这项工作是由RO1-AG028614到JM，RO1-AR063084to NLW和RO1-AR057404到JZ支持。

1。设置渗透胁迫灌注系统 图1是钙的示意性协议火花评估在正常肌肉纤维。 <ol><li>设置了一个三轴（XYZ）显微操作器能够定位含有至少两个通道的灌注系统的出口端部。此可以作出与一次性鲁尔注射器筒与附3 -路鲁尔 - 乐旋塞打开和/或关闭灌注液的流动。这些灌注通道应能够输送的溶液&gt; 1毫升/分钟通过一个单一的灌注尖与&gt;直径0.2mm的。 </li><li>加载两个通道灌注系统，一个与等渗台氏液和其他与低渗台氏液中。 </li></ol> 2。从鼠标准备完好的单趾短屈肌（FDB）肌纤维<ol><li>从鼠标欧盟取出脚thanized以下使用对重解剖剪通过上面的踝关节的腿切NIH和IACUC准则。 </li><li>脚脚到一个夹层腔充满了最小的Ca 2 +台氏液与跖面朝上。 </li><li>解剖FDB通过切割肌腱并轻轻上肌腱拉起到肌肉从周围组织中分离出来。 </li><li>通过切割所述远端肌腱在那里分支成各个单独的数字在肌肉的深层完成清扫。 </li><li>拿起通过其筋钳，以避免对肌肉纤维的额外伤害并放入胶原酶消化液的解冻等分管转移FDB肌肉预热至37°C。 </li><li>孵育在轨道摇床上于37℃下进行60-90分钟为160 rpm的转速含有FDB直立的管。此肌肉束解离协议需要通过实验来检查动物的不同菌株，特别是对那些病/突变体纤维易受膜损伤。 </li><li>转移消化FDB到管700微升等渗的Tyrode溶液并轻轻磨碎，通过一系列的通过用干净的刀片切割逐渐减小的直径200毫升微量移液器尖端拉伸肌肉数次，以释放纤维以除去提示200毫升微量。为了避免疾病特别弱的肌肉纤维损害，确保针尖正好大到足以使纤维束通过不沾。通过太小开口不要强行捆绑。 </li><li>积垢的FDB纤维轻轻拍打并重新悬浮FDB纤维在试管中，然后绘制出70毫升（总纤维的1/10）用切200毫升微量成含有1ml等渗的35毫米德尔塔TPG培养皿的中心台氏液，以降低整个盘子扩散。 </li><li>测定完整的单纤维在D的数量十岁上下用解剖显微镜。添加FDB纤维额外等分，得到3-4完整纤维的菜。 </li><li>存储额外的隔离肌纤维在4℃，并在6小时使用。 </li></ol> 3。荧凌晨4点染料荷载和Ca 2 +成像（火花测量） <ol><li>转让500毫升等渗台氏液从菜FDB镀成纤维用10毫升荧光 - 凌晨4点股票（1毫米）的管道，混合，并添加回菜10毫米的最终荧光 - 凌晨4点浓度。可替换地，聚醚酸可用于增加染料装载效率。 </li><li>加载60分钟，在室温下在黑暗中。 </li><li>通过仔细地绘制出500毫升荧光4含AM-等渗台氏液从菜用一个完整无缺的200毫升塑料枪头洗纤维和更换新鲜台氏等渗溶液等体积。 </li><li>重复此过程3次以上。 </li><li>为了形象化线粒体功能，传输500毫升Isoto的NIC台氏液从菜FDB纤维成筒用5毫升TMRE股（1毫米），混合，并添加回菜50nm的终浓度。 </li><li>在室温下孵育10分钟。 </li><li>通过仔细地绘制出500毫升洗纤维TMRE含等渗台氏液从与未切割200mL的塑料枪头菜，换上新鲜台氏等渗溶液等体积。 </li><li>重复此过程3次以上。 </li><li>转让菜的共聚焦显微镜，并选择一个完整的纤维条纹清晰和平滑细胞膜的膜进行实验。 </li><li>用显微操作器的控制地定位灌注系统400-500微米的前端远离目标肌肉纤维和偏离中心。 </li><li>开始流动的等渗台氏液，以确保FDB纤维留在地方。 </li><li>从荧光4上午从40X油浸物镜记录荧光信号，并激发荧光 - 凌晨4点与氩激光（激发波长488 nm）和创纪录的发射信号（波长510-580纳米）。荧光信号的强度正比于细胞内Ca 2 +浓度（[的Ca 2 +] i）的相对水平。 </li><li>通过改变胞外溶液中，以产生渗透胁迫对纤维的渗透压诱导的Ca 2 +瞬变。最初灌注纤维等渗的Tyrode溶液（290毫渗透摩尔浓度）（60秒基线集合），然后用低渗的Tyrode溶液（170毫渗透摩尔浓度）为100秒，以诱导肿胀。灌注FDB纤维回用等渗台氏液。通常，只有一个渗透压休克被施加到一个单一的完整的纤维在一个增量TPG菜和火花事件最后10分钟进行数据采集。 </li><li>的Ca 2 +的火花（ 图2），使用时间序列的xy二维图像与LPS 166（每秒行，和EA扫描速度的记录空间定位通道线的等渗（每次1 min条件包括512像素造成3.08秒/帧）），低渗应激（100秒）和后低渗应激（5分钟）。用于离线分析存储的信号来评估的Ca 2 +火花下列实验完成的分布和频率。 </li><li>找到一道新菜新纤维并遵循相同的渗透休克协议诱发的Ca 2 +瞬变。使用共焦线扫描（XT）模式（以长度为512个像素，2毫秒/行扫描的采样率与激光共聚焦扫描线直接置于下方肌膜膜记录的Ca 2 +瞬变一分钟（ 即 30000线）（ 图3中 ）行扫描变更为不同的焦平面记录线扫描单个钙火花的幅度和动力学分析的三个连续的系列（在1分钟间隔）。 </li></ol> 4。数据分析<ol><li>收集了大​​量的XT线扫描轨迹来评估个体的Ca 2 +火花参数的形态和动力学， 即振幅（F / F 0，其中F 0是静止的Ca 2 +荧光），上升时间，时间到了高峰，持续时间（FDHM ，充分停留时间的一半大小），并且所记录的火花的空间宽度（FWHM，半幅度全宽）。这些参数代表兰尼的Ca 2 +通道，如钙离子通量（振幅）的基本门控特性，和RyRs的（持续时间）的门控动力学。 </li><li>以将其用图像处理语言的IDL（交互式数据语言）创建的，如前面11,16中记载的自定义开发的，半自动的算法分析的数字图像数据。由于Ca 2 +的独特的动力学释放的钙离子通过渗透胁迫诱导的FDB纤维火花的情况下，最好是结合钙的手动和自动功能2 +引发的事件识别和处理这些自定义生成的图像分析程序11。该算法是非常有用的，当它需要手动识别的ROI（感兴趣区域）的一些肌肉疾病模型。一旦投资回报率的定义，算法可以自动导出，然后可以导出到Excel文件中的上述火花参数。可选地，公共可获得的图像处理软件， 如 SparkMaster在ImageJ的，可以用来定义钙火花的动力学性质和它们在骨骼肌17的空间分布。 </li></ol>

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没有利益冲突的声明。

评估在正常肌肉的Ca 2 +的火花此方法对于肌肉的生理和疾病研究的有用工具。我们发现，在Ca 2 +的火花响应被改变在不同条件，包括肌营养不良症11，老化18，19，以及在肌萎缩性侧索硬化症20。我们最近的研究还发现IP 3受体和RyR的代表，有助于钙在骨骼肌肉纤维21 2火花激活的关键组成部分之间的功能耦合。其他几个研究小组也采?...

细胞内游离Ca 2 +（内[Ca 2 +] i的）是监管的兴奋细胞，如神经元，心肌，骨骼肌和平滑肌多种细胞功能（综述见Stutzmann和马特森1）灵活的和重要的第二信使。调控的Ca 2 +动员和串扰肌浆网（SR）和T型肾小管间（TT）膜是肌肉生理学的基本监管。此外，改变的Ca 2 +信号已被证明是收缩功能障碍的各种肌肉疾病的潜在机制。 钙火花的本地化基本的Ca 2 +释放事件从肌浆网（SR）膜（2）开放的兰尼碱受体（Ryanodine受体）通道的起源。在心肌，火花发生自发地通过由一个的Ca 2 +诱导的Ca 2 +勒开口的RyR2的信道的ASE（CICR）机制3-5。在骨骼肌，RYR1是严格的TT膜6,7电压传感器控制。因此，Ca 2 +的火花被抑制，很少发现在正常肌肉纤维的静息状态。直到最近，需要细胞膜的膜通过各种化学或机械的"换肤"方法被打乱，以脱开的RYR1电压传感器的抑制和允许的Ca 2 +引发骨骼肌8,9被检测到的事件。一个方法之前描述的肌纤维由皂素洗涤剂10膜的透化需要。 2003年，我们发现，无论是短暂的低渗应激或高渗应激可引起外周血Ca 2 +的火花毗邻的完整肌肉纤维11细胞膜的膜。这种方法已经被修改，以研究的Ca 2 +的分子机制和调制</suP&gt;释放和动态12-16。在这里，我们勾勒出我们的实验方法对Ca 2 +的火花正常肌肉感应，检测和分析的细节。我们还提出了可以用来量化个体的Ca 2 +火花在骨骼肌， 如火花的频率和幅度（ΔF/F0，它反映的RyR通道的开放概率的元素的属性我们的定制火花分析程序和的Ca 2 +的负荷对SR内）;达峰时间（上升时间）和持续时间（FDHM，充分停留时间的火花在半最大振幅），以及Ca 2 +的火花的空间分布。此外，我们目前的证据表明链接改变的Ca 2 +火花的各种病理生理状态的骨骼肌，如肌肉萎缩症和肌萎缩性侧索硬化症。 这种技术的优点涉及到测量的Ca 2 +的相对undam的能力老化细胞，而不是剥离的肌肉纤维，使记录的Ca 2 +火花在条件更接近生理，。此外，我们的定制设计方案提供与肌纤维火花的特性更精确的计算。

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		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td style="width:367px;">Dissect out fibers and check muscle integrity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Dissection tools -scissors, and fine tip forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>DOW CORNING</td>
			<td style="width:119px;">184 SIL ELAST KIT</td>
			<td style="width:367px;">&#160;Make ahead of time for fiber dissection. Mix 5 ml liquid Sylgard components and pour into a 60-mm glass Petri dish and waiting 48 hr to let the Sylgard compound become clear, colorless solid substrate.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm glass petri dish&#160;</td>
			<td style="width:111px;">Pyrex</td>
			<td style="width:119px;">3160</td>
			<td>Fiber dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isotonic Tyrode Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minimal Ca2+ Tyrode Solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypotonic Tyrode Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">collagenase type I&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C-5138</td>
			<td>Fiber digestion&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fluo-4 AM&#160;</td>
			<td style="width:111px;">Invitrogen</td>
			<td>F14201</td>
			<td>Fluorescent Ca2+ imaging dyes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMRE</td>
			<td style="width:111px;">Invitrogen</td>
			<td>T669</td>
			<td>mitochondrial membrane potential fluorescent dyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delta TPG dishes&#160;</td>
			<td>Bioptechs</td>
			<td style="width:119px;">0420041500C</td>
			<td>35 mm glass bottom dish for imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Spark Fit software</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>data analysis software</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Radiance 2100 laser scanning confocal microscope or equivalent microscope</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">3 axis micromanipulator</td>
			<td style="width:111px;">Narishige International</td>
			<td style="width:119px;">MHW-3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">temperature controllable orbital shaker</td>
			<td style="width:111px;">New Brunswick scientific</td>
			<td style="width:119px;"></td>
			<td>Fiber dissociation</td>
		</tr>
	</tbody>
</table>

assessment of calcium sparks in intact skeletal muscle fibers

维持内环境稳定的Ca 2 +信号传导是一个基本的生理过程中的活细胞。 钙火花是钙的基本单元+中显示为高度本地化由兰尼碱受体介导的钙释放事件（碱受体）的Ca 2 +对肌浆网（SR）膜释放渠道横纹肌纤维的信号。正确评估肌肉的钙火花可以提供有关的健康和疾病横纹肌细胞内Ca 2 +的处理性能信息。尽管Ca 2 +的火花事件是常见于静息心肌细胞，它们很少在静息骨骼肌纤维观察到，因此有必要对方法来生成和分析在骨骼肌纤维产生火花。 这里详细是一个实验性协议，用于测量Ca 2 +的火花在孤立的屈肌digitorm短（FDB）肌纤维取fluorescent Ca 2 +的指示器和激光扫描共聚焦显微镜。在这种方法中，隔离的FDB纤维暴露到瞬时低渗应力之后返回到等渗生理溶液。在这些条件下，一个强大的Ca 2 +火花响应被检测到相邻的年轻健康的FDB肌纤维的细胞膜膜。改变Ca 2 +的响应火花在营养不良或老年骨骼肌纤维检测。这种方法最近已证实，膜分隔的信令涉及受体（IP 3 R）肌醇之间的串扰（1,4,5） -三磷酸和碱受体贡献的Ca 2 +火花激活骨骼肌。总之，使用渗透胁迫我们的研究引起Ca 2 +的火花表明，该细胞内的反应反映了肌肉的信令机制，在生理和衰老/疾病状态，包括肌肉萎缩症的小鼠模型（mdx小鼠）或肌萎缩侧索硬化症（ALS模型）。

早期的研究表明，瞬态低渗胁迫引起的周围的Ca 2 +火花相邻的完整的肌肉纤维的肌膜膜11。 图1示出的完整单肌纤维具有光滑肌膜膜和特征明确的条纹图像， 图2示出了典型的Ca 2 +火花（如XY图像）由瞬态（100秒）与低渗的解决方案，从膨胀年轻，健康小鼠的FDB肌肉纤维治疗引起的。由于肌纤维收缩回原来的体积，强大的Ca 2 +的火花?...

Watch this Scientific Journal Video about Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers at JoVE.com

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

这里描述的是一种方法来直接测量钙火花的Ca 2 +的基本单元从正常肌肉纤维肌质网释放。该方法利用渗透压应激介导的触发的Ca 2 +释放在孤立的肌纤维兰尼碱受体。动力学和细胞内Ca 2 +信号的稳态容量可以被用来评估在健康和疾病中的肌肉功能。

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 ...

assessment-of-calcium-sparks-in-intact-skeletal-muscle-fibers

Research

JoVE Journal

Biology

15.1K 次观看.  The Ohio State University Wexner Medical Center. 这里描述的是一种方法来直接测量钙火花的Ca 2 +的基本单元从正常肌肉纤维肌质网释放。该方法利用渗透压应激介导的触发的Ca 2 +释放在孤立的肌纤维兰尼碱受体。动力学和细胞内Ca 2 +信号的稳态容量可以被用来评估在健康和疾病中的肌肉功能。 

视频: Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

科研

生物学

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Respirometric皂甙通透心脏纤维的氧化磷酸化的评估

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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, ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

将质粒DNA电穿孔到小鼠骨骼肌中

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

马骨骼肌的高分辨率氟呼吸测定法

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...