High-resolution Optical Mapping Research Articles

Origin of ventricular fibrillation triggers in a model of localized repolarization heterogeneity

BackgroundHeterogeneities in ventricular repolarization contribute significantly to the genesis of ventricular fibrillation (VF). Although clinical arrhythmias are spontaneously triggered by premature ventricular complexes, these triggers are difficult to document and little is known about their site of origin. ObjectiveTo characterize spontaneous VF initiation in an experimental model of repolarization heterogeneity and to identify the origin of triggers in relation to the spatial dispersion of repolarization. MethodsSpatially limited repolarization heterogeneity was created in isolated perfused porcine right ventricles (N =16) by local administration of pinacidil (20 μM) into a terminal branch of the right coronary artery. High-resolution optical mapping and pseudo-ECG were performed in control conditions and after pinacidil perfusion. ResultsNo arrhythmia occurred at baseline, but 74 VF episodes were observed in 13 hearts (82%) after pinacidil perfusion and were most often initiated by a ventricular trigger with short coupling interval (297±66ms). Sixteen VF initiations were optically mapped in four hearts. Mapping showed triggers originating in all cases from the border zone between altered and normal repolarization areas where local action potential duration and repolarization time gradients were steep (15.9 and 15.8 ms/mm versus 1.5 and 3.0 ms/mm in non-trigger sites). Optical action potential traces were compatible with a phase-two reexcitation mechanism. The subsequent VF cycles were driven by activities located in the same region. ConclusionsThis model of localized repolarization heterogeneity is able to produce spontaneous VF initiation. Our study demonstrates that VF triggers originate consistently from the border zone of repolarization dispersion.

Chronic angiotensin-converting enzyme inhibition attenuates frailty and protects against atrial fibrillation in aging mice

BackgroundAging is a major risk factor for atrial fibrillation (AF); however, not all individuals age at the same rate. Frailty, which is a measure of susceptibility to adverse health outcomes, can be quantified with a frailty index (FI). ObjectiveThis study aimed to determine the effects of angiotensin-converting enzyme (ACE) inhibition on AF and atrial remodeling in aging and frail mice. MethodsAging mice were treated with the ACE inhibitor enalapril for 6 months beginning at 16.5 months of age and frailty was quantified. AF susceptibility and atrial structure and function were assessed by intracardiac electrophysiology in anesthetized mice, high-resolution optical mapping in intact atrial preparations, patch clamping in isolated atrial myocytes, and histology and molecular biology in atrial tissues. ResultsEnalapril attenuated frailty in aging mice with larger effects in females. AF susceptibility was increased in aging mice but attenuated by enalapril. AF susceptibility and duration also increased as a function of FI score. P-wave duration was increased and atrial conduction velocity was reduced in aging mice and improved after enalapril treatment. Furthermore, P-wave duration and atrial conduction velocity were strongly correlated with FI score. Atrial action potential upstroke velocity (Vmax) and Na+ current (INa) were reduced whereas atrial fibrosis was increased in aging mice. Action potential Vmax, INa, and fibrosis were improved by enalapril and also correlated with FI scores. ConclusionACE inhibition with enalapril attenuates frailty and reduces AF susceptibility in aging mice by preventing atrial electrical and structural remodeling.

Detection of an 8p23.1 Inversion Using High-Resolution Optical Genome Mapping

Abstract Objective To evaluate the performance of optical genome mapping (OGM) in identifying an inversion located in the short arm of chromosome 8 (8p, 8p23.1), flanked by regions of complex segmental duplication (SD), using the GRCh38 and telomere-to-telomere (T2T) genome references. Methods We investigated a couple suspected of carrying the 8p23.1 inversion due to a terminal deletion combined with an interstitial duplication of 8p found in their abortus. OGM was performed on both individuals. The data were mapped to the current GRCh38 and the updated T2T genome references, respectively. Results The 8p23.1 inversion was observed in the female when mapping OGM data to the T2T assembly. In contrast, under the GRCh38 reference, the orientation between the suspected breakpoints within the SD regions could not be distinguished. Additional variants of uncertain significance were also identified in both individuals. Conclusion Our findings highlight the superiority of the T2T reference in recognizing structural variations involving SD regions. The enhanced SV detection using the T2T reference may contribute to a better understanding of genome instability and human diseases.

The smooth-walled human RVOT contains trabeculations that cause conduction delay

Abstract Funding Acknowledgements None. Background The right ventricular outflow tract (RVOT) is the outlet from the right ventricle and is the initiating substrate of life-threatening arrhythmias. While the luminal wall of the RVOT is often assumed to be without the complex trabecular meshwork that characterizes the right ventricular free wall, the anatomy of the RVOT is an understudied subject. Aim to investigate whether trabeculations occur in the RVOT and to assess whether this impacts electrical propagation. Methods Human hearts were investigated using high-resolution MRI, histology, RNA-sequencing, and optical and electrical mapping. Results We used high-resolution MRI and serial sectioning to reconstruct the macroscopic details of the human RVOT and identified cases exhibiting much trabeculation. The smooth lumen of the RVOT varied between 9% and 23% of the total RV anterior surface (N=11). Histological analysis on additional six hearts indicated that the RVOT compact layer is thinner when trabeculations are present. RNA sequencing of four human donor hearts revealed enrichment in the subendocardial region of 88 genes associated with cardiac conduction and trabeculations (P adjusted<0.05). Finally, we selected two human donor hearts showing trabeculations in the RVOT from which we generated wedge preparation and performed optical and electrical mapping. The trabecular regions demonstrated high degree of fractionation when compared to non-trabeculated regions, which coincided with delayed activation. Conclusion Trabeculations are found in the RVOT, and their extent varies among individuals. This impacts on the thickness of the compact wall in the RVOT, restricting the depth of tissue at which clinical interventions can be performed, as well as influencing electrical propagation and possible arrhythmogenicity.

Self-Assembly of Ionic Superdiscs in Nanopores.

Discotic ionic liquid crystals (DILCs) consist of self-assembled superdiscs of cations and anions that spontaneously stack in linear columns with high one-dimensional ionic and electronic charge mobility, making them prominent model systems for functional soft matter. Compared to classical nonionic discotic liquid crystals, many liquid crystalline structures with a combination of electronic and ionic conductivity have been reported, which are of interest for separation membranes, artificial ion/proton conducting membranes, and optoelectronics. Unfortunately, a homogeneous alignment of the DILCs on the macroscale is often not achievable, which significantly limits the applicability of DILCs. Infiltration into nanoporous solid scaffolds can, in principle, overcome this drawback. However, due to the experimental challenges to scrutinize liquid crystalline order in extreme spatial confinement, little is known about the structures of DILCs in nanopores. Here, we present temperature-dependent high-resolution optical birefringence measurement and 3D reciprocal space mapping based on synchrotron X-ray scattering to investigate the thermotropic phase behavior of dopamine-based ionic liquid crystals confined in cylindrical channels of 180 nm diameter in macroscopic anodic aluminum oxide membranes. As a function of the membranes' hydrophilicity and thus the molecular anchoring to the pore walls (edge-on or face-on) and the variation of the hydrophilic-hydrophobic balance between the aromatic cores and the alkyl side chain motifs of the superdiscs by tailored chemical synthesis, we find a particularly rich phase behavior, which is not present in the bulk state. It is governed by a complex interplay of liquid crystalline elastic energies (bending and splay deformations), polar interactions, and pure geometric confinement and includes textural transitions between radial and axial alignment of the columns with respect to the long nanochannel axis. Furthermore, confinement-induced continuous order formation is observed in contrast to discontinuous first-order phase transitions, which can be quantitatively described by Landau-de Gennes free energy models for liquid crystalline order transitions in confinement. Our observations suggest that the infiltration of DILCs into nanoporous solids allows tailoring their nanoscale texture and ion channel formation and thus their electrical and optical functionalities over an even wider range than in the bulk state in a homogeneous manner on the centimeter scale as controlled by the monolithic nanoporous scaffolds.

Abstract 18867: A Novel Light-Sensitive Ex-Vivo Rat Atrial Anatomical Model for Studying Atrial Arrhythmias and Cardioversion

Introduction: Atrial arrhythmias, including atrial fibrillation (AF), contribute to significant morbidity and mortality. However, the lack of suitable tissue models in small animals hampers our understanding of the underlying mechanisms. Optogenetic actuators offer precise and reversible stimulation with high spatiotemporal resolution, presenting a unique opportunity to investigate atrial arrhythmias. Hypothesis: Our aim was to develop a light-sensitive ex-vivo anatomical model in rats for studying atrial arrhythmias and to utilize this model for optimizing optogenetic stimulation and cardioversion strategies. Methods: We injected purified AAV particles containing the transgenic light-sensitive channel (CoChR) into 5-day-old Wistar rats, followed by an 8-10 week period for atrial growth. The atria were then isolated, flattened, and affixed to a custom-made silicon plate. Superfusion with oxygenated Tyrode's solution and loading with Di-4-ANBDQBS allowed for high-resolution optical mapping. Atrial arrhythmias were induced through targeted tachypacing combined with carbamylcholine treatment. Additionally, we compared different optical and pharmacological treatment strategies to evaluate their efficacy in modulating atrial arrhythmias. Results: Immunostaining confirmed atrial expression of CoChR. Optical mapping revealed a functional syncytium with sinus node activation and propagation throughout the atria. Pacing at increasing frequencies resulted in a relatively flattened action potential duration restitution curve. Successful induction of reentrant arrhythmias was achieved. Additionally, we demonstrated the feasibility of optogenetic actuators for atrial pacing, modulation of spontaneous activity, and optical termination of arrhythmias. Conclusions: The developed optogenetic superfused atria model provides a valuable platform for studying atrial electrophysiology and modeling reentrant arrhythmias. Furthermore, it enables the optimization of optogenetic stimulation and cardioversion strategies. This integrated approach enhances our understanding of atrial arrhythmias and holds promise for the development of novel therapeutic interventions.

Natriuretic Peptide Receptor B Protects Against Atrial Fibrillation by Controlling Atrial cAMP Via Phosphodiesterase 2.

β-AR (β-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca2+ homeostasis via cAMP-dependent mechanisms; however, enhanced β-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca2+ homeostasis, and atrial arrhythmogenesis is incompletely understood. Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B+/-) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca2+ imaging, and in mouse and human atrial tissues using molecular biology. Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B+/- mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B+/- mice leading to larger increases in atrial cAMP in the presence of the β-AR agonist isoproterenol. NPR-B+/- mice displayed larger increases in action potential duration and L-type Ca2+ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca2+ release events and delayed afterdepolarizations in NPR-B+/- atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B+/- atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca2+ current through PDE2 in mouse and human atrial myocytes. NPR-B protects against AF by preventing enhanced atrial responses to β-adrenergic receptor agonists.

BS-452682-4 EFFECTS OF CHRONIC ANGIOTENSIN CONVERTING ENZYME INHIBITION ON ATRIAL FUNCTION AND ARRHYTHMOGENESIS IN AGED AND FRAIL MICE

Aging is associated atrial remodeling and increased susceptibility to atrial fibrillation (AF); however, aging-dependent changes can be variable. Frailty measures health status independent of chronological age, can be quantified using a frailty index (FI) and can account for heterogeneity in aging individuals. Angiotensin converting enzyme inhibition (ACEi) can impact frailty however its effects on AF and atrial remodeling are unknown. To determine the effects of chronic ACEi on atrial structure, function, and arrhythmogenesis in aged and frail mice. Wildtype mice were treated with the ACEi enalapril (20 mg/kg/day) or placebo for 6 months starting at 75 weeks of age. Mice aged 10-15 weeks were used as young controls. Mice were used in intracardiac electrophysiology, high resolution optical mapping, patch-clamping, and histology. ACEi reduced FI in aged mice (0.24 vs 0.36; n=27-31; P<0.05) without altering blood pressure. AF susceptibility was increased in aged vs young mice (100 vs 17%; n=6; P<0.05) and reduced in ACEi vs aged mice (43 vs 100%; n=6-7; P<0.05). P wave duration was reduced in ACEi vs aged mice (27.4 vs 32.5 ms; n=7; P<0.05), showed variability within age/treatment groups, and was correlated and graded by FI (R=0.91; n=20; P<0.05). Optical mapping revealed an age dependent reduction in atrial conduction velocity (CV; 35.3 vs 40.2 cm/s; n=6-9; P<0.05) while ACEi improved CV in aged mice (37.7 vs 35.3 cm/s; n=6-9; P=0.05). CV was also correlated with FI score (R=0.72; n=24; P<0.05). Patch-clamp studies in isolated left atrial (LA) myocytes identified a reduction in action potential upstroke velocity (Vmax; 150.8 vs 165.4 V/s; n=18-32; P<0.05) that was associated with a reduction in peak sodium current (INa; -38.3 vs -52.1 pA/pF; n=7-19; P<0.05) in aged vs young mice. INa was increased in ACEi vs aged (-47.2 vs -38.3 pA/pF; n=14–19; P=0.09) and not different (P=0.62) vs young mice. Importantly, Vmax (R=0.32; n=73; P<0.05) and INa (R=0.5; n=36; P < 0.05) were graded by FI. LA fibrosis was increased in aged mice vs young (10.8 vs 3.9%; n=5-8; P<0.05), reduced in ACEi vs aged mice (8.9 vs 10.8%; n=5-8; P<0.05), and strongly correlated with FI (R=0.89; n=19; P<0.05). ACEi reduced frailty in aging mice and attenuated age-related AF and atrial remodeling. Aging was associated with substantial variability within groups. Frailty was a strong predictor of changes atrial function in aging mice including after ACEi treatment, independent of chronological age.

Electrophysiological characterization of a 19-years-old male marathon runner suffering a sudden cardiac death

1 in 50,000 of athletes suffers a cardiac student death (SCD) per year, more of them under 35 years of age. Around 25% of these patients had normal structure, implying that other electrical disorder cause these SCD. Determine the electrophysiological causes of a SCD of a young 19-years-old occurred during a marathon with any heart structural diseases. Pathologic left ventricle (PLV) (n = 1, male, 19-years-old) and control left ventricle (CLV) (n = 1, female, 56-years-old) were perfused and imaged using high resolution optical mapping and Di-4-ANEPPS like a dye. Endocardial and endocardial free wall and his bundle were paced to obtain activation time (AT), repolarization time (RT) and action-potential duration (APD). Purkinje-muscle junctions (PMJs) were determine like activation origins far from the his bundle pacing. Effective refractory period (ERP) was determined using S1S2S3S4 short-coupled pacing. The protocol was repeated after isoplenalie injection [500 nM] like β-adrenergic receptor agonist. His-ventricle activation at high frequency pacing was higher in PLV (143ms) than in control (84ms) and decrease in both at similar levels under ISO (≈60ms). APD gradient increase was observed in PLV near PMJs under ISO (from 3ms/mm to 40ms/mm) and any gradient was observed in the control one. Ectopic episodes were observed in PLV under ISO and 82% of the origins had less that 4 mm of distance with the PMJs. In PLV, the ERP in His-pacing is always lower (320, 260, 250 ms) than endocardial (350, 315, 285 ms) pacing but in CLV, the His-pacing ERP was always higher (320, 260, 240 ms) that endocardial pacing (280, 240, 220 ms). Ventricular fibrillation (VF) was observed in PLV after his bundle pacing at high frequency (more than 330 ms) and any VF was observed in CLV. During this VF, 76% of the focal activities had less than 4 mm of distance with the PMJs. Conduction system (CS) of the PLV shows high sensitivity to high frequency pacing and to presence of ISO. Ectopic episodes and APD gradient near PMJs may show the role of Purkinje network like arrhythmic source. Indeed, the shorter ERP during His-pacing than in the endocardium, give the possibility to produce a macro-re-entry in the conduction system and maintain the arrhythmia with focal activities. CS abnormalities likely played a critical role in arrhythmogenicity and maintenance of VF in this patient during sudden cardiac death.

PO-02-212 TWISTED FIBROTICALLY-INSULATED MYOBUNDLES CREATE THE COMMON TRACK AND PIVOTING POINTS FOR REENTRANT DRIVERS MAINTAINING PERSISTENT AF IN-VIVO

Localized reentrant drivers have been suggested to maintain persistent atrial fibrillation (perAF). However, their existence, electrophysiological mechanisms, and structural substrates in-vivo are controversial. Define AF driving mechanism in-vivo and ex-vivo in a perAF canine model. A clinically relevant canine model (n=5) of self-sustained (4 months) perAF was developed to study AF maintaining mechanisms both in-vivo and ex-vivo by integrated 3D imaging, including high-resolution near-infrared optical mapping (NIOM, 0.3-0.5mm resolution), contrast-enhanced magnetic resonance imaging (CE-MRI, 0.15mm resolution). Transmural fibrosis was validated with histology. CE-MRI was used to quantitatively analyze 3D atrial structure, including transmural fiber orientation and fibrosis content in the sub-epi, intramural, and sub-endocardial layers. In all canine models (n=5), sequential in-vivo to ex-vivo sub-surface NIOM and 3D CE-MRI revealed that the self-sustained perAF was primarily maintained by localized reentrant drivers with temporal stability 53±17% (n=10), which recurrent on the same arrhythmogenic hubs. The 3D structure of the arrhythmogenic driver hubs includes a common intramural reentry track and multiple return paths formed by fibrotically-insulated bundles. The in-vivo spatio-temporal activation patterns of reentrant drivers were preserved ex-vivo (Figure A), including driver AFCL (91±7 ms vs 96±12 ms, p>0.05) and temporal driver stability (51±8% vs 52±6%, n=4, p>0.05). Targeted ablation of the driver reentrant tracks terminated or converted AF to slower tachycardia. Quantitative CE-MRI analysis of 3D myofiber orientations revealed that the transmural fiber twist along the common reentry tracks and pivoting points is greater than in non-driver regions (Figure B-C). The driver reentrant tracks had increased interstitial fibrotic strands and significantly higher intramural fibrosis than the neighboring non-driver regions (Figure D-E). Our results provide in-vivo proof-of-concept that spatially stable reentrant drivers are a mechanism of perAF maintenance. Intramural fibrotic strands and fibrotically-insulated myobundles in regions with the highest transmural myofiber twist which may form the common tracks and pivoting points for return paths of the spatially-stable reentrant AF driver.

Abstract 3703: Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma

Abstract Radiation resistance can halt a well-curated treatment plan in its tracks, but it may take weeks to realize this lack of efficacy resulting in lost time and unnecessary side effects. In this study, we sought to elucidate novel biomarkers of radiation resistance to predict treatment response in head and neck squamous cell carcinoma (HNSCC) at much earlier timepoints. We grew tumor xenografts from radiation-resistant UM-SCC-47 (n=25) and -sensitive UM-SCC-22B (n=37) cells in nude athymic mice. Each group was randomly divided into treatment and control groups, which were further subdivided into four timepoints for tumor excision: pre-treatment baseline, 1-, 24-, and 48-hours post-treatment. Prior to excision, we injected pimonidazole to quantify tumor hypoxia. We determined the two-photon excited fluorescence intensity and lifetime of NADH and FAD and quantified the optical redox ratio, ratio of free to bound NADH, and mean NADH lifetime of frozen sections of each tumor. We observed spatial and temporal heterogeneity in these high-resolution optical maps that indicated different metabolic programs in radiation-resistant and -sensitive tumors. A histogram-based heterogeneity analysis revealed time dependent shifts toward glycolysis in the radiation-resistant UM-SCC-47 treatment group, while the sensitive UM-SCC-22B treatment group showed the opposite trend. Proteomics performed on both cell lines identified multiple dysregulated proteins that are often implicated in HNSCC and associated with metabolic reprogramming — specifically, EGFR and SMAD4. We also found elevated levels of hypoxia and HIF-1 in the UM-SCC-47 tumors compared with the UM-SCC-22B tumors prior to and post-radiation. Using spatially co-registered optical imaging and immunohistochemical maps of the same field of view, we will present correlations between our optical markers and EGFR, SMAD4, and HIF-1. These studies will provide evidence of how protein dysregulation causes changes to optical metabolic imaging markers. Citation Format: Jesse D. Ivers, Sina Dadgar, Narasimhan Rajaram. Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3703.

Critical Link between Calcium Regional Heterogeneity and Atrial Fibrillation Susceptibility in Sheep Left Atria.

Atrial fibrillation is the most sustained form of arrhythmia in the human population that leads to important electrophysiological and structural cardiac remodeling as it progresses into a chronic form. Calcium is an established key player of cellular electrophysiology in the heart, yet to date, there is no information that maps calcium signaling across the left atrium. The aim of this study is to determine whether calcium signaling is homogenous throughout the different regions of the left atrium. This work tests the hypothesis that differences across the healthy left atrium contribute to a unique, region-dependent calcium cycling and participates in the pro-arrhythmic activity during atrial fibrillation. An animal model relevant to human cardiac function (the sheep) was used to characterize both the electrical activity and the calcium signaling of three distinct left atrium regions (appendage, free wall and pulmonary veins) in control conditions and after acetylcholine perfusion (5 μM) to induce acute atrial fibrillation. High-resolution dual calcium-voltage optical mapping on the left atria of sheep was performed to explore the spatiotemporal dynamics of calcium signaling in relation to electrophysiological properties. Action potential duration (at 80% repolarization) was not significantly different in the three regions of interest for the three pacing sites. In contrast, the time to 50% calcium transient decay was significantly different depending on the region paced and recorded. Acetylcholine perfusion and burst pacing-induced atrial fibrillation when pulmonary veins and appendage regions were paced but not when the free wall region was. Dantrolene (a ryanodine receptor blocker) did not reduce atrial fibrillation susceptibility. These data provide the first evidence of heterogenous calcium signaling across the healthy left atrium. Such basal regional differences may be exacerbated during the progression of atrial fibrillation and thus play a crucial role in focal arrhythmia initiation without ryanodine receptor gating modification.

Abstract P3052: Dysregulation Of The Ampk-drp1 Axis Promotes Pathophysiological Remodeling Of Mitochondrial Ultrastructure And Ventricular Electrophysiological Function

AMP-activated protein kinase (AMPK), a master metabolic sensor, plays a central role in the regulation of mitochondrial (mito) function. AMPK activation inhibits mito fission potentially by modulating mito dynamics proteins, including dynamin-related protein 1 (DRP1). The role of the AMPK-DRP1 axis in electrophysiological (EP) function is unknown. Methods: We examined the impact of myocardial AMPK inactivation on mito ultrastructure by electron microscopy, mito dynamics proteins by western blot, and EP function by optical mapping in mice expressing an AMPK kinase dead isoform (AMPK-KD, N=3-7) vs littermate controls (Ctrl, N=3-6). Results: Quantification of mito morphology revealed increased mito area consistent with swelling (0.53 μm 2 vs 0.80 μm 2 , p&lt;0.0001) and decreased mito count (0.68 mito/μm 2 vs 0.44 mito/μm 2 , p=0.13) in AMPK-KD vs Ctrl. Immunoblots revealed decreased DRP1 phosphorylation at S637 (a fission inhibiting site) with no major change in total DRP1, S616 phospho-DRP1, MFN1/2 or OPA1 levels in AMPK-KD. High resolution optical mapping of ex vivo perfused hearts uncovered marked (by 39.5%, p&lt;0.001) conduction slowing ( Figure ) and a moderate increase in AP duration (43.8 ms vs 49.7 ms, p&lt;0.05) reflecting widespread ventricular EP remodeling in AMPK-KD. Of note, discrete lines of functional conduction block were observed in 4/7 AMPK-KD but not Ctrl hearts consistent with pro-arrhythmic remodeling. Conclusions: Myocardial AMPK pathway inactivation causes mito ultrastructural abnormalities and pathological EP remodeling. The AMPK-DRP1 axis may be a novel target for arrhythmia suppression in metabolic disorders with defective AMPK downstream signaling.

Detection of cryptic balanced chromosomal rearrangements using high-resolution optical genome mapping

BackgroundChromosomal rearrangements have profound consequences in diverse human genetic diseases. Currently, the detection of balanced chromosomal rearrangements (BCRs) mainly relies on routine cytogenetic G-banded karyotyping. However, cryptic BCRs are hard...

A case study: High resolution optical mapping of a 19 year old male victim of sudden cardiac death during a marathon

Fifty thousand athletes suffer sudden cardiac death every year due to ventricular fibrillation (VF). Most sudden cardiac deaths of young patients have unexplained origin due to the absence of structural or electrical disease. Purkinje network is essential for a normal heart function, however, its role during electrical pathologies remains unclear. A case study to investigate the electrophysiological properties and the Purkinje network role in a 19 years old male after suffering a fatal sudden cardiac arrest during a marathon event. A coronary-perfused left ventricular preparation was imaged using high resolution optical mapping of epicardial and endocardial surfaces using a voltage-sensitive dye, Di-4-ANEPPS. Activation time, repolarization time and action potential duration were assessed during paced activity through the His bundle and endocardial and epicardial surfaces. Conduction system activation origins were identified as distant endocardial origins during His bundle pacing. Short-coupled S1S2S3S4 pacing was used to determine effective refractory period (ERP). Pacing protocols were repeated in the presence of isoprenaline (ISO) at 100 nM, 500 nM and 1 μM to mimic stress conditions. No changes in total activation time are observed during myocardial pacing cycle lengths of 667ms in control (63ms) and under ISO (up to 64ms). In contrary, His-ventricle delay during conduction system pacing was shorted from 61ms in control to 42ms, 44ms and 16ms under 100 nM, 500 nM and 1 μM of ISO respectively. Under His pacing, local APD gradients are observed near Purkinje-muscle junctions under ISO (from 3ms/mm and 3 ms/mm to 40ms/mm and 41ms/mm). ERP under His pacing was 55ms shorter than endocardial pacing. 82% of the ectopy origins observed have less than 4 mm distance from the PMJs. Repeated focal activations were observed during VF and 76% of them had less than 4 mm distance from PMJs. No structural abnormalities were identified under 9.4 T MRI. Conduction system electrical abnormalities play a role in arrhythmogenicity and maintenance of VF in this patient during sudden cardiac death.

Real-Time Optical Mapping of Contracting Cardiac Tissues With GPU-Accelerated Numerical Motion Tracking.

Optical mapping of action potentials or calcium transients in contracting cardiac tissues are challenging because of the severe sensitivity of the measurements to motion. The measurements rely on the accurate numerical tracking and analysis of fluorescence changes emitted by the tissue as it moves, and inaccurate or no tracking can produce motion artifacts and lead to imprecise measurements that can prohibit the analysis of the data. Recently, it was demonstrated that numerical motion-tracking and -stabilization can effectively inhibit motion artifacts, allowing highly detailed simultaneous measurements of electrophysiological phenomena and tissue mechanics. However, the field of electromechanical optical mapping is still young and under development. To date, the technique is only used by a few laboratories, the processing of the video data is time-consuming and performed offline post-acquisition as it is associated with a considerable demand for computing power. In addition, a systematic review of numerical motion tracking algorithms applicable to optical mapping data is lacking. To address these issues, we evaluated 5 open-source numerical motion-tracking algorithms implemented on a graphics processing unit (GPU) and compared their performance when tracking and compensating motion and measuring optical traces in voltage- or calcium-sensitive optical mapping videos of contracting cardiac tissues. Using GPU-accelerated numerical motion tracking, the processing times necessary to analyze optical mapping videos become substantially reduced. We demonstrate that it is possible to track and stabilize motion and create motion-compensated optical maps in real-time with low-resolution (128 x 128 pixels) and high resolution (800 x 800 pixels) optical mapping videos acquired at 500 and 40 fps, respectively. We evaluated the tracking accuracies and motion-stabilization capabilities of the GPU-based algorithms on synthetic optical mapping videos, determined their sensitivity to fluorescence signals and noise, and demonstrate the efficacy of the Farnebäck algorithm with recordings of contracting human cardiac cell cultures and beating hearts from 3 different species (mouse, rabbit, pig) imaged with 4 different high-speed cameras. GPU-accelerated processing provides a substantial increase in processing speed, which could open the path for more widespread use of numerical motion tracking and stabilization algorithms during routine optical mapping studies.

High resolution optical mapping of cardiac electrophysiology in pre-clinical models

Optical mapping of animal models is a widely used technique in pre-clinical cardiac research. It has several advantages over other methods, including higher spatial resolution, contactless recording and direct visualisation of action potentials and calcium transients. Optical mapping enables simultaneous study of action potential and calcium transient morphology, conduction dynamics, regional heterogeneity, restitution and arrhythmogenesis. In this dataset, we have optically mapped Langendorff perfused isolated whole hearts (mouse and guinea pig) and superfused isolated atria (mouse). Raw datasets (consisting of over 400 files) can be combined with open-source software for processing and analysis. We have generated a comprehensive post-processed dataset characterising the baseline cardiac electrophysiology in these widely used pre-clinical models. This dataset also provides reference information detailing the effect of heart rate, clinically used anti-arrhythmic drugs, ischaemia-reperfusion and sympathetic nervous stimulation on cardiac electrophysiology. The effects of these interventions can be studied in a global or regional manner, enabling new insights into the prevention and initiation of arrhythmia.

Loss of Natriuretic Peptide Receptor C Enhances Sinoatrial Node Dysfunction in Aging and Frail Mice.

Heart rate (HR) is controlled by the sinoatrial node (SAN). SAN dysfunction is highly prevalent in aging; however, not all individuals age at the same rate. Rather, health status during aging is affected by frailty. Natriuretic peptides regulate SAN function in part by activating natriuretic peptide receptor C (NPR-C). The impacts of NPR-C on HR and SAN function in aging and as a function of frailty are unknown. Frailty was measured in aging wild-type and NPR-C knockout (NPR-C-/-) mice using a mouse clinical frailty index (FI). HR and SAN structure and function were investigated using intracardiac electrophysiology in anesthetized mice, high-resolution optical mapping in intact atrial preparations, histology, and molecular biology. NPR-C-/- mice rapidly became frail leading to shortened life span. HR was reduced and SAN recovery time was increased in older versus younger mice, and these changes were exacerbated in NPR-C-/- mice; however, there was substantial variability among age groups and genotypes. HR and SAN recovery time were correlated with FI score and fell along a continuum regardless of age or genotype. Optical mapping demonstrates impairments in SAN function that were also correlated with FI score. SAN fibrosis was increased in aged and NPR-C-/- mice and was graded by FI score. Loss of NPR-C results in accelerated aging and rapid decline in health status in association with impairments in HR and SAN function. Frailty assessment was effective and better able to distinguish aging-dependent changes in SAN function in the setting of shortened life span due to loss of NPR-C.

High-Throughput Characterization of Cytogenomic Heterogeneity of MDS Using High-Resolution Optical Genome Mapping

IntroductionIntroduction of next-generation sequencing has defined the somatic mutational landscape in MDS. Comprehensive high-throughput structural variant profiling (SVP) is as important as mutation profiling in characterizing MDS clonal architecture since these large genomic aberrations have already shown to be critical for diagnosis and risk-stratification of MDS. A subset (MECOM, KMT2A rearrangements) are therapeutic targets in clinical trials. At this time, technical advances in SVP for copy number alterations (CNAs) and fusions have not been congruent with mutation profiling due to the inability of short-read (150bp) NGS to detect SVs. Currently available long-read (10-20Kbp) and whole genome sequencing cannot detect all SVs due to the presence of repeat sequences. Hence, conventional karyotyping (CK) remains the gold standard. Optical genome mapping (OGM) is a novel single-platform technique that measures ultra-long-range sequence patterns (>300Kbp), thereby unaffected by repeat sequences, enabling unbiased evaluation of all types of SVs at a high resolution. Here, we performed comprehensive SVP and mutation profiling in a large well-characterized cohort of MDS.MethodsWe selected samples with available fresh/frozen BM cells from consecutive treatment-naïve MDS pts who also underwent standard-of-care tests (CK, FISH, targeted 81-gene NGS for mutations). For OGM, ultra-high-molecular-weight-DNA was extracted, followed by labeling, linearization and imaging of DNA (Saphyr, Bionano) [median coverage:>300X]. The results were analyzed using de novo (>500 bp), rare variant (>5000 bp) and copy number (>500,000 bp) pipelines. The data was compared against 200 healthy controls to exclude common germline SVs. Clinical significance of the SVs was determined based on the location/overlap with the coding region of myeloid malignancy associated genes. The detection sensitivity was 10%.ResultsThere were 76 treatment naïve MDS patients. Baseline characteristics, comprehensive cytogenetic scoring system (CCSS) and R-IPPS risk categories and somatic mutations are in Fig 1.OGM identified all clonal abnormalities detected by CK [CNAs, inversions, inter/intra-chromosomal translocations, dicentric, complex derivative chromosomes]. Precise mapping of SVs by OGM at gene-level allowed determining the status of clinically informative biomarkers such as TET2, MECOM, TP53 and KMT2A, without the need for confirmatory assays. Detailed gene-level characterization of different SVs included KMT2A-ELL [t(11;19)] in MDS with WT1 mut, t(9;11) with SYTL2 fusion (and not KMT2A), der(1;7) leading to del(7q) in MDS with GATA2 mut/IDH2 mutand t(1;3)(p36;q21) rearrangements with potential PRDM16 disruption in SF3B1 mut/RUNX1 mutMDS, among others. Using OGM, we mapped the sequence patterns in both samples with IM with high level of confidence.Additionally, OGM identified 23 cryptic, clinically significant SVs in 14 (18%) of 76 pts. These included deletions of TET2, KMT2A, and del(5q), KMT2A amplification in MDS with FLT3-ITD/DNMT3A mut/RAS mut, NUP98-PRRX2, MECOM rearrangement in TET mut mutated NK-MDS. In addition, there were SVs of uncertain significance: duplications of chr1 (PDE41P), deletions of chr21 (involving RUNX1), chr2 (DNMT3A, ASXL2), chr12 (ETV6) and chr22 (EP300) and der(16)t(12;16)(q21.1;q12.1). These cryptic SVs were noted across all R-IPSS risk categories (highest yield in very-low and low R-IPSS) and across all cytogenetic risk-groups (very-good to very-poor).In complex karyotype setting, OGM could resolve the markers and additional genetic material, and in most cases, showed a much higher the degree of complexity within the genome than was apparent by CK. Four pts showed SV patterns typical of chromothripsis/chromoplexy.The median number of mutations per pt was 1 (0-6). When compared to mutation subsets, cryptic SVs were only identified in pts with ≤3 mutations. Majority represented either MDS with TP53 mut (6, 29%) or SF3B1 mut/TET mut (deletions of TET2, KMT2A, NOTCH1 and EP300 genes).ConclusionsUnbiased, high-throughput whole genome SVP revealed cryptic, clinically significant SVs in ~18% of MDS pts. OGM is a single-platform cytogenomic tool that can facilitate SVP at a gene-level resolution. This study provides strong support for further validation in expanded cohorts to guide clinical implementation and integration of SVP for routine work-up. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Kantarjian: Ipsen Pharmaceuticals: Honoraria; Amgen: Honoraria, Research Funding; Astellas Health: Honoraria; Astra Zeneca: Honoraria; AbbVie: Honoraria, Research Funding; KAHR Medical Ltd: Honoraria; NOVA Research: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Isolated superfused rats atrial model for the investigation of atrial fibrillation mechanisms and treatment

Abstract Background One of the major barriers to an improved mechanistic understanding of atrial fibrillation (AF), and thus in the pipeline of drug development, has been a lack of appropriate tissue models, especially in small animals. Aim We propose an advanced anatomical ex-vivo model based on rat atria for acute assessment of AF susceptibility. This novel model could yield a better understanding of arrhythmia mechanisms as well as the development of potential therapeutic strategies for the prevention or termination of atrial arrhythmias. Methods Wistar rats atria (N=25) were isolated, flattened and pinned to a custom-made silicon plate. Atria were superfused with an oxygenized Tyrode's solution. Tissues were then loaded with a voltage-sensitive dye and mapped using a high-resolution optical mapping system. AF was induced with 1uM carbamylcholine (N=23) coupled with pacing maneuvers and treated with 30uM Vernakalant (N=10) or 10uM Flecainide (N=10). Finally, the feasibility of a new ablation technique (electroporation) was evaluated. Results Optical mapping results suggested that the superfusion procedure led to a fast atrial recovery. Sinus activity was conserved for all atria for a long period. All the anatomical landmarks were clearly visualized. The acquired optical signals were analyzed during sinus rhythm and pacing, which allowed the creation of detailed activation maps and measurements of action potential duration (APD) and conduction velocity (CV) at different pacing rates. The resulting APD restitution curves revealed electrical excitation at high pacing rates (cycle length between 50ms and 300ms) with a relatively flattened curve. AF was successfully induced and optically mapping confirmed the presence of reentrant activity. AF was successfully treated using Vernacalant and Flecainide. Finally, we demonstrated the feasibility of a new ablation approach (electroporation) for creation of a continuous linear lesion serving as a functional block. Conclusion The isolated superfused atria model, coupled with voltage-sensitive dyes, can be utilized for long-term high-resolution functional imaging of the atria during sinus rhythm, pacing and arrhythmogenic activity. This allows the study of the atrial electrophysiological properties, the mechanisms involved in AF initiation, perpetuation, and termination as well as the study of drug and new ablation modalities. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC) Spontaneous activation of isolated atria