This work was supported by the following funding sources: R01-AR063649, AG-020591, F31-AR035931.

所有涉及动物或人类受试者的程序应按照相关准则，法规和监管机构来执行。密歇根委员会对动物的使用和保养密歇根大学医学中心机构审查委员会的大学大学（UCUCA），并批准了所有的动物在本文中描述的人类手续。 1.解剖和存储解决方案股票注:在下面的指令中的最后一个卷可以放大或缩小的期望。 <ol><li>在一个1000毫升烧杯中，加800毫升去离子水（ASTM类型1）。保持温和搅拌，都在表1中列出的化合物添加到去离子水中，并允许他们溶解。 <table><tbody><tr><td> 复合 </td><td> 期望中的浓。 （M）的 </td><td> 式重量（g / mol）的 </td><td> 加至1升（克） </td> &lt;/ TR&gt; <tr><td> K-丙酸</td><td> 0.250 </td><td> 112.17 </td><td> 28.040 </td></tr><tr><td>咪唑</td><td> 0.040 </td><td> 68.08 </td><td> 2.720 </td></tr><tr><td> EGTA </td><td> 0.010 </td><td> 380.40 </td><td> 3.800 </td></tr><tr><td>氯化镁2•6H 2 O </td><td> 0.004 </td><td> 203.31 </td><td> 0.813 </td></tr></tbody></table></li></ol> 表1:解剖和存储原液组件。 <ol start="2"><li>带来至1000毫升去离子水的最终体积。注意，这是不必要至pH的溶液在这个时候。储存原液于4℃。 </li></ol> 2.存储解决方案<ol><li>为了使将200ml的存储解决方案，开始用100毫升解剖和储存原液在250毫升烧杯中。添加足够的Na 2 H 2的ATP带来最终腺苷三磷酸（ATP）的浓度为2毫摩尔。带来至200毫升与甘油的最终体积。调节至pH 7.00与氢氧化钾（KOH）。储存在-20℃下贮存溶液。 注意:由于甘油的粘性性质，它可能难以准确地按体积分配。由于这个原因，我们一般按重量计添加甘油（100毫升的丙三醇的重量大约为126克）。 </li></ol> 3.解剖解决方案<ol><li>使200毫升解剖溶液，开始用100毫升的解剖和储存原液在250毫升烧杯中。 </li><li>添加足够的Na 2 H 2的ATP使最终ATP浓度为2毫摩尔。调节pH值至7.00用KOH。使终体积为200毫升去离子水。等分试样中以2.5ml体积并储存在-80℃。 </li></ol> 4.进行剖析与解决方案58的Brij 注意:的Brij 58是一种非离子型洗涤剂是破坏（permeabilizes）脂质双层。 &lt;OL&gt; <li>使200毫升解剖用的Brij 58的溶液，开始用200毫升中的250毫升烧杯中的解剖溶液。保持温和搅拌，添加1克的Brij 58（0.5％重量/体积）的解剖溶液，并允许它以溶解。等分试样中以2.5ml体积并储存在-80℃。 </li></ol> 5.测试解决方案注:以下是改编自Moisescu和Thieleczek 1978年（6）。见讨论有关准备测试解决方案的补充意见。 <ol><li>制备三个独立千毫升烧杯标记为"放松"，"预活化"和"激活"。加入400毫升去离子水到各烧杯中。 </li><li>在表2所示的化合物添加到适当的烧杯中，然后加热该溶液至之间70℃和80℃。保持70-80℃的溶液温度下进行30分钟，同时连续搅拌。 注:70-80℃助攻的ELIMI温度碳酸的国家由碳酸钙用EGTA在活化溶液中的反应而形成的。放松溶液和预活化溶液处理以相同的方式作为活化溶液，以保持一致性。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2" style="border:0px #fff solid"></td><td colspan="2"> RELAXING解决方案 </td><td colspan="2"> PRE-活性溶液 </td><td colspan="2"> 活化溶液 </td></tr><tr><td>复合</td><td>式重量（g / mol）的</td><td>所需浓度（mM） </td><td>必需的海量（G） </td><td>所需浓度（mM） </td><td>必需的海量（G） </td><td>所需浓度（mM） </td><td>必需的海量（G） </td></tr><tr><td> HEPES（酸） </td><td> 238.30 </td><td> 90.0 </td><td> 107.24 </td><td> 90.0 </td><td> 107.24 </td><td> 90.00 </td><td> 107.24 </td></tr><tr><td> 氧化镁 </td><td> 40.31 </td><td> 10.3 </td><td> 0.208 </td><td> 8.5 </td><td> 0.171 </td><td> 8.12 </td><td> 0.164 </td></tr><tr><td> EGTA（酸） </td><td> 380.40 </td><td> 52.0 </td><td> 9.890 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td> 52.00 </td><td> 9.890 </td></tr><tr><td> HDTA（酸） </td><td> 348.36 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td> 50.0 </td><td> 8.709 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td></tr><tr><td> 碳酸钙 </td><td> 100.10 </td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td bgcolor="#A4A4A4"></td><td> 50.00 </td><td> 2.503 </td></tr></tbody></table> 表2:重新laxing，预活化和活化溶液组成。 <ol start="3"><li>冷却该溶液至室温，并添加足够的NaN 3 / KOH，使其终NaN 3的浓度为1mM。 注意:NaN 3（和叠氮化钠）是有毒的。请参阅之前处理这种化学物质的化学MSDS。 <ol><li>至100ml 100mM的叠氮化钠溶液中，添加0.65克的NaN 3至10ml的1N KOH。调整至100ml去离子水的最终体积。 </li></ol></li><li>将pH调节至约7.10使用的KOH。 </li><li>以下步骤5.4添加足够的Na 2 H 2的ATP，使最终ATP浓度至8毫和足够的Na 2 CRP以使终肌酸phosophate（CRP）浓度至10mM。 </li><li>带来每种溶液的使用去离子水500毫升最终体积。寒意或加热溶液到在该实验将执行的温度，然后用氢氧化钾使pH至7.10，同时保持该温度。 </li><li>添加放松溶液至包含预活化溶液的烧杯，使得最终的预活化溶液为1份放松在500预活化溶液溶液。等分试样中以2.5ml体积并储存在-80℃。 </li></ol> 6.进行缝合循环<ol><li>开始的非无菌的USP 10-0单线尼龙缝合线料。 </li><li>使用镊子来创建与使用双上手结技术的链环。减少大小的结约750微米的直径。环直径可在显微镜下用目镜刻度标记进行评估。 </li><li>使用microdissecting剪刀以除去多余的缝线只留下在任一侧的环和小的（500微米）的尾巴。的成品环的例子示于图1。 </li><li>直到6.2-6.3可用4回路已重复步骤。商店循环在硅氧烷弹性体镀彼得我菜供将来使用。 注意:四缝合环所需要的每根纤维进行测试。 </li></ol> 7.束样本注意:下面的步骤描述的过程解剖原始样本成更小的实验&#39;捆&#39;从其中单纤维将最终被提取和测试。在任何时候都应该样品小心对待。对于这个描述的目的，说明将给出如果研究者是右撇子。 <ol><li>获得的感兴趣的样品，并将其转移到设备，在夹层将发生。 注:组织活检的方法将根据实验模型和研究设计有所不同。在可能的情况，肌肉灌注应保持，直到活检时。 </li><li>如果样品是收获站点和解剖部位之间传送，传送它在含有冷冻解剖溶液中，同时保持在冰上的小瓶。</li><li>准备5cm的硅弹性体镀培养皿冷冻解剖溶液和两到三个昆虫安装管脚（直径100微米，不锈钢）。 </li><li>样品转移到菜。确保样品仍浸没通过增加更多的解剖解决方案，如果有必要的。 </li><li>检查在显微镜下的样品和操纵它对齐的纤维的纵向轴线朝向所述研究者（ 图2）的右肩。然后，通过钉扎在角锚定样品的菜。 注意:请使用任何剩余的结缔组织如在这个时候，因为这将最大限度地提高样品的使用和保护纤维的完整性锚固点。束可以固定在小的张力，以帮助限定纤维间边距。 </li><li>与在左手镊子和在右边的microdissecting剪刀，开始沿纤维之间的纵向边缘轻轻解剖束 注:根据其总长度，进一步剖析捆绑成无数个较小的包。 </li><li>确保束尺寸测量约0.5-1毫米宽和≥3毫米长。 ，或者将一个统治者名下的菜评估使用在目镜刻度标记显微镜的尺寸。 </li><li>取下并丢弃任何由镊子或销作为解剖过程的结果受损组织。 </li><li>重复该过程，直到束的足够数量的被解剖或直到样品已经用完。 注意:可以得到将取决于很多因素，包括大小和初始样品的情况下，肌肉的形态，和研究者的技能束的数量。 </li></ol> 8.纤维透<ol><li>转移从解剖溶液束到含有2.5新鲜，冷冻，用非离子型洗涤剂"的Brij 58&#39;解剖溶液的小瓶中加入（0.5％，重量/体积）。在冰上孵育30分钟，偶尔，轻轻摇动。确保束保持整个孵化淹没。 </li><li>在30分钟温育结束时，捆转移到含有新鲜解剖溶液（无的Brij 58）的小瓶中，并轻轻搅动，并简要以除去任何残留的洗涤剂。 </li></ol> 9.准备捆绑的存储<ol><li>转移捆含冷藏存储解决方案小瓶和孵化一夜之间在4℃。 </li></ol> 10.商店捆绑<ol><li>第二天，准备一个收纳盒，能够承受-80℃，有足够的个人0.5毫升旋盖锥形管，以适应在解剖过程中（每管一个捆绑）获得的所有包。每个锥形管应充满200-400微升新鲜的存储解决方案。 </li><li>转移捆绑成单独标记锥形管。确保束不坚持锥形管或浮在溶液表面上的一侧。盖住锥形管和储存样品在-80℃直至测试日。 </li></ol> 11.准备实验装置注:自定义装置由容纳长度控制器和力传感器，运动室系统和10X解剖显微镜的一个阶段。千分尺驱动装置允许精准操控的纤维附着表面。激光衍射图案被用于估计小节的长度。实验过程中所产生的数据被记录在个人计算机上。参考图3，对本实验的建立注释的图像。 <ol><li>每个解冻放松，预激活和激活解决方案之一小瓶和冰上维护。需要注意的是ATP和CRP是应保持在低温下不稳定的化合物。 </li><li>准备显微镜，测试设备和相关联的计算机使用。 </li&gt; <li>填写第一个试验室放松的解决方案。在我们的装置中，第一腔室含有棱镜允许研究者拍摄从侧光纤。填第二试验室具有预活化溶液和第三与活化溶液。 </li><li>调节温度，以使在腔温度计显示读数15℃。螺纹两人准备缝合环上同时从力传感器和长度控制器（ 图5A）延伸的不锈钢连接表面。 </li></ol> 12.提取透单纤<ol><li>解冻感兴趣的纤维束，并转移到有机硅弹性体镀培养皿中的新鲜，冰鲜轻松的解决方案。固定捆绑销在任一端，并确保它被浸没。 </li><li>使用镊子，把握光纤在一端和顺利开始沿其纵轴提取它。 注:为压缩破坏引起的镊子纤维到底是不是在此时，因为在这方面的收缩特性不会测试一个关注的问题。护理应，但是，可以提取束的纤维时，由于纤维与细胞外基质之间的粘连会导致过度的张力，最终导致拉伸诱导的损伤采取。需要注意的是相当大的可变性存在于程度肌肉其中纤维粘附到周围的细胞外基质中。被怀疑已损坏由这样的拉伸的纤维应被丢弃。 </li><li>用剃刀刀片或手术刀来修改一个吸管尖所示（ 图4）。光纤引入连同少量放松溶液的尖端。从硅氧烷弹性体镀培养皿的单纤维转移到试验室中，其中包含了轻松的溶液。 </li></ol> 13.安装单纤维注:一步一步depicti上可以在图5中看到。 <ol><li>用镊子轻轻引导，从吸管尖取出​​纤维，并将其固定的长度控制器（左）使用第一缝合线环。 <ol><li>紧缩与镊子循环时使用一个单一的，平滑的运动。确保相等且相反的张力被施加到缝线的任一端。 </li><li>第一环路应从长度控制器安装面的端部捆绑约1至2mm。 </li></ol></li><li>操纵朝向的力换能器的光纤的另一端（右），并使用相同的方法固定在纤维上。使用microdissecting剪刀（ 图5C）删除多余的缝线。 </li><li>通过增加使用的x坐标微米驱动器（ 图3A）的长度控制器和力传感器臂之间的距离放置下少量张力的纤维。 </li><li>螺纹的第二环在第一和锚定纤维在在0.2毫米的力换能器安装面（ 图5D）的端部的一个点。 <ol><li>使用microdissecting剪刀去除多余的缝线。 </li></ol></li><li>纤维附着过程可导致从腔室的溶液的损失。如果必要的话，加入更多的放松溶液以确保溶液的表面是平坦的（既不凹也不凸）。评估采用激光衍射小节的长度，当一个平坦的表面是非常重要的。 </li><li>通过调整在y轴方向上的长度控制器的位置对齐平行于试验室的侧壁上的纤维。 </li><li>使用棱镜侧视图勘测纤维和调整长度控制器在z轴的方向上的位置，直到该纤维是平行于腔室的地板上。 注:定位平行于腔室的地板上的纤维可以不室棱镜由第一聚焦在纤维和叔一端来实现母鸡，不调整显微镜聚焦，利用其z轴千分尺驱动使光纤的另一端成为焦点。 </li><li>如果纤维是以任何方式扭曲，扭曲或损坏作为安装过程的结果，该纤维应当被丢弃，一个新的光纤连接的。 </li></ol> 14.设置最佳小节的长度<ol><li>当纤维已在腔室中被正确地对准，插入校准目标屏幕到显微镜的前方面和对齐在第一实验室。 注意:目标屏是采用标准的光栅方程校准，λ= SLSINθ，其中SL是小节的长度，θ为0°和1°衍射光束和λ之间的衍射角是激光的波长。 </li><li>打开激光器和调整，使得激光通过光纤的中心的舞台的位置。 注意:浓缩的激光光可以破坏牛逼Ø视力。不要试图通过显微镜以可视化的光纤时，激光器上。 </li><li>相对于光纤定位到激光束衍射激光和观察的干涉图案的校准目标屏幕上（ 图6）。关灯更清晰地显现了这种模式。 注意:如果，用激光束的正确定位，没有干涉图案可以看到的，这表明，在纤维的肌原纤维组件是异常/损坏的，而且纤维应更换用新鲜。 </li><li>要设置小节的长度，增加或减少有关使用长度控制器x轴千分尺驱动器，直至衍射光的期望间距目标屏幕上观察到的纤维的张力。 注:纤维的最佳小节的长度将依赖于动物的从其获得样品的物种。 2.7微米的肌节长度一般认为驴的时候是最佳唱从人体组织7,8的纤维。 </li><li>后最佳小节的长度已定，测量两个最内缝线之间的距离。这是通过使用其控制腔室的x轴运动的微米驱动器上的数字读出最容易完成。定位腔，使得所述目镜的垂直横毛发上最里面的缝合线的最内边界对齐和置零微米驱动器上的数字读出。 </li><li>平移沿x轴相对于所述显微镜的阶段，直到到达另一最内缝合。数字显示将指示所述光纤的长度。此值应记录为纤维长度L F。 注意:应该理解的是，这两个最内缝线之间的距离将决定收缩组织的功能长度被评估。研究者应当争取在一个在这方面的一致性（ 即 L F）一系列的实验。 </li></ol> 15.估计截面面积（CSA）的<ol><li>保持光纤为 L f和使用显微镜载相机来同时从顶视图和侧视图捕获纤维的中央部分的高放大倍率的图像。侧视图的图片可使用嵌入在腔室的一侧的棱镜被捕获。 注意:当顶视图和侧视图之间移动来移动所述显微镜仅在y方向上，以确保这两个图像是"寄存器"，因此显示了纤维的同一部分的两个不同的观点是重要的。 </li><li>获得的测量此时在研究后正常化的绝对力。该技术用于获得这些测量后述和在图7A和7B所示。 </li></ol> 16.引导学生回答等长收缩注意:虽然这些实验过程中所产生的数据之间可以收集和解释，而无需使用一台计算机，软件，允许对力的响应的获取，显示，存储和分析是有利的。由我们实验室建立的定制LabVIEW软件允许这些功能，以及有能力设计&#39;运动列车"支配的实验期间的长度控制器的动作。 <ol><li>确认放松，预活化的温度和激活溶液是稳定在15℃。 </li><li>使用腔室控制软件对纤维移动到包含预活化溶液的腔室，并培育有3分钟。 注意:预活化溶液是弱缓冲以供的Ca 2+，导致引入光纤到活化溶液中时非常迅速激活和力的发展。 </li><li>用10秒中剩余的预活化溶液，并保持为L f显示纤维长度，建立零FO在实验记录RCE水平。 注:长度控制器的"查找强制零&#39;运动表明对应于零力的力换能器的水平（ 即纤维简要地变得松弛作为移动的结果）。被动力是零，并且力水平之前到换能器调零运动之间的差异。 </li><li>在3分钟的末尾，将纤维以含有所述活化溶液的腔室，并允许最大等距力发展就证明了在力的高原是前面有一个快速上升。 </li><li>达到最大等长收缩力后，使用长度控制器，以识别对应于零力在含有活化溶液腔室中的力换能器的输出。 注意:这是必要的，因为这对应于零力的力传感器的输出是，在一般情况下，对于每个溶液填充腔的不同。 </li><li>继实现第二个力PLateau，返回该纤维含有该放松溶液的腔室。测试完成。任何一个会话期间吸测试多种纤维所有的解决方案，并添加新的，冷冻的解决方案。 注:以上的长时间引发宫缩最大布伦纳时的循环程序应予以考虑。此协议已被证明在最大限度活化纤维9保存结构和机械性能。 </li></ol>

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的透单骨骼肌纤维的收缩特性评估用在各种各样的环境中，调查肌肉功能。例子包括已老化评估12的影响的研究，锻炼10,13,14，航天15，伤害2,3,16，药物治疗17,18，疾病和19对纤维的结构和功能基因操纵20,21。由于直接评估其天然构肌原纤维的收缩性能的能力，这种技术提供了从中形成潜在的混杂影响是存在的肌原纤维功能缺失的理解，一个有吸引力的平台时，神经肌肉信号传输和激励引起的钙释放被包括在所研究的系统。另外，单纤维的功能性测试可用于补充收缩蛋白鉴定结果如通过免疫组织化学或凝胶电泳+免疫印迹获得22。 之一的骨骼肌的主要功能是产生力。因此的sF O，一个收缩系统的内在动力发电能力的措施，是非常感兴趣的肌肉生理学家。的sF 邻可靠的估计需要两个纤维CSA和FØ准确的措施。因为纤维是，在一般情况下，在横截面中在沿其长度的CSA既不圆形，也不均匀，非常小心，应推定的CSA时服用。为此，测量是在沿光纤的长度的几个位置制成，并在每个位置，从两个角度90°分开。 ˚F 邻可靠的措施，需要注意几个细节，包括占被动力，调整肌节的长度，最大限度地厚，薄丝重叠，采用与钙浓度的测定活化液帽子结果在最大活化，保持所需实验温度，并保持现有的实验当天纤维的最佳储存条件（温度和时间）。 而此处列出的步骤描述了用于评估最大等长收缩力的方法，它是经常需要评估的骨骼肌纤维其它重要的功能特性。这可以通过延伸实验方案以包括纤维的额外的机械操作来实现。例如，在其中纤维缩短针对一系列不同负载的速度的测量允许确定的力-速度关系，从中力功率和速度-功率关系可以计算10,23,24。此外，卸载的缩短的速度可以通过采用"松弛测试"25，其由应用一系列松弛诱导缩短的步骤和measuri来确定纳克由光纤所需要的时间，以消除松弛。即经常报告另一个动力学参数k 潮流 ，以下的机械扰动暂时分离所有的速率常数力重建crossbridges 26。最后，钙离子浓度和活性力产生（"强制-PCA关系"）之间的关系是感兴趣18常，并且可以通过使纤维的一系列与钙浓度范围从低于所述阈值用于激活收缩溶液来确定系统对那些足以引起最大活化，因此最大的力（F O）。 虽然大部分提到设备的需要，以评估单纤维的收缩，其他设备不是绝对必要的。长度控制器，例如，为任何实验方案，需要快速或准确加长纤维或缩短必要，但不是评估最大等长收缩力（虽然在力创纪录零水平力仍然必须通过某种手段来识别）绝对必要的。允许观察从侧面的纤维，而评估横截面积有用的棱镜，实验室中定位的纤维时并不是绝对必要的。此外，另外的装置，用于暴露纤维的各种实验方案可以采用，其中包括制定一个手工操作的腔室或单腔室，其允许快速填充系统和排空的解决方案。最后，虽然子生理实验的温度如15℃下通常用于改善机械测量1,2,3,5,8,12,17,27的再现性，就可以产生在其他温度23有效数据，28只要温度对溶液性质（钙浓度，pH值等 ）的影响考虑在内。 </P&gt; 测试溶液的组合物中的这里所描述的通透纤维技术的最关键的方面。关于解决方案组成的考虑很复杂，超出了本文的范围。在协议部分的步骤5中描述的解决方案的设计重点放在了透化纤维在从预激活到激活解决方案，同时保持恒定的离子强度，阳离子组合物，和摩尔渗透压浓度6,29其传输快速活化。其他的方法来解决成分已受聘具有显着的成功由其他研究小组，并且通常利用出版结合常数和计算工具27,30,31的。钙离子在各种活化溶液中的浓度是在涉及次最大活化如力-PCA评价研究尤为重要。对于实验，其中纤维被完全激活，例如那些描述ð这里，在活化溶液中的钙浓度通常超过由一个舒适的余量，需要以达到最大的力，使得它的精确知识不那么重要。另外磷酸肌酸是用于缓冲，否则将与收缩活动相关联的intramyofibrillar的ATP和ADP的波动很重要的。肌酸激酶是需要从催化的磷酸肌酸磷酸转移到ADP。根据该导致高的ATP周转率，包括在高温下工作或测量高速缩短在快纤维32的实验条件下，肌酸激酶必须加入到该溶液中，以补充其保持结合到纤维中的内源性肌酸激酶。对于要求较低的实验条件下，ATP的再生系统是不太关键27。 的渗透性作用单纤维技术的局限性包括以下内容。通过这些试验产生的数据定义附于实验装置，该特定的肌原纤维单元的收缩性能。因此，该捕获来自该段得到，这反过来，代表了肌肉内纤维的总数的一小部分，整个多核光纤的仅一小部分。研究者因此应该仔细考虑支持从实验中得出的任何结论所需要的采样。此外，评估对纤维功能的运动训练干预的影响假定评估纤维在培训期间确实招募。虽然该协议试图模仿纤维的天然细胞内环境，肌膜透过程是非特异性的，必然允许可溶性胞内组分，以自由地扩散到洗澡的解决方案。细胞膜的通透性的另一后果是渗透平衡由肿胀纤维体积33证据发生变化。该纤维肿胀增加导致肌丝系统34,35的减少钙敏感性的肌动蛋白和肌球蛋白纤丝之间的距离，但可以通过引进大，渗透活性化合物34逆转。最后限制要考虑的是用于将纤维附着到实验装置的技术的结果。这总是需要在附近的附着点歪曲丝系统内部的空间关系，与主治功能缺损。具体而言，纤维在和相邻的连接点的区域在功能上受到影响，从而有助于人为的系列弹性的测量系统。 总之，我们已经描述了通过该评估体外化学透骨骼肌纤维的力产生能力的装置。尽管本文的重点是最大等长收缩力generatin评估人骨骼肌纤维的克容量，实验方法可以修改和扩展，以确定在一系列物种，哺乳动物或其他的各种动力学参数和关系。

骨骼肌的主要功能是产生力。肌肉力通过事件的复序列，其中包括运动神经动作电位，神经肌肉传递，肌肉纤维的动作电位，细胞内钙的释放，活化的监管和收缩蛋白的系统引起体内 。因为力产生是该序列的最终结果是，在力的赤字可以由一个单独的步骤的一个或多个的故障引起的。透化纤维制备的关键属性是它消除了大部分的体内所需力产生的步骤，以仅与肌原纤维装置其余相关的法规和收缩功能。研究者假定控制激活钙和能量（ATP）的交付，并奖励了简化系统，使孤立的监管和收缩的结构评估其天然的合作nfiguration。评估体内观察到的肌肉功能的改变，当使用透骨骼肌纤维的力测量因此价值。例如，我们已经使用此技术来纤维的力发电量从氯化筒箭毒碱缺陷小鼠1表征并评估持续肌无力表现出以下慢性肩袖撕裂2,3的原因。 现代透纤维的方法，可以追溯到早期有影响力的研究和4,5是当前正在使用由多个研究小组。虽然该技术已在文献中进行了描述，它们尚未被呈现在视频格式。本文的目的是要说明一个更新，有效和可靠的技术，用于测量单纤维从化学透化骨骼肌样品的最大的力的发电量。为了做到这一点，一个单个纤维段（在此被称为一个̶0;纤维"）是从纤维的预通透束抽出并置于含有一个轻松溶液的实验腔室中，限定功能，它是一种钙离子浓度即&lt;10纳米。然后使光纤在一端连接到一个力传感器，并在另一端连接到一个长度控制器。与在最佳小节的长度保持的纤维，将其转移到活化溶液，其具有钙浓度足以引起最大活化，从而最大等长收缩力。力数据采集，储存和使用个人计算机进行分析。

<table><tbody><tr><td>Polystyrene culture test tube with cap</td><td>Fisher Scientific&amp;nbsp;</td><td>14-956-3D</td><td></td></tr><tr><td>0.5 ml screw cap microcentrifuge</td><td>Fisher Scientific&amp;nbsp;</td><td>02-681-334</td><td></td></tr><tr><td>0.5 ml microcentrifuge caps with o-ring</td><td>Fisher Scientific&amp;nbsp;</td><td>02-681-358</td><td></td></tr><tr><td>Microcentrifuge cryobox</td><td>Fisher Scientific&amp;nbsp;</td><td>5055-5005</td><td></td></tr><tr><td>pH meter</td><td>Mettler-Toledo</td><td>FE20</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific&amp;nbsp;</td><td>08-757-11YZ</td><td></td></tr><tr><td>Nonsterile-suture 10-0 monofilament</td><td>Ashaway Line Twine</td><td>S30002</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Forceps - Dumont #5</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Microdissecting scissors</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>MZ8</td><td></td></tr><tr><td>Micrometer drives</td><td>Parker Hannifin</td><td>3936M</td><td></td></tr><tr><td>Thermometer</td><td>Physitemp</td><td>BAT-12</td><td></td></tr><tr><td>Water bath circulator&amp;nbsp;</td><td>Neslab Instruments</td><td>RTE-111</td><td></td></tr><tr><td>Temperature controller</td><td>Aplha Omega Instruments</td><td>Series 800</td><td></td></tr><tr><td>LabVIEW software</td><td>National Instruments</td><td>-</td><td></td></tr><tr><td>Computer</td><td>Varied</td><td>-</td><td></td></tr><tr><td>Chamber system</td><td>Aurora Scientific</td><td>802D</td><td></td></tr><tr><td>Length-controller</td><td>Aurora Scientific</td><td>312C</td><td></td></tr><tr><td>Force-transducer</td><td>Aurora Scientific</td><td>403A</td><td></td></tr><tr><td colspan="4">Reagents</td></tr><tr><td>K-proprionate</td><td>TCI America</td><td>P0510</td><td></td></tr><tr><td>Imadizole</td><td>Sigma-Aldrich</td><td>I0125</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Brij 58</td><td>Sigma-Aldrich</td><td>P5884</td><td></td></tr><tr><td>EGTA (acid)</td><td>Sigma-Aldrich</td><td>E0396</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;ATP&amp;bull;0.56H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>A7699</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279</td><td></td></tr><tr><td>HEPES (acid)</td><td>Sigma-Aldrich</td><td>H7523</td><td></td></tr><tr><td>MgO</td><td>Sigma-Aldrich</td><td>529699</td><td></td></tr><tr><td>HDTA (acid)</td><td>TCI America</td><td>D2019</td><td></td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4830</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>KOH (1 N)</td><td>Sigma-Aldrich</td><td>35113</td><td></td></tr><tr><td>HCL (1 N)</td><td>Sigma-Aldrich</td><td>318949</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CrP&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>P7936</td><td></td></tr><tr><td>pH 10 standard</td><td>Fisher Scientific</td><td>SB115</td><td></td></tr><tr><td>pH 7 standard</td><td>Fisher Scientific</td><td>SB107</td><td></td></tr></tbody></table>

measurement of maximum isometric force generated by permeabilized skeletal muscle fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

健康，透化学纤维单丝应该出现均匀的形状，并具有一致的条纹间距在高倍率下观察时。当纤维用钳子操纵是不灵活或有明显的结构性损坏应丢弃。 在步骤15采取高倍率数字图像进行5成对直径测量沿光纤的中部进行分析。估计纤维CSA假设椭圆形横截面，平均5个独立的CSA测量值作为图7A的所示 。 图7B还用于说明如何在一个视图纤维尺寸与在其它视图（即，交叉配对尺寸相比可以显著不同部分都没有，在一般情况下，圆形）。 从人类慢和快纤维代表力迹线示于图8A和8B分别。 Voltag在测试过程中被采集和转换用数据采集和分析软件（LabVIEW中），以强制（MN）的力换能器电子输出。 图9示出了用于评估最大活性力（F o）就方法，它是通过减去计算出的以保持纤维在最佳小节的长度所需要的力，而在松弛状态（无源力F P），从在最大纤维活化开发的最大等长收缩力（总力F T）。由于力传感器，对应于零力的输出，在一般情况下，对于每一个不同的沐浴室不同，我们简要放松光纤在两个预活化和活化的解决方案来捕获在零力水平实验记录。由纤维CSA最大的主动力正常化是用来生成比力（SF O）的更多的信息价值。因为它考虑到光纤的CSA，顺丰Ø </s的ub&gt;提供了纤维的收缩装置的固有的力发电能力的量度，从而允许不同尺寸的纤维之间的功能比较。它应该然而，应当注意，CSA测量不能够区分由收缩长丝与由其他亚细胞结构所占的比例占据的纤维的比例。 的健康典型特征，克拉夫林等2011 10为人类，Mendias 等2011 1从成年纤维鼠标和Gumucio 等人 2012 2大鼠都在表3中详细描述。在表3中的所有数据使用生成在这篇文章中描述的技术。 <table border="0" cellpadding="0" cellspacing="0" style="text-align:center"><tbody><tr><td rowspan="3" style="border:0px #fff solid"></td><td colspan="4"> 人的 （股外侧） </strong&gt;的</td><td> 鼠标 （EDL） </td><td nowrap="nowrap"> 鼠 （冈） </td></tr><tr><td colspan="2">男</td><td colspan="2">女</td><td>男</td><td>男</td></tr><tr><td>类型1 </td><td> 2A型</td><td>类型1 </td><td> 2A型</td><td> （没有输入） </td><td> （没有输入） </td></tr><tr><td> CSA（微米2） </td><td> 4880 - 6900 </td><td> 5270 - 8380 </td><td> 3870 - 5470 </td><td> 4010 - 5610 </td><td> 1850 - 3080 </td><td> 5290 - 8010 </td></tr><tr><td> ˚FO（MN） </td><td> 0.79 - 1.17 </td><td> 1.02 - 1.54 </td><td> 0.64 - 0.97 </td><td> 0.71 - 1.07 </td><td> 0.14 - 0.25 </td><td> 0.55 - 0.97 </td></tr><tr><td> 的sF O（千帕） </td><td> 142 - 182 </td><td> 165 - 210 </td><td> 156 - 193 </td><td> 172 - 214 </td><td> 67 - 94 </td><td> 102 - 131 </td></tr><tr><td> ñ </td><td> 129 </td><td> 160 </td><td> 149 </td><td> 207 </td><td> 37 </td><td> 94 </td></tr></tbody></table> 表3.典型的人类股健康成年纤维的特性外侧10，鼠标趾长伸肌1和大鼠冈2肌肉。肌节的最佳长度分别设定为2.7微米的纤维人类7,8,9 2.5微米的两个鼠标（36， 37）和大鼠纤维（38）。实验L˚F范围（25 次和第 75 个四分位数）为1.39-1.73毫米，1.17-1.53 ​​毫米，1.32-1.59毫米，用于分别人类，小鼠和大鼠。所示范围表示第 25和第 75位数和n是纤维测试的次数。 在测试过程中遇到的最常见的问题包括一个缝合线套环滑动，这导致了力response为一个"抓"，如在图10A中示出，并且该纤维，这导致力响应返回突然朝向或到（中断）的部分或全部厚度的撕裂零而纤维仍浸在活化溶液（ 图10B）。如果打滑，撕裂或断裂的实验过程中发生，该纤维应当被丢弃，数据排除在外，虽然保持了光纤故障的记录也可以是信息11。另一个负面结果可能遇到是纤维的过早活化，而 ​​在预活化的溶液（ 图10C）。部分活化在预活化溶液表明显著交叉以及污染（ 即，活化的预孔的无意增加钙离子浓度的）。在这种情况下，所有的缸应吸出并用离子交换水充分漂洗。干燥室之间的分隔表面也recommenDED湿气或冷凝在这些领域可能会导致排汗浴场之间的解决方案。该决定包括或排除数据最终将取决于实验的重点和设计研究时，因此应该予以考虑。 <img alt="图1" src="/files/ftp_upload/52695/52695fig1.jpg" /> 图1:缝合环（10-0单线尼龙缝线）。 <img alt="图2" src="/files/ftp_upload/52695/52695fig2.jpg" /> 图2:捆绑解剖钳是在左手，显微切割剪刀在右边。红线表示手腕和剪刀与纤维的纵向轴的有利方向。 <img alt="图3" src="/files/ftp_upload/52695/52695fig3.jpg" /> 图3: </strong&gt;的（A）测试设备与部件标记。 （a）在透明底的试验室中。 （二）长度控制器。 （三）强制换能器。 （四）光源。 （五 ）长控制器XYZ微米驱动器，数字显示。 （ 六）舞台千分尺驱动器，数字显示。 （G）强制换能器XYZ微米的车程。 （H）平台校准激光衍射目标屏幕。 （ 一）隔振表。 （B）的实验室特写图。 （j）条不锈钢安装面从长度控制器延伸。 （k）的不锈钢连接表面的力换能器延伸。 （L）侧视棱镜。 （M）为住房热电冷却模块。 （N）热电偶报告室TEmperature。 <img alt="图4" src="/files/ftp_upload/52695/52695fig4.jpg" /> 图4:修改用于传输从解剖菜光纤到实验室100微升枪头。 <img alt="图5" src="/files/ftp_upload/52695/52695fig5.jpg" /> 图5:安装单纤维到实验装置（A）中准备的缝合线套环拧到不锈钢附连表面。 （B）的纤维转移至实验室。由第一对缝合线套环用过量的缝合除去（C）的纤维锚定到不锈钢附连表面。 （D）的第二对缝合环拧在第一缝合线环的顶部和并列代替。 <img alt="图6"SRC ="/文件/ ftp_upload / 52695 / 52695fig6.jpg"/&gt; 图6:肌节长度由激光干涉图案的投影分摊到校准目标屏幕 （ 一 ）激光源。 （二）镜像。 （三）目标屏幕。 （四）激光干涉图案。 <img alt="图7A" src="/files/ftp_upload/52695/52695fig7A.jpg" /><img alt="图7B" src="/files/ftp_upload/52695/52695fig7B.jpg" /> 图7:（A）测定，纤维的横截面面积的最佳小节的长度（人= 2.7微米）。假定一个椭圆形横截面，CSA计算每个的沿着光纤中部五个地点和五个单独测量的平均值报告为纤维的CSA。 2a表示俯视直径为<html>椭圆的一个轴，图2b表示侧视图直径和是椭圆的另一个轴。 （B）的代表性的纤维的图像示出各在顶部和侧面视图采取的五个相应直径测量。 <img alt="图8" src="/files/ftp_upload/52695/52695fig8.jpg" /> 图8:从健康人股代表力的痕迹外侧肌纤维（A）1型纤维（CSA:5710微米2，F O型:0.89万和SF○:156千帕）。 （ 二）2A型纤维（CSA:9510微米2，F O型:1.66万和SF○:174千帕）。纤维肌球蛋白重链的类型是通过利用电泳分离和银染色技术22确定。 OAD / 52695 / 52695fig9.jpg"/&gt; 图9:最大活性力（F O）计算的纤维力响应在预活化溶液引发长度控制器的松弛诱导运动期间（ 一 ）扩大图。 在 fp是保持2.7微米的小节的长度与纤维在休息所需的力。 （二）长度控制器松弛引起的运动展开视图。注意了f O = F T - 在 fp。 <img alt="图10" src="/files/ftp_upload/52695/52695fig10.jpg" /> 图10:（A）缝合循环支路，掘起力过程中证明有效跟踪一个"抓"。检查，以确保循环是激活光纤之前安全。激活过程中（B）光纤断裂。在缝线环的地方可能是由于不良的纤维完整性或腐蚀性纤维处理换货。 （ 三）部分过早激活纤维由于预活化室与钙污染2+。

Watch this Scientific Journal Video about Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers at JoVE.com

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

化学剥皮，或透化，骨骼肌纤维的收缩特性的分析提供了强有力的手段，其中在单个肌细胞的水平来评估肌肉功能。在这篇文章中，我们勾勒出一个有效和可靠的技术准备和测试透骨骼肌纤维体外 。

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

measurement-maximum-isometric-force-generated-permeabilized-skeletal

Research

JoVE Journal

Bioengineering

25.4K 次观看.  University of Michigan Medical School. 化学剥皮，或透化，骨骼肌纤维的收缩特性的分析提供了强有力的手段，其中在单个肌细胞的水平来评估肌肉功能。在这篇文章中，我们勾勒出一个有效和可靠的技术准备和测试透骨骼肌纤维体外 。 

视频: Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

生物工程

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

神经科学

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

对大鼠的过程<em在原位</em骨骼肌收缩性能

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

生物学

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

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

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

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

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, ...

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. ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

最大等轴泰塔尼奇力测量大鼠的蒂比亚利斯前肌肉

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

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 ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

体内 猪后肢背屈等距扭矩的测量

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...

最大等长收缩力的测量通过透骨骼肌纤维生成

摘要

探索更多视频

离体骨骼肌肌的力学分析

中的应用

测量神经肌肉接头功能

对大鼠的过程

体外评估对离体骨骼肌的收缩，疲劳性和交替“

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

最大等轴泰塔尼奇力测量大鼠的蒂比亚利斯前肌肉

使用皮肤心肌细胞进行心脏功能的体外评估

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

体内猪后肢背屈等距扭矩的测量

最大等长收缩力的测量通过透骨骼肌纤维生成

摘要

探索更多视频

离体骨骼肌肌的力学分析

中的应用

测量神经肌肉接头功能

对大鼠的过程

体外评估对离体骨骼肌的收缩，疲劳性和交替“

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

最大等轴泰塔尼奇力测量大鼠的蒂比亚利斯前肌肉

使用皮肤心肌细胞进行心脏功能的体外评估

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

体内 猪后肢背屈等距扭矩的测量

体内猪后肢背屈等距扭矩的测量