Name: assessment of calcium sparks in intact skeletal muscle fibers
Uploaded: 2014-02-24T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers at JoVE.com

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><l...

<ol>
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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。

<table border="0" cellpadding="0" cellspacing="0" width="790">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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 (Video) | JoVE

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

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Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

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成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

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Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers (Video) | JoVE

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, ...

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

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

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

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles (Video) | JoVE

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, ...

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

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements (Video) | JoVE

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 ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System (Video) | JoVE

使用基于光学的系统对水凝胶嵌入的完整小鼠肌纤维进行高通量收缩测量

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability (Video) | JoVE 

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration (Video) | JoVE 

基于脱细胞的骨骼肌脂肪浸润定量

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Construct Design for Transfecting Cysteine-Engineered Voltage-Gated Ca2+ Channels in Murine Isolated Skeletal Muscle Fibers

Construct Design for Transfecting Cysteine-Engineered Voltage-Gated Ca2+ Channels in Murine Isolated Skeletal Muscle Fibers (Video) | JoVE 

在小鼠分离的骨骼肌纤维中转染半胱氨酸工程电压门控CA2+ 通道的构建设计

Decellularization of Skeletal Muscle

Decellularization of Skeletal Muscle  (Video) | JoVE 

骨骼肌脱细胞 