Name: preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices
Uploaded: 2020-06-16T13:30:00
Description: Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

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.

All methods described have been performed in compliance with all institutional, national, and international guidelines for human welfare. Research was approved by the Institution Review Board (IRB) at The George Washington University.
NOTE: Donor human hearts were acquired from Washington Regional Transplant Community as deidentified discarded tissue with approval from the George Washington University IRB. Explanted hearts are cardioplegically arrested by flushing the heart with a solution of ...

<ol>
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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 ...

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 ...

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.

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 ...

This protocol is significant because it provides researchers with the skills to employ donor human hearts for preclinical physiological drug testing. This tissue utilizes both voltage and calcium dyes for the simultaneous characterization of cardiac action potentials and intracellular calcium transients in human tissue slices. To set up the optical mapping system, attach a tissue bath with a bottom polydimethylsiloxane gel layer to a perfusion system and circulate one liter of recovery solution at 37 degrees Celsius oxygenated with 100%oxygen through the perfusion system at a flow rate fast enough to maintain the temperature and to prevent the accumulation of bath perfusate.Then use a target to adjust the focus and alignment of the two CMOS cameras and use a green LED light source with a 520 plus or minus five nanometer wavelength to excite the voltage sensitive and calcium indicator dyes simultaneously. Before obtaining tissue sections, mix frozen and liquid cardioplegia solution and place the heart tissue into the ice cold cardioplegia bath and identify the left ventricular free wall. Glue the pre-made agarose gel to the back of the metal tissue holders of the vibratome.Cut one centimeter cubed blocks of tissue in cold cardioplegic solution and use topical skin adhesive to quickly mount the tissue blocks onto the metal tissue holders with the endocardial surfaces facing up. Transfer the metal holders into the vibratome bath of ice cold oxygenated slicing solution so that the tissues are completely submerged and move the blade to the front edge of the tissue. Turn on the vibratome to begin slicing with the preset parameters, discarding the first several slices until the blade reaches beyond the trabeculae into the smooth endocardial tissue.When the experimental tissue has been reached, carefully transfer each slice into individual 100 micrometer nylon mesh cell strainers in a culture dish containing room temperature oxygenated Tyrode's recovery solution and cover the slices with mesh washers to keep the tissue slices from curling. When all of the slices have been collected, carefully transfer each slice into individual wells of a six-well plate containing three milliliters of PBS per well. Rinse the slice with gentle rocking and repeat this process three times with fresh sterile PBS before transferring the samples into individual wells of a six-well plate containing three milliliters of 37 degree Celsius culture medium per well.Then place the plate onto a orbital shaker at 20 revolutions per minute in a humidified incubator at 37 degrees, 30%oxygen, and 5%carbon dioxide. After 20 minutes of incubation in recovery solution for freshly cut slices or immediately following culturing, carefully transfer the slice of interest to the perfusion system tissue bath and pin down the four corners to the gel layer while applying minimal stretch to the tissue. Allow the slices to rest in this bath with circulating recovery solution for approximately 10 minutes before adding 0.3 to 0.5 microliters of stock Blebbistatin to the reservoir of recovery solution.While the slice is incubating in the Blebbistatin, reconstitute 30 microliters of the stock voltage sensitive dye in one milliliter of recovery solution at 37 degrees. After 10 minutes of Blebbistatin treatment, turn off the pumps and slowly load 0.2 to 0.3 milliliters of working dye solution onto the surface of the slice over a period of 30 seconds. After 90 seconds, turn on the pumps to wash out any excess dye.After dying the slice with stock calcium indicator as just demonstrated, focus the cameras onto the slice and pace the slice at one hertz with a two millisecond pulse width duration at 1.5 times the amplitude of the predetermined pacing threshold. Place a coverslip over the tissue slice and illuminate the slice with the LED excitation light source. Then record the emitted voltage and calcium signals with the cameras at 1, 000 frames per second.To condition the voltage and calcium optical mapping data, in the appropriate analysis software, click condition parameters and remove background. Then adjust the signal conditioning parameters to obtain optimal action potential and calcium transient traces. To calculate the conduction velocity, select activation map and enter a start and end time to encompass a single action potential in the trace.Then click regional map and select the region of interest to display the activation map of the selected region. Next, select conduction velocity map and select the start and end times. Adjust the inter-pixel resolution values based on the setup as necessary.Click generate vector map to select a region of interest and display the conduction velocity vectors within that region. The mean, median, standard deviation, and number of vectors included in the analysis as well as the average angle of propagation of the conduction velocity vectors in the selected region will be displayed. To calculate the action potential duration, select action potential duration, calcium transient duration map and select the start and end times to encompass one full action potential.Then click regional action potential duration calculation to select a region of interest and to generate the action potential duration map. To determine the rise time, select rise time and select the start and end times to select the upstroke of one single action potential or calcium transient. Enter the start and end percentage values and click calculate to select the region of interest and to determine the mean, median, standard deviation, and number of pixels included in the analysis of rise time.To determine the calcium decay, select calcium decay and enter the start and end times to encompass the entire decay portion of a single calcium transient signal. Then click calculate tau to select the region of interest and to determine the mean, median, standard deviation, and number of pixels included in the analysis of calcium decay time constant. In this representative experiment, the action potential and calcium transient traces were signal conditioned.The activation times were determined for each pixel and isochronal maps of the activation times were plotted from the conditioned voltage and calcium traces. Conduction velocity vectors were calculated using the activation times and the known inter-pixel resolution. The calcium transient decay constant was measured by fitting a polynomial to the decaying portion of the calcium traces.The action potential duration and calcium transient duration were measured as the time duration between the activation time and a specified percent of the re-polarization or calcium removal from the cytoplasm respectively. The rise times of the voltage and calcium traces were also measured and mapped. In this representative analysis, the effect of doxorubicin on cardiac conduction was tested resulting in a reduction of the transverse conduction velocity.It is important to make sure that the slices are in complete cardioplegic arrest to minimize damage to the tissue during the slicing procedure and to obtain viable slices. At the end of the protocol, the slices can be frozen or fixed for molecular assessment. The mechanistic pathways involved in the electrophysiological phenomenon of interest can then be determined.Development of this human cardiac slice model has allowed the study of the responses to treatments and disease in human heart tissue bridging the gap between animal and clinical studies. When working with human tissues while performing this technique, be sure to wear the appropriate PPE and to take any other necessary safety precautions.

Research

JoVE Journal

Bioengineering

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. ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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 ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...