<meta charset="utf8"></head><body>实时定量肌肉纤维中的线粒体呼吸

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#181;L Hamilton Syringe (glass syringe)</td>
			<td>ThermoFisher</td>
			<td>14-813-125</td>
			<td>For respiration assay titration</td>
		</tr>
		<tr>
			<td>25 &#181;L Hamilton Syringe (glass syringe)</td>
			<td>ThermoFisher</td>
			<td>14-813-133</td>
			<td>For respiration assay titration</td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>Merck</td>
			<td>117105</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>Black Glass Dissection Dish</td>
			<td>Scintica</td>
			<td>DD-90-S-BLK</td>
			<td>For sample preparation</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Sigma</td>
			<td>B0560</td>
			<td>Working MiR05 Solution</td>
		</tr>
		<tr>
			<td>BSA, fatty acid free</td>
			<td>Sigma</td>
			<td>A6003</td>
			<td>MiR05 Solution</td>
		</tr>
		<tr>
			<td>Calcium Carbonate</td>
			<td>Sigma</td>
			<td>C4830</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>Creatine</td>
			<td>Sigma</td>
			<td>27900</td>
			<td>Working MiR05 Solution</td>
		</tr>
		<tr>
			<td>Cytochrome C</td>
			<td>Sigma</td>
			<td>C7752</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>DatLab</td>
			<td>Oroboros Instruments</td>
			<td>N/A</td>
			<td>Respirometry Software</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)&#160;</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>D-Sucrose</td>
			<td>Sigma</td>
			<td>84097</td>
			<td>MiR05 Solution</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td>BIOPS &#38; MiR05 Solution</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma</td>
			<td>C2920</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma</td>
			<td>G1626</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7523</td>
			<td>MiR05 Solution</td>
		</tr>
		<tr>
			<td>Imidazole&#160;</td>
			<td>Sigma</td>
			<td>56750</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>KH2PO4&#160;</td>
			<td>Sigma</td>
			<td>P5379</td>
			<td>MiR05 Solution</td>
		</tr>
		<tr>
			<td>Lactobionic acid</td>
			<td>Sigma</td>
			<td>153516</td>
			<td>MiR05 Solution</td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>Sigma</td>
			<td>M1000</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>MES hydrate&#160;</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>MgCl2 - 6 H2O&#160;</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>BIOPS &#38; MiR05 Solution</td>
		</tr>
		<tr>
			<td>Oroboros Oxygraph-2K (O2K) System</td>
			<td>Oroboros Instruments</td>
			<td>10203-03</td>
			<td>High resolution respirometer</td>
		</tr>
		<tr>
			<td>Palmitoyl-Carnitine</td>
			<td>Sigma</td>
			<td>P4509</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Sigma</td>
			<td>P1767</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>Precision Tweezers</td>
			<td>Fisher</td>
			<td>17-467-168</td>
			<td>For sample preparation</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S2149</td>
			<td>For Fiber Permeabilization</td>
		</tr>
		<tr>
			<td>Sodium ATP</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>Sodium Phosphocreatine</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td>BIOPS Solution</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma</td>
			<td>S2378</td>
			<td>Respirometry Assay</td>
		</tr>
		<tr>
			<td>Taurine&#160;</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td>BIOPS &#38; MiR05 Solution</td>
		</tr>
	</tbody>
</table>

measurement of mitochondrial respiration in human and mouse skeletal muscle fibers by high-resolution respirometry

Mitochondria are the cornerstone of cellular energy production, making them indispensable for maintaining optimal cellular and organismal homeostasis. These double-membraned organelles are primarily responsible for oxidative phosphorylation. This process efficiently converts nutrients, such as sugars and fatty acids, into adenosine triphosphate (ATP), the cellular currency for energy. Beyond their role in energy metabolism, mitochondria are also key regulators of various cellular processes, including apoptosis, calcium homeostasis, and reactive oxygen species (ROS)1,2. Because of their pivotal role in maintaining cellular and organismal homeostasis, disruptions in mitochondrial function often have detrimental effects on tissue and organismal health. In skeletal muscle, mitochondrial dysfunction is associated with numerous disease states, including metabolic disorders (e.g., obesity, diabetes, and cardiovascular disease), sarcopenia, and muscular dystrophy3,4,5,6,7,8.
Mitochondrial dysfunction can primarily manifest as altered mitochondrial content, number, and morphology, as well as disrupted metabolism. Thus, achieving a comprehensive understanding of mitochondrial dysfunction requires an integrative and holistic approach. Initial characterization studies involve examining expression levels of respiratory chain protein complexes as a readout of mitochondrial content, quantifying mitochondrial DNA and markers of biogenesis as a measure of mitochondrial biogenesis, and sophisticated electron microscopy imaging to assess mitochondrial morphology9,10. Additional assessments of mitochondrial function include evaluating cellular ROS and ATP production and mitochondrial membrane potential9.
Because mitochondria are essential for cellular energy production and homeostasis, a hallmark for assessing mitochondrial function is to measure mitochondrial oxidative phosphorylation (OXPHOS). High-resolution respirometry of permeabilized muscle fibers allows for the measurement of real-time changes in mitochondrial oxygen consumption rate as a readout for the dynamic changes in mitochondrial OXPHOS respiratory chain activity9,11,12. The application of selective chemical modulators and inhibitors allows one to measure the activity of different respiratory complexes directly and sequentially. Although isolated mitochondria may be used in respirometry, the use of fresh, permeabilized muscle fibers maintains endogenous mitochondrial networks and membrane integrity - thus allowing for more physiologically relevant measurements. Additionally, because different muscle fiber types have different substrate preferences and rates of respiration, this system allows one to measure changes in fuel preference and metabolic flexibility based on fiber type13.
Here, we describe a comprehensive protocol for mitochondrial OXPHOS measurements using either human or mouse skeletal muscle fibers in a high-resolution respirometer system. Included are methods to quantify mitochondrial oxygen respiration in oxidative or glycolytic fibers using either pyruvate or palmitoyl-carnitine as a substrate. This protocol allows for the use of other fuel substrates to address specific metabolic questions pertaining to defects in substrate utilization and fuel preference.

<meta charset="utf8"></head><body>作者聚焦：揭示代谢疾病中的线粒体功能和细胞代谢适应

通过高分辨率呼吸测定法测量人和小鼠骨骼肌纤维中的线粒体呼吸

Our research focuses on the intersection of metabolism and stress responses in metabolic disease, a major theme of the Washington University NORD program. Through synergisms between our NORD cores, we are able to tackle complex questions in nutritional metabolism and disease through an interdisciplinary and collaborative approach. Traditional mitochondrial respiration assays examined isolated mitochondria.Isolating mitochondria is time-consuming and can compromise outer mitochondrial membrane integrity. Additionally, mitochondria do not perform alone, but are part of a dynamic network of cellular metabolic adaptation. Our protocol maintains this network to provide a physiologically relevant assessment of mitochondrial function.Our focus has extended to identifying regulators of metabolic tailoring that are important in health and disease. Key questions we are asking include, are these regulators, like site-1 protease, equally important across different tissue types? And what is their physiological significance?

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

measurement-mitochondrial-respiration-human-mouse-skeletal-muscle

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Research

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

JoVE Journal

Biology

729 次观看.  Washington University School of Medicine. 我们的研究重点是代谢疾病中代谢和应激反应的交叉点，这是华盛顿大学 NORD 项目的一个主要主题。通过我们 NORD 核心之间的协同作用，我们能够通过跨学科和协作的方法解决营养代谢和疾病中的复杂问题。传统的线粒体呼吸测定检查了孤立的线粒体。分离线粒体非常耗时，并且会损害线粒体外膜的完整性。此外，线粒体不单独执行，而是细胞...