Name: preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices
Uploaded: 2020-06-16T13:30:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices at JoVE.com

Funding by NIH (grants R21 EB023106, R44 HL139248, and R01 HL126802), by Leducq foundation (project RHYTHM) and an American Heart Association Postdoctoral Fellowship (19POST34370122) are gratefully acknowledged.

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The authors have nothing to disclose.

Here, we present step-by-step methods to obtain viable cardiac slices from cardioplegically arrested human hearts and to functionally characterize the slices using dual optical mapping of transmembrane potential and intracellular calcium. With preserved extracellular environment and native cell-cell coupling, human cardiac slices can be used as an accurate model of the human heart for fundamental scientific discovery and for efficacy and cardiotoxicity testing of pharmacological agents and gene therapies. The technology also allows for structural-functional mapping of specific regions of the heart, such as the sinoatrial node, atrioventricular node, and Purkinje fibers. The protocol described here lists basic steps for cardiac slice generation, imaging, and analyses that are best for short-term studies. Static culture of tissue slices have been shown to induce changes in cardiac tissue over prolonged periods of time, so for longer-term studies, we encourage the reader to incorporate more physiological culture conditions, such as electromechanical stimulation12,13,14.
Care should be taken for a number of steps to maintain tissue health. For example, the time between heart harvesting and tissue slicing should be minimized. As shown previously21, extended length of cardioplegic arrest can lead to altered electrophysiology. Also, to preserve the integrity of the extracellular matrix and cell-cell coupling, excessive handling and stretching of the slices should be avoided during slice collection, culturing, and functional studies. Excessive stretching of the slices could result in conduction blocks. Additionally, all solutions described in this protocol should be kept at a pH of 7.4. The Tyrode&#8217;s solution described here utilizes HEPES to maintain the proper pH using 100% O2. Sodium bicarbonate may be used to control pH instead, but this solution should be bubbled with 95% O2 and 5% CO2. Finally, during transportation and slicing of the heart, one should ensure that the cardioplegia and slicing solution are kept as close to freezing temperature as possible to preserve tissue viability.

Animal models have been a valuable tool used for understanding the underlying mechanisms of human physiology and pathophysiology, as well as a platform for preliminary testing of therapies to treat various diseases1. Great strides have been taken in the field of biomedical research based on these animal studies2. However, significant interspecies differences exist between human and animal physiologies, including mice, rats, guinea pigs, rabbits, sheep, pigs, and dogs3,4. As a result, there have been numerous drug, gene, and cell therapies that showed promise during the animal testing stage but failed to live up to the results in clinical trials5. To bridge this gap, isolated cardiac myocytes and human induced pluripotent stem cells (iPSCs) were developed as models to test the response of human physiology to various drugs and diseases6. Stem cell-derived cardiomyocytes have been widely used in organ-on-a-chip systems as a surrogate of the heart6,7,8. However, the usefulness of iPSC-derived cardiomyocytes (iPSC-CMs) is impeded by their relatively immature phenotype and the lack of representation of the cardiomyocyte subpopulation; the mature myocardium is a complex structure comprised of several coexisting cell types such as fibroblasts, neurons, macrophages, and endothelial cells. On the other hand, isolated human cardiomyocytes are electrically mature, and different cardiomyocyte subpopulations can be obtained by altering culturing parameters9. Still, these myocytes generally exhibit altered action potential morphologies due to the lack of cell-cell coupling, rapid de-differentiation, and occurrence of proarrhythmic behavior in vitro10,11. Some of the limitations were addressed by 3D cell culture models of iPSC-CMs and cardiac myocytes. These models, which include spheroids, hydrogel scaffold encapsulated 3D cultures, engineered heart tissues (EHTs), and heart-on-a-chip systems, use multiple cardiac cell populations such as cardiomyocytes, fibroblasts, and endothelial cells. They either self-assemble or assemble along a scaffold to form 3D structures, and some even reproduce the complex anisotropic nature of the myocardium. These models have been reported to have cells of mature phenotypes, contractile properties, and molecular profiles similar to cardiac tissue. The heart-on-a-chip system also allows the study of systemic effects in drug testing and disease models. However, in vitro cell-based models lack the native extracellular matrix and therefore cannot accurately mimic organ level electrophysiology. Human cardiac slices, by contrast, have an intact extracellular matrix and native cell-to-cell contacts, making them useful for more accurately examining arrhythmogenic properties of the human myocardium.
Researchers have developed human cardiac organotypic slices as a physiological preclinical platform for acute and chronic drug testing and to study cardiac electrophysiology and cardiac disease progression12,13,14,15,16,17,18,19. When compared with iPSC-derived cardiomyocytes, human cardiac slices more faithfully replicate adult human cardiac electrophysiology with a mature cardiomyocyte phenotype. When compared with isolated human cardiomyocytes, cardiac slices exhibit physiological action potential durations because of the well-preserved cell-cell coupling and the intrinsic existence of their native intra- and extracellular environments.
This protocol describes the process of generating human cardiac slices from whole donor hearts, performing acute (i.e., hours-long) and chronic (i.e., days-long) studies to test cardiac electrophysiology parameters via optical mapping. While this protocol describes only the use of the left ventricular (LV) tissue, it has been successfully applied to other regions of the heart as well as other species such as mice, rats, guinea pigs, and pigs14,20,21,22. Our laboratory uses whole human donor hearts that have been rejected for transplantation for the last 5 years, but it is feasible for these same procedures to be carried out on any donor heart sample tissues obtained by alternative means (e.g., left ventricular assist device [LVAD] implantations, biopsies, myectomies) as long as the tissues have the ability to be sectioned into cubes. Optical mapping is employed for analysis in this study due to its capacity to simultaneously map optical action potentials and calcium transients with high spatial (100 x 100 pixels) and temporal (&#62;1,000 frames/s) resolution. Alternative methods can also be used, such as multielectrode arrays (MEAs) or microelectrodes, but these techniques are limited by their relatively low spatial resolutions. Additionally, MEAs were designed for use with cell cultures, and sharp microelectrodes are more easily managed for use with whole hearts or large tissue wedges.
The goal of the article is to enable more researchers to use human cardiac tissues for cardiac electrophysiology studies. It should be noted that the technology described in this article is relatively simple and beneficial for short-term studies (on the order of several hours to days). More physiological biomimetic culture for longer-term studies (on the order of weeks) has been discussed and described by a number of other studies12,18,23. Electrical stimulation, mechanical loading, and tissue stretching are advantageous conditioning mechanisms that can help limit the onset of in vitro tissue remodelling12,18,23.

<table><tbody><tr><td>1mL BD Syringe</td><td>Thomas Scientific</td><td>309597</td><td></td></tr><tr><td>2,3-butanedione monoxime</td><td>Sigma-Aldrich</td><td>B0753</td><td></td></tr><tr><td>6 well culture plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Biosafety cabinet</td><td>ThermoFisher Scientific</td><td>1377</td><td></td></tr><tr><td>Blebbistatin</td><td>Cayman</td><td>13186</td><td></td></tr><tr><td>Bubble Trap</td><td>Radnoti</td><td>130149</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>Corning Cell Strainers</td><td>Fisher Scientific</td><td>07-201-432</td><td></td></tr><tr><td>Di-4-ANEPPS</td><td>Biotium</td><td></td><td>stock solution at 1.25 mg/mL in DMSO</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dumont #3c Forceps</td><td>Fine Science Tools</td><td>11231-20</td><td></td></tr><tr><td>Emission dichroic mirror</td><td>Chroma</td><td>T630LPXR-UF1</td><td></td></tr><tr><td>Emission filter (RH237)</td><td>Chroma</td><td>ET690/50m</td><td></td></tr><tr><td>Emission Filter (Rhod2AM)</td><td>Chroma</td><td>ET590/33m</td><td></td></tr><tr><td>Excitation dichroic mirror</td><td>Chroma</td><td>T550LPXR-UF1</td><td></td></tr><tr><td>Excitation Filter</td><td>Chroma</td><td>ET500/40x</td><td></td></tr><tr><td>Falcon 50mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-49A</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Heat exchanger</td><td>Radnoti</td><td>158821</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Incubator</td><td>ThermoFisher Scientific</td><td>50145502</td><td></td></tr><tr><td>Insulin Transferrin Selenium (ITS)</td><td>Sigma-Aldrich</td><td>I3146</td><td></td></tr><tr><td>LED excitation light source</td><td>Prizmatix</td><td>UHP-Mic-LED-520</td><td></td></tr><tr><td>Magnessium chloride hexahydrate</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>Medium 199</td><td>ThermoFisher Scientific</td><td>11150059</td><td></td></tr><tr><td>Micam Ultima L type CMOS camera</td><td>Scimedia</td><td>N/A</td><td></td></tr><tr><td>Minutien Pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Pennicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Cole Parmer</td><td>EW-07522-20</td><td></td></tr><tr><td>Platinum pacing wire</td><td>Alfa Aesar</td><td>43275</td><td></td></tr><tr><td>Pluronic F127</td><td>ThermoFisher Scientific</td><td>P6867</td><td>nonionic, surfactant polyol</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Powerlab data acquisition and stimulator</td><td>AD Instruments</td><td>Powerlab 4/26</td><td></td></tr><tr><td>RH237</td><td>Biotium</td><td>61018</td><td></td></tr><tr><td>Rhod2AM</td><td>ThermoFisher Scientific</td><td>R1245MP</td><td></td></tr><tr><td>Rhod-2AM</td><td>Invitrogen, Carlsbad, CA</td><td></td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Sterilizer, dry bead</td><td>Sigma-Aldrich</td><td>Z378550</td><td></td></tr><tr><td>Stone Oxygen Diffuser</td><td>Waterwood</td><td>B00O0NUVM0</td><td></td></tr><tr><td>TissueSeal - Histoacryl Topical Skin Adhesive</td><td>gobiomed</td><td>AESCULAP</td><td></td></tr><tr><td>UltraPure Low Melting Point Agarose</td><td>Thermo Fisher Scientific</td><td>16520100</td><td></td></tr><tr><td>Ultrasound sonicator</td><td></td><td>Branson 1800</td><td></td></tr><tr><td>Vibratome</td><td>Campden Instruments</td><td>7000 smz</td><td></td></tr></tbody></table>

preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

Human organotypic slices were collected from the left ventricle of a donor human heart according to the protocol detailed above and illustrated in Figure 1. A dual camera optical mapping system like that in Figure 2 was used in the upright imaging configuration to perform simultaneous optical mapping of voltage and calcium about 1 h after the completion of the slicing protocol. Data were analyzed using RHYTHM1.2 (Figure 3), an open source optical mapping data analysis tool previously published by our laboratory and freely available on Github (https://github.com/optocardiography/Rhythm-1.2). The electrophysiological parameters measured are illustrated in Figure 4. The action potential and calcium transient traces were signal conditioned and representative traces used in further analysis are illustrated in Figure 4A. Activation times were determined for each pixel and isochronal maps of activation times determined from voltage and calcium traces are illustrated in Figure 4B,C. Note that activation in the calcium isochrone lags behind that of the voltage as expected. CV vectors plotted in Figure 4D were calculated using the activation times and the known interpixel resolution. The average CV in the transverse direction was determined to be 21.2 cm/s in this slice. This CV value is comparable to previously reported ventricular transverse CV measured from explanted whole human hearts (24 &#177; 4 and 28 &#177; 7 cm/s in hearts with diffuse and patchy fibrosis)28. The calcium transient decay constant was measured by fitting a polynomial to the decaying portion of the calcium traces and average decay constant was determined to be 105.3 ms in this slice (map in Figure 4E). Next, APD and CaTD were measured as the time duration between activation time and a specified percent of repolarization/calcium removal form the cytoplasm. Average APD80 and CaTD80 were determined to be 343.1 ms and 442.6 ms, respectively (Figure 4F,G). Previous in vivo human studies report the activation-recovery interval (ARI) measured from unipolar electrograms recorded from in vivo human hearts during steady state pacing at 1 Hz as a surrogate for APD. ARI values in these studies range from 250&#8211;450 ms29,30. The APD values from human cardiac slices reported here are comparable to the previous ARI values. Regions with motion artifacts were removed from APD and CaTD calculations. Finally, the rise times (i.e., the duration of the upstroke) of the voltage and calcium traces were measured and mapped. These are shown in Figure 4H and Figure 4I, respectively. The average values were determined to be 10.2 ms and 13.3 ms, respectively. The artifacts in the maps to the right of the point of pacing are due to the pacing wire within the field of view. Finally, the use of this slice model in acute drug testing was demonstrated when slices were cultured for 24 h with doxorubicin (DOX), a chemotherapeutic agent known to have cardiotoxic effects. Treatment of slices with 50 &#956;M DOX resulted in a reduction of transverse conduction velocity from 19.4 &#177; 3.4 cm/s to 9.6 &#177; 3.2 cm/s, as illustrated by the activation maps in Figure 5A, CV vector maps in Figure 5B, and summary data in Figure 5C. The smaller sample size in the DOX group was due to a higher number of nonviable slices after DOX treatment. Increased motion artifacts in DOX-treated slices prevented the calculation of accurate APD values. Another important parameter that can be measured by dual voltage and calcium imaging is Vm-Ca delay, to determine robust excitation-contraction coupling in the tissue31. Delays or negative values of this parameter could be detrimental.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60781/60781fig01.jpg" /> 
Figure 1: Human cardiac organotypic slice preparation.&#160;(A) Human hearts were stored in an ice-cold cardioplegic bath (mixture of cardioplegia solution and cardioplegia ice) upon retrieval. (B) The 1 cm3 cubes of left ventricular tissue were cut and mounted onto a metal tissue holder with 4% agarose gel glued to the back wall of the holder and transferred to an ice-cold bath of oxygenated slicing solution. (C) Once cut, slices were transferred to oxygenated recovery solution at RT in individual 100 &#181;m nylon mesh cell strainers. Meshed washers covered the tissues to keep the slices from curling. (D) For long term culture, slices were washed in PBS and cultured in 6 well plates with 3 mL of tissue medium at 37 &#176;C. (E) Mason&#8217;s trichrome stained slice section showing fiber orientation. (F) Representative optical action potential recorded from a slice. (G) Representative activation map of a slice paced at the center (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60781/60781fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60781/60781fig02.jpg" /> 
Figure 2: Dual camera optical mapping system.&#160;Dual camera optical mapping system in the upright imaging configuration. System parts include: 1) master and slave cameras for dual mapping; 2) tissue bath with PDMS gel; 3) filter cubes that house the excitation and emission filters and dichroic mirrors; 4) lens holders and lenses; and 5) excitation light source (520 nm green LED). Inset on right details filter and dichroic mirror combinations for dual optical mapping of Vm and calcium using RH237 and Rhod-2-AM, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60781/60781fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60781/60781fig03.jpg" /> 
Figure 3: RHYTHM1.2 graphical user interface (GUI) of the open source optical mapping data analysis tool for single parameter and multiparameter analysis.&#160;The data file loading options and file list are displayed in the red box. Data analysis options are listed in the data analysis dropdown menu indicated in green. Display windows for displaying data maps are indicated in dark blue. Checkboxes to link dual mapping data files for simultaneous analysis of dual camera data are indicated in purple. The waveform window to plot action potential and calcium transient traces is displayed in yellow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60781/60781fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60781/60781fig04.jpg" /> 
Figure 4: Transmembrane potential and calcium transients mapping from a human organotypic cardiac slice preparation.&#160;(A) Representative optical action potential (black) and calcium transient (burgundy). (B,C) Activation maps obtained from Vm and calcium recordings, respectively. (D) Conduction velocity (CV) vector map. (E) Calcium transient decay constant (Tau) map. (F,G) Action potential duration (APD80) and calcium transient duration (CaTD80) map, respectively. (H,I) Maps of the rise time of the upstrokes of action potential and the calcium transient, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60781/60781fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60781/60781fig05.jpg" /> 
Figure 5: Doxorubicin treatment slows transverse conduction velocity.&#160;(A) Activation maps from slices cultured for 24 hours without (control) and with doxorubicin at 50 &#956;M (DOX). (B) Conduction velocity vector maps from control and DOX slices. (C) Average transverse conduction velocity calculated from control and DOX-treated slices. *P &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60781/60781fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

preclinical-cardiac-electrophysiology-assessment-dual-voltage-calcium

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.7K Views.  The George Washington University. Dit protocol is belangrijk omdat het onderzoekers de vaardigheden biedt om donormensenharten in dienst te nemen voor preklinische fysiologische drugstesten. Dit weefsel maakt gebruik van zowel spanning en calcium kleurstoffen voor de gelijktijdige karakterisering van cardiale actie mogelijkheden en intracellulaire calcium transiënten in menselijk weefsel plakjes. Om het optische kaartsysteem op te zetten, bevestigt u een weefselbad met een bodem polydimethylsiloxaangellaag aan een perfusiesy...

Video: Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biology

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Medicine

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

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