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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100&#8211;1000 &#181;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 &#181;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 (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera&#160;</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&#34; OD x 0.030&#34; 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 (MgCl2)</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&#38;adgroupid 
			=116963199831&#38;keyword=ix73%20 
			microscope&#38;gclid=EAIaIQobChMIl 
			qjyiMWP-AIVVx-tBh2JoQ85EAA 
			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 (KH2PO4)</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 ...

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1.9K 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

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. After isolation and retrograde perfusion of the heart according to the Langendorff technique, the heart is inverted into an upside-down position and is mechanically stabilized by a balloon catheter inserted into the left ventricle. Then, a thin latex cap &#8211; previously cast to match the average size of the rat heart &#8211; is placed over the epicardial surface. The outlet of the latex cap is connected to silicon tubing, with the distal opening 10 cm below the base level of the heart, creating slight suction. CT continuously produced on the epicardial surface is collected in ice-cooled vials for further analysis. The rate of CT formation ranged from 17 to 147 &#181;L/min (n = 14) in control and infarcted hearts, which represents 0.1-1% of the coronary venous effluent perfusate. Proteomic analysis and high performance liquid chromatography (HPLC) revealed that the collected CT contains a wide spectrum of proteins and purinergic metabolites.

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

The present protocol describes a unique approach that enables the collection of cardiac transudate (CT) from the isolated, saline-perfused rat heart. ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...