Optical mapping of contracting hearts

Optical mapping is a widely used tool to record and visualize the electrophysiological properties in a variety of myocardial preparations such as Langendorff-perfused isolated hearts, coronary-perfused wedge preparations, and cell culture monolayers. Motion artifact originating from the mechanical contraction of the myocardium creates a significant challenge to performing optical mapping of contracting hearts. Hence, to minimize the motion artifact, cardiac optical mapping studies are mostly performed on non-contracting hearts, where the mechanical contraction is removed using pharmacological excitation–contraction uncouplers. However, such experimental preparations eliminate the possibility of electromechanical interaction, and effects such as mechano-electric feedback cannot be studied. Recent developments in computer vision algorithms and ratiometric techniques have opened the possibility of performing optical mapping studies on isolated contracting hearts. In this review, we discuss the existing techniques and challenges of optical mapping of contracting hearts

Optical mapping and optogenetics in cardiac electrophysiology research and therapy:a state-of-the-art review

State-of-the-art innovations in optical cardiac electrophysiology are significantly enhancing cardiac research. A potential leap into patient care is now on the horizon. Optical mapping, using fluorescent probes and high-speed cameras, offers detailed insights into cardiac activity and arrhythmias by analysing electrical signals, calcium dynamics, and metabolism. Optogenetics utilizes light-sensitive ion channels and pumps to realize contactless, cell-selective cardiac actuation for modelling arrhythmia, restoring sinus rhythm, and probing complex cell–cell interactions. The merging of optogenetics and optical mapping techniques for ‘all-optical’ electrophysiology marks a significant step forward. This combination allows for the contactless actuation and sensing of cardiac electrophysiology, offering unprecedented spatial–temporal resolution and control. Recent studies have performed all-optical imaging ex vivo and achieved reliable optogenetic pacing in vivo, narrowing the gap for clinical use. Progress in optical electrophysiology continues at pace. Advances in motion tracking methods are removing the necessity of motion uncoupling, a key limitation of optical mapping. Innovations in optoelectronics, including miniaturized, biocompatible illumination and circuitry, are enabling the creation of implantable cardiac pacemakers and defibrillators with optoelectrical closed-loop systems. Computational modelling and machine learning are emerging as pivotal tools in enhancing optical techniques, offering new avenues for analysing complex data and optimizing therapeutic strategies. However, key challenges remain including opsin delivery, real-time data processing, longevity, and chronic effects of optoelectronic devices. This review provides a comprehensive overview of recent advances in optical mapping and optogenetics and outlines the promising future of optics in reshaping cardiac electrophysiology and therapeutic strategies

Genetically Encoded Voltage Indicators in Circulation Research

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided

Novel optics-based approaches for cardiac electrophysiology: a review

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research

Cardiac Electrophysiological Changes during High Intensity Focused Ultrasound Ablation.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is characterized by disorganized electrical activities that cause atrial quivering and uncoordinated contraction. AF significantly affects the quality of life for patients and increases the risk of stroke. Ultrasound ablation surgery has been proposed a decade ago as a treatment for AF. By focusing ultrasound energy at a narrow spot, rapid temperature rises along with tissue necrosis are generated. In this thesis, we investigated high-intensity focused ultrasound (HIFU), an ablation technology being used to eliminate arrhythmogenic foci for treatment of AF. During HIFU ablation, little is known regarding the detailed characteristics of cellular electrophysiological (EP) changes. The first part of the thesis aims to characterize EP changes during HIFU corresponding with temperature increases. Langendorff-perfused intact rabbit heart model stained with di-4-ANEPPS, a fluorescent dye sensitive to the membrane voltage changes, was used. Simultaneous optical mapping and infrared imaging were employed to measure epicardial EP and temperature during HIFU application. The results revealed the temperature-dependent spatiotemporal characteristics of HIFU-induced EP changes including changes of action potential (AP) amplitude, duration, and electrical activation. Temperature dosage criterion for generating irreversible tissue physical and AP changes were obtained. Intra-procedural imaging is important for guiding cardiac ablation for AF. However, it is difficult to obtain intra-procedural correlation of thermal lesion with AP changes in tissue transmural plane. The second part is to develop parametric ultrasound imaging techniques for transmural lesion and AP detection during ablation. Perfused canine ventricular wedge was used. Simultaneous optical mapping and high frequency ultrasound imaging of the same tissue trasnsmural plane were performed during HIFU. Tissue transmural EP changes were characterized and the AP changes were spatiotemporally correlated between optical and ultrasound images. The results show that parametric ultrasound imaging using cumulative extrema of ultrasound parameters (log-normal and Rayleigh) can detect HIFU lesions and surrounding AP amplitude changes. Overall, the information obtained from this thesis enhances our understanding of the EP mechanisms of HIFU ablation and can help promote the development of effective HIFU ablation strategies. Ultrasound parametric imaging provides a promising technique to identify lesion transmurality which is important in clinical ablations.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/100027/1/ziqiwu_1.pd

Electrophysiological and cellular analysis of filamin-C mutations causing cardiomyopathy using human iPSC-derived cardiomyocytes

Background: Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disease resulting from different mutations within proteins constituting the intercalated disc, including desmosomal and nondesmosomal proteins. Recent studies have revealed that mutations in filamin-C (FLNC) may lead to AC. The arrhythmogenesis and electrophysiological effects of FLNC-related AC are incompletely understood. Therefore, the aim of this study is to assess the potential electrophysiological consequences of FLNC loss as occurs in AC in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Specifically, I aimed to characterise abnormal electrical activity and the expression and function of key proteins in cardiac electrical activity such as gap junction protein connexin 43 (Cx43).// Methods: hiPSC-CMs were differentiated and observed by immunofluorescence microscopy. Small interfering RNA (siRNA) transfection was utilised to knockdown the expression of FLNC in hiPSC-CMs. Protein analysis was performed using western blotting to confirm the knockdown efficiency. Electrophysiological properties were recorded using a multielectrode array and manual patch clamping. Optical recording of membrane potential and calcium activity from hiPSC-CMs were also carried out using parameter sensitive dyes.// Results: Silencing of FLNC led to markedly decreased immunofluorescence signals of FLNC, Cx43, desmoplakin, and junctional plakoglobin. No significant reductions were noted in the immunofluorescence signals of voltage-gated sodium channel (Nav1.5) and plakophilin-2 compared with control hiPSC-CMs. Western blotting showed the reduction of FLNC and Cx43 expression following silencing of FLNC. Knockdown of FLNC resulted in disturbances to the recorded action and field potential signals of hiPSC-CMs and arrhythmic likeevents. Transfected hiPSC-CMs with siRNA-FLNC were associated with prolongation of calcium transient durations, optical action potential duration, and action potentials measured with patch clamping.// Conclusion: The current findings indicated that loss of FLNC resulted in a complex arrhythmogenic phenotype in hiPSC-CM

Marker-Free Tracking for Motion Artifact Compensation and Deformation Measurements in Optical Mapping Videos of Contracting Hearts

Optical mapping is a high-resolution fluorescence imaging technique, which provides highly detailed visualizations of the electrophysiological wave phenomena, which trigger the beating of the heart. Recent advancements in optical mapping have demonstrated that the technique can now be performed with moving and contracting hearts and that motion and motion artifacts, once a major limitation, can now be overcome by numerically tracking and stabilizing the heart's motion. As a result, the optical measurement of electrical activity can be obtained from the moving heart surface in a co-moving frame of reference and motion artifacts can be reduced substantially. The aim of this study is to assess and validate the performance of a 2D marker-free motion tracking algorithm, which tracks motion and non-rigid deformations in video images. Because the tracking algorithm does not require markers to be attached to the tissue, it is necessary to verify that it accurately tracks the displacements of the cardiac tissue surface, which not only contracts and deforms, but also fluoresces and exhibits spatio-temporal physiology-related intensity changes. We used computer simulations to generate synthetic optical mapping videos, which show the contracting and fluorescing ventricular heart surface. The synthetic data reproduces experimental data as closely as possible and shows electrical waves propagating across the deforming tissue surface, as seen during voltage-sensitive imaging. We then tested the motion tracking and motion-stabilization algorithm on the synthetic as well as on experimental data. The motion tracking and motion-stabilization algorithm decreases motion artifacts approximately by 80% and achieves sub-pixel precision when tracking motion of 1–10 pixels (in a video image with 100 by 100 pixels), effectively inhibiting motion such that little residual motion remains after tracking and motion-stabilization. To demonstrate the performance of the algorithm, we present optical maps with a substantial reduction in motion artifacts showing action potential waves propagating across the moving and strongly deforming ventricular heart surface. The tracking algorithm reliably tracks motion if the tissue surface is illuminated homogeneously and shows sufficient contrast or texture which can be tracked or if the contrast is artificially or numerically enhanced. In this study, we also show how a reduction in dissociation-related motion artifacts can be quantified and linked to tracking precision. Our results can be used to advance optical mapping techniques, enabling them to image contracting hearts, with the ultimate goal of studying the mutual coupling of electrical and mechanical phenomena in healthy and diseased hearts