OptiDiff: structural variation detection from single optical mapping reads

Detecting structural variation (SV) in eukaryotic genomes is of broad interest due to its often dramatic phenotypic effects, but remains a major, costly challenge based on DNA sequencing data. A cost-effective alternative in detecting large-scale SV has become available with advances in optical mapping technology. However, the algorithmic approaches to identifying SVs from optical mapping data are limited. Here, we propose a novel, open-source SV detection tool, OptiDiff, which employs a single molecule based approach to detect and classify homozygous and heterozygous SVs at coverages as low as 20x, showing better performance than the state of the art.

Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to establish an atrial filtration (AF) model in our laboratory, we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB1 KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined may not reflect human AF well, and raise a cautionary note for use of those murine models to study AF.

Rotor Localization and Phase Mapping of Cardiac Excitation Waves Using Deep Neural Networks

The analysis of electrical impulse phenomena in cardiac muscle tissue is important for the diagnosis of heart rhythm disorders and other cardiac pathophysiology. Cardiac mapping techniques acquire local temporal measurements and combine them to visualize the spread of electrophysiological wave phenomena across the heart surface. However, low spatial resolution, sparse measurement locations, noise and other artifacts make it challenging to accurately visualize spatio-temporal activity. For instance, electro-anatomical catheter mapping is severely limited by the sparsity of the measurements, and optical mapping is prone to noise and motion artifacts. In the past, several approaches have been proposed to create more reliable maps from noisy or sparse mapping data. Here, we demonstrate that deep learning can be used to compute phase maps and detect phase singularities in optical mapping videos of ventricular fibrillation, as well as in very noisy, low-resolution and extremely sparse simulated data of reentrant wave chaos mimicking catheter mapping data. The self-supervised deep learning approach is fundamentally different from classical phase mapping techniques. Rather than encoding a phase signal from time-series data, a deep neural network instead learns to directly associate phase maps and the positions of phase singularities with short spatio-temporal sequences of electrical data. We tested several neural network architectures, based on a convolutional neural network (CNN) with an encoding and decoding structure, to predict phase maps or rotor core positions either directly or indirectly via the prediction of phase maps and a subsequent classical calculation of phase singularities. Predictions can be performed across different data, with models being trained on one species and then successfully applied to another, or being trained solely on simulated data and then applied to experimental data. Neural networks provide a promising alternative to conventional phase mapping and rotor core localization methods. Future uses may include the analysis of optical mapping studies in basic cardiovascular research, as well as the mapping of atrial fibrillation in the clinical setting.

Loss of natriuretic peptide receptor C enhances sinoatrial node dysfunction in aging and frail mice

Heart rate 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 wildtype (WT) 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 lifespan. HR and SAN recovery time were increased in older vs. younger mice and this was 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 strongly 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 due to a rapid decline in health status in association with impairments in HR and SAN function. Frailty assessment was effective and often better able to distinguish aging-dependent changes in SAN function in the setting of shorted lifespan due to loss of NPR-C.

Panoramic Endocardial Optical Mapping Demonstrates Serial Rotors Acceleration and Increasing Complexity of Activity During Onset of Cholinergic Atrial Fibrillation

Background Activation during onset of atrial fibrillation is poorly understood. We aimed at developing a panoramic optical mapping system for the atria and test the hypothesis that sequential rotors underlie acceleration of atrial fibrillation during onset. Methods and Results Five sheep hearts were Langendorff perfused in the presence of 0.25 µmol/L carbachol. Novel optical system recorded activations simultaneously from the entire left and right atrial endocardial surfaces. Twenty sustained (>40 s) atrial fibrillation episodes were induced by a train and premature stimuli protocol. Movies obtained immediately (Initiation stage) and 30 s (Early Stabilization stage) after premature stimulus were analyzed. Serial rotor formation was observed in all sustained inductions and none in nonsustained inductions. In sustained episodes maximal dominant frequency increased from (mean±SD) 11.5±1.74 Hz during Initiation to 14.79±1.30 Hz at Early Stabilization ( P <0.0001) and stabilized thereafter. At rotor sites, mean cycle length (CL) during 10 prerotor activations increased every cycle by 0.53% ( P =0.0303) during Initiation and 0.34% ( P =0.0003) during Early Stabilization. In contrast, CLs at rotor sites showed abrupt decreases after the rotors appearances by a mean of 9.65% ( P <0.0001) during both stages. At Initiation, atria‐wide accelerations and decelerations during rotors showed a net acceleration result whereby post‐rotors atria‐wide minimal CL (CLmin) were 95.5±6.8% of the prerotor CLmin ( P =0.0042). In contrast, during Early Stabilization, there was no net acceleration in CLmin during accelerating rotors (prerotor=84.9±11.0% versus postrotor=85.8±10.8% of Initiation, P =0.4029). Levels of rotor drift distance and velocity correlated with atria‐wide acceleration. Nonrotor phase singularity points did not accelerate atria‐wide activation but multiplied during Initiation until Early Stabilization. Increasing number of singularity points, indicating increased complexity, correlated with atria‐wide CLmin reduction ( P <0.0001). Conclusions Novel panoramic optical mapping of the atria demonstrates shortening CL at rotor sites during cholinergic atrial fibrillation onset. Atrial fibrillation acceleration toward Early Stabilization correlates with the net result of atria‐wide accelerations during drifting rotors activity.

Distinct Effects of Ibrutinib and Acalabrutinib on Mouse Atrial and Sinoatrial Node Electrophysiology and Arrhythmogenesis

Background Ibrutinib and acalabrutinib are Bruton tyrosine kinase inhibitors used in the treatment of B‐cell lymphoproliferative disorders. Ibrutinib is associated with new‐onset atrial fibrillation. Cases of sinus bradycardia and sinus arrest have also been reported following ibrutinib treatment. Conversely, acalabrutinib is less arrhythmogenic. The basis for these different effects is unclear. Methods and Results The effects of ibrutinib and acalabrutinib on atrial electrophysiology were investigated in anesthetized mice using intracardiac electrophysiology, in isolated atrial preparations using high‐resolution optical mapping, and in isolated atrial and sinoatrial node (SAN) myocytes using patch‐clamping. Acute delivery of acalabrutinib did not affect atrial fibrillation susceptibility or other measures of atrial electrophysiology in mice in vivo. Optical mapping demonstrates that ibrutinib dose‐dependently impaired atrial and SAN conduction and slowed beating rate. Acalabrutinib had no effect on atrial and SAN conduction or beating rate. In isolated atrial myocytes, ibrutinib reduced action potential upstroke velocity and Na + current. In contrast, acalabrutinib had no effects on atrial myocyte upstroke velocity or Na + current. Both drugs increased action potential duration, but these effects were smaller for acalabrutinib compared with ibrutinib and occurred by different mechanisms. In SAN myocytes, ibrutinib impaired spontaneous action potential firing by inhibiting the delayed rectifier K + current, while acalabrutinib had no effects on SAN myocyte action potential firing. Conclusions Ibrutinib and acalabrutinib have distinct effects on atrial electrophysiology and ion channel function that provide insight into the basis for increased atrial fibrillation susceptibility and SAN dysfunction with ibrutinib, but not with acalabrutinib.

KairoSight: Open-Source Software for the Analysis of Cardiac Optical Data Collected From Multiple Species

Cardiac optical mapping, also known as optocardiography, employs parameter-sensitive fluorescence dye(s) to image cardiac tissue and resolve the electrical and calcium oscillations that underly cardiac function. This technique is increasingly being used in conjunction with, or even as a replacement for, traditional electrocardiography. Over the last several decades, optical mapping has matured into a “gold standard” for cardiac research applications, yet the analysis of optical signals can be challenging. Despite the refinement of software tools and algorithms, significant programming expertise is often required to analyze large optical data sets, and data analysis can be laborious and time-consuming. To address this challenge, we developed an accessible, open-source software script that is untethered from any subscription-based programming language. The described software, written in python, is aptly named “KairoSight” in reference to the Greek word for “opportune time” (Kairos) and the ability to “see” voltage and calcium signals acquired from cardiac tissue. To demonstrate analysis features and highlight species differences, we employed experimental datasets collected from mammalian hearts (Langendorff-perfused rat, guinea pig, and swine) dyed with RH237 (transmembrane voltage) and Rhod-2, AM (intracellular calcium), as well as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) dyed with FluoVolt (membrane potential), and Fluo-4, AM (calcium indicator). We also demonstrate cardiac responsiveness to ryanodine (ryanodine receptor modulator) and isoproterenol (beta-adrenergic agonist) and highlight regional differences after an ablation injury. KairoSight can be employed by both basic and clinical scientists to analyze complex cardiac optical mapping datasets without requiring dedicated computer science expertise or proprietary software.

Open-Source Low-Cost Cardiac Optical Mapping System

Fluorescent imaging with voltage- or calcium-sensitive dyes, i.e. optical mapping, is one of the indispensable modern techniques to study cardiac electrophysiology, unsurpassed by temporal and spatial resolution. High-speed CMOS cameras capable of optical registration of action potential propagation are in general very costly. We present a complete solution priced below US$1,000 (including camera and lens) at the moment of publication with an open-source image acquisition and processing software. We demonstrate that the iDS UI-3130CP rev.2 camera we used in this study is capable of 200x200 977 frames per second (FPS) action potential recordings from rodent hearts. The signal-to-noise-ratio of a conditioned signal was 16 ± 10 for rodent hearts. A comparison with a specialized MiCAM Ultimate-L camera has shown that signal-to-noise ratio (SNR) is sufficient for accurate measurements of AP waveform, conduction velocity (± 0.04 m/s) and action potential duration (± 7ms) in mouse and rat hearts. We measured the action potential prolongation during 4-aminopyridine administration in mouse heart, showing that proposed system signal quality is adequate for drug studies.