这项工作是由美国心脏协会SDG 10SDG2630086赵X，RO1-AR061385马静和GO资助RC2AR05896 Brotto M.

解决方案组成： 2.5毫摩尔的Ca 2 + Tyrode溶液：140 mM氯化钠，5mM的氯化钾，10mM的HEPES，2.5毫摩尔的CaCl 2，2mM的MgCl 2和10mM葡萄糖 0毫摩尔的Ca 2 + Tyrode溶液：140 mM氯化钠，5mM的氯化钾，10mM的HEPES，2毫摩尔MgCl 2，0.1mM的乙二醇四乙酸（EGTA），10mM葡萄糖应注意：沐浴液与100％的O 2饱和，如果使用上述的解决方案，但用95％O 2与5％CO 2，如果使用基于碳酸氢盐缓冲液保持pH恒定。 2.5mM的Ca 2 +的添加到泳季缓冲区，扼要重述水平的Ca 2 +在细胞外空间中找到和10mM葡萄糖线粒体是很重要的，因为仍然发挥作用，在这些肌肉以连续生产在葡萄糖存在下的ATP。 1。设置了体外收缩试验使用ADI PowerLab系统<ol><li>甲一个4通道的体外收缩系统的示意图示出在图1中。一个计算机控制刺激产生的方波脉冲，这是过滤隔离病房，以达到对铂丝周围的每一个孤立的肌肉 - 肌腱复杂的。这也被称为电场刺激。响应的刺激的肌肉收缩的力传感器感测到的，所产生的信号被放大，滤波和调回到该计算机上的A / D转换器（信号调理）。然后，该信号被数字化，并且可以保存购买分析。 </li><li>为了准备体外收缩试验，第一回合在球场上的刺激，A / D转换器（在这种情况下，ADI电源实验室）或类似的软件（ 例如 ，LABVIEW），其次是电脑。 </li><li>打开Chart4软件（或其他软件的版本与系统兼容）和始发EA 4通道的任务，启动硬件配置的软件和硬件的正常通信表示，在软件上的START按钮开始运作（直播）4通道功能，可以同时对肌肉的测量的野生型和突变小鼠，或2的EDL和2从相同的鼠标的比目鱼肌，并且它可以用于其他如隔膜（整个膜片，半隔膜，或隔膜肌条）和胫骨前肌;它也可以被用于心脏肌肉制剂和肌肉束，尤其是当使用的是大鼠肌肉。在这种情况下肌肉的大小可能会限制氧气的扩散，使肌肉束是一个更好的选择。然而，专家解剖技术，需要为肌束准备。 </li><li>力传感器的校准：这是为了确保在不同的信道，并在不同的时间产生的数据集的可比性。首先开始录音，sequentiaLLY挂在样品上的力传感器的树丛1克，2克和5克重量，停止记录，计算相应的变化示出在mV在每个通道Chart4软件中，绘制ΔMV和重量来检查的线性度和确定mV和克的力之间的转换系数。我们建议执行这些无论是在开始或在每个实验结束校准，以保证特定的校准为每个特定的实验中得到。 </li><li>的Teflon管检查正确的连接，排出的液体中存在的任何的组织浴管，洗涤三次或更多次的腔室与DDH 2 O，20〜25毫升2.5毫摩尔的Ca 2 + Tyrode溶液填充。的Ca 2 +和葡萄糖在泳季溶液中，以帮助维持细胞膜的完整性，最优的Ca 2 +的加载的SR，和一个由肌肉本身的ATP生成现成的能量来源，由于线粒体功能仍然完全在这些制剂中。 </li><li>检查正确的连接的氧供给，打开氧气罐，调整氧气流提供的腔室的扩散和均匀的鼓泡。的氧含量可以容易地确定作为收缩力随时间变化的函数。也可以安装流量计，尤其是如果研究人员感兴趣的研究低氧或高氧的影响。如果肌肉缺氧，力自然会降低。虽然高氧也损坏肌肉，但其影响可能会对更难被检测到，因为大多数实验在人工高氧条件下进行的补偿的情况下的正常的血液供应到肌肉。在大多数系统中，它是能够简单地通过控制氧流量的每个室常数用相同量的氧气气泡的不同的腔室。 </li><li>不饱和的通道，所有通道的灵敏度调整，以确保最大力量解决。信道的灵敏度有很大的不同功能的d龄期的动物，实验温度，基因操作，肌肉的体积，小鼠品系，实验者的能力，正确地剖析肌肉免费的损害赔偿责任。根据我们的经验，当测量比目鱼肌收缩力，灵敏度的变化为0.5〜10毫伏/厘米，而在EDL的箱子，灵敏度可能会有所不同从1-20毫伏/厘米。 </li><li>建立一种协议的疲于奔命，刺激的需要。在Chart4软件的宏都可以使用。设定新的宏：在Chart4软件，单击“开始测量”，然后单击“宏”，宏命令，开始录音，开始重复，选择所需的刺激频率，然后结束重复。一个平衡的协议，刺激，每分钟为30分钟，并，疲于奔命协议，刺激重复每2秒5分钟。中的这些变化的周期性的刺激，直接影响，这也是一个函数的刺激持续时间的占空比。这些参数可以改变，以测试不同的ASPECTS的收缩力和疲劳。 </li></ol> 2。准备完整的肌束<ol><li> EDL夹层小鼠牺牲后，美国国立卫生研究院的指导方针和IACUC机构动物的协议。鼠标断颈处死。鼠标，然后安排在横向位置。 EDL肌是一个快速糖酵解的肌肉，淡淡的粉红色，白色。这是约长10-13毫米，体重8-11毫克野生型C57BL / 6小鼠。的EDL的延伸脚趾2-5，和后背成脚在脚踝弯曲的功能。腓总神经支配。解剖EDL，作一粗浅的皮肤切口，切开筋膜之间的胫骨前和后的肌肉群，和本地化的起源（近端），韧带连接的前向外侧髁，胫骨和卓越的3/4表面腓骨（骨间保证金）。切细腻的眼科剪刀远侧的肌肉韧带，这再修改Ë释放近端EDL肌肉的起源。用钝头镊子和拉韧带保持缓慢释放的EDL。它削减一些肌束周围的EDL EDL和其他周围的肌肉，是至关重要的，因为可能会损坏EDL肌肉在这种微妙的步骤一个步骤，可能有必要。下一步，将插入区的四个远端肌腱插入的数字2-5的中段和远端指骨。尽可能切筋的肌肉。 </li><li>孤立的EDL肌转移到一个解剖菜含有等渗台氏液。某些突变体的转基因和基因敲除的动物模型有非常脆弱的肌肉和利用的Ca 2 + -自由Tyrode溶液是必要的，以防止的Ca 2 +诱导的肌肉损伤前收缩力测量。接下来，使用外科结两端的EDL肌扎紧从肌肉尽可能向远侧。一个很好的措施是为配合略高于中旬-P韧带或肌腱OINT纵向。使用6-0大小缝合此过程以及传输的EDL肌肉的O 2饱和的组织浴室，安装在样品上的力传感器的槽和浴室的底部上的信纸钩肌肉，重复换另一条腿的过程。我们也开始利用一种新方法，用夹子固定，而不是缝合持有的肌肉。如果主要的目标是研究动力学性质和/或获得的肌肉力量，但建议，缝合线尽可能的短，或缝合线被替换为一个金属棒。 </li><li>比目鱼肌的解剖：比目鱼主要是一个缓慢的氧化具有丰富的红色肌肉。是短比EDL〜1毫米，但重量稍多于的EDL。它由胫骨神经支配和足底弯曲脚它会执行的动​​作。要解剖比目鱼，请访问后外侧的腿，拨开腓肠肌，通常包括soleu，并确定与暗红色的肌肉。在原点（近端），切断连接到后胫骨的近端半沿soleal线和近端三分之一后腓骨韧带;下，在插入（远端），切断跟腱插入到后跟骨。仔细游离正确安装了比目鱼肌和比目鱼肌在洗澡室的EDL。 </li></ol> 3。测量的离体骨骼肌收缩<ol><li>一旦肌肉被安装在单独的组织浴室，开始录制。最相似的系统允许的基线力记录归零。此函数通常是与放大器相关联，在PowerLab系统的特定情况下，作为桥式放大器的功能。它有利于观察基线的变化，并进行分析，从零提供了一个便捷的方式，所有的肌肉收缩。孤立的肌肉，然后与方波脉冲刺激范围广泛的腔室的大小的功能，铂丝厚度，导线之间的距离，和甚至实验溶液的组合物。我们已聘请电流为60 mA（我们注意到，超过350毫安的电流似乎是不利于肌肉的准备），刺激列车，350，500，和1000毫秒，这取决于特定的协议的目标。个别的方波脉冲，应该为期0.3-1毫秒。 </li><li>下一个步骤是选择一个频率能够生产的稠强直性刺激（刺激前：〜100 Hz至允许的最大的力的生产中的EDL肌肉和比目鱼肌〜60赫兹），而缓慢而小心地拉伸肌肉识别这些肌肉的最佳长度。仔细的肌肉拉长，等待30秒，在100 Hz的刺激，等待30秒，伸展，和重复的刺激，直到点的力不增加了。 </li><li>这些肌肉有undergon的Ë环境的显着变化。建议肌肉，以适应新的环境，我们的协议中的一个步骤称为“平衡”。这些肌肉的平衡过程中使用的拉伸阶段，100赫兹为20-30分钟，在相同的频率的刺激，直到至少5个连续的强直收缩是完全稳定（未减少，而不是增加，稳定的基线）。期间的平衡的刺激列车（100赫兹，单独的脉冲持续时间为500毫秒，1毫秒）的周期是1分钟，相当于1.66％的占空比，非疲劳刺激。在此背景下，占空比为1.66％，是指，超过总量​​为100％，肌肉工作的时间的1.66％，通过增加占空比肌肉最终可以诱导疲劳。另有约定的肌肉的健康，野生型小鼠表现出疲劳的这个平衡期间的档案，它是可能的，他们在解剖损坏，缺氧发生在室/肌肉，或过度元素ctrolysis通过刺激电极的产生自由基。先前已建议westerblad电流大于400毫安的诱导自由基23的形成。显然，最优质的铂建议，因为其他金属肯定会导致自由基的形成与肌肉毒性。 </li><li>获取的力对频率的关系（FF）通过刺激肌肉与以下刺激频率：1-140赫兹（5-10赫兹的增量），进行实验时，在25℃下具有周期为30-60 s进行实验，在37°C时，延长FF更高的频率高达300赫兹膈肌180-200 Hz的比目鱼，和220-250赫兹的EDL。其次，确定频率的刺激，产生最大强直收缩力（T MAX）及约½最大强直收缩力（1/2 T MAX），有时是很难得到确切的½T 最大 EDL和唯一我们肌肉正在被使用，如果只有一个激励源，因为这些肌肉的特性之间的差异。一种可行的解决方案是，以确定的频率，产生30-70％的T max的。这些频率的刺激的理由是T MAX提供了重要的信息，而½T 最大收缩的机械活动/调制提供更多相关的Ca 2 +调节和ECC过程中的信息。要确定最大的力量已经达到10-20毫米，咖啡因可以添加到沐浴液，同时刺激肌肉。如果已达到最大力量，力量会增加在咖啡因存在的。 FF是右移和力量倾向于在较高的实验温度要稍高。使用本系统时，任何其他的刺激频率，如果需要的话，可以进行。明显的收缩更新的快速糖酵解的EDL的肌肉和缓慢氧化比目鱼肌如图2中所示。 </li><li>平衡后，疲劳的肌肉在½T max的为5分钟，刺激间隔2 s和25％的占空比（ 图3）。这种特殊的疲劳，协议被认为是肌质网释放Ca 2 +肌肉收缩31，以更好地反映了贡献。 </li><li>恢复肌肉½T max的30分钟或直到力是稳定的，以1分钟的间隔。现在使用的T max的刺激，这被认为是反映收缩机器31的状态，可以进行一个额外的疲劳，协议。另一种选择是延长疲劳协议地嵌入刺激列车生成期间T MAX和½T max的疲劳和恢复与相同类型的刺激肌肉。 </li><li>重复在步骤3.4中所描述的FF，其理由是，表型之间的差异的差异erent菌株，疾病模型，或药物治疗，可以观察到通过分析之前和之后的FF疲劳。我们以前报道例如，FF年轻的野生型肌肉疲劳后移位到左边，而从中年肌肉肌肉，它被移动到右侧，作为一个功能暗示骨骼肌疲劳差调节作用老化。 </li><li>要探测的胞外Ca 2 +在肌肉收缩的条目的贡献，洗澡的解决方案可以被改变成溶液不含有Ca 2 +的 ，但0.1mM的EGTA（0 mM的Ca 2 +的溶液）的Tyrode 32。可替换地，可以应用在不同商店的操作通道阻滞剂泳季溶液。的几个例子是：2 -氨基乙基diphenylborinate（2-APB），SKF96365，3,5 -双（三氟甲基）吡唑2（BTP-2）和阿珠莫林，等33，34。咖啡因可以用来探测兰尼碱受体的功能，氯化钾可用于评估整体这些准备去极化性能。其他药物也可用于研究的重要调节力在疲劳的Na +，K +和Na +-K +泵35。这些药物具有相对小的尺寸和似乎很快扩散到这些肌肉，证明他们的切身影响。由任何给定的药物的效果的缺乏并不一定意味着这种药物是无效的，和剂量使用远高于在单肌纤维实验所用的额外的测试，有时是必要的。 </li><li>本体外系统的一个独特的应用，导致了最近发现的机械交替TRIC-A - / -肌肉4，30。交替“被定义为波动突发事件的收缩力在下降阶段的疲劳的配置文件。在这些活动中的收缩力可以随时增加超过以前的水平力疲劳的，因为这无论是较多的Ca 2 +被释放或收缩机械已成为更为敏感的Ca 2 +的刺激。爆发的收缩力高50％，比以前的力量和爆发应该看到至少有10次在5分钟疲劳性刺激过程。在骨骼肌中不常见的技工交替从野生型小鼠中，但可以看出，在一些突变与扰动的细胞内Ca 2 +信号过程，如三聚体的细胞内阳离子通道A型（TRIC一个） - / -肌肉肌肉4。机械交替通过疲劳的刺激引起的，治疗与咖啡因和环匹阿尼酸（CPA），有代表性的记录，这些机械的交替， 见图3。这种现象的本质是非常有趣的，而一个肌肉，疲惫不堪，似乎能够瞬间产生更多的力量。在我们以前出版，组合与单细胞的Ca 2 +分析结果显示交替的外观是一个结果SR的Ca 2 +过载和不稳定的SR。我们相信，一个更深入的了解交替可能会导致更好地了解ECC过程。 </li><li>在实验结束时，测量使用校准卡钳和分析天平的各个肌肉的长度和重量，闪光灯在液氮中冷冻肌肉，并储存在-80℃，因为可以进行生化分析，在这些肌肉。这些肌肉也可以是机械剥皮详细探测的ECC过程，或通过化学剥皮的测定必需的收缩性能的情况下的ECC调控机制。 </li><li>记录下来的肌肉力量（毫伏）首先被转化为克强制校准结果的基础上，然后归一化到的生理的横截面面积（PCSA），使用下面的公式：肌肉力量（N /厘米2）=（力（g）×肌肉lengt（CM）×1.06）/（肌肉重量（g）×0.00981）5,36。另外，肌肉力量可以被归到总蛋白或使用Bradford蛋白分析/ Commossie的蓝染色量化的个别肌肉的肌动蛋白含量。肌肉质量，而在某些情况下可能会受到严重影响，由于疾病，衰老，药物治疗，力正常化肌肉质量，蛋白质和/或肌动蛋白含量的基础上，可以提供更稳定的读出。 </li></ol> 4。代表性的成果典型的室温EDL和比目鱼肌收缩力的低，中和高频刺激响应示于图2。 5 Hz的刺激诱 ​​导的EDL收缩仍然作为个别的SERCA的Ca 2 + ATP酶和本征的Ca 2 +收缩机械的灵敏度特性，而比目鱼收缩5赫兹开始熔化（较慢的ATP酶活性的快速动作由于抽搐ð更高的灵敏度的Ca 2 +的收缩机械的），但仍然是单独的峰值力。在20 Hz刺激，EDL收缩部分融合，而，比目鱼形成一个完全融合的强直收缩力。在频率的刺激，产生刺激T max的，它可以在室温的温度而有所不同，从EDL和60-90赫兹比目鱼80-110赫兹，快速上行程和EDL强直力量快速弛豫指出，这是违背缓慢的特性的比目鱼肌。 图2B表明的力-频率曲线右移的EDL肌相比，比目鱼肌，表明比目鱼肌更敏感Ca 2 +释放的，在任何给定频率由于慢肌球蛋白和肌钙蛋白亚型的存在下刺激。此外，收缩机械具有相对更大的力的比目鱼肌中在较低的频率响应， 图3示出正常疲劳的EDL（上图）和比目鱼肌（中间面板）的档案。请注意更快的收缩力下降的EDL肌肉疲劳的刺激下，有效减少和较高的结束时的5分钟疲劳的协议。最后，一个典型的机械alternan档案中的突变的肌肉被示于图3（下面板），这被定义为在肌肉疲劳的档案中的下降阶段期间的瞬态力爆发。爆发的收缩力高50％，比以前的力量和爆发应该看到至少有10次在5分钟疲劳性刺激过程。 <img alt="图1" src="/files/ftp_upload/4198/4198fig1.jpg" /> 图1。一个4通道的体外收缩系统的示意图。方波脉冲产生由一个计算机控制的Grass刺激。刺激隔离过滤来自电刺激Trical公司刺激单元，以除去任何的电信号中的波动，并建立了稳定的方波信号。该滤波后的电信号被发送到4沐浴室含有铂丝电极周围的每个隔离的肌肉。最终，它是当前的跨越两个电极（称为字段刺激），产生一个动作电位，诱导的肌肉收缩。这种收缩是由一个特定的力传感器，发送到桥式放大器检测，过滤，平均（信号调理）通过A / D转换器和记录由计算机软件。 <img alt="图2" src="/files/ftp_upload/4198/4198fig2.jpg" /> 图2。代表EDL和比目鱼肌的收缩力（A）诱导的收缩力由5赫兹（上面板），20赫兹（中间面板）和最大强直力（T 最大 ）（下面板）;入口示出了跟踪的收缩力受损的肌肉;（B）一种代表个人收缩力与频率的关系EDL（FF，上图）和曲线，从FF（下图） <a href="http://www.jove.com/files/ftp_upload/4198/4198fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4198/4198fig3.jpg" /> 图3。代表疲惫不堪，配置文件和机械交替。EDL肌肉（上图）和缓慢下降的比目鱼肌疲劳的个人资料（中）一个典型的快速下降，疲惫不堪的档案。疲劳运动刺激导致的外观机械交替一个TRIC一个- / -肌肉干扰Ca 2 +的处理性能（下面板）。

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Cell Biol. 4, 379-383 (2002).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azumolene+inhibits+a+component+of+store-operated+calcium+entry+coupled+to+the+skeletal+muscle+ryanodine+receptor.">Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor.</a> J. Biol. Chem. 281, 33477-33486 (2006).</li><li>Renaud, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+force+development+by+Na+,+K+,+Na++K++pump+and+KATP+channel+during+muscular+activity.">Modulation of force development by Na+, K+, Na+ K+ pump and KATP channel during muscular activity.</a> Can. J. Appl. Physiol. 27, 296-315 (2002).</li><li>Brotto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+biochemical+modifications+in+skeletal+muscles+from+malarial+mice.">Functional and biochemical modifications in skeletal muscles from malarial mice.</a> Exp. Physiol. 90, 417-425 (2005).</li><li>Brotto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+fatigue-induced+modification+of+function+and+proteins+in+intact+and+skinned+murine+diaphragm+muscle.">Hypoxia and fatigue-induced modification of function and proteins in intact and skinned murine diaphragm muscle.</a> Pflugers Arch. 440, 727-734 (2000).</li><li>Smith, M. A., Reid, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+modulation+of+contractile+function+in+respiratory+and+limb+skeletal+muscle.">Redox modulation of contractile function in respiratory and limb skeletal muscle.</a> Respir Physiol Neurobiol. 151, 229-241 (2006).</li><li>Bagni, M. A., Cecchi, G., Colomo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofilament+spacing+and+force+generation+in+intact+frog+muscle+fibres.">Myofilament spacing and force generation in intact frog muscle fibres.</a> J. Physiol. 430, 61-75 (1990).</li></ol>

没有利益冲突的声明。

测量的收缩力和疲劳骨骼肌功能的总体评价是非常重要的。此法的主要目的是确定在肌肉力量和疲劳性能，在某些病理条件下的变化，如肌肉减少症和肌肉疲劳，肌肉收缩力的药物/试剂测试效果。由于肌肉力量是密切相关的细胞内Ca 2 +释放，细胞外Ca 2 +进入和这两者之间的串扰，我们还可以收集信息，使用这种方法对Ca 2 +信号在骨骼肌中的地位。在这里，我们展示了一个...

骨骼肌附加到骨头的骨架和中枢神经系统的控制下，产生收缩力。励磁收缩耦合（ECC）的是指转换的电刺激的机械响应的过程。 Ca 2 +信号是骨骼肌的收缩功能的一个必要组成部分。有效的Ca 2 +动员从肌质网（SR）是一个重要组成部分是用于ECC在肌肉细胞中1，2，和变化，细胞内Ca 2 +信号用在一个数量的肌肉疾病3-5相应的收缩功能障碍的基础。肌肉收缩的正确评估是必要的，免费的Ca 2 +影像和其他实验获得的见解骨骼肌功能，不只是在收缩，而且在动力学水平。力和速度也可以得到通知的重要属性肌肉力量的ECC过程中不同的生理和病理条件下的状态。 这肥沃的研究领域有非常丰富的历史超过两千年6和肌肉的收缩出现的许多理论。现代的肌肉可能在1674年至1682年开始研究用显微镜观察肌纤维的横条纹和肌原纤维中的列文虎克6。将近一个世纪之后，路易伽伐尼指出，青蛙肌肉收缩，大力的神经被触动时，用解剖刀在火花放电从一个遥远的电机7-9。收缩，也可以通过金属导体连接的腿部的肌肉的神经。复杂的电气信号转导机制的细 ​​节所倡导的伽伐尼的最终制定的霍奇金，赫胥黎和Katz在其著名的公式10，11，成为电的基础。显着的观察结果凛蒙古包的影响外Ca 2 +的收缩的青蛙心脏和骨骼肌肉的12-15代表第一个重要步骤识别的Ca 2 +作为一个关键的调节肌肉的收缩16，17。从1980年到现在实现了一阵，发现在肌肉收缩领域，由于引进肌肉的收缩和疲劳的协议，在小鼠的骨骼肌18。琼斯和爱德华兹是第一个表明，低频率间歇性疲劳（有效减少运动引起的）19与变化的ECC机制，而不是收缩装置。的迟到1980年的和1990年代初，Kolkeck 等[20]，Kolbeck提供和Nosek 21，和里德22使用膈肌从啮齿类动物模型，以研究茶碱，cortiosterone，和自由基的影响骨骼肌肉的收缩，而布鲁克斯和Faulkn呃就在快和慢肌的小鼠22的重复力和功率测量的测量报告。此外，Lannegren，Westerblad，羊肉，Westerblad的第一个直接链接与细胞内Ca 2 +调控的离体收缩，并开始质疑23日，24肌肉疲劳性酸中毒的作用。 我们的实验室有显着贡献自2000年初，对了解新基因的调节和调控作用在肌肉ECC重要的角色，在肌肉收缩，易疲劳，和老化，使用完整的小鼠肌肉收缩研究的结合，细胞内的Ca 2 +监控中完整肌肉和皮肤的纤维和分子遗传操作3-5，25-29。 在这里，我们详细介绍了实验协议，用于测量小鼠孤立的比目鱼肌和趾长伸肌收缩（EDL）的肌肉，这对应于一个多缓慢氧化（Ⅰ型和Ⅱa族的肌纤维）和大多快速glyocolytic肌（IIb型和IIx肌纤维）与不同的收缩性能。在这个协议中，完整的肌肉-肌腱配合物的分离和沐浴在ADI PowerLab系统Radnotti室系统，无论是纯的氧或氧的混合物（95％）和CO 2（5％）供给。从基层刺激所产生的电刺激收缩力和力传感器与ADI公司的PowerLab/400系统集成，允许自定义宏例程来控制数据的采集，收集，数字化和存储检测。这种设置可以测量肌肉力量，肌肉力量，以及力与频率的关系，肌肉疲劳，恢复肌肉疲劳，肌肉的收缩速度和整体动力学特性。此外，药物对肌肉收缩的影响，可以通过这些实验中监测。 </p&gt; 这种方法的优点在于从骨骼肌肉去除神经和血管成分，可直接评估肌肉收缩的内在属性。此外， 体外收缩分析允许操纵周围的孤立的肌肉细胞外环境中，这使得能够使用，以定义其生理作用骨骼肌功能的药理操纵各种离子渗透通道和转运。 体外系统，使我们最近发现一个独特的alternan行为在某些突变的肌肉，这与改变细胞内Ca 2 +处理性能 。交替“被定义为波动突发事件的收缩力在下降阶段的疲劳的配置文件。在这些活动中收缩力暂时增加超过以前的水平力D姚小萍Yao Xiaoping疲劳的刺激，也许是因为无论较多的Ca 2 +的释放或收缩机械已成为更为敏感的Ca 2 + 30。环匹阿尼酸（CPA），肌浆内质网钙ATP酶（SERCA），咖啡因，激动剂的ryanodine通道（RyR的）的可逆阻断剂的治疗，和反复疲劳刺激都可以诱导机械交替4，表明交替有直接关系的EC耦合过程的调节。示范的方法，机械交替在设置在体外收缩，诱发并记录作为一个例子来展现多元化的实验参数，可以得到系统或类似的，根据个人的研究兴趣。 这种方法可能是肌肉生理学研究的兴趣。类似的设置也可用于从其他孤立的骨骼muscle-tendon/ligament的复合物解剖位置，以及用于单纤维和肌条。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 2-APB </td><td> TOCRIS </td><td> 1224 </td><td>拦截器的数量的Ca 2 +进入渠道，包括SOC和TRP等。 </td></tr><tr><td> SKF96365 </td><td>西格玛</td><td> SKF-96365 </td><td>拦截器的数量的Ca 2 +进入渠道包括SOC和受体介导的Ca 2 +进入等</td></tr><tr><td> BTP-2 </td><td>密理博</td><td> 203890-5MG </td><td>比较具体的SOC阻滞剂</td></tr><tr><td> CPA </td><td>西格玛</td><td> C1530 </td><td>可逆SERCA受体阻滞剂</td></tr><tr><td>咖啡因</td><td>西格玛</td><td> C0750 </td><td>快速行动碱受体激动剂</td></tr><tr><td> Radnoti四单位组织器官水浴系统</td><td> Radnoti </td><td> 159920 </td><td></td></tr><tr><td>结合组织的支持/刺激电极</td><td> Radnoti </td><td> 160151 </td><td>垂直的Zig Zag型组织的支持</td></tr><tr><td>四桥式放大器</td><td>的ADInstruments </td><td> FE224 </td><td></td></tr><tr><td> PowerLab/400 </td><td>的ADInstruments </td><td></td><td>该产品是不再可用。选择其他版本的数据采集系统。 </td></tr><tr><td>力换能器（5 MG - 25克） </td><td>的ADInstruments </td><td> MLT0201/RAD </td><td></td></tr><tr><td>图V4.02 </td><td>的ADInstruments </TD&gt; <td></td><td> LabChart 7.3是图软件的最新版本。 </td></tr><tr><td> S8800双脉冲数字刺激</td><td> GRASS技术</td><td></td><td>该产品是不再可用。 S88X双输出方波脉冲刺激是一个较新的刺激。 </td></tr><tr><td> RF变压器隔离单位</td><td> GRASS技术</td><td>型号SIU5 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table>

ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles

这里所描述的方法来衡量的离体骨骼肌的收缩。参数，如肌肉力量，肌肉力量，收缩动力学，耐疲劳性，和恢复疲劳后，可以得到以评估具体方面的兴奋收缩偶联（ECC）的过程中，如兴奋性，收缩机械和Ca 2 +的处理能力。这种方法消除了神经和血液供应，并着重对离体骨骼肌本身。我们经常使用这种方法，虽然调制的Ca 2 +信号转导通路，从而改变骨骼肌的收缩特性，以确定遗传成分。在这里，我们描述了一种新发现的骨骼肌表型， 即 ，机械师交替，作为一个实施例的各种和丰富的方式获得的信息，可以使用在体外肌的收缩力测定。此法单细胞分析，遗传方法和生化组合斯特雷检测到ECC在骨骼肌的机制提供了重要的见解。

Watch this Scientific Journal Video about Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles at JoVE.com

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

我们描述了一种方法来直接测量肌肉力量，肌肉力量，收缩动力学和离体骨骼肌疲劳性的<em在体外</em系统使用领域的刺激。有价值的信息，对Ca<sup&gt; 2 +</sup&gt;处理性和机械的肌肉收缩，可以使用不同的刺激协议获得。

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

ex-vivo-assessment-contractility-fatigability-alternans-isolated

Research

JoVE Journal

Biology

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

23.6K 次观看.  UMDNJ-Robert Wood Johnson Medical School. 我们描述了一种方法来直接测量肌肉力量，肌肉力量，收缩动力学和离体骨骼肌疲劳性的

视频: Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

等距和骨骼肌产生偏心力评估隔离肌营养不良症的小鼠模型

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

测量小鼠分离和孵育成熟骨骼肌中胰岛素和收缩刺激的葡萄糖摄取

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...