Name: in vitro assessment of myocardial protection following hypothermia-preconditioning in a human cardiac myocytes model
Uploaded: 2020-10-27T14:45:00
Description: Watch this Scientific Journal Video about In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model at JoVE.com

这项工作部分由中国国家自然科学基金委员会（81970265，81900281，81700288）、中国博士后科学基金会（2019M651904） 资助：以及中国国家重点研究发展计划（2016年YFC1101001，2017YFC1308105）。

有关商业试剂和仪器的信息列在 材料表中。
AC16人类心肌细胞系源自成人心室心脏组织的主要细胞与SV40转化的人类成纤维细胞17的融合，这些细胞是从BLEFBI（中国上海）购买的。细胞系具有心肌细胞的许多生化和形态特征。此外，细胞系被广泛用于评估心肌损伤和心肌功能体外18，19。
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<ol>
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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=Hsp90aa1:+a+novel+target+gene+of+miR-1+in+cardiac+ischemia/reperfusion+injury.">Hsp90aa1: a novel target gene of miR-1 in cardiac ischemia/reperfusion injury.</a> Sci Rep. 6, 24498 (2016).</li><li>Castedo, E., 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=Influence+of+hypothermia+on+right+atrial+cardiomyocyte+apoptosis+in+patients+undergoing+aortic+valve+replacement.">Influence of hypothermia on right atrial cardiomyocyte apoptosis in patients undergoing aortic valve replacement.</a> Journal of Cardiothoracic Surgery. 2, 7 (2007).</li><li>Krech, 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=Moderate+therapeutic+hypothermia+induces+multimodal+protective+effects+in+oxygen-glucose+deprivation/reperfusion+injured+cardiomyocytes.">Moderate therapeutic hypothermia induces multimodal protective effects in oxygen-glucose deprivation/reperfusion injured cardiomyocytes.</a> Mitochondrion. 35, 1-10 (2017).</li><li>Cooper, W. A., 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=Hypothermic+circulatory+arrest+causes+multisystem+vascular+endothelial+dysfunction+and+apoptosis.">Hypothermic circulatory arrest causes multisystem vascular endothelial dysfunction and apoptosis.</a> ANNALS OF THORACIC SURGERY. 69 (3), 696-702 (2000).</li><li>Kajimoto, 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=Selective+cerebral+perfusion+prevents+abnormalities+in+glutamate+cycling+and+neuronal+apoptosis+in+a+model+of+infant+deep+hypothermic+circulatory+arrest+and+reperfusion.">Selective cerebral perfusion prevents abnormalities in glutamate cycling and neuronal apoptosis in a model of infant deep hypothermic circulatory arrest and reperfusion.</a> JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM. 36 (11), 1992-2004 (2016).</li><li>Liu, Y., 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=Deep+Hypothermic+Circulatory+Arrest+Does+Not+Show+Better+Protection+for+Vital+Organs+Compared+with+Moderate+Hypothermic+Circulatory+Arrest+in+Pig+Model.">Deep Hypothermic Circulatory Arrest Does Not Show Better Protection for Vital Organs Compared with Moderate Hypothermic Circulatory Arrest in Pig Model.</a> Biomed Research International. 2019, 1420216 (2019).</li><li>Davidson, M. 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=Novel+cell+lines+derived+from+adult+human+ventricular+cardiomyocytes.">Novel cell lines derived from adult human ventricular cardiomyocytes.</a> JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY. 39 (1), 133-147 (2005).</li><li>Khan, K., Makhoul, G., Yu, B., Schwertani, A., Cecere, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytoprotective+impact+of+yes-associated+protein+1+after+ischemia-reperfusion+injury+in+AC16+human+cardiomyocytes.">The cytoprotective impact of yes-associated protein 1 after ischemia-reperfusion injury in AC16 human cardiomyocytes.</a> EXPERIMENTAL BIOLOGY AND MEDICINE. 244 (10), 802-812 (2019).</li><li>Pan, J. A., 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=miR-146a+attenuates+apoptosis+and+modulates+autophagy+by+targeting+TAF9b/P53+pathway+in+doxorubicin-induced+cardiotoxicity.">miR-146a attenuates apoptosis and modulates autophagy by targeting TAF9b/P53 pathway in doxorubicin-induced cardiotoxicity.</a> Cell Death Discovery. 10 (9), 668 (2019).</li><li>Schmitt, K. R., 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=S100B+modulates+IL-6+release+and+cytotoxicity+from+hypothermic+brain+cells+and+inhibits+hypothermia-induced+axonal+outgrowth.">S100B modulates IL-6 release and cytotoxicity from hypothermic brain cells and inhibits hypothermia-induced axonal outgrowth.</a> NEUROSCIENCE RESEARCH. 59 (1), 68-73 (2007).</li><li>Tong, G., 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=Deep+hypothermia+therapy+attenuates+LPS-induced+microglia+neuroinflammation+via+the+STAT3+pathway.">Deep hypothermia therapy attenuates LPS-induced microglia neuroinflammation via the STAT3 pathway.</a> Neuroscience. 358, 201-210 (2017).</li><li>Yu, Z. P., 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=Troxerutin+attenuates+oxygenglucose+deprivation+and+reoxygenationinduced+oxidative+stress+and+inflammation+by+enhancing+the+PI3K/AKT/HIF1alpha+signaling+pathway+in+H9C2+cardiomyocytes.">Troxerutin attenuates oxygenglucose deprivation and reoxygenationinduced oxidative stress and inflammation by enhancing the PI3K/AKT/HIF1alpha signaling pathway in H9C2 cardiomyocytes.</a> Molecular Medicine Reports. 22 (2), 1351-1361 (2020).</li><li>Drescher, C., Diestel, A., Wollersheim, S., Berger, F., Schmitt, K. 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S., Schaible, N., Tveita, T., Sieck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discontinued+stimulation+of+cardiomyocytes+provides+protection+against+hypothermia-rewarming-induced+disruption+of+excitation-contraction+coupling.">Discontinued stimulation of cardiomyocytes provides protection against hypothermia-rewarming-induced disruption of excitation-contraction coupling.</a> EXPERIMENTAL PHYSIOLOGY. 103 (6), 819-826 (2018).</li><li>Yarbrough, W. 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=Caspase+inhibition+attenuates+contractile+dysfunction+following+cardioplegic+arrest+and+rewarming+in+the+setting+of+left+ventricular+failure.">Caspase inhibition attenuates contractile dysfunction following cardioplegic arrest and rewarming in the setting of left ventricular failure.</a> Journal of cardiovascular pharmacology. 44 (6), 645-650 (2004).</li><li>Egorov, Y. V., Glukhov, A. V., Efimov, I. R., Rosenshtraukh, L. 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完整动物的复杂性，包括不同类型的细胞之间的相互作用，往往妨碍对I/R损伤特定成分的详细研究。因此，有必要建立一个体外细胞模型，能够准确反映体内缺血后的分子变化。关于OGD模型的研究已经报告13，22，许多复杂的方法已经建立23，24。OGD 模型的制备过程包括两个关键步骤：缺氧和葡萄糖剥夺。在本...

缺血/输液衍生心肌功能障碍是患者心脏手术后常见的临床方案1，2。在非脉动低流量灌注和总循环阻滞期间，仍发生涉及所有类型心脏细胞的损伤。特别是，心肌细胞对缺血性损伤的敏感性高于其他细胞群体。目前，治疗性体温过低（TH）为接受心脏手术的病人提供了实质性的保护，防止预期的缺血性侮辱。TH 被定义为 14-34 °C 的核心体温，尽管对于心脏手术5、6、7期间冷却的定义没有共识。2013年，一个国际专家小组提出了一个标准化的报告系统，对系统性低温循环抑制8的各种温度范围进行分类。根据脑电图和大脑的新陈代谢研究，他们把体温过低分为四个层次：深度体温过低（≤14°C）、深度体温过低（14.1-20°C）、中度体温过低（20.1-28°C）和轻度体温过低（28.1-34°C）。专家共识提供了一个明确和统一的分类，使研究更具可比性，并提供更临床相关的结果。TH提供的这种保护是基于其降低细胞代谢活性的能力，进一步限制了细胞高能磷酸盐消耗9、10的速率。然而，TH在心肌保护中的作用是有争议的，并可能具有多种影响，这取决于体温过低的程度。
心肌 I/R 是众所周知的伴随着增加的细胞凋亡11.最近的报告观察到，在开放心脏手术期间，计划心肌细胞死亡增加，并可能与坏死同时发生，从而增加死亡心肌细胞的数量12。因此，减少心肌细胞凋亡是临床实践中一种有用的治疗方法。在小鼠心房HL-1心肌细胞模型中，治疗性体温过低被证明可减少13年输液期间细胞色素c和凋亡诱导因子（AIF）的线粒体释放。然而，温度在调节凋亡方面的影响是有争议的，似乎取决于体温过低的程度。Cooper及其同事观察到，与正常热心肺旁路对照组相比，患有深温低循环阻滞的猪心肌组织凋亡率提高了14。此外，一些研究结果表明，深度体温过低可能激活凋亡通路，而不太积极的体温过低似乎抑制通路12，15，16。造成这一结果的原因可能是与缺血性损伤相关的混淆效应和对温度影响心肌组织的机制缺乏了解。因此，应准确定义增强或衰减凋亡的温度限制。
为了更好地了解与体温过低的疗效相关的机制，并为在人类中实施这种机制提供合理的基础，必须确定一种类似于体内疾病的文化状况，这种状况能够产生类似于以可重复方式观察到的临床状况的损害。实现这一目标的一个重要步骤是建立诱导心肌细胞凋亡的最佳条件。因此，在本研究中，我们探索了有关培养细胞的缺氧-葡萄糖剥夺实验的方法细节，这是缺血-输液的一种外体模型。此外，我们评估了不同缺氧时间对心肌细胞凋亡的影响，并验证了我们关于不同温度条件对体外细胞凋亡影响的假设。

<table>
	<tbody>
		<tr>
			<td>Annexin V-FITC cell apoptosis detection kit</td>
			<td>Bio-Technology,China</td>
			<td>C1062M</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac myocyte growth supplement</td>
			<td>Sciencell,USA</td>
			<td>6252</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 3 activity assay kit</td>
			<td>Bio-Technology,China</td>
			<td>C1115</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 8 activity assay kit</td>
			<td>Bio-Technology,China</td>
			<td>C1151</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, no glucose</td>
			<td>Gibco,USA</td>
			<td>11966025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium</td>
			<td>Gibco,USA</td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco,USA</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry</td>
			<td>CytoFLEX,USA</td>
			<td>B49007AF</td>
			<td></td>
		</tr>
		<tr>
			<td>Human myocardial cells</td>
			<td>BLUEFBIO,China</td>
			<td>BFN60808678</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial membrane potential assay kit with JC-1</td>
			<td>Bio-Technology,China</td>
			<td>C2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin solution</td>
			<td>Gibco,USA</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive oxygen species assay kit</td>
			<td>Bio-Technology,China</td>
			<td>S0033S</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-gas incubator</td>
			<td>Memmert,Germany</td>
			<td>ICO50</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco,USA</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assessment of myocardial protection following hypothermia-preconditioning in a human cardiac myocytes model

缺血/输液衍生心肌功能障碍是心脏手术后患者常见的临床方案。特别是，心肌细胞对缺血性损伤的敏感性高于其他细胞群体。目前，体温过低为预期的缺血性侮辱提供了相当大的保护。然而，对复杂的体温过低引起的分子变化的调查仍然有限。因此，必须确定一种类似于体内条件的文化条件，这种病症可以诱发类似于临床状况中以可重复的方式观察到的损伤。为了模拟体外类似缺血的条件，这些模型中的细胞通过缺氧/葡萄糖剥夺（OGD）进行治疗。此外，我们还应用了心脏手术中使用的标准时间温度协议。此外，我们提出了一种方法，使用简单但全面的方法对心肌损伤进行定量分析。通过流动细胞测量和使用 ELISA 套件对凋亡相关蛋白质的凋亡和表达水平进行评估。在这个模型中，我们测试了一个关于不同温度条件对体外心肌细胞凋亡的影响的假设。该模型的可靠性取决于严格的温度控制、可控的实验程序和稳定的实验结果。此外，该模型还可用于研究体温过低心脏保护的分子机制，这可能对开发用于体温过低的补充疗法有重要影响。

OGD 暴露对高致公箱生存能力的影响由 CCK-8 检测决定。与对照组观察到的细胞存活率相比，细胞存活率在时间依赖性方面显著降低（图2A）。输液后不同时间的氟氯化物凋亡率呈特定趋势，从0至16小时，凋亡率逐渐升高，达到16小时时点的最高率（图2B）。由于OGD 12小时将细胞活性降低约50%，12 h OGD在随后的实验中用于诱导细胞损伤。
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In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

不同程度的体温过低对心肌保护的明显影响尚未得到彻底评估。本研究的目标是量化人类心肌细胞基模型中不同体温过低治疗后细胞死亡水平，为未来深入的分子研究奠定基础。

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

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