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Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm 2 ) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.
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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 2018 1 , 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.
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- Award ID(s):
- 1830941
- PAR ID:
- 10356491
- Date Published:
- Journal Name:
- Frontiers in Physiology
- Volume:
- 12
- ISSN:
- 1664-042X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Since the neurobiological inception of optogenetics, light‐controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light‐sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle‐specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under:Metabolic Diseases > Molecular and Cellular Physiologymore » « less
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Abstract Bioelectronic devices that allow simultaneous accurate monitoring and control of the spatiotemporal patterns of cardiac activity provide an effective means to understand the mechanisms and optimize therapeutic strategies for heart disease. Optogenetics is a promising technology for cardiac research due to its advantages such as cell‐type selectivity and high space‐time resolution, but its efficacy is limited by the insufficient number of modulation channels and lack of simultaneous spatiotemporal mapping capabilities in current implantable cardiac optogenetics tools available for in vivo investigations. Here, soft implantable electro‐optical cardiac devices integrating multilayered highly uniform arrays of transparent microelectrodes and multicolor light‐emitting diodes in thin, flexible platforms are designed for mechanically compliant high‐content high‐precision electrical mapping and single‐/multi‐site optogenetics and electrical stimulation without light‐induced artifacts. Systematic benchtop characterizations, together with ex vivo and in vivo evaluations on healthy and diseased small animal hearts and human cardiac slices demonstrate their functionalities in real‐time spatiotemporal mapping and control of cardiac rhythm and function, with broad applications in basic and ultimately clinical cardiology.more » « less
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null (Ed.)Abstract Intercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and non-excitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g., cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells (e.g., cardiomyocytes) that are light-insensitive. Cell–cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modelling and proper calibration. Lastly, the sensitivity of OptoGap to intrinsic cell-scale excitability is robustly characterized via computational analysis.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fphys.2021.769586
