作者想感谢她的支持，林恩斯宾塞在核磁共振光谱实验的部分。这项工作是由国家卫生基金R01 HL059246研究院，R01 HL067970，R01 HL088634（田博士）和F32 HL096284（博士Kolwicz）的赠款支持。

<ol><li>对于这些实验，同时利用两个独立的系统。收购31个P谱，布鲁克14T磁铁与AVANCE III控制台和与Topspin V2.1的软件配备计算机接口。评估心脏功能，自定义的内置心脏灌流系统接口与PowerLab系统4 / 30数据采集与数据分析软件的LabChartPro 6配备，。 </li><li>在实验的当天，1公升克雷布斯- Henseleit缓冲准备如下：0.5MM EDTA，5.3mM氯化钾，1.2mm 的硫酸镁，118mM氯化钠， 碳酸氢钠 3和25mm 。然后将混合物冒泡5％CO 2 / 95％直径为2mm的氯化钙此外前10-15分钟2。最后，基板，在10毫米的葡萄糖和0.5mm丙酮酸形式，添加。 </li><li>在实验过程中的温度调节是至关重要的。加热循环器是用来保持温度之间的37.0 - 37.5 ° C，而心内的磁铁。温度监控使用光纤温度探测器实验的持续时间。 </li><li>灌注压和左心室压力，通过压力传感器连接到一个数据采集系统，并使用附带的软件显示监测。这些都与实验前的标准血压计校准。此外，压线刷新充分，以消除所有气泡。 </li><li> 150 mM磷酸钠（这是等同的KH缓冲液的离子强度）的一个标准样本是使用“校准”前探头插入心脏。这有利于信号，并降低所需的时间开始收购期间，一旦心脏内的探针定位。 </li><li>为了减少凝血，鼠标注入200个单位的肝素的IP 5分钟后，戊巴比妥钠（175毫克/公斤）的IP。 </li><li>心是迅速切除（完好的肺和胸腺），在冰冷的KH缓冲被捕。 </li><li>虽然放在冰上，迅速取出肺。胸腺叶，轻轻去皮暴露主动脉。胸腺是删除。主动脉，然后小心删除任何周围组织分离。 </li><li>微缝合钳轻轻托起主动脉两个墙壁暴露管腔。主动脉小心放置到0.965毫米外径的聚乙烯管（PE50）的套管。而缝线快速主动脉周围捆绑主动脉微血管钳举行到位。剪辑被删除，使用镊子仔细检查，主动脉根部以上的套管。其他关系添加必要时举行的地方心。 </li><li>使用镊子和microscissors删除任何额外的组织。一个小切口进入左耳廓。通过左心房，左室腔，并通过心尖同时轻轻地捧着心，仔细插入一个0.61毫米外径的聚乙烯管（PE10）。修剪多余的油管。 </li><li>泄了气的充满水的气球是通过心房插入到LV，是在使用胶带或缝合的地方举行。蠕动泵的速度正在逐步增加，以提供足够的流向心脏。直到心入NMR探头的地方，心将继续与恒定流量相当于约2毫升/分钟灌注。 LV气球充气使用微米注射器验证，LV压力传感器运作的一个小体积。 </li><li>仔细插入心脏是一个10毫米的核磁共振管。一个大口径的“微调”，是用来帮助引导到适当的位置内的探头管。整个装置是牢固地附着在“脐带”，用胶带。 </li><li>脐带慢慢降低到磁铁上孔，直到心/核磁共振管内10毫米NMR探头的线圈。 </li><li>一旦心脏是在探头内的适当位置，蠕动泵，流量调整，从而实现了灌注压80mmHg。 （请记住，到这一点心了约2毫升/分钟的流量恒定灌注）。然后灌注压维持使泵的控制器上的“持有”机制。然后让心脏是一个15-20分钟的平衡时间。那段时间，LV的气球的体积是调整，以达到8-10毫米汞柱，舒张末期压力。 </li><li>在平衡期，这是必要的优化谱仪参数，以获得最好的磷信号。这是通过设置在磷原子核产生共鸣的频率（“调整”）的无线电脉冲磁场同质（“垫片”）。 </li><li>平衡时间后，多个31个P NMR谱可以得到。收购期间每个频谱是依赖于T他的磁铁磁场强度的样本大小，和一个特定的实验所需的信噪比的信号。谱得到使用14 Telsa磁铁平均获得256 60度翻转角和2.0秒的延迟20微秒的无线电频率脉冲信号。本实验将需要大约10分钟。 </li></ol>代表性的成果从数据采集的硬件和LabChart软件，可以测量心脏功能的几个参数，在整个实验的协议。典型的测量心脏功能，左心室发展压（LVDevP），是通过减去舒张末压（EDP）的收缩压（图1）。这项措施可以改变鼠标的应变和心脏的条件（即压力超负荷）而定。然而，在一个正常C57BL6小鼠心脏LVDevP通常固定舒张末期8-10毫米汞柱的压力在100-110毫米汞柱之间。此外，LabChart程序允许基于LV压力波循环测量心率的测量。同样，这一措施可以有所不同，但典型值350-400 BPM时，心中都允许击败内在率。然而，心可使用心率保持在420 BPM的起搏系统标准化。此外，收缩（+ DP / DT）和放松（-dP/dt）的措施，可使用的LV压力波的一阶导数估计。在实验的协议，很容易评估纳入一个压力 - 体积关系德裕机制。这是通过在LV气球体积逐渐增加，并注意到LVDevP以及电算化。这些值可以被绘制成图2所示。虽然德裕曲线是最优的，注意到量要达到8-10毫米汞柱的EDP可以给一个低压室尺寸的间接的想法。这可以用来肥大心中绑扎主动脉模型中，通常需要一个气球体积较小，而扩张型心需要一个体积较大，与对照组相比。表1显示了代表心脏功能灌注协议中获得的数据。 31个P核磁共振波谱仪将提供信号的磷酸（PCR）和ATP（γ- ATP，α- ATP和β- ATP）以及无机磷（PI），如图2所示的三个磷酸盐。每个峰的分析提供了价值曲线下的面积。 ATP的量估计平均γ- ATP和β- ATP的地区。 （α- ATP不使用NAD分子作出贡献，因为一个总信号的未知部分）。 PCR和ATP地区（PCR：ATP的比例）的商数，是由心脏的充满活力的状态。该值通常为1.5 - 1.7葡萄糖作为主基板提供了一个小鼠心脏。 31 P NMR虽然不提供直接ATP或PCR措施，峰的面积是含有样品中的化合物磷的量成正比。这些信号的估计值可以通过使用其他方法。例如，可以产生ATP的直接措施，高性能液体色谱法（HPLC）在心中的队列的平均浓度。这个值可以被用来校正光谱中观察到的平均ATP领域。 PCR的浓度可以计算基于PCR的ATP区面积的相对。它也可以通过分析信号（PI）的无机磷的相对化学位移PCR信号估计的pH值。使用不同的无线电脉冲序列，肌酸激酶反应速度或ATP的合成反应速度还可以进行测量2。 <img src="/files/ftp_upload/2069/2069tab1.jpg" alt="表1" /> 表1。基准从离体心脏的心脏功能。 LVDevP：左心室发展压力; LVEDP：左室舒张末压; HR：心率; RPP：利率的压力产品; + DP / DT：一阶导数的LV压力积极; -dP/dt：第一个衍生LV的压力负，PP灌注压; CF：冠脉流量。 <img src="/files/ftp_upload/2069/2069fig1.jpg" alt="图1" /> 图1。代表LV LabChart Pro软件的压力波。 <img src="/files/ftp_upload/2069/2069fig2.jpg" alt="图2" /> 图2：从控制（实线）和主动脉带状（虚线）小鼠的代表德裕曲线。一）确定的LV气球的体积收缩功能，提高左室容积LVDevP代表。二）舒张功能，提高左室容积电算化代表由LV的气球的体积决定。 LVDevP：左心室发展压（收缩压减去舒张压pressure）;电算化：舒张末压。 <img src="/files/ftp_upload/2069/2069fig3.jpg" alt="图3" /> 图3，代表31个P NMR谱的离体小鼠心脏。请注意相对较小的PI峰。除了葡萄糖丙酮酸或脂肪酸提供的有氧灌注心脏，这个峰值应该不大。在缺血期间，此峰的增加，而PCR高峰下降。注意肩膀α- ATP的峰权。这是NAD分子的贡献。 PI：无机磷; PCR：磷酸，三磷酸腺苷三磷酸腺苷。

<ol>
	<li>Nascimben, L., Ingwall, J. S., Lorell, B. H., Pinz, I., Schultz, V., Tornheim, K., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+increased+glycolysis+in+the+hypertrophied+rat+heart.">Mechanisms for increased glycolysis in the hypertrophied rat heart.</a> Hypertension. 44, 662-667 (2004).</li><li>Spindler, M., Saupe, K. W., Tian, R., Ahmed, S., Matlib, M. A., Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+creatine+kinase+enzyme+kinetics+in+diabetic+cardiomyopathy.+A(31)P+NMR+magnetization+transfer+study+of+the+intact+beating+rat+heart.">Altered creatine kinase enzyme kinetics in diabetic cardiomyopathy. A(31)P NMR magnetization transfer study of the intact beating rat heart.</a> J Mol Cell Cardiol. 31, 2175-2189 (1999).</li><li>Ingwall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+nuclear+magnetic+resonance+spectroscopy+of+cardiac+and+skeletal+muscles.">Phosphorus nuclear magnetic resonance spectroscopy of cardiac and skeletal muscles.</a> Am J Physiol. 242, H729-H744 (1982).</li><li>Ingwall, J. S., Javadpour, M. M., Miao, W., Hoit, B. D., Walsh, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=31P+NMR+spectroscopy+of+the+mouse+heart.">31P NMR spectroscopy of the mouse heart.</a> Cardiovascular physiology in the genetically engineered. , 151-163 (2002).</li><li>Luptak, I., Balschi, J. A., Xing, Y., Leone, T. C., Kelly, D. P., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+contractile+and+metabolic+reserve+in+peroxisome+proliferator-activated+receptor-alpha-null+hearts+can+be+rescued+by+increasing+glucose+transport+and+utilization.">Decreased contractile and metabolic reserve in peroxisome proliferator-activated receptor-alpha-null hearts can be rescued by increasing glucose transport and utilization.</a> Circulation. 112, 2339-2346 (2005).</li><li>Yan, J., Young, M. E., Cui, L., Lopaschuk, G. D., Liao, R., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glucose+uptake+and+oxidation+in+mouse+hearts+prevent+high+fatty+acid+oxidation+but+cause+cardiac+dysfunction+in+diet-induced+obesity.">Increased glucose uptake and oxidation in mouse hearts prevent high fatty acid oxidation but cause cardiac dysfunction in diet-induced obesity.</a> Circulation. 119, 2818-2828 (2009).</li><li>Luptak, I., Shen, M., He, H., Hirshman, M. F., Musi, N., Goodyear, L. J., Yan, J., Wakimoto, H., Morita, H., Arad, M., Seidman, C. E., Seidman, J. G., Ingwall, J. S., Balschi, J. A., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+activation+of+AMP-activated+protein+kinase+remodels+metabolic+network+in+favor+of+cardiac+glycogen+storage.">Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage.</a> J Clin Invest. 117, 1432-1439 (2007).</li><li>Xing, Y., Musi, N., Fujii, N., Zou, L., Luptak, I., Hirshman, M. F., Goodyear, L. J., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+and+energy+homeostasis+in+mouse+hearts+overexpressing+dominant+negative+alpha2+subunit+of+AMP-activated+protein+kinase.">Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase.</a> J Biol Chem. 278, 28372-28377 (2003).</li><li>Luptak, I., Yan, J., Cui, L., Jain, M., Liao, R., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+increased+glucose+entry+on+mouse+hearts+during+normal+aging+and+ischemic+stress.">Long-term effects of increased glucose entry on mouse hearts during normal aging and ischemic stress.</a> Circulation. 116, 901-909 (2007).</li><li>Tian, R., Abel, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+GLUT4-deficient+hearts+to+ischemia+underscore+the+importance+of+glycolysis.">Responses of GLUT4-deficient hearts to ischemia underscore the importance of glycolysis.</a> Circulation. 103, 2961-2966 (2001).</li><li>Liao, R., Jain, M., Cui, L., D'Agostino, J., Aiello, F., Luptak, I., Ngoy, S., Mortensen, R. M., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac-specific+overexpression+of+GLUT1+prevents+the+development+of+heart+failure+attributable+to+pressure+overload+in+mice.">Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice.</a> Circulation. 106, 2125-2131 (2002).</li><li>Tian, R., Musi, N., D'Agostino, J., Hirshman, M. F., Goodyear, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+adenosine+monophosphate-activated+protein+kinase+activity+in+rat+hearts+with+pressure-overload+hypertrophy.">Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy.</a> Circulation. 104, 1664-1669 (2001).</li></ol>

31个P核磁共振光谱的Langendorff灌注的离体小鼠心脏提供可靠和可重复性的数据。3，4然而，当务之急是完成主动脉插管和LV的球囊插入正确等，让心脏功能稳定，而里面的核磁共振管。此外，温度调节，以达到适当的基线功能是最重要的的。取得了良好的，分析的核磁共振光谱中的一个重要因素是提高信噪比。要做到这一点，确保最佳的“调整”和“垫片”的样本。在协议文本中提到，使用的标准样品前插入心脏，可以促进这项工作。这也有利于有足够规模的“样品”。心称量小于100毫克，通常提供较低的PCR和ATP的信号，所以采集时间的增加，将要获得良好的磷谱。 有几种方法可以修改现有的协议，以争取更多的信息，对心脏功能和能量。在我们的实验室，我们灌注混合衬底缓冲区，其中可以包括不同的组合（低，高浓度的）的脂肪酸，乳酸，酮体，和胰岛素的存在心中。稳定同位素在灌注缓冲区的使用（即，13 C标记的基板），我们所拥有的能力估计基质利用TCA循环标记的乙酰辅酶A的相对贡献 。5-7对于这种应用，我们执行isotopomer 13的C3 - 13的C4 -谷氨酸与13 C - NMR谱分析。这就要求冻结夹紧在灌注协议结束的心脏和执行冰冻组织提取。这将是一个额外的实验分析，需要使用不同的探头具有独立设置参数。其他应用包括缓冲区脱氧葡萄糖替代，同时监测随时间变化的2 -脱氧葡萄糖磷酸中使用的31 P核磁共振光谱的心脏的积累。这种方法允许7 8此外，我们的实验室分析心脏功能和能量灌注缺血/再灌注和高工作量的挑战组成的协议。，6，8-10心肌对葡萄糖的摄取测量。 总之，31 P NMR光谱在小鼠离体心脏是一个技术上具有挑战性的过程，需要使用先进设备。然而，它产生的数据是非常宝贵的研究人员希望分析生物工程小鼠模型的功能和能量。我们的实验室，这些技术已在心肌功能，能量和新陈代谢的各种压力所带来的后果的认识至关重要。1，11 ，12

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>
<tr>
<td>Magnesium Sulfate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>M7506</td>
</tr>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>E1644</td>
</tr>
<tr>
<td>Potassium chloride</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P4505</td>
</tr>
<tr>
<td>Sodium bicarbonate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S6297</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S7653</td>
</tr>
<tr>
<td>Calcium chloride dihydrate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>C5080</td>
</tr><tr>
<td>D-Glucose</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>G7528</td>
</tr><tr>
<td>Sodium Pyruvate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P2256</td>
</tr><tr>
<td>Bruker Ultrashield 600WB Plus</td>
<td>Equipment</td>
<td>Bruker</td>
<td>&nbsp;</td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab 4/30</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">ML866/P</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 6 Pro</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">MLS260/6</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/FE224/" target="_blank">Quad Bridge Amp</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/FE224/" target="_blank">ML224</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/research/product/ML175/" target="_blank">STH Pump Controller</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/research/product/ML175/" target="_blank">ML175</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML172/" target="_blank">Minipuls 3 Peristaltic Pump</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML172/" target="_blank">ML172</a></td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0699/" target="_blank">Disposable BP Transducer</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT0699/" target="_blank">MLT0699</a></td>
</tr><tr>
<td>10mm NMR Sample Tube</td>
<td>Equipment</td>
<td>Wilmad LabGlass</td>
<td>513-7PP-7</td>
</tr><tr>
<td>Polyethylene tubing PE10</td>
<td>Equipment</td>
<td>Becton-Dickinson</td>
<td>427401</td>
</tr><tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT844/" target="_blank">Physiological Pressure Transducer</a></td>
<td>Equipment</td>
<td><a href="http://www.adinstruments.com" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/MLT844/" target="_blank">MLT844</a></td>
</tr><tr>
<td>Polyethylene tubing PE50</td>
<td>Equipment</td>
<td>Becton-Dickinson</td>
<td>427411</td>
</tr><tr>
<td>Micrometer syringe</td>
<td>Equipment</td>
<td>Gilmont Instruments</td>
<td>GS-1101</td>
</tr><tr>
<td>McPherson Forceps</td>
<td>Equipment</td>
<td>Miltex Inc.</td>
<td>18-949</td>
</tr><tr>
<td>Castraviejo microscissors</td>
<td>Equipment</td>
<td>Roboz Surgical Instruments</td>
<td>RS-5650</td>
</tr><tr>
<td>Neoptix Signal Conditioner</td>
<td>Equipment</td>
<td>Neoptix, Inc.</td>
<td>Reflex - 1</td>
</tr>	
</tbody>
</table>

assessment of cardiac function and energetics in isolated mouse hearts using 31p nmr spectroscopy

生物工程小鼠模型已成为强大的研究工具，在确定分子改变和心血管疾病的模型之间的因果关系。虽然分子生物学识别信号转导通路中的关键变化是必要的，它是不是功能意义的替代品。虽然生理功能的问题提供答案，在完整的生理生化代谢物评估相结合，跳动的心脏，使心脏功能和能量的一个完整的画面。多年来，我们的实验室已利用离体心脏灌注核磁共振（NMR）谱相结合来完成这项任务。左室功能评估的Langendorff模式离体心脏灌注，而心脏的能量是通过执行计量<sup&gt; 31</sup&gt;灌注心中的P磁共振波谱。有了这些技术，在心脏功能相结合，与磷酸肌酸和ATP水平的指标可以同时测量跳动的心。此外，这些参数可以被监控，生理或病理应激而。例如，可以采用缺血/再灌注或高工作量的挑战协议。绑扎主动脉瓣或其他心肌病理模型的使用以及APT。无论在协议的变种，分子的转基因小鼠模型的修改功能和充满活力的意义，可以充分说明，领导到相关的酶和代谢途径的新见解。因此，<sup&gt; 31</sup&gt; P核磁共振光谱在离体心脏是一个有价值的研究方法，在心血管疾病的动物模型。

Watch this Scientific Journal Video about Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy at JoVE.com

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

离体心脏灌流的Langendorff -模式，配合<sup&gt; 31</sup&gt; P核磁共振光谱，结合成一个实验生物化学和生理学等领域。该协议允许动态测量高能磷酸含量和心的同时监测生理功能的营业额。当正确执行，这是一个有价值的技术评估心脏能量。

在分离的小鼠使用红桃心功能和能量学的评估 31 P核磁共振

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

assessment-cardiac-function-energetics-isolated-mouse-hearts-using

Research

JoVE Journal

Medicine

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

26.1K 次观看.  University of Washington School of Medicine. 离体心脏灌流的Langendorff -模式，配合 31 P核磁共振光谱，结合成一个实验生物化学和生理学等领域。该协议允许动态测量高能磷酸含量和心的同时监测生理功能的营业额。当正确执行，这是一个有价值的技术评估心脏能量。

视频: Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH的荧光成像隔离双心室工作离体兔心

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

科研

医学

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

左室壁应力和改进功能衰竭心脏使用Algisyl LVR减少

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

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 ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

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 ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

工作模式下的心功能和代谢评估

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

成人大鼠心脏解剖与功能的超声心动图评估

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

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 ...

Methods Article

在分离的小鼠使用红桃心功能和能量学的评估³¹ P核磁共振