<meta charset="utf8"></head><body>Quantificazione in tempo reale della respirazione mitocondriale nelle fibre muscolari

<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>Autore in primo piano: Svelare la funzione mitocondriale e l'adattamento metabolico cellulare nelle malattie metaboliche

Misurazione della respirazione mitocondriale nelle fibre muscolari scheletriche umane e di topo mediante repirometria ad alta risoluzione

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

Without Section

Research

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

JoVE Journal

Biology

711 Views.  Washington University School of Medicine. La nostra ricerca si concentra sull'intersezione tra metabolismo e risposte allo stress nelle malattie metaboliche, un tema importante del programma NORD della Washington University. Grazie ai sinergismi tra i nostri nuclei NORD, siamo in grado di affrontare questioni complesse nel metabolismo nutrizionale e nelle malattie attraverso un approccio interdisciplinare e collaborativo. I saggi tradizionali di respirazione mitocondriale hanno esaminato i mitocondri...