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>
	<li>Reihle, C., Bauersachs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+of+heart+failure.">Small animal models of heart failure.</a> Cardiovascular Research. 115 (13), 1838-1849 (2019).</li><li>Peter, A. K., Bjerke, M. A., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+the+cardiac+myocyte+in+heart+disease.">Biology of the cardiac myocyte in heart disease.</a> Molecular Biology of the Cell. 27 (14), 2149-2160 (2016).</li><li>Rossman, E. I., 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=Abnormal+frequency-dependent+responses+represent+the+pathophysiologic+signature+of+contractile+failure+in+human+myocardium.">Abnormal frequency-dependent responses represent the pathophysiologic signature of contractile failure in human myocardium.</a> Journal of Molecular and Cellular Cardiology. 36 (1), 33-42 (2004).</li><li>McDonald, K. S., Hanft, L. M., Robinett, J. C., Guglin, M., Campbell, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+myofilament+contractile+function+in+human+donor+and+failing+hearts.">Regulation of myofilament contractile function in human donor and failing hearts.</a> Frontiers in Physiology. 11, 468 (2020).</li><li>Ravichandran, V. S., Patel, H. J., Pagani, F. D., Westfall, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+contractile+dysfunction+and+protein+kinase+C-mediated+myofilament+phosphorylation+in+disease+and+aging.">Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging.</a> The Journal of General Physiology. 151 (9), 1070-1080 (2019).</li><li>Chaudhary, K. 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=Altered+myocardial+Ca2++cycling+after+left+ventricular+assist+device+support+in+failing+human+heart.">Altered myocardial Ca2+ cycling after left ventricular assist device support in failing human heart.</a> Journal of the American College of Cardiology. 44 (4), 837-845 (2004).</li><li>Walker, C. A., Spinale, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+function+of+the+cardiac+myocyte:+A+review+of+fundamental+concepts.">The structure and function of the cardiac myocyte: A review of fundamental concepts.</a> The Journal of Thoracic and Cardiovascular Surgery. 118 (2), 375-382 (1999).</li><li>Lang, S. E., Westfall, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+cardiac+myocytes.">Gene transfer into cardiac myocytes.</a> Methods in Molecular Biology. 1299, 177-190 (2015).</li><li>Davis, J., 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=Designing+heart+performance+by+gene+transfer.">Designing heart performance by gene transfer.</a> Physiological Reviews. 88 (4), 1567-1651 (2008).</li><li>Sweitzer, N. K., Moss, 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=Determinants+of+loaded+shortening+velocity+in+single+cardiac+myocytes+permeabilized+with+a-hemolysin.">Determinants of loaded shortening velocity in single cardiac myocytes permeabilized with a-hemolysin.</a> Circulation Research. 73 (6), 1150-1162 (1993).</li><li>Yasuda, S., 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=Unloaded+shortening+increases+peak+of+Ca2++transients+but+accelerates+their+decay+in+rat+single+cardiac+myocytes.">Unloaded shortening increases peak of Ca2+ transients but accelerates their decay in rat single cardiac myocytes.</a> American Journal of Physiology-Heart and Circulatory Physiology. 285 (2), 470-475 (2003).</li><li>Racca, A. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+antibody+effects+on+cellular+function+of+isolated+cardiomyocytes.">Measurement of antibody effects on cellular function of isolated cardiomyocytes.</a> Journal of Visualized Experiments. (73), e4237 (2013).</li><li>Robbins, H., Koch, S. E., Tranter, M., Rubinstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+future+of+probenecid.">The history and future of probenecid.</a> Cardiovascular Toxicology. 12 (1), 1-9 (2012).</li><li>Sakthivel, S., 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=In+vivo+and+in+vitro+analysis+of+cardiac+troponin+I+phosphorylation.">In vivo and in vitro analysis of cardiac troponin I phosphorylation.</a> Journal of Biological Chemistry. 280 (1), 703-714 (2005).</li><li>Qi, M., 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=Downregulation+of+sarcoplasmic+reticulum+Ca2+-ATPase+during+progression+of+left+ventricular+hypertrophy.">Downregulation of sarcoplasmic reticulum Ca2+-ATPase during progression of left ventricular hypertrophy.</a> The American Journal of Physiology. 272 (5), 2416-2424 (1997).</li><li>Sonnenblick, E. 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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1.3K ビュー.  University of Michigan, Ann Arbor. このアプローチは、心不全の発症中の収縮機能の変化を検出します。神経ホルモンおよび潜在的な治療薬に対する筋細胞における機能的応答もスクリーニングすることができる。筋細胞の位置決めには練習が必要ですが、一貫したサルコメア長イメージングを得るために不可欠です。手順を実演するのは、研究所の准教授兼主任研究者であるマーガレットウェスト?...

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

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...