Name: in vitro assessment of myocardial protection following hypothermia-preconditioning in a human cardiac myocytes model
Uploaded: 2020-10-27T14:45:00.000+00:00
Duration: 8 min 22 s
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。
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>

<ol>
	<li>Kim, B. 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=Myocardial+Ischemia+Induces+SDF-1alpha+Release+in+Cardiac+Surgery+Patients.">Myocardial Ischemia Induces SDF-1alpha Release in Cardiac Surgery Patients.</a> Journal of Cardiovascular Translational Research. 9 (3), 230-238 (2016).</li><li>Klein, 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=Less+invasive+ventricular+reconstruction+for+ischaemic+heart+failure.">Less invasive ventricular reconstruction for ischaemic heart failure.</a> EUROPEAN JOURNAL OF HEART FAILURE. 21 (12), 1638-1650 (2019).</li><li>Otto, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+hypothermia+applicable+to+cardiac+surgery.">Therapeutic hypothermia applicable to cardiac surgery.</a> VETERINARY ANAESTHESIA AND ANALGESIA. 42 (6), 559-569 (2015).</li><li>Wang, X., 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=Safety+of+Hypothermic+Circulatory+Arrest+During+Unilateral+Antegrade+Cerebral+Perfusion+for+Aortic+Arch+Surgery.">Safety of Hypothermic Circulatory Arrest During Unilateral Antegrade Cerebral Perfusion for Aortic Arch Surgery.</a> CANADIAN JOURNAL OF CARDIOLOGY. 35 (11), 1483-1490 (2019).</li><li>Leshnower, B. 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=Moderate+Versus+Deep+Hypothermia+With+Unilateral+Selective+Antegrade+Cerebral+Perfusion+for+Acute+Type+A+Dissection.">Moderate Versus Deep Hypothermia With Unilateral Selective Antegrade Cerebral Perfusion for Acute Type A Dissection.</a> ANNALS OF THORACIC SURGERY. 100 (5), 1563-1568 (2015).</li><li>Vallabhajosyula, 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=Moderate+versus+deep+hypothermic+circulatory+arrest+for+elective+aortic+transverse+hemiarch+reconstruction.">Moderate versus deep hypothermic circulatory arrest for elective aortic transverse hemiarch reconstruction.</a> ANNALS OF THORACIC SURGERY. 99 (5), 1511-1517 (2015).</li><li>Keeling, W. B., 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=Safety+of+Moderate+Hypothermia+With+Antegrade+Cerebral+Perfusion+in+Total+Aortic+Arch+Replacement.">Safety of Moderate Hypothermia With Antegrade Cerebral Perfusion in Total Aortic Arch Replacement.</a> ANNALS OF THORACIC SURGERY. 105 (1), 54-61 (2018).</li><li>Yan, T. D., 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=Consensus+on+hypothermia+in+aortic+arch+surgery.">Consensus on hypothermia in aortic arch surgery.</a> Annals of Cardiothoracic Surgery. 2 (2), 163-168 (2013).</li><li>Zhou, J., Empey, P. E., Bies, R. R., Kochanek, P. M., Poloyac, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+arrest+and+therapeutic+hypothermia+decrease+isoform-specific+cytochrome+P450+drug+metabolism.">Cardiac arrest and therapeutic hypothermia decrease isoform-specific cytochrome P450 drug metabolism.</a> DRUG METABOLISM AND DISPOSITION. 39 (12), 2209-2218 (2011).</li><li>Sharp, W. 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=Inhibition+of+the+mitochondrial+fission+protein+dynamin-related+protein+1+improves+survival+in+a+murine+cardiac+arrest+model.">Inhibition of the mitochondrial fission protein dynamin-related protein 1 improves survival in a murine cardiac arrest model.</a> CRITICAL CARE MEDICINE. 43 (2), 38-47 (2015).</li><li>Zhu, W. 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. 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&#039;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

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

科研

医学

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

心肌顿抑相关的小鼠离体心脏模型心脏停搏

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

无输血或强心剂剂的大鼠心肺旁路移植模型

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

免疫与感染

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

设计生物反应器以改善工程心脏组织的数据采集和模型通量

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

生物工程

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循环死亡后心脏捐献的临床前模型

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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 ...

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Langendorff 模型中离 体 小鼠双心室功能的评估

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

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

摘要

探索更多视频

此视频中的章节

闭胸鼠心肌缺血再灌注模型

心肌顿抑相关的小鼠离体心脏模型心脏停搏

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

无输血或强心剂剂的大鼠心肺旁路移植模型

设计生物反应器以改善工程心脏组织的数据采集和模型通量

循环死亡后心脏捐献的临床前模型

研究小鼠心肌梗死的低温损伤模型

使用皮肤心肌细胞进行心脏功能的体外评估

Langendorff 模型中离体小鼠双心室功能的评估

人体心室心肌细胞与颤音-切切心肌切片分离

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

摘要

探索更多视频

此视频中的章节

闭胸鼠心肌缺血再灌注模型

心肌顿抑相关的小鼠离体心脏模型心脏停搏

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

无输血或强心剂剂的大鼠心肺旁路移植模型

设计生物反应器以改善工程心脏组织的数据采集和模型通量

循环死亡后心脏捐献的临床前模型

研究小鼠心肌梗死的低温损伤模型

使用皮肤心肌细胞进行心脏功能的体外评估

Langendorff 模型中离 体 小鼠双心室功能的评估

人体心室心肌细胞与颤音-切切心肌切片分离

Langendorff 模型中离体小鼠双心室功能的评估