths towards the near-infrared. Within this family, three VSDs, with variations in the hydrophilic tail and charged carrier, were tested. Results showed that these VSDs have peak excitation above 500 nm and peak emission around 730 nm. Although two of these dyes showed a low potentiometric sensitivity (<1%), one VSD had a ΔF/F similar to conventional dyes (>5%) with a stable signal for over 2 hours after a bolus injection in the hearts. In mechanically arrested hearts, the VSDs had no impact on electrophysiological parameters. However, in beating hearts, the VSDs significantly increased diastolic pressure. We have developed new red-shifted VSDs and validate their use in cardiac applications, which might allow optimizing optical mapping of cardiac arrhythmia dynamics in deeper tissue layers and/or in blood-perfused tissues.

Evaluation of Multiscale Frequency Approach for Visualizing Rotors in Patients with Atrial Fibrillation.

Atrial Fibrillation (AF) is most common cardiac arrhythmia. It is associated with increased risk of stroke, heart failure and sudden cardiac death. Catheter ablation is a treatment used to control AF and has had suboptimal success for patients with persistent AF, which is primarily maintained by rotors outside of the pulmonary veins (PV) region. The pivot point (core) of the rotor is considered an efficient target for ablation. Currently available electro-anatomical mapping systems cannot accurately predict the exact location of the pivot point of rotors outside of the PV region, so there is a need for novel approaches to accurately identify and distinguish sites for ablation. Recently, a multiscale frequency (MSF) technique was developed for accurate identification of the pivot point of rotors and validated using optical mapping experiments in exvivo rabbit hearts, where electrical activity can be directly visualized. However, the nature of optical signals and its spatial resolution are very different from clinical intracardiac electrograms (iEGM). Here we extend the MSF approach to 3D iEGM and compare its prediction with the traditional dominant frequency (DF) approach, using Pearson's correlation and earth mover's distance methods. Our results demonstrate that the similarity between MSF and DF are high in some regions, but very low in other spatial regions of the human atria. This indicates the inconsistency in the traditional DF approach in identifying pivot points and identifying such low similarity regions can be used to find sites for successful ablation.

CaMKII-dependent late Na+ current increases electrical dispersion and arrhythmia in ischemia-reperfusion.

The mechanisms underlying Ca2+/calmodulin-dependent protein kinase II (CaMKII)-induced arrhythmias in ischemia-reperfusion (I/R) are not fully understood. We tested the hypothesis that CaMKII increases late Na+ current ( INa,L) via phosphorylation of Nav1.5 at Ser571 during I/R, thereby increasing arrhythmia susceptibility. To test our hypothesis, we studied isolated, Langendorff-perfused hearts from wild-type (WT) mice and mice expressing Nav channel variants Nav1.5-Ser571E (S571E) and Nav1.5-Ser571A (S571A). WT hearts showed a significant increase in the levels of phosphorylated CaMKII and Nav1.5 at Ser571 [p-Nav1.5(S571)] after 15 min of global ischemia (just before the onset of reperfusion). Optical mapping experiments revealed an increase in action potential duration (APD) and APD dispersion without changes in conduction velocity during I/R in WT and S571E compared with S571A hearts. At the same time, WT and S571E hearts showed an increase in spontaneous arrhythmia events (e.g., premature ventricular contractions) and an increase in the inducibility of reentrant arrhythmias during reperfusion. Pretreatment of WT hearts with the Na+ channel blocker mexiletine (10 μM) normalized APD dispersion and reduced arrhythmia susceptibility during I/R. We conclude that CaMKII-dependent phosphorylation of Nav1.5 is a crucial driver for increased INa,L, arrhythmia triggers, and substrate during I/R. Selective targeting of this CaMKII-dependent pathway may have therapeutic potential for reducing arrhythmias in the setting of I/R. NEW & NOTEWORTHY Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of Nav1.5 at Ser571 leads to a prolongation of action potential duration (APD), increased APD dispersion, and increased arrhythmia susceptibility after ischemia-reperfusion in isolated mouse hearts. Genetic ablation of the CaMKII-dependent phosphorylation site Ser571 on Nav1.5 or low-dose mexiletine (to inhibit late Na+ current) reduced APD dispersion, arrhythmia triggers, and ventricular tachycardia inducibility.

Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.

Role of Purkinje network in maintaining ventricular fibrillation before and after focal catheter ablation of Purkinje fibers in sheep left ventricle

Ventricular Fibrillation (VF) is one of the most frequent causes of sudden cardiac death. It is known that Purkinje fibers (PF) play an important role of arrhythmia trigger and recent studies suggest a potential role of these fibers as a substrate maintaining VF. The goal of this study was to define the role of Purkinje-muscle junctions (PMJ) in maintaining VF and to determine the effect of catheter ablation of these junctions. Optical mapping experiments were performed in coronary-perfused LV wedge preparations from sheep ( n = 8), stained with Di-4-ANBDQBS and using LED illumination at 660 nm. CMOS camera (MiCAM, BrainVision) was used to obtain endocardial recordings. PMJ were localized through PF stimulation and were compared to late activation zones (LAZ). Endocardial burst pacing (10 Hz) was applied to trigger 10 minutes of VF, before and after PMJ ablation (30 W, 15 s), and recordings were acquired every 2 minutes. Phase mapping was used to quantify rotor and focal dynamics. No dominant frequency differences between PMJ and LAZ were found for all VF episodes, before ( P = 0.81) and after ( P = 0.79) PMJ ablation. No change in focal activity was observed during this initial phases of VF. However, a tendency for colocalization for distances between PMJs and rotors was observed before ablation. Distances increased significantly (20 ± 7 mm vs. 24 ± 7 mm; P = 0.009) after ablation. Moreover, a decrease in mean rotor duration and a significant increase in rotor number for all VF episodes (6 ± 4 vs. 12 ± 11; P = 0.026) were also found after ablation. These results suggest that the PMJ is involved in maintaining VF by playing a role in rotor dynamics.

Real-Time Closed Loop Diastolic Interval Control Prevents Cardiac Alternans in Isolated Whole Rabbit Hearts.

Cardiac alternans, a beat-to-beat alternation in action potential duration (APD), can lead to fatal arrhythmias. During periodic pacing, changes in diastolic interval (DI) depend on subsequent changes in APD, thus enhancing cardiac instabilities through a 'feedback' mechanism. Recently, an anti-arrhythmic Constant DI pacing protocol was proposed and shown to be effective in suppressing alternans in 0D and 1D in silico studies. However, previousexperimental validation of Constant DI pacingin the heart has been unsuccessful due to the spatio-temporal complexity of 2D cardiac tissue and the technical challenges in its real-time implementation. Here, we developed a novel closed loop system to detect T-waves from real-time ECG data, enabling successful implementation of Constant DI pacing protocol, and performed high-resolution optical mapping experiments on isolated whole rabbit hearts to validate its anti-arrhythmic effects. The results were compared with: (1) Periodic pacing (feedback inherent) and (2) pacing with heart rate variability (HRV) (feedback modulation) introduced by using either Gaussian or Physiological patterns. We observed that Constant DI pacingsignificantly suppressed alternans in the heart, while maintaining APD spatial dispersion and flattening the slope of the APD restitution curve, compared to traditional Periodic pacing. In addition, introduction of HRV in Periodicpacing failed to prevent cardiac alternans, and was arrhythmogenic.

Causality analysis of leading singular value decomposition modes identifies rotor as the dominant driving normal mode in fibrillation.

Cardiac fibrillation is a major clinical and societal burden. Rotors may drive fibrillation in many cases, but their role and patterns are often masked by complex propagation. We used Singular Value Decomposition (SVD), which ranks patterns of activation hierarchically, together with Wiener-Granger causality analysis (WGCA), which analyses direction of information among observations, to investigate the role of rotors in cardiac fibrillation. We hypothesized that combining SVD analysis with WGCA should reveal whether rotor activity is the dominant driving force of fibrillation even in cases of high complexity. Optical mapping experiments were conducted in neonatal rat cardiomyocyte monolayers (diameter, 35 mm), which were genetically modified to overexpress the delayed rectifier K+ channel IKr only in one half of the monolayer. Such monolayers have been shown previously to sustain fast rotors confined to the IKr overexpressing half and driving fibrillatory-like activity in the other half. SVD analysis of the optical mapping movies revealed a hierarchical pattern in which the primary modes corresponded to rotor activity in the IKr overexpressing region and the secondary modes corresponded to fibrillatory activity elsewhere. We then applied WGCA to evaluate the directionality of influence between modes in the entire monolayer using clear and noisy movies of activity. We demonstrated that the rotor modes influence the secondary fibrillatory modes, but influence was detected also in the opposite direction. To more specifically delineate the role of the rotor in fibrillation, we decomposed separately the respective SVD modes of the rotor and fibrillatory domains. In this case, WGCA yielded more information from the rotor to the fibrillatory domains than in the opposite direction. In conclusion, SVD analysis reveals that rotors can be the dominant modes of an experimental model of fibrillation. Wiener-Granger causality on modes of the rotor domains confirms their preferential driving influence on fibrillatory modes.

Regulation of cardiac excitation-contraction coupling by fibroblasts in health and disease

Introduction: Cardiomyocyte structure and function are influenced directly via cell-cell communication, the ubiquitous extra-cellular matrix (ECM), and paracrine interactions. These interactions modulate cardiomyocytes in health and the changes that occur in disease. Clear mechanisms in these interactions are still lacking, particularly how fibroblasts may affect myocyte excitation-contraction coupling in a human model. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were co-cultured with human cardiac fibroblasts, isolated from heart failure patients, at a ratio of 2:1 fibroblasts:hiPSC-CMs for 24 hours in three different conditions: (1) hiPSC-CMs in fibroblast-conditioned medium, (2) co-culture with fibroblasts in tissue culture inserts, and (3) fibroblasts in direct contact with hiPSC-CMs. These conditions differentiate the various aspects of cardiomyocyte-fibroblast interaction. Optical mapping experiments were conducted to visualise changes in excitation-contraction coupling. Results: Human fibroblasts-conditioned medium altered excitation-contraction coupling by decreasing calcium transient amplitude and prolonging time to 80% decay vs baseline (p<0.001, -30±2.6% and +6±2.6%). Direct contact with fibroblasts further decreased calcium transient amplitude (p<0.001 vs. baseline, -46±4.3%) and further prolonged time to 80% decay (p<0.001 vs. baseline, +47±8.7%). Soluble Arginine-Glycine-Aspartic acid (RGD) motif-containing peptides present in the attachment site of many ECM proteins reduce the duration of transients and cause hiPSC-CM clustering and detachment from the glass surface substrate. These effects are not attenuated in the presence of anti-β1 or anti-β3-integrin receptor antibodies, but are also likely to prevent integrin receptor activation. Discussion:Human fibroblasts isolated from the ventricles of cardiomyopathy patients modulate calcium transients in hiPSC-CMs. The effects are dependent on whether the interaction is paracrine or direct, and unidirectional or bidirectional. Although it is unclear which interactions play the greatest role in modulating cardiomyocytes, it is accepted that multiple pathways and factors are involved. Further work is needed to identify mechanisms and understand their subsequent consequences in health and in disease.

Computational assessment of the functional role of sinoatrial node exit pathways in the human heart.

AimThe human right atrium and sinoatrial node (SAN) anatomy is complex. Optical mapping experiments suggest that the SAN is functionally insulated from atrial tissue except at discrete SAN-atrial electrical junctions called SAN exit pathways, SEPs. Additionally, histological imaging suggests the presence of a secondary pacemaker close to the SAN. We hypothesise that a) an insulating border-SEP anatomical configuration is related to SAN arrhythmia; and b) a secondary pacemaker, the paranodal area, is an alternate pacemaker but accentuates tachycardia. A 3D electro-anatomical computational model was used to test these hypotheses.MethodsA detailed 3D human SAN electro-anatomical mathematical model was developed based on our previous anatomical reconstruction. Electrical activity was simulated using tissue specific variants of the Fenton-Karma action potential equations. Simulation experiments were designed to deploy this complex electro-anatomical system to assess the roles of border-SEPs and paranodal area by mimicking experimentally observed SAN arrhythmia. Robust and accurate numerical algorithms were implemented for solving the mono domain reaction-diffusion equation implicitly, calculating 3D filament traces, and computing dominant frequency among other quantitative measurements.ResultsA centre to periphery gradient of increasing diffusion was sufficient to permit initiation of pacemaking at the centre of the 3D SAN. Re-entry within the SAN, micro re-entry, was possible by imposing significant SAN fibrosis in the presence of the insulating border. SEPs promoted the micro re-entry to generate more complex SAN-atrial tachycardia. Simulation of macro re-entry, i.e. re-entry around the SAN, was possible by inclusion of atrial fibrosis in the presence of the insulating border. The border shielded the SAN from atrial tachycardia. However, SAN micro-structure intercellular gap junctional coupling and the paranodal area contributed to prolonged atrial fibrillation. Finally, the micro-structure was found to be sufficient to explain shifts of leading pacemaker site location.ConclusionsThe simulations establish a relationship between anatomy and SAN electrical function. Microstructure, in the form of intercellular gap junction coupling, was found to regulate SAN function and arrhythmia.

AF Ablation Guided by Spatiotemporal Electrogram Dispersion Without Pulmonary Vein Isolation: A Wholly Patient-Tailored Approach

BackgroundThe use of intracardiac electrograms to guide atrial fibrillation (AF) ablation has yielded conflicting results. ObjectivesThe authors evaluated the usefulness of spatiotemporal dispersion, a visually recognizable electric footprint of AF drivers, for the ablation of all forms of AF. MethodsThe authors prospectively enrolled 105 patients admitted for AF ablation. AF was sequentially mapped in both atria with a 20-pole PentaRay catheter. The authors tagged and ablated only regions displaying electrogram dispersion during AF. Results were compared to a validation set in which a conventional ablation approach was used (pulmonary vein isolation/stepwise approach). To establish the mechanism underlying spatiotemporal dispersion of AF electrograms, the authors conducted realistic numerical simulations of AF drivers in a 2-dimensional model and optical mapping of ovine atrial scar-related AF. ResultsAblation at dispersion areas terminated AF in 95% of the 105 patients. After ablation of 17 ± 10% of the left atrial surface and 18 months of follow-up, the atrial arrhythmia recurrence rate was 15% after 1.4 ± 0.5 procedures per patient versus 41% in the validation set after 1.5 ± 0.5 procedures per patient (arrhythmia free-survival: 85% vs. 59%; log-rank p < 0.001). Compared with the validation set, radiofrequency times (49 ± 21 min vs. 85 ± 34.5 min; p = 0.001) and procedure times (168 ± 42 min vs. 230 ± 67 min; p < 0.0001) were shorter. In simulations and optical mapping experiments, virtual PentaRay recordings demonstrated that electrogram dispersion is mostly recorded in the vicinity of a driver. ConclusionsThe clustering of intracardiac electrograms exhibiting spatiotemporal dispersion is indicative of AF drivers. Their ablation allows for a nonextensive and patient-tailored approach to AF ablation. (Substrate Ablation Guided by High Density Mapping in Atrial Fibrillation [SUBSTRATE HD]; NCT02093949)

Spontaneous initiation of premature ventricular complexes and arrhythmias in type 2 long QT syndrome.

The occurrence of early afterdepolarizations (EADs) and increased dispersion of repolarization are two known factors for arrhythmogenesis in long QT syndrome. However, increased dispersion of repolarization tends to suppress EADs due to the source-sink effect, and thus how the two competing factors cause initiation of arrhythmias remains incompletely understood. Here we used optical mapping and computer simulation to investigate the mechanisms underlying spontaneous initiation of arrhythmias in type 2 long QT (LQT2) syndrome. In optical mapping experiments of transgenic LQT2 rabbit hearts under isoproterenol, premature ventricular complexes (PVCs) were observed to originate from the steep spatial repolarization gradient (RG) regions and propagated unidirectionally. The same PVC behaviors were demonstrated in computer simulations of tissue models of rabbits. Depending on the heterogeneities, these PVCs could lead to either repetitive focal excitations or reentry without requiring an additional vulnerable substrate. Systematic simulations showed that cellular phase 2 EADs were either suppressed or confined to the long action potential region due to the source-sink effect. Tissue-scale phase 3 EADs and PVCs occurred due to tissue-scale dynamical instabilities caused by RG and enhanced L-type calcium current (ICa,L), occurring under both large and small RG. Presence of cellular EADs was not required but potentiated PVCs when RG was small. We also investigated how other factors affect the dynamical instabilities causing PVCs. Our main conclusion is that tissue-scale dynamical instabilities caused by RG and enhanced ICa,L give rise to both the trigger and the vulnerable substrate simultaneously for spontaneous initiation of arrhythmias in LQT2 syndrome.

Nonalternans repolarization variability and arrhythmia – the calcium connection

Repolarization alternans precedes certain types of ventricular arrhythmias and its presence may be prognostically useful, but some ventricular arrhythmias are not preceded by repolarization alternans.Nonalternans changes of ventricular repolarization occur in patients with coronary artery disease and in subjects with congenital long QT syndrome. In animal experiments, nonalternans repolarization lability occurs in a canine model of chronic AV block with propensity to polymorphic ventricular tachycardia, in the perfused rabbit heart exposed to IKr block, and in a murine model of catecholaminergic polymorphic ventricular tachycardia. Optical mapping experiments indicate that heterogeneity of intracellular calcium handling underlies nonalternans repolarization variability.Detection of nonalternans repolarization lability poses specific challenges, since signal averaging does not increase the signal to noise ratio. Nonalternans repolarization lability may be erroneously reported as repolarization alternans by non-spectral methods applied to short data segments. Additional research will be needed to determine the role of nonalternans repolarization variability in mechanism of ventricular arrhythmias.

Pro-arrhythmic effect of heart rate variability during periodic pacing.

Clinically, healthy hearts have been associated with a high ventricular heart rate variability (HRV) while diseased hearts have been known to exhibit low ventricular HRV. Hence, low HRV is suggested to be a marker of cardiac ventricular arrhythmias. Over the past few years, there has been considerable amount of interest in incorporating HRV in pacing to emulate healthy heart conditions and re-stabilize the electrical activity in diseased hearts. Recently, we used single cell numerical simulations to demonstrate that HRV incorporated into periodic pacing promotes alternans formation and thus, can be pro-arrhythmic. Here, we performed high-resolution optical mapping experiments on Langendorff perfused, healthy whole mice hearts to validate our numerical findings. Our results indeed demonstrate that HRV promoted the onset of cardiac alternans, which is believed to be a precursor of fatal cardiac rhythms. Hence, our present study suggests that incorporating HRV into periodic pacing while addressing several clinical needs may not be safe. There is a pressing need to better understand paced cardiac dynamics and develop anti-arrhythmic pacing techniques that would prevent cardiac arrhythmias.

Voltage-Sensitive Fluorescence of Indocyanine Green in the Heart

So far, the optical mapping of cardiac electrical signals using voltage-sensitive fluorescent dyes has only been performed in experimental studies because these dyes are not yet approved for clinical use. It was recently reported that the well-known and widely used fluorescent dye indocyanine green (ICG), which has FDA approval, exhibits voltage sensitivity in various tissues, thus raising hopes that electrical activity could be optically mapped in the clinic. The aim of this study was to explore the possibility of using ICG to monitor cardiac electrical activity. Optical mapping experiments were performed on Langendorff rabbit hearts stained with ICG and perfused with electromechanical uncouplers. The residual contraction force and electrical action potentials were recorded simultaneously. Our research confirms that ICG is a voltage-sensitive dye with a dual-component (fast and slow) response to membrane potential changes. The fast component of the optical signal (OS) can have opposite polarities in different parts of the fluorescence spectrum. In contrast, the polarity of the slow component remains the same throughout the entire spectrum. Separating the OS into these components revealed two different voltage-sensitivity mechanisms for ICG. The fast component of the OS appears to be electrochromic in nature, whereas the slow component may arise from the redistribution of the dye molecules within or around the membrane. Both components quite accurately track the time of electrical signal propagation, but only the fast component is suitable for estimating the shape and duration of action potentials. Because ICG has voltage-sensitive properties in the entire heart, we suggest that it can be used to monitor cardiac electrical behavior in the clinic.

Forced fusion of human ventricular scar cells with cardiomyocytes suppresses arrhythmogenicity in a co-culture model.

Fibrosis increases arrhythmogenicity in myocardial tissue by causing structural and functional disruptions in the cardiac syncytium. Forced fusion of fibroblastic cells with adjacent cardiomyocytes may theoretically resolve these disruptions. Therefore, the electrophysiological effects of such electrical and structural integration of fibroblastic cells into a cardiac syncytium were studied. Human ventricular scar cells (hVSCs) were transduced with lentiviral vectors encoding enhanced green fluorescent protein alone (eGFP↑-hVSCs) or together with the fusogenic vesicular stomatitis virus G protein (VSV-G/eGFP↑-hVSCs) and subsequently co-cultured (1:4 ratio) with neonatal rat ventricular cardiomyocytes (NRVMs) in confluent monolayers yielding eGFP↑- and VSV-G/eGFP↑-co-cultures, respectively. Cellular fusion was induced by brief exposure to pH = 6.0 medium. Optical mapping experiments showed eGFP↑-co-cultures to be highly arrhythmogenic [43.3% early afterdepolarization (EAD) incidence vs. 7.7% in control NRVM cultures, P < 0.0001], with heterogeneous prolongation of action potential (AP) duration (APD). Fused VSV-G/eGFP↑-co-cultures displayed markedly lower EAD incidence (4.6%, P < 0.001) than unfused co-cultures, associated with decreases in APD, APD dispersion, and decay time of cytosolic Ca(2+) waves. Heterokaryons strongly expressed connexin43 (Cx43). Also, maximum diastolic potential in co-cultures was more negative after fusion, while heterokaryons exhibited diverse mixed NRVM/hVSC whole-cell current profiles, but consistently showed increased outward Kv currents compared with NRVMs or hVSCs. Inhibition of Kv channels by tetraethylammonium chloride abrogated the anti-arrhythmic effects of fusion in VSV-G/eGFP↑-co-cultures raising EAD incidence from 7.9 to 34.2% (P < 0.001). Forced fusion of cultured hVSCs with NRVMs yields electrically functional heterokaryons and reduces arrhythmogenicity by preventing EADs, which is, at least partly, attributable to increased repolarization force.