This work was supported by the AnaBios Corporation and an NIH Small Business Innovation Research (SBIR) grant (1R44TR003162-01).

All methods were carried out in accordance with relevant guidelines and regulations. All human hearts used for the studies were non-transplantable and ethically obtained by informed legal consent (first person or next-of-kin) from cadaveric organ donors in the United States (US). The recovery protocols and in vitro experimentation were pre-approved by Institutional Review Boards (IRBs ) at transplant centers within the US Organ Procurement Transplant Network (OPTN). Furthermore, all transfers of the donor hearts are fully traceable and periodically reviewed by US federal authorities.
NOTE: Apply all necessary safety procedures during the execution of this protocol, including wearing appropriate personal protective equipment (e.g., laboratory coats, safety glasses, gloves).
1. Isolation of cardiomyocytes (1 day before measuring contractility)
<ol>
	<li>Re-perfuse donor hearts immediately on arrival at the laboratory with ice-cold proprietary cardioplegic solution6 and isolate enzymatically adult human primary myocytes from the ventricles11,12,13,14,15.</li>
	<li>Then, maintain the cardiomyocytes in suspension and store them with 10 mL of solution A (110 mM sucrose, 0.005 mM CaCl2, 3 mM MgCl2, 70 mM KOH, 60 mM lactobionic acid, 10 mM KH2PO4, 20 mM taurine, 20 mM L-histidine, 20 mM HEPES, 2 mM L-glutamic acid, 2 mM L-(-)-malic acid, pH 7.4 with KOH) in 20 mL Wheaton vials (Table of Materials) at a refrigerated temperature until they are experimentally interrogated. 
	&#8203;NOTE: Store 200,000 cells per vial.</li>
</ol>
2. Laminin coating preparation (1 day before measuring contractility)
<ol>
	<li>Place a single glass coverslip (25 mm x 25 mm square, Table of Materials) in each well of an eight-well culture plate (Table of Materials).</li>
	<li>Then, coat the coverslips with laminin at a 5 &#181;g/mL concentration. To do so, add 800 &#181;L of the human recombinant Laminin 521 stock (Table of Materials, stored at &#8722;20 &#176;C) to 7.2 mL of solution B (145 mM NaCl, 4 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 11.1 mM glucose, and 10 mM HEPES, pH 7.4 with NaOH) and mix well.</li>
	<li>Drop 200 &#181;L of the diluted laminin solution into the center of each coverslip. Then, cover the plate and stack it in a 4 &#176;C fridge. 
	NOTE: Prepare multiple eight-well culture plates to ensure there are enough laminin-coated coverslips.</li>
</ol>
3. Preparation of Ca2+-tolerant cardiomyocytes (on the day of measuring contractility)
<ol>
	<li>Remove a vial of cells from the fridge, aspirate solution A from the vial and add a refrigerated 10 mL of solution B. 
	NOTE: Ensure the cells are not suctioned and disperse solution B into the vial by placing the pipette on the inside of the top of the vial wall.</li>
	<li>Cap the vial of cells and then gently swirl to ensure the cells are dispersed throughout solution B. Finally, allow the cells to settle for 1 h at room temperature (RT).</li>
</ol>
4. Preparation of the recording system (on the day of measuring contractility)
NOTE: The recording system includes a temperature control box, stimulator, computer-controlled pressure-driven perfusion, pressure-driven perfusion bottles, in-house acquisition software permitting the selection of regions of interest (ROIs) and display of contractility transients, inverted microscope, cell chamber, and camera (Figure 1).
<ol>
	<li>Turn on the suction vacuum system. Then, remove the microscope cover, turn it on, connect the camera (Table of Materials, Figure 1), and finally, confirm the fan is turned on to ensure the camera does not overheat.</li>
	<li>Then, turn on the microscope heating plate (Table of Materials) and temperature control box (Table of Materials, Figure 1), and set them to the proper operating temperature. Next, turn on the field stimulator (Table of Materials, Figure 1) and the pressure system. Finally, connect the solution&#39;s tubing (Table of Materials) to the recording chamber (Table of Materials, Figure 1).</li>
</ol>
5. Preparation of test compounds (on the day of measuring contractility)
<ol>
	<li>Dilute dimethyl sulfoxide (DMSO; Table of Materials) 1,000-fold in solution C (145 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 11.1 mM glucose, and 10 mM HEPES, pH 7.4 with NaOH) to make a 0.1% DMSO vehicle solution.</li>
	<li>To test a compound at 1-fold, 10-fold, 100-fold, and 1000-fold of its therapeutic exposure of 0.001 &#181;M, dissolve the test compound in DMSO at a concentration of 1 mM.</li>
	<li>Dilute this solution serially with DMSO to produce three further DMSO stocks (e.g., 0.001 mM, 0.01 mM, and 0.1 mM). Finally, dilute each test compound stock 1,000-fold in 100 mL of solution C to obtain final test concentrations (0.001 &#181;M, 0.01 &#181;M, 0.1 &#181;M, and 1 &#181;M).</li>
	<li>Add the vehicle solution and solutions of final micromolar concentrations of the compound to 100 mL glass bottles (Table of Materials, Figure 1) and then connect the bottles to the pressure-driven perfusion system (Table of Materials, Figure 1).</li>
	<li>Then, prime the perfusion system with a computer-driven program (Table of Materials) using a pressure of 200-300 mbar to deliver a constant perfusion flow at a rate of 1.8 mL/min. 
	NOTE: Do not conduct the experiment if the test compound is found to be associated with a solubility issue. Also, formulate the compound test solutions from stock solutions within 30 min prior to experimental application to the cells.</li>
</ol>
6. Plating of cardiomyocytes (on the day of measuring contractility)
<ol>
	<li>Take an eight-well plate containing laminin-coated coverslips out of the 4 &#176;C fridge and carefully place one coverslip in a very well-cleaned recording chamber (Figure 1). Then, return the eight-well plate to the 4 &#176;C fridge until the next plating. 
	NOTE: Use a lint-free wipe (Table of Materials) to wipe up the coated coverslip while keeping the coverslip coated in a thin layer of laminin. Also, ensure to dispose of used coverslips in a sharps container.</li>
	<li>Then, take an escalated vial of cells (step 3.2) and aspirate solution B from the vial to reach as small a volume as possible without losing cells (~200 &#181;L). Next, dispense the 200 &#181;L of cells into the recording microscope chamber mounted on the stage of an inverted microscope (Table of Materials).</li>
	<li>Let the cells settle on the coverslip for 5 min. While waiting for the cells to fully settle, open the field of view on the microscope and determine if the cell density is adequate (~70%) for the experimental run to begin. 
	&#8203;NOTE: Ensure the suction does not aspirate all the cells away if more than 200 &#181;L are dispensed.</li>
</ol>
7. Recording of cardiomyocyte contractility
<ol>
	<li>After plating is completed, equilibrate the cells for 5 min by continuously perfusing them with solution C using the pressure-driven perfusion system (Table of Materials). Adjust the suction correctly, turn on both the temperature control box and heating plate, and set them to deliver ~35 &#176;C.</li>
	<li>Then, stimulate the cells with supra-threshold voltage at a 1 Hz pacing frequency (bipolar pulse of 3 ms duration) on a field stimulator with a pair of platinum wires placed on opposite sides of the chamber connected to a field stimulator.</li>
	<li>Set the amplitude of the stimulating pulse at 1 V, and then increase it until the cardiomyocytes start generating contraction-relaxation cycles. Use a value of 1.5x threshold throughout the experiment. 
	NOTE: Select healthy cells with rod-shaped morphology and clear striations. Then, adjust the field of view and focus on bringing as many contracting cells as possible into view (Figure 2A).</li>
	<li>Next, display the digitized images of the cells within the acquisition software of the optical contractility recording system. This software utilizes a high-resolution, high-frame rate camera with a rate of 150 FPS (Table of Materials, Figure 1) to record a video stream containing multiple myocytes in the same frame. 
	NOTE: This provides sufficient temporal and spatial resolution to capture and measure the sarcomere dynamics of several healthy myocytes simultaneously. Modern high-core-count processors are leveraged to enable the parallel calculation of changes in sarcomere length from the video stream in real time (optical density data are collected from user-defined regions of interest [ROI] placed over the images of healthy cardiomyocytes and parallel to the axis of contraction, Figure 2B). The optical intensity data show bright and dark bands corresponding to the Z-lines of the cardiomyocytes (Figure 1, Figure 2C). Finally, these data are displayed to the user for monitoring purposes and saved to a disk for later analysis (Figure 2C).
	<ol>
		<li>Avoid areas of the cells that are not in focus. Also, do not position the ROIs close to the edge of the cells, as the changes in the cells&#39; contraction during the application of drugs can cause the ROIs to be unable to read the cells&#39; contraction.</li>
	</ol>
	</li>
	<li>When the selection of ROIs is completed and the contractions meet the necessary standards for use (e.g., sarcomere shortening &#62;1%; rhythmic contraction upon application of a 1 Hz pacing frequency, absence of arrhythmic events), start the experiment to assess the effects of a compound. The stimulation protocol and the sequence of the compound&#39;s application of test concentrations are shown in Table 1. The acquisition software will manage, in an automated fashion, the acquisition and display of data and labeling of test concentrations and treatment time. 
	NOTE: Apply test concentrations if the contraction remains at a stable amplitude throughout the entirety of the baseline vehicle period. Disqualify cells displaying either a run-up or run-down. Also, ensure perfusion is switched to the different test concentrations throughout the experiment.</li>
	<li>Upon completion of the experiment and data storage on the server, switch off the perfusion and heater, stop the stimulation, clean the microscope chamber with distilled water, then remove the glass coverslip, and dry the chamber thoroughly. 
	NOTE: Clean the chamber thoroughly as this is critical to avoid exposing the next set of cells to any compound prematurely, thus invalidating any data collected.</li>
	<li>Next, perform a new plating of cardiomyocytes and perform an additional experiment to obtain enough data for completing the testing of the compound or testing a new compound.</li>
</ol>
8. Turning off the optical contractility recording system
<ol>
	<li>When daily testing of the compound(s) is completed, first switch off all equipment that is not necessary to clean the system and copy the data for offline analysis.</li>
	<li>Next, remove the 100 mL glass bottles (Table of Materials) containing solutions of final test concentrations and replace them with glass bottles halfway filled with RBS 25 Concentrate solution (Table of Materials). Perfuse RBS solution through the microscope chamber for 5 min, then disconnect the perfusion tubing from the chamber, clean it with distilled water, and finally, dry it thoroughly. 
	NOTE: Ensure the vacuum is working well to prevent any possible flooding.</li>
	<li>Next, connect the perfusion tubing to the drainage tubing. Using the software of the pressure-driven perfusion system (Table of Materials), run the RBS solution for 10 min while ensuring the pressure and flow rate are adequately set. Then, perfuse 1% DMSO for 5 min and then distilled water for 15-20 min to complete the cleaning of the perfusion system.</li>
	<li>Turn off the microscope light and put its cover. Then, unplug the camera wires from the box on the side of the microscope and turn off the fan. Ensure that all used bottles are sent for cleaning and autoclave. Finally, ensure all drainage buckets are 1/4 full or less. 
	NOTE: Make sure there are enough laminin-coated coverslips for use the following day.</li>
	<li>Finally, copy the files in the acquisition folder of all the experiments done on each recording system and paste them into a secure backup server for additional offline analysis. Next, delete all the data files from the acquisition folder.</li>
</ol>
9. Analysis of contractility data
<ol>
	<li>Perform offline analysis using the analysis software and a custom-made macro to average the data. The analysis software calculates and reports various metrics from the sarcomere dynamics data produced by the acquisition software. A detailed list of these parameters is provided in Figure 2D. 
	NOTE: The application of low-level software techniques allows many runs of recordings to be analyzed in 5 s. The principal parameter to assess drug-induced changes in contractility is the contractility amplitude (sarcomere shortening % = change in sarcomere length). It is calculated as the sarcomere length at peak contraction/resting sarcomere length and averaged over the last 20 contractility transients for each test concentration.</li>
	<li>Quantify the test compound effects on the averaged contractility amplitude relative to each cardiomyocyte&#39;s specific baseline vehicle control condition.</li>
	<li>Express the average results as mean &#177; SEM and produce a graph plotting the concentration effect of the test compound on the contractility amplitude. Next, fit the concentration-response curve to the Hill equation using analysis software (Table of Materials) to derive IC50 (concentration producing a 50% inhibition of contractility amplitude) and EC50 (concentration producing a 50% increase in contractility amplitude) values.</li>
	<li>In addition to the contractility amplitude, you can also calculate other parameters:
	<ol>
		<li>Aftercontraction: Identify aftercontraction as a spontaneous secondary contraction transient of the cardiomyocyte that occurs before the next regular contraction and produces an abnormal and unsynchronized contraction.</li>
		<li>Contraction failure: Identify contraction failure as the inability of the electrical stimulus to induce a contraction.</li>
		<li>Short-term variability (STV) and alternans: Visualize these two parameters in Poincar&#233; plots of contraction amplitude variability.
		<ol>
			<li>Calculate the STV (&#8721;|CAn + 1 &#8722; CAn| (20 &#215; &#8730;2)&#8722;1) with the last 20 transients of each control and test article concentration period. Identify alternans as repetitive alternating short and long contractility amplitude transients.</li>
			<li>To calculate the incidence of pro-arrhythmia, normalize the STV values to the vehicle control value of each cell, plot aftercontraction, contraction failure, STV, and alternans, and express them as % of the incidence of cells exhibiting each of the signals.</li>
		</ol>
		</li>
		<li>Complete the multiparametric mechanistic profiling with the calculation of time Ato peak, decay to 30% relaxation (Decay 30), decay to 90% relaxation (Decay 90), time to 90% relaxation (TR90) (Figure 2D), baseline sarcomere length, time to 50% peak, peak height, sarcomere length at peak contractility, maximum contraction velocity, and maximum relaxation velocity12. Like the contractility amplitude, express these parameters relative to each cardiomyocyte&#39;s specific baseline control condition and graph them in concentration-response plots.</li>
	</ol>
	</li>
</ol>

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All authors are employees of the AnaBios Corporation and declare no competing interests.

This manuscript provides a detailed protocol for the adult human cardiomyocyte contractility-based optical system for a simplified medium-throughput method that enables the testing of the acute efficacy and cardiotoxicity of novel compounds. This optical contractility recording system is easy to use, allows recordings from multiple cells in parallel, enables the simultaneous assessment of cell health, physiology, and pharmacology, comes with automated and rapid data analysis (a run of multiple cells is analyzed in 5 s), and permits rapid data collection (concentration-response curve every 30 min/compound/device). Taking into consideration these attributes, the recording system can be used not only to detect the effects of drugs on cardiomyocyte contractility but also to provide structure-activity relationship data for supporting medicinal chemistry efforts during the early phases of drug discovery16. Since tens of millions of cells can be obtained from a cardiomyocyte isolation protocol, the application of the optical contractility recording system-cardiomyocyte platform is currently being explored to achieve increased testing capacity (with the use of well-based plates) with reduced cost. Moreover, the assessment of drug effects on the systolic and relaxation parameters measured with the recording system can provide multiparametric mechanistic profiling of inotropic drugs12. Additionally, cardiomyocyte contractility data can be used to rank novel drugs from most to least cardiotoxic (e.g., safety margin) and from least to most efficacious (e.g., potency margin). Follow-up cardiomyocyte contractility studies can also be conducted to support development programs that have been associated with a clinical decrease in myocardial contractility12.
Another significant advantage of using the human cardiomyocyte contractility optical recording system is its alignment with the 3Rs concept (replacement, reduction, and refinement)17 since it can be considered as an alternative method that avoids or replaces the use of animals for data generation within the pharmaceutical industry. This 3Rs benefit can also be extended to academic cardiac research. The entirety of current knowledge of cardiomyocyte physiology and pharmacology comes from academic research studies conducted with cells isolated from animal hearts18. Thus, the human cardiomyocyte optical contractility model opens the possibility for critical translational studies to be performed. To perform these studies, protocols for the preservation and shipment of human adult cardiomyocytes must be developed (currently under evaluation in AnaBios&#39; laboratory), and the contractility system must have the ability to record changes in sarcomere length from non-human cardiomyocytes (this is the case with the optical contractility recording system since sarcomeres are well conserved among species).
The human cardiomyocyte contractility system can emulate several physiological conditions (e.g., electromechanical coupling, pacing frequency mimicking heart rate, body temperature, the integration of all human cardiac targets) and has demonstrated translational value as a key component in drug discovery11,12,13,14, although it cannot mimic the changes in mechanical load and shear stress seen during the cardiac contractile cycle. The structure and function of cardiac extracellular matrices are now better understood19, the development of such matrices can potentially help overcome the mechanical load limitation, and matrices with different heart-like stiffnesses are currently being evaluated in AnaBios&#39; laboratory. Another limitation of the human cardiomyocyte optical contractility system is the absence of the network of nerves that supplies the heart (e.g., sympathetic and parasympathetic fibers)20. This neuro-cardiac contact can be re-established with the co-application of neurotransmitters (e.g., isoproterenol, an agonist of &#946;-adrenoceptor receptors; acetylcholine, an agonist of M2 muscarinic receptors), with the compound being assessed for its potential effects on cardiomyocyte contractility. Furthermore, the contractility transients are recorded with no simultaneous measurements of action potentials and Ca2+ transients, which are also essential when evaluating drug effects on the electrocardiogram and Ca2+ handling. Although this omission can be considered a limitation of the system, it is not too critical to have since the recordings of action potential signals (with the current-clamp method or voltage-sensitive dyes) and Ca2+ transients (with Ca2+ indicators/dyes) can be associated with cytotoxicity. Such cytotoxic effects can impact the assessment of novel drugs to modulate heart contractility. On the contrary, the use of a non-invasive optical method that preserves the health, physiology, and pharmacology of the cardiomyocytes, like the recording system described in this protocol, would not only ensure that the highest quality of contractility data is obtained but also provide data that can predict well the contractile effects of novel drugs in humans.

Myocardial contractility (inotropy), which represents the natural capacity of the heart muscle to contract, is a key property of cardiac function and depends on the dynamics of the electro-mechanical coupling. Drug-induced changes in myocardial contractility are desired to treat heart disease (e.g., heart failure) and unsought in the context of cardiotoxicity (e.g., reduction in left ventricular ejection fraction). Therefore, preclinical contractility models must be associated with accurate predictivity to make sure novel drugs can succeed during clinical development. However, current preclinical strategies, which rely on reductionist artificial cellular models (e.g., genetically engineered immortalized cell lines that overexpress specific cardiac targets of interest) and non-human animal models, have shown significant limitations and have been found to be associated with high drug attrition rates (i.e., a high rate of false signals)1,2,3,4. Accordingly, it is imperative to establish new and reliable human cellular heart contractility models that are associated with high power (i.e., high rate of true signals) to predict drug outcomes in humans and, hence, help to accelerate the launch of new therapies5.
The groundbreaking methods recently established for the recovery of human donor hearts for research6,7,8,9,10 and in cardiomyocyte isolation techniques11,12,13,14,15 have provided a unique opportunity for conducting human-based studies during preclinical development. To this end, adult human primary cardiomyocytes have already shown utility in assessing drug-induced changes in human heart contractility11,12,13,14. The current article details the protocol for investigating the contractility effects of novel compounds in adult human cardiomyocytes.

<table><tbody><tr><td>100&amp;ndash;1000 &amp;micro;L Filtered, Wide Orifice, Sterile tips</td><td>Pipette</td><td>UF-1000W</td><td></td></tr><tr><td>100 mL, Duran pressure plus bottles</td><td>DWK Life Sciences</td><td>218102406</td><td></td></tr><tr><td>1 L, 0.22 &amp;micro;m Vacuum Filter system</td><td>VWR</td><td>567-0020</td><td></td></tr><tr><td>290 mmol/kg Osmolarity Standard</td><td>Wescor</td><td>OA-029</td><td></td></tr><tr><td>Benchtop pH Meter</td><td>Mettler Toledo</td><td></td><td>https://www.mt.com/us/en/home/products/Laboratory_Analytics_Browse/pH-meter/pH-meters.html</td></tr><tr><td>Calcium Chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C3881</td><td></td></tr><tr><td>Camera&amp;nbsp;</td><td>Optronis GmbH</td><td>Cyclone-25-150-M</td><td>https://optronis.com/en/products/cyclone-25-150/</td></tr><tr><td>Corning 25 mm x25 mm Square #1 Cover Glass</td><td>Corning</td><td>2845-25</td><td></td></tr><tr><td>Cyclone-25-150</td><td>Optronis</td><td></td><td>https://optronis.com/en/products/cyclone-25-150/</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Digital Timer/Stopwatch</td><td>Fisher Scientific</td><td>14-649-17</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Eight-well rectangular polystyrene sterile culture plate</td><td>Thermo Fisher Scientific</td><td>73521-426</td><td>https://us.vwr.com/store/product/4679368/nunclontm-delta-rectangular-dishes-polystyrene-sterile-thermo-scientific</td></tr><tr><td>FHD Microscope Chamber System</td><td>IonOptix</td><td></td><td></td></tr><tr><td>Flow EZ, Modular pressure-based flow controller with a computer driven program version 1.1.0.0.</td><td>Fluigent OxyGEN</td><td></td><td></td></tr><tr><td>Heavy Duty Vacuum Bottles</td><td>VWR</td><td>16211-080</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Human Recombinant Laminin 521</td><td>BioLamina</td><td>LM521-05</td><td></td></tr><tr><td>Idex Chromatography Tubing, Natural FEP, 1/16&quot; OD x 0.030&quot; ID</td><td>Cole-Palmer</td><td>1520L</td><td></td></tr><tr><td>Kimberly-Clark Professional Kimtech Science Kimwipes</td><td>Fisher Scientific</td><td>06-666</td><td></td></tr><tr><td>L-(-)-Malic acid</td><td>Sigma-Aldrich</td><td>112577</td><td></td></tr><tr><td>Lactobionic acid</td><td>Sigma-Aldrich</td><td>153516</td><td></td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>49449</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H8000</td><td></td></tr><tr><td>Magnesium Chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>Microscope Temperature Control Stage Warmer</td><td>AmScope</td><td>TCS-100</td><td></td></tr><tr><td>MyoPacer Field Stimulator</td><td>IonOptix</td><td></td><td></td></tr><tr><td>Nunc Rectangular Dishes</td><td>Thermo Scientific</td><td>267062</td><td></td></tr><tr><td>Olympus IX83P1ZF Ixplore Standard microscope</td><td>Olympus</td><td></td><td>https://www.olympus-lifescience.com/en/microscopes/inverted/ixplore-standard/?campaignid=657680540&amp;amp;adgroupid&lt;br/&gt; =116963199831&amp;amp;keyword=ix73%20&lt;br/&gt; microscope&amp;amp;gclid=EAIaIQobChMIl&lt;br/&gt; qjyiMWP-AIVVx-tBh2JoQ85EAA&lt;br/&gt; YASAAEgLp3fD_BwE</td></tr><tr><td>pH 4.01, 7.00, and 10.01 Standards</td><td>Oakton</td><td>WD-05942-10</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>746436</td><td></td></tr><tr><td>Potassium Hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>P4494</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>795488</td><td></td></tr><tr><td>Prism Software</td><td>GraphPad Software - Dotmatics</td><td></td><td>https://www.graphpad.com/</td></tr><tr><td>RBS 25 Liquid Detergent</td><td>Sigma-Aldrich</td><td>83460</td><td></td></tr><tr><td>Sharps Container</td><td>Uline</td><td>S-15307</td><td></td></tr><tr><td>SigmaPlot analysis software</td><td>Systat Software Inc.</td><td></td><td>https://systatsoftware.com/</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>Student Dumont #5 Forceps</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr><tr><td>Temperature Control Box</td><td>Warner Insturments</td><td>TC-324C</td><td></td></tr><tr><td>Vapor Pressure Osmometer</td><td>ELITechGroup</td><td>Model 5600</td><td></td></tr><tr><td>Wheaton 20 mL Vials</td><td>DWK Life Sciences</td><td>225288</td><td></td></tr></tbody></table>

measurement of heart contractility in isolated adult human primary cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

Described in this protocol is a procedure for measuring contractility in isolated adult human primary cardiomyocytes from organ donors and evaluating acute changes in contractility parameters induced by a test compound. The measurement of the contractility transient is accomplished using a video-based cell geometry system, the MyoBLAZER, that measures sarcomere dynamics, and the adult heart condition is emulated by inducing contractility with electrical stimulation at the physiological pacing frequency (1 Hz). The functional viability of the cardiomyocytes is confirmed by assessing their excitation-contraction coupling (i.e., cellular processing linking electrical excitation [the sarcolemmal action potential] with contraction). There are no spontaneous contractility transients in human cardiomyocytes, and the cardiomyocytes respond to external electrical stimulation with contractility/relaxation cycles (Figure 2C, E), as well as to isoproterenol, a &#946;-adrenergic agonist (Figure 2F,G). Isoproterenol causes a concentration-dependent increase in contractility (Figure 2G), and its effects on the kinetics of contractility transient can also be characterized (Figure 2D)12.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64394/64394fig01.jpg" /> 
Figure 1: Experimental setup of the optical contractility recording system. Bright-field contractility recordings are made from adult human primary cardiomyocytes as previously described11,12,13,14. The setup includes a (A) temperature control box, (B) field stimulator, (C,D) pressure-driven perfusion system, (E) computer pressure-driven program, (F) pressure plus bottles, (G) acquisition software, (H) selection of ROIs with acquisition software, (I) display of contractility transients with acquisition software, (J) inverted microscope, (K) microscope chamber, and (L) camera. All features of the equipment are given in the Table of Materials. <a href="https://www.jove.com/files/ftp_upload/64394/64394fig01large.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/64394/64394fig02.jpg" /> 
Figure 2: Validation of human cardiomyocyte contractility measurement and the effect of &#946;-adrenergic stimulation. (A) Phase contrast microscopy image of a representative adult human primary cardiomyocyte, (B) user-defined regions of interest (ROI) placed over the images of healthy cardiomyocytes and parallel to the axis of contraction, (C) display of contractility transients obtained from the same ROIs in (B) with the acquisition software, (D) parameters related to the contractility transient measured with the acquisition software: contractility amplitude, time to peak, Decay 30, Decay 90, TR90. Additional parameters can also be measured: baseline sarcomere length, time to 50% peak, peak height, sarcomere length at peak contractility, maximum contraction velocity, and maximum relaxation velocity. (E) Typical contractility transients recorded from an adult human primary ventricular myocyte at a pacing frequency of 1 Hz in the presence of vehicle control (F) and after exposure to 30 nM isoproterenol, a non-selective &#946;-adrenergic agonist. The contractility transients demonstrated in panels E and F were collected from the same cardiomyocyte. Isoproterenol from human cardiomyocytes paced at 1 Hz pacing frequency was used to generate the potency information. (G) Typical cumulative concentration-response curve generated by human cardiomyocyte contractility measurement with isoproterenol (n = 8 cells). <a href="https://www.jove.com/files/ftp_upload/64394/64394fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Perfusion Sequence</td>
			<td>Vehicle solution</td>
			<td>Concentration 1 (&#181;M)</td>
			<td>Concentration 2 (&#181;M)</td>
			<td>Concentration 3 (&#181;M)</td>
			<td>Concentration 4 (&#181;M)</td>
			<td>Wash</td>
		</tr>
		<tr>
			<td>Treatment Time</td>
			<td>120 s</td>
			<td>300 s</td>
			<td>300 s</td>
			<td>300 s</td>
			<td>300 s</td>
			<td>300 s</td>
		</tr>
		<tr>
			<td>Stimulation Frequency</td>
			<td>1 Hz</td>
			<td>1 Hz</td>
			<td>1 Hz</td>
			<td>1 Hz</td>
			<td>1 Hz</td>
			<td>1 Hz</td>
		</tr>
	</tbody>
</table>
Table 1: Stimulation protocol and test compound application sequence. The stability of contractility transients is assessed by continuous recording for 120 s in solution C, establishing the vehicle control (typically in 0.1% DMSO). Subsequently, the test article concentration is applied for a minimum of 300 s or until a steady-state effect is attained. Four ascending test article concentrations are used, providing cumulative concentration-effect (C-E) curves. At the end of the cumulative additions of the test article, a 300-s washout period can be implemented.

Watch this Scientific Journal Video about Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes at JoVE.com

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

measurement-heart-contractility-isolated-adult-human-primary

Nanyang Technological University

Research

JoVE Journal

Medicine

2.0K Views.  AnaBios Corporation. This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

Video: Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

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 procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

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

Bioengineering

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Biology

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

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 ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...