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

这项工作得到了美国国立卫生研究院（NIH）拨款R01 HL144777（MVW）的支持。

对啮齿动物进行的研究遵循公共卫生服务关于人道护理和使用实验动物的政策，并获得了密歇根大学机构动物护理和使用委员会的批准。在这项研究中，从体重≥200g5的3-34个月大的Sprague-Dawley和F344BN大鼠中分离出肌细胞。使用男性和女性比率。
1. 肌细胞起搏用于收缩功能研究
<ol><li>为每组分离的肌细胞制作新鲜的基于M199的培养基（材料?...

<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. 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>

步骤1中概述的慢性起搏方案延长了研究分离的肌细胞和评估长期治疗影响的有用时间。在我们的实验室中，当使用慢性起搏的肌细胞上的肌节长度测量收缩功能时，隔离后长达 4 天获得了一致的结果。然而，当使用超过 1 周的培养基来调整心肌细胞时，肌细胞收缩功能会迅速恶化。
对于收缩功能研究，数据在37°C下收集，这接近大鼠的体温。为了优化肌细胞活力并获得每个?...

心脏泵功能分析通常需要一系列方法来获得足够的洞察力，特别是对于心力衰竭（HF）的动物模型。超声心动图或血流动力学测量可深入了解体内心功能不全1，而体外方法通常用于确定功能障碍是否由肌丝和/或负责将激发或动作电位与收缩功能偶联的 Ca2+ 瞬变（例如，激发-收缩 [E-C] 耦合）引起的。体外方法还提供了在寻求昂贵和/或费力的体内治疗策略之前筛选对神经激素、载体诱导的遗传改变以及潜在治疗剂的功能反应的机会2。
有几种方法可用于研究体外收缩功能，包括完整小梁3 或透化肌细胞4 中的力测量，以及在存在和不存在 HF5，6 的情况下完整肌细胞中的卸载缩短和 Ca2+ 瞬变。这些方法中的每一种都侧重于心肌细胞收缩功能，它直接负责心脏泵功能2，7。然而，收缩和E-C偶联的分析最常通过测量分离的Ca 2+耐受成年肌细胞中的肌肉长度缩短和Ca2+瞬变来进行。实验室利用详细的已发布方案从大鼠心脏中分离出肌细胞进行此步骤8。
Ca2+ 瞬时和肌丝都有助于完整肌细胞的缩短和再延长，并可能导致收缩功能障碍2，7。因此，当体外功能分析需要含有Ca2+循环机制和肌丝的完整肌细胞时，建议使用这种方法。例如，完整的分离肌细胞对于通过基因转移修饰肌丝或Ca2+循环功能后研究收缩功能是理想的9。此外，在研究下游第二信使信号通路的影响和/或对治疗剂的反应时，建议使用完整的肌细胞方法来分析神经激素的功能影响2。单个肌细胞中负荷依赖性力的替代测量最常在低温（≤15°C）膜透化（或蒙皮）后进行，以去除Ca2+瞬时贡献并关注肌丝功能10。完整肌细胞中负载依赖性力加上Ca2+瞬变的测量很少见，这主要是由于该方法11的复杂性和技术挑战，特别是当需要更高的通量时，例如测量对神经激素信号的反应或作为治疗剂的筛选。心脏小梁的分析克服了这些技术挑战，但也可能受到非肌细胞、纤维化和/或细胞外基质重塑的影响2。上述每种方法都需要含有成人肌细胞的制剂，因为新生儿肌细胞和来源于诱导多能干细胞（iPSC）的肌细胞尚未表达成人肌丝蛋白的全部补体，并且通常缺乏成人杆状肌细胞中存在的肌丝组织水平2。迄今为止，iPSCs的证据表明，在培养物中，向成体亚型的完全过渡超过134天12。
鉴于本系列的重点是HF，这些协议包括区分衰竭与非衰竭完整肌细胞收缩功能的方法和分析。代表性的例子来自在肾上缩窄后18-20周研究的大鼠肌细胞，如前所述5，13。然后与假处理的大鼠的肌细胞进行比较。
此处描述的协议和成像平台用于分析和监测HF发展过程中杆状心肌细胞中缩短和Ca2+ 瞬变的变化。对于该分析，将2 x 104 Ca 2+耐受的杆状肌细胞接种在22mm 2 层粘连蛋白涂层玻璃盖玻片（CSs）上并培养过夜，如前所述8。材料 表中提供了为该成像平台组装的组件，以及用于最佳成像的培养基和缓冲液。此处还提供了使用软件进行数据分析的指南和代表性结果。整个方案分为单独的子部分，前三个部分侧重于分离的大鼠肌细胞和数据分析，然后是细胞Ca 2 + 瞬时实验和肌细胞数据分析。

<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

收缩功能障碍和Ca2+ 瞬变通常在细胞水平上进行分析，作为心脏诱导损伤和/或重塑综合评估的一部分。评估这些功能改变的一种方法是在原发性成人心肌细胞中使用卸载缩短和Ca2+ 瞬时分析。对于这种方法，通过胶原酶消化分离成体肌细胞，使其具有Ca 2+ 耐受性，然后粘附在层粘连蛋白包被的盖玻片上，然后在无血清培养基中进行电起搏。一般方案利用成年大鼠心肌细胞，但可以很容易地调整来自其他物种的原代肌细胞。受伤心脏肌细胞的功能改变可以与假心肌细胞和/或 体外 治疗进行比较。该方法包括肌细胞起搏所需的基本要素，以及细胞室和平台组件。这种方法的详细协议包括通过肌节长度检测测量空载缩短和使用比率指示剂Fura-2 AM测量的细胞Ca2+ 瞬变以及原始数据分析的步骤。

从分离后的第二天（第2天）开始至隔离后4天对大鼠肌细胞进行收缩功能研究。虽然可以在分离后的第二天（即第2天）记录肌细胞，但在基因转移或处理后通常需要更长的培养时间以改变收缩功能8。对于分离后培养超过18小时的肌细胞，第1节中描述的起搏方案有助于维持T小管和一致的缩短和重新延长结果。
图 1A显示了含有用于缩短研...

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

提出了一组协议，描述了通过肌节长度检测以及钙（Ca 2+）瞬时测量在分离的大鼠肌细胞中测量收缩功能。这种方法在心力衰竭动物模型中的应用也包括在内。

啮齿动物心肌细胞心脏收缩功能障碍和Ca2+ 瞬变分析

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

这种方法检测心力衰竭发展过程中收缩功能的变化。还可以筛选肌细胞对神经激素和潜在治疗药物的功能反应。定位肌细胞可能需要练习，但对于获得一致的肌节长度成像至关重要。演示该程序的是实验室副教授兼首席研究员Margaret Westfall。在开始实验之前，将凝胶包和培养基在37摄氏度的培养箱中预热30分钟。打开稿件中提到的收缩功能平台组件，将管道组装到蠕动泵中。然后，将预热的凝胶包转移到管支架中，并在细胞刺激器的电源中打开真空。将带有介质的 50 毫升管放入绝缘管支架中，以每分钟 0.5 毫升的速度开始通过蠕动泵管灌注。接下来，将两毫升预热的培养基加入一个小称重舟中，并将一个含有肌细胞的盖玻片从刺激室转移到称重船上。将刺激室在37摄氏度和5%二氧化碳下返回培养箱。从秤船上取下盖玻片并轻轻擦拭底面，然后将其转移到刚涂过油的盖玻片室中，并在盖玻片上添加一滴介质。将铂电极支架放在盖玻片上，然后用镊子在顶部放置一个新的盖玻片，并将顶部支架放在盖玻片三明治上。安装两个或四个零号盘头螺钉以完成腔室的组装。在蠕动泵中，当介质存在于整个管道中时，将管道连接到预热器，将预热器连接到盖滑室。然后，以每分钟0.5毫升的速度开始灌注，并在腔室下方放置纸巾擦拭以确保没有泄漏。将盖玻片室放入显微镜的载物台适配器后，打开加热系统并使组件平衡5至10分钟，以在室中心实现37摄氏度的恒定介质温度。观察在腔室另一端收集的灌注介质，管道连接到真空系统。在平衡期间，在显微镜上使用40倍水浸物镜观察肌细胞。要激活起搏刺激器，请将电压设置为 35 至 40 伏，并将刺激频率调整为 0.2 赫兹，以优化收缩功能和肌细胞存活。要收集肌节缩短痕迹，请在计算机上打开软件并选择确定，文件和新选项卡。通过选择迹线、编辑用户限制和肌节长度来准备屏幕模板。使用文件和新选项卡记录肌节长度轨迹，识别收缩的肌细胞并沿相机的纵轴定位肌细胞，使条纹图案垂直。使用计算机鼠标将感兴趣区域或ROI框放在肌细胞上。选择收集并开始记录收缩功能。记录每个肌细胞在0.5赫兹的低刺激频率下缩短60秒。记录肌节缩短，从每张盖玻片 5 个细胞缩短到 10 个细胞。为了测量一系列频率的缩短，请在记录之前将肌细胞放置在每个频率以获得稳态缩短。将灌注速率加倍，并在刺激后 15 至 20 秒以 0.2 至 2 赫兹范围内的新频率开始记录，并且每个盖玻片记录不超过两个肌细胞，以便在给定的刺激频率范围内缩短。记录每个细胞至少七次收缩，以获得可靠的信号平均数据。选择文件，选择记录的跟踪，然后选择“打开”。选择基本跟踪的一个选项卡，然后在跟踪顶部设置黄色面板以获取 sarc 长度。准备一个分析模板，其中包含操作单调瞬态分析选项和 TTL 事件标记，在菜单中的必要选项中的 T 零定义框中。通过选择模板保存这些分析选项，并使用标识符保存分析模板。在分析每个跟踪之前加载分析模板。要分析数据，请选择标记，门控，添加瞬态，然后从事件标记和分析范围转换。将肌细胞的时间范围设置为负 0.01 到 1.20 秒，步调为 0.2 赫兹。为以较高频率刺激的肌细胞选择较短的时间范围。选择操作和平均事件以在原始基本迹线下方生成信号平均记录。接下来，选择信号平均迹线的选项卡之一，然后转到上方菜单上的标记，然后添加瞬态，选择操作和单调瞬态分析以在信号平均显示面板中显示基线肌节参数的信号平均值。选择导出单调瞬态分析和剪贴板电流，并将瞬态肌节分析传输到电子表格，以便对多个肌细胞进行复合分析。要复制信号平均迹线，请依次选择导出、当前迹线，然后选择剪贴板和选项选项卡，并将小数位数设置为 5。选择制表符分隔符，然后单击确定。将每个心肌细胞记录的信号平均迹线放入第二个电子表格中。研究表明，与假组相比，压力超负荷或PO降低了肌细胞的收缩功能。然而，在心肌肌钙蛋白IT 144D或cTnIT144D基因转移4天后，PO肌细胞的收缩能力恢复到假水平，表明肌细胞功能可以恢复。在最初的研究中，与CTnI相比，cTnIT144D的基因转移增强了峰值缩短和舒张期钙水平升高。通常需要微调心肌细胞的位置以获得清晰的缩短痕迹。该方案也可以对装载有钙敏感荧光染料（如Fura-2 AM）的心肌细胞进行，以检测钙瞬变以及缩短。

Research

JoVE Journal

Medicine

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

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

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

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

科研

医学

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

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

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

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

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

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

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

从成年小鼠分离心膜肌细胞

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

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

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

人类心脏肌细胞模型中体温过低预科后心肌保护体外评估

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

严重卵巢功能障碍患者的生育保护

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

An Intact Pericardium Ischemic Rodent Model

完整的心包缺血性啮齿动物模型

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

罗斯手术的啮齿动物模型：全身位置的同源肺动脉移植植入

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