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。
1. 细胞培养
注:基础培养介质由无血清杜尔贝科改良鹰介质（DMEM）、10%胎儿牛血清（FBS）、1%心脏肌细胞生长补充剂和1%青霉素/链霉素溶液组成。在使用前将介质存储在 4 °C 和预热至 37 °C。
<ol><li>从液氮中取出低温保存的人类心肌细胞（HCM），并在37°C的水浴中解冻。</li>
	<li>在 37 °C 的水浴中轻轻摇动小瓶（+lt:1 分钟），直到小瓶中仅剩一小块冰。</li>
	<li>将小瓶转移到无菌层流罩中。用浸入 70% 酒精的棉球擦拭小瓶的外部。</li>
	<li>将4mL预热完全生长中下垂到含有解冻细胞的离心机管中。</li>
	<li>将细胞悬浮在200×g下10分钟。 离心后，丢弃超自然物，用5mL的全中等介质重新喷射颗粒。</li>
	<li>将细胞保持在37°C的潮湿孵化器下，空气为95%，二氧化碳为5%。 注意:在采集细胞进行实验之前，细胞可以生长，直到达到大约60-70%的汇流。</li>
</ol>2. 建立缺氧（OGD）模型
注:在研究期前两小时，用无血清介质取代生长介质，细胞在潮湿的孵化器中再孵化2小时，在37°C的大气中含有5%的二氧化碳。
<ol><li>从6井板中吸气介质，用磷酸盐缓冲盐水（PBS）轻轻清洗细胞三次。</li>
	<li>每口井添加 2 mL 的新鲜无糖 DMEM。</li>
	<li>在大气层下的三气体孵化器中以恒定温度培养细胞，混合温度为 95% N2、5%CO2和 0.1% O2， 温度为 37 °C，持续 1、2、4、8、12 或 16 小时。</li>
	<li>缺氧治疗后，从6井板吸气介质，用PBS（pH 7.4）清洗细胞三次。</li>
	<li>每口井添加2毫升完整的DMEM。</li>
	<li>将细胞保持在37°C的潮湿孵化器下，空气为95%，二氧化碳为5%。</li>
</ol>3. 时间温度协议
注:在心脏手术中使用标准的时间温度协议，如其他人之前描述的20，21。根据以下协议（图 1） 处理 HCM : 时间点 1 （T1） 表示感应结束，时间点 2 （T2） 表示维护结束，时间点 3 （T3） 表示回暖结束。分析在连续正常热条件下保持的控制单元（37 °C）。温度条件使用三气体孵化器创建，从而允许精确的温度调节。
<ol><li>在实验前两小时，从6井板中吸气培养介质，每口井添加2毫升的新鲜无血清DMEM。</li>
	<li>在37°C的大气中以5%的二氧化碳在潮湿的孵化器中重新孵化细胞2小时。</li>
	<li>2小时后，从6井板吸气介质，用PBS（pH 7.4）清洗细胞三次。</li>
	<li>每口井添加 2 mL 的新鲜无血清 DMEM。</li>
	<li>在三气体孵化器中重新孵化细胞。 注意:更换介质以去除未连接的单元和碎屑。</li>
	<li>立即将培养皿放入三气孵化器，从而改变温度。</li>
	<li>冷却 1 小时后，快速从 6 井板中吸气介质，每口井添加 2 mL 的新鲜无糖 DMEM。</li>
	<li>培养大气下三气体孵化器中的细胞，包括95%N 2、5%CO2和0.1%O2，在37°C下12小时建立缺氧。</li>
	<li>设置下面描述的温度。 注:协议从10小时的低温治疗开始，然后是2小时至37°C的回暖阶段和24小时的正常热度（37°C）。在每个时间点，三个培养皿被移除进行分析。</li>
	<li>经过低温处理后，从6井板吸气介质，用PBS（pH 7.4）洗三次。</li>
	<li>每口井添加2毫升完整的DMEM。</li>
	<li>将细胞保持在37°C的潮湿孵化器下，空气为95%，二氧化碳为5%。</li>
</ol>4. CCK-8 可行性测定
<ol><li>使用前将 0.25% 的三聚氰胺-乙酰胺乙酰氨基酸 （EDTA） 溶液和 PBS 加热至 37 °C。</li>
	<li>吸气介质，用 1 mL 的 PBS 冲洗细胞，然后沿井壁加入 0.5 mL 的 0.25% trypsin。孵化在37°C，直到几乎所有的HCM分离（约1分钟）。</li>
	<li>将 1 mL 的 DMEM 完整介质添加到井中，以中和肌氨脂。</li>
	<li>将细胞悬架转移到 15 mL 离心机管中，通过离心机在 500 × g 下颗粒 HCM 3 分钟。在不干扰颗粒的情况下吸气超自然人。</li>
	<li>将细胞悬架（5000 个细胞/井）的 100 μL 等离子分配到 96 井板中。在加湿的孵化器中预孵化板 24 小时（在 37 °C.）</li>
	<li>使用 96 井板中的培养细胞生成不同的治疗组。</li>
	<li>在孵化器中孵化板的适当时间长度（16 h）。</li>
	<li>在板的每个井中加入 10 μL 的 CCK-8 溶液。</li>
	<li>在孵化器中孵化盘子1小时。</li>
	<li>使用微板读卡器测量 450 nm 的吸收度。 注意:小心不要将气泡引入井中，因为它们会干扰OD读数测量。</li>
</ol>5. 用于凋亡分析的流动细胞测量
<ol><li>按照步骤 4.2-4.4 执行尝试化和离心步骤。 注:细胞在没有EDTA的情况下用肌氨辛收获。为了评估凋亡，收集浮动细胞和粘附细胞。</li>
	<li>在1000×克时将细胞离心5分钟。丢弃超自然物，在 1 mL 的 PBS 中重新喷射颗粒。</li>
	<li>使用血细胞计数数细胞。使用移液器，将 100 μL 的 Trypan 蓝色处理细胞悬架转移到血细胞计中。使用手计数计数计数器，在一组 16 平方中计算活的未受污染的细胞，然后使用 PBS 以 1×107 细胞/毫升生成细胞悬架。</li>
	<li>获得 200 μL 的细胞悬浮（5 × 105 -1 × 106 细胞）。</li>
	<li>离心机在 1000 × g 下 5 分钟，并在附件 V-FITC 绑定解决方案中重新注入颗粒。</li>
	<li>加入 5μL 的附件 V-FITC 来染色细胞。</li>
	<li>在细胞悬架中加入 10 μL 的碘化物丙酸。</li>
	<li>在黑暗中的室温下轻轻混合细胞并孵育20分钟。 注意:细胞在孵化过程中被再吸收2-3次，以改善染色。</li>
	<li>启动流量细胞仪，并确保软件正常工作。</li>
	<li>打开流细胞测量软件中的两个点图窗口。</li>
	<li>选择 Y 轴 X 轴上的向前散射光 （FSC） 和侧散光 （SSC），分别从大小和粒度上排除细胞碎片和/或团块。</li>
	<li>选择 PE （590 mm） 检测通道和 FITC （530 mm），用于测量细胞的荧光强度。</li>
	<li>将空白（未染色的HCM）样品管放在流动细胞仪上。</li>
	<li>单击 "记录" 从空白样品管中的悬浮物中收集粒子，然后对细胞群进行门控，以便在第一个点图中进行进一步分析。</li>
	<li>将单染色样品放在管支撑臂上。单击 "记录" 从悬架中收集粒子，然后对细胞群进行门控，以便在第一个点图中进行进一步分析。</li>
	<li>收集其他样品管中的丙肝，并通过调整荧光通道的电压来优化测量。 注:在实验过程中，未染色和单染色样品用作补偿控制。</li>
	<li>显示每个样本管的统计数据，并计算每个样本的凋亡率。</li>
</ol>6. 线粒体去极化评估
<ol><li>按照步骤 4.2-4.4 执行尝试式脱脂和离心。</li>
	<li>将细胞在 500 μL 的完整介质中恢复，然后调整到 1 × 105 - 6 × 106 个单元格的细胞密度</li>
	<li>为每个管添加 0.5 mL 的 JC-1 工作解决方案。</li>
	<li>在37°C的细胞孵化器中孵化细胞20分钟。 注意:在孵化期间，将 1 mL 的 5 × JC-1 染色缓冲器添加到 4 mL 蒸馏水中，以准备 JC-1 染色缓冲。</li>
	<li>孵化后，在4°C下以600×克的离心机3分钟。</li>
	<li>丢弃超自然物，在 JC-1 染色缓冲器的 1 mL 中重新吸收颗粒。</li>
	<li>重复步骤 6.5 到 6.6 三次。</li>
	<li>在 1.5 mL 离心管中将高温计重新注入 1 mL 的冰冷染色缓冲器中，并在 30 分钟内使用电池进行分析。</li>
	<li>选择 PE （590 nm） 检测通道和 FITC （530 nm） 来测量细胞中 JC-1 染料的荧光强度。 注意:按照步骤 5.10-5.17 设置流量细胞计。</li>
</ol>7. 活性氧物种检测
<ol><li>按照步骤 4.2-4.4 执行尝试式脱脂和离心。</li>
	<li>用 10μM DCFDA 在培养介质中染色细胞，并将细胞密度调整为 1 × 106 - 1 × 107 细胞</li>
	<li>在37°C孵育30分钟。</li>
	<li>每 3-5 分钟轻轻将细胞向上/向下移液。</li>
	<li>孵育后，用无血清细胞培养介质清洗细胞三次。</li>
	<li>按照步骤 5.10-5.17 分析流动细胞仪上的细胞。 注:DCF 在 488 nm 时兴奋不已，排放强度为 530 nm。</li>
</ol>8. 测量卡斯帕斯 3/ 卡斯帕斯 8 活动
<ol><li>按照步骤 4.2-4.4 执行尝试个性化。</li>
	<li>通过离心收集细胞，在 600 ×克，在 4 °C 5 分钟。</li>
	<li>每 2 个× 106 个细胞添加 100 μL 的利萨特缓冲器。</li>
	<li>在冰上将细胞酶化15分钟。</li>
	<li>在冰浴中孵育 15 分钟后，将样品在 1.6 × 104 x g 在 4 °C 下浸泡 15 分钟。</li>
	<li>将超母体转移到冰冷离心管中使用。 注:样品中的蛋白质浓度应至少为1-3毫克/升。</li>
	<li>取出交流-DEVD-pNA（2 mM），并将其放在冰浴中使用。</li>
	<li>准确地将 40 μL 的缓冲溶液添加到酶标记板中，加入 80 μL 的样品，最后添加 10 μL 的 Ac-DEVD-pNA （2 mM）。</li>
	<li>在 37 °C 下孵化样品 120 分钟。</li>
	<li>根据制造商的说明测量微板读卡器上的 A405 值。 注:通过从样品中减去空白控制的 A405 值来计算 caspase-3/caspase-8 产生的 pNA 产生的吸收。</li>
</ol>

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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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+hypothermia+protect+cardiomyocytes+during+cardioplegic+ischemia.">How does hypothermia protect cardiomyocytes during cardioplegic ischemia.</a> European journal of cardiothoracic surgery. 40 (2), 352-359 (2011).</li><li>Diestel, A., Drescher, C., Miera, O., Berger, F., Schmitt, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia+protects+H9c2+cardiomyocytes+from+H2O2+induced+apoptosis.">Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis.</a> Cryobiology. 62 (1), 53-61 (2011).</li><li>Zhang, 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=HIF-1alpha/BNIP3+signaling+pathway-induced-autophagy+plays+protective+role+during+myocardial+ischemia-reperfusion+injury.">HIF-1alpha/BNIP3 signaling pathway-induced-autophagy plays protective role during myocardial ischemia-reperfusion injury.</a> BIOMEDICINE &amp; PHARMACOTHERAPY. 120, 109464 (2019).</li><li>An, 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=Exogenous+IL-19+attenuates+acute+ischaemic+injury+and+improves+survival+in+male+mice+with+myocardial+infarction.">Exogenous IL-19 attenuates acute ischaemic injury and improves survival in male mice with myocardial infarction.</a> BRITISH JOURNAL OF PHARMACOLOGY. 176 (5), 699-710 (2019).</li><li>Han, Y. 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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia-induced+spatially+discordant+action+potential+duration+alternans+and+arrhythmogenesis+in+nonhibernating+versus+hibernating+mammals.">Hypothermia-induced spatially discordant action potential duration alternans and arrhythmogenesis in nonhibernating versus hibernating mammals.</a> AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY. 303 (8), 1035-1046 (2012).</li><li>Bobi, 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+Hypothermia+Modifies+Coronary+Hemodynamics+and+Endothelium-Dependent+Vasodilation+in+a+Porcine+Model+of+Temperature+Management.">Moderate Hypothermia Modifies Coronary Hemodynamics and Endothelium-Dependent Vasodilation in a Porcine Model of Temperature Management.</a> Journal of the American Heart Association. 9 (3), 014035 (2020).</li><li>Dietrichs, E. S., Tveita, T., Myles, R., Smith, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ECG-biomarker+for+cardiac+arrest+during+hypothermia.">A novel ECG-biomarker for cardiac arrest during hypothermia.</a> Scandinavian Journal of Trauma Resuscitation &amp; Emergency Medicine. 28 (1), 27 (2020).</li></ol>

作者没有什么可透露的。

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。
<p class="jove_content...

Watch this Scientific Journal Video about In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model at JoVE.com

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

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

in-vitro-assessment-myocardial-protection-following-hypothermia

Research

JoVE Journal

Medicine

3.0K 次观看.  Children's Hospital of Nanjing Medical University. 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。

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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

科研

医学

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

使用小动物看更衣室反转恢复（SALLI）的MRI大鼠心脏功能和心肌形态学评估

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心肌梗死的猪模型主要成果评估

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

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.

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 ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

几内亚猪模型心肌损伤后 hiPSC-derived 衍生心肌贴片的植入

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

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.

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 ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...