Name: analysis of cardiac contractile dysfunction and ca2+ transients in rodent myocytes
Uploaded: 2022-05-25T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes at JoVE.com

This work is supported by National Institutes of Health (NIH) grant R01 HL144777 (MVW).

Studies performed on rodents followed the Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the University of Michigan Institutional Animal Care and Use Committee. For this study, myocytes were isolated from 3-34-month-old Sprague-Dawley and F344BN rats weighing &#8805; 200 g5. Both male and female rates were used.
1. Myocyte pacing for contractile function studies
<ol>
	<li>Make fresh M199-based media for ...

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+developing+human+fetal+cardiac+muscle.">Contractile properties of developing human fetal cardiac muscle.</a> The Journal of Physiology. 594 (2), 437-452 (2016).</li><li>Kim, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+expression+and+basal+lamina+remodeling+in+human+heart+failure.">Differential protein expression and basal lamina remodeling in human heart failure.</a> Proteomics Clinical Applications. 10 (5), 585-596 (2016).</li><li>Eckerle, L. G., Felix, S. B., Herda, L. 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H., Spiro, D., Spotnitz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultrastructure+basis+of+Starling's+law+of+the+heart:+the+role+of+the+sarcomere+is+determining+ventricular+size+and+stroke+volume.">The ultrastructure basis of Starling's law of the heart: the role of the sarcomere is determining ventricular size and stroke volume.</a> American Heart Journal. 68 (3), 336-346 (1964).</li><li>Kabaeva, Z., Zhao, M., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+extends+culture+life+of+adult+mouse+cardiac+myocytes+and+allows+efficient+and+stable+transgene+expression.">Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.</a> American Journal of Physiology-Heart and Circulatory Physiology. 294 (4), 1667-1674 (2008).</li><li>Zak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+myofibrillar+proteins+in+the+normal+and+hypertrophic+heart.">Metabolism of myofibrillar proteins in the normal and hypertrophic heart.</a> Basic Research in Cardiology. 72 (2-3), 235-240 (1977).</li><li>Palmer, B. M., Thayer, A. M., Snyder, S. M., Moore, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shortening+and+[Ca2+]+dynamics+of+left+ventricular+myocytes+isolated+from+exercise-trained+rats.">Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats.</a> Journal of Applied Physiology. 85 (6), 2159-2168 (1998).</li></ol>

The chronic pacing protocol outlined in step 1 extends the useful time for studying isolated myocytes and assessing the impact of longer treatments. In our lab, consistent results were obtained up to 4 days post isolation when measuring contractile function using sarcomere length on chronically paced myocytes. However, myocyte contractile function deteriorates quickly when using media older than 1 week to pace myocytes.
For contractile function studies, the data are collected at 37 &#176;C, wh...

The analysis of cardiac pump function often requires a range of approaches to gain adequate insight, especially for animal models of heart failure (HF). Echocardiography or hemodynamic measurements provide insight into in vivo cardiac dysfunction1, while in vitro approaches are often employed to identify whether dysfunction arises from changes in the myofilament and/or the Ca2+ transient responsible for coupling excitation, or the action potential, with contractile function (e.g., excitation-contraction [E-C] coupling). In vitro approaches also provide an opportunity to screen the functional response to neurohormones, vector-induced genetic alterations, as well as potential therapeutic agents2 prior to pursuing costly and/or laborious in vivo treatment strategies.
Several approaches are available to investigate in vitro contractile function, including force measurements in intact trabeculae3 or permeabilized myocytes4, as well as unloaded shortening and Ca2+ transients in intact myocytes in the presence and absence of HF5,6. Each of these approaches focuses on cardiac myocyte contractile function, which is directly responsible for cardiac pump function2,7. However, the analysis of both contraction and E-C coupling together is most often performed by measuring shortening of the muscle length and Ca2+ transients in isolated, Ca2+ tolerant adult myocytes. The laboratory utilizes a detailed published protocol to isolate myocytes from rat hearts for this step8.
Both the Ca2+ transient and myofilaments contribute to shortening and re-lengthening in intact myocytes and can contribute to contractile dysfunction2,7. Thus, this approach is recommended when in vitro functional analysis requires an intact myocyte containing the Ca2+ cycling machinery plus the myofilaments. For example, intact isolated myocytes are desirable for studying contractile function after modifying the myofilament or Ca2+ cycling function via gene transfer9. In addition, an intact myocyte approach is suggested for analyzing the functional impact of neurohormones when studying the impact of downstream second messenger signaling pathways and/or response to therapeutic agents2. An alternative measurement of load-dependent force in single myocytes is most often performed after membrane permeabilization (or skinning) at low temperatures (&#8804;15 &#176;C) to remove the Ca2+ transient contribution and focus on myofilament function10. The measurement of load-dependent force plus Ca2+ transients in intact myocytes is rare due largely to the complex and technical challenge of the approach11, especially when higher throughput is needed, such as for measuring responses to neurohormone signaling or as a screen for therapeutic agents. The analysis of cardiac trabeculae overcomes these technical challenges but also may be influenced by non-myocytes, fibrosis, and/or extracellular matrix remodeling2. Each of the approaches described above requires a preparation containing adult myocytes because neonatal myocytes and myocytes derived from inducible pluripotent stem cells (iPSCs) do not yet express the full complement of adult myofilament proteins and usually lack the level of myofilament organization present in the adult rod-shaped myocyte2. To date, evidence in iPSCs indicates that the full transition to adult isoforms exceeds more than 134 days in culture12.
Given the focus of this collection on HF, the protocols include approaches and analysis to differentiate contractile function in failing versus non-failing intact myocytes. Representative examples are provided from rat myocytes studied 18-20 weeks after a supra-renal coarctation, described earlier5,13. Comparisons are then made to myocytes from sham-treated rats.
The protocol and imaging platform described here are used to analyze and monitor changes in shortening and Ca2+ transients in rod-shaped cardiac myocytes during the development of HF. For this analysis, 2 x 104 Ca2+-tolerant, rod-shaped myocytes are plated on 22 mm2 laminin-coated glass coverslips (CSs) and cultured overnight, as described earlier8. The components assembled for this imaging platform, along with the media and buffers used for optimal imaging, are provided in the Table of Materials. A guide for data analysis using a software and the representative results are also provided here. The overall protocol is broken down into separate sub-sections, with the first three sections focusing on isolated rat myocytes and data analysis, followed by cellular Ca2+ transient experiments and data analysis in myocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MEDIA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma (Roche)</td>
			<td>3117057001</td>
			<td>Final concentration = 0.2% (w/v)</td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma</td>
			<td>G-6529</td>
			<td>Final concentration = 10 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-7006</td>
			<td>Final concentration = 15 mM</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>Sigma</td>
			<td>M-2520</td>
			<td>1 bottle makes 1 L; pH 7.45</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S-8875</td>
			<td>Final concentration = 4 mM</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Fisher</td>
			<td>15140122</td>
			<td>Final concentration = 100 U/mL penicillin, 100 &#956;g/mL streptomycin</td>
		</tr>
		<tr>
			<td>REAGENTS SPECIFICALLY FOR Ca2+ IMAGING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma&#160;</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2AM</td>
			<td>Invitrogen (Molecular Probes)</td>
			<td>F1221</td>
			<td>50 &#956;g/vial;&#160; Prepare stock solution of 1 mM Fura-2AM + 0.5 M probenicid in DMSO;&#160; Final Fura2-AM concentration in media is&#160; 5 &#956;M</td>
		</tr>
		<tr>
			<td>Probenicid</td>
			<td>Invitrogen (Fisher)</td>
			<td>P36400</td>
			<td>Add 7.2 mg probenicid (0.5 M) to 1 mM Fura-2AM stock; Final concentration in media is 2.5 mM</td>
		</tr>
		<tr>
			<td>MATERIALS FOR RAT MYOCYTE PACING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>#1 22&#160; mm2 glass coverslips</td>
			<td>Corning</td>
			<td>2845-22</td>
			<td></td>
		</tr>
		<tr>
			<td>3 x 36 inch cables with banana jacks</td>
			<td>Pomona Electronics</td>
			<td>B-36-2</td>
			<td>Supplemental Figure 1, panel C</td>
		</tr>
		<tr>
			<td>37oC Incubator&#160; with 95% O2:5% CO2&#160;</td>
			<td>Forma</td>
			<td>3110</td>
			<td>Supplemental Figure 1, panel E.&#160; Multiple models are appropriate</td>
		</tr>
		<tr>
			<td>Class II A/B3 Biosafety cabinet with UV lamp</td>
			<td>Forma</td>
			<td>1286</td>
			<td>Multiple models are appropriate</td>
		</tr>
		<tr>
			<td>Forceps - Dumont #5 5/45</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>1800-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Low magnification inverted microscope</td>
			<td>Leica</td>
			<td>DM-IL&#160;</td>
			<td>Position this microscope adjacent to the incubator to monitor paced myocytes for contraction at the start of pacing and after media changes; &#160;4X and 10X objectives recommended</td>
		</tr>
		<tr>
			<td>Pacing chamber</td>
			<td>Custom</td>
			<td></td>
			<td>Supplemental Figure 1, panel A. The Ionoptix C-pace system is a commercially available alternative or see 22</td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Ionoptix</td>
			<td>Myopacer</td>
			<td>Supplemental Figure 1, panel D.</td>
		</tr>
		<tr>
			<td>MATERIALS FOR CONTRACTILE FUNCTION and/or Ca2+ IMAGING ANALYSIS</td>
			<td></td>
			<td></td>
			<td>ID in Supplemental Figure 2 &#38; Alternatives/Recommended Options</td>
		</tr>
		<tr>
			<td>Additional components for Ca2+ imaging analysis</td>
			<td>Ionoptix</td>
			<td>Essential system components: -- Photon counting system&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; --&#160; Xenon power supply with dual excitation light source&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; --&#160; Fluorescence interface</td>
			<td>&#160;- The photon counting system contains a photomultiplier (PMT) tube and dichroic mirrorand is installed adjacent to the CCD camera&#160; (panel A #4).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; - The power supply for the xenon bulb light source (see panel A #5 and panel C, left) is integrated with a dual excitation interface (340/380 nm&#160; excitation and 510 nm emission) shown in panel A #6.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; - The fluorescence interface between the computer and&#160; light source is shown in panel B, #12.</td>
		</tr>
		<tr>
			<td>CCD camera with image acquisition&#160; hardware and software (240 frames/s)</td>
			<td>Ionoptix&#160;</td>
			<td>Myocam with CCD controller&#160;</td>
			<td>Myocam and CCD controller are shown in Supplemental Fig. 2, panel A #4&#160; and&#160; panel A #5 &#38; panel C #5 (right), respectively.&#160; The controller is integrated with a PC computer system (panel B #14).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>&#160;Chamber stimulator</td>
			<td>Ionoptix&#160;</td>
			<td>Myopacer</td>
			<td>Panel B, #13; Alternative:&#160; Grass model S48</td>
		</tr>
		<tr>
			<td>Coverslip mounted perfusion chamber</td>
			<td>Custom chamber for 22 mm2 coverslip with silicone adapter and 2-4 Phillips pan-head #0 screws&#160; (arrow, panel F)</td>
			<td></td>
			<td>Panel A #10 &#38; panel F;&#160;&#160; Chamber temperature is calibrated to 37oC using a TH-10Km probe and the TC2BIP temperature controller (see temperature controller).&#160; Commercial alternatives:&#160; Ionoptix FHD or C-stim cell&#160; chambers; Cell MicroControls culture stimulation system</td>
		</tr>
		<tr>
			<td>Dedicated computer &#38; software for data collection and analysis of function/Ca2+ transients</td>
			<td>Ionoptix</td>
			<td>PC with Ionwizard PC board and software</td>
			<td>Panel B, #14; Contractile function is measured using either SarcLen (sarcomere length) or SoftEdge (myocyte length) acquisition modules of the IonWizard software. The Ionwizard software also includes PMT acquisition software for ratiometric&#160; Ca2+ imaging in Fura-2AM-loaded myoyctes. 
			- A 4 post electronic rack mount cabinet and shelves are recommended for housing the somputer and cell stimulator.&#160; The fluorescence interface for Ca2+ imaging also is housed in this cabinet (see below).</td>
		</tr>
		<tr>
			<td>Forceps - Dumont #5 TI</td>
			<td>Fine Science Tools</td>
			<td>11252-40</td>
			<td>Panel F</td>
		</tr>
		<tr>
			<td>Insulated tube holder for media</td>
			<td>Custom</td>
			<td></td>
			<td>Panel A #9; This holder is easily assembled using styrofoam &#38; a pre-heated gel pack to keep media warm</td>
		</tr>
		<tr>
			<td>Inverted brightfield microscope</td>
			<td>Nikon</td>
			<td>TE-2000S</td>
			<td>Install a rotating turret for epi-fluorescence (Panel A #2) for Ca2+ imaging.&#160; &#160;A deep red (590 nm) condenser filter also is recommended to minimize fluorescence bleaching during Ca2+ imaging.</td>
		</tr>
		<tr>
			<td>Isolator Table</td>
			<td>TMC Vibration Control</td>
			<td>30 x 36 inches</td>
			<td>Panel A, #1; Desirable:&#160; elevated shelving, Faraday shielding&#160;&#160;</td>
		</tr>
		<tr>
			<td>Microscope eyepieces &#38; objective</td>
			<td>Nikon</td>
			<td>10X CFI eyepieces 40X water CFI Plan Fluor objective</td>
			<td>Panel A #3; 40X objective:&#160; n.a. 0.08; w.d. 2 mm.&#160; A Cell MicroControls HLS-1 objective heater is mounted around the objective (see temperature controller below).&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;NOTE:&#160; water immersion dispensers also are now available for water-based objectives.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Minipuls 3</td>
			<td>Panel A #8 and panel E</td>
		</tr>
		<tr>
			<td>small weigh boat</td>
			<td>Fisher&#160;</td>
			<td>08-732-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Cell MicroControls</td>
			<td>TC2BIP</td>
			<td>Panel A #7; Panel D. This temperature controller heats the coverslip chamber to 37oC.&#160;&#160;&#160; A preheater and objective heater are recommended for this platform.&#160; A Cell MicroControls HPRE2 preheater and&#160; HLS-1 objective heater are controlled&#160; by the TC2BIP temperature controller for our studies.</td>
		</tr>
		<tr>
			<td>Under cabinet LED light with motion sensor&#160;</td>
			<td>Sylvania</td>
			<td>#72423 LED light</td>
			<td>Recommended for data collection during Ca2+ transient imaging under minimal room light..&#160; Alternative:&#160; A clip on flashlight/book light with flexible neck - multiple suppliers are available.</td>
		</tr>
		<tr>
			<td>Vacuum line with in-line Ehrlenmeyer flask &#38; protective filter</td>
			<td>Fisher</td>
			<td>Tygon tubing - E363;&#160; polypropylene Ehrlenmeyer flask - 10-182-50B; Vacuum filter - 09-703-90</td>
			<td>Panel A #11</td>
		</tr>
	</tbody>
</table>

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.

Contractile function studies are performed on rat myocytes starting the day after isolation (day 2) up to 4 days post isolation. Although myocytes can be recorded the day after isolation (i.e., day 2), longer culture times are often required after gene transfer or treatments to modify contractile function8. For myocytes cultured for more than 18 h after isolation, the pacing protocol described in section 1 helps maintain t-tubules and consistent shortening and re-lengthening results.
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Watch this Scientific Journal Video about Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes at JoVE.com

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.

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 ...

This approach detects changes in contractile function during the development of heart failure. The functional response in myocytes to neurohormones and potential therapeutic agents can also be screened. Positioning the myocyte may take practice but is essential in obtaining consistent sarcomere length imaging.Demonstrating the procedure is Margaret Westfall, associate professor and principal investigator of the laboratory. Before starting the experiment, preheat a gel pack and media in an incubator at 37 degrees Celsius for 30 minutes. Turn on the contractile function platform components as mentioned in the manuscript and assemble tubing into the peristaltic pump.Then, transfer the preheated gel pack into the tube holder and turn on the vacuum in the power to the cell stimulator. Place a 50 milliliter tube with media in the insulated tube holder to begin perfusion at 0.5 milliliters per minute through the peristaltic pump tubing. Next, add two milliliters of preheated media to a small weigh boat and transfer one cover slip containing myocytes from the stimulation chamber to the weigh boat.Return the stimulation chamber to the incubator at 37 degrees Celsius and 5%carbon dioxide. Remove the cover slip from the weigh boat and gently wipe the underside before transferring it to a freshly greased cover slip chamber and adding a drop of media onto the cover slip. Place a platinum electrode mount over the cover slip, then lay a new cover slip over the top with forceps and place a top mount over the cover slip sandwich.Install two or four number zero panhead screws to finish assembling the chamber. In the peristaltic pump when the media is present throughout the tubing, attach the tubing to the preheater and the preheater to the cover slip chamber. Then, begin perfusion at 0.5 milliliters per minute and place a tissue wipe below the chamber to ensure no leaks.After placing the cover slip chamber in the stage adapter of the microscope, turn on the heating system and equilibriate the assembly for five to 10 minutes to achieve a constant media temperature of 37 degrees Celsius in the center of the chamber. Observe the perfused media being collected at the opposite end of the chamber with tubing connected to a vacuum system. During the equilibration time, visualize myocytes with the 40x water immersion objective on the microscope.To activate the pacing stimulator, set the voltage to 35 to 40 volts and adjust the stimulation frequency to 0.2 hertz to optimize contractile function and myocyte survival. To collect sarcomere shortening traces, open the software on the computer and select okay, file and new tabs. Prepare a screen template by selecting traces, edit user limits and sarcomere length.Record sarcomere length traces with the file and new tabs, identify a contracting myocyte and position the myocyte along the camera's longitudinal axis so the striation pattern is vertical. Use the computer mouse to place the region of interest or ROI box over the myocyte. Select collect and start to record contractile function.Record shortening for 60 seconds from each myocyte at low stimulation frequencies of 0.5 hertz. Record sarcomere shortening from five to 10 cells per cover slip. To measure shortening over a range of frequencies, place the myocytes at each frequency to obtain steady state shortening prior to recording.Double the perfusion rate and begin recording 15 to 20 seconds after stimulation at a new frequency in the range of 0.2 to two hertz and record no more than two myocytes per cover slip for shortening across the given range of stimulation frequencies. Record at least seven contractions per cell to obtain reliable signal averaged data. Select the file, choose a recorded trace and then select open.Select tab one of the base trace and then set the yellow panel at the top of the trace to obtain sarc length. Prepare an analysis template with operations monotonic transient analysis options and TTL event mark in the definition of T Zero box in the necessary options from the menu. Save these analysis options by selecting templates and save analysis template with an identifier.Load the analysis template before analyzing each trace. To analyze the data, select marks, gate, add transient then convert from event mark and analysis range. Set the time range from minus 0.01 to 1.20 seconds for myocytes paced at 0.2 hertz.Select a shorter time range for myocytes stimulated at higher frequencies. Select operations and average events to produce a signal averaged recording below the original base trace. Next, select tab one of the signal averaged trace and then go to marks on the upper menu followed by add transient, choose operations and monotonic transient analysis to display the signal averaged values for baseline sarcomere parameters in the signal averaged display panel.Select export monotonic transient analysis and clipboard current and transfer transient sarcomere analysis to a spreadsheet for the composite analysis of multiple myocytes. To copy the signal averaged trace, select export, current trace, then clipboard and options tabs in order and set the decimal places to five. Choose tabs delimiter and click okay.Pace the signal averaged traces for each myocyte recording into a second spreadsheet. The study showed that the pressure overload or PO, reduced the contractile function of the myocytes when compared to the sham group. However, four days after the gene transfer of cardiac troponin IT 144D or cTnIT144D, the contractile ability in the PO myocytes returned towards sham levels, indicating myocyte function could be restored.In the initial studies, the gene transfer of cTnIT144D enhanced peak shortening and elevated diastolic calcium levels compared to CTnI. Fine adjustments in positioning of the cardiac myocyte are often needed to obtain clear shortening traces. This protocol can also be performed on cardiac myocytes loaded with calcium sensitive fluorescent dyes such as Fura-2 AM to detect calcium transients in addition to shortening.

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