<meta charset="utf8"></head><body>从啮齿动物中分离线粒体以进行高质量的生物能量研究

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL glass beaker</td>
			<td></td>
			<td></td>
			<td>For organ disection and mincing</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td></td>
			<td></td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-diphosphate monopotassium salt dihydrate</td>
			<td>Sigma&#160;</td>
			<td>A5285</td>
			<td>Respirometry assays</td>
		</tr>
		<tr>
			<td>BSA Protein Assay Kit</td>
			<td>Thermo Scentific</td>
			<td>PI23225</td>
			<td>Mitochondrial protein quantification</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma&#160;</td>
			<td>DX0145</td>
			<td>For buffer (CB)</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-amino-ethylether)-N,N,N&#39;,N&#39;-tetraacetic acid&#160;</td>
			<td>Sigma&#160;</td>
			<td>E4378</td>
			<td>For buffers (CB and IB) and respirometry assays</td>
		</tr>
		<tr>
			<td>Glass cannula</td>
			<td>Radnoti</td>
			<td></td>
			<td>Guinea pig and rat heart perfusion</td>
		</tr>
		<tr>
			<td>Heparin sodium porcine mucosa</td>
			<td>Sigma&#160;</td>
			<td>SRE0027-250KU</td>
			<td>Animal IP injection</td>
		</tr>
		<tr>
			<td>High-resolution respirometer</td>
			<td></td>
			<td></td>
			<td>Clark-type electrode; oxygraph with 2 mL chambers</td>
		</tr>
		<tr>
			<td>Induction chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma&#160;</td>
			<td>792632</td>
			<td>Anesthetic&#160;</td>
		</tr>
		<tr>
			<td>L-malic acid</td>
			<td>Sigma&#160;</td>
			<td>02288-50G</td>
			<td>Respirometry assays</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma&#160;</td>
			<td>M9272</td>
			<td>For buffer (RB)</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma&#160;</td>
			<td>MX0214</td>
			<td>For buffer (IB)</td>
		</tr>
		<tr>
			<td>Microliter syringes</td>
			<td></td>
			<td></td>
			<td>Sizes ranging from 5&#8211;50 &#181;L</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td></td>
			<td></td>
			<td>Must be able to incubate at 37 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma&#160;</td>
			<td>475898</td>
			<td>For all buffers</td>
		</tr>
		<tr>
			<td>O2 tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OMNI THQ Homogenizer</td>
			<td>OMNI International&#160;</td>
			<td>12-500</td>
			<td>Similar rotor stator homogenizers will work</td>
		</tr>
		<tr>
			<td>pipettes</td>
			<td></td>
			<td></td>
			<td>Volumes of&#160; 2&#8211;20 &#181;L; 20&#8211;200 &#181;L; 200&#8211;1000 &#181;L</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma&#160;</td>
			<td>P3911</td>
			<td>For buffers (RB and CB)</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma&#160;</td>
			<td>795496</td>
			<td>For buffers (IB and RB)</td>
		</tr>
		<tr>
			<td>Protease from Bacillus licheniformis</td>
			<td>Sigma&#160;</td>
			<td>P5459</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma&#160;</td>
			<td>S9888</td>
			<td>For buffer (CB)</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Fisher bioreagents</td>
			<td>BP356-100</td>
			<td>Respirometry assays</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma&#160;</td>
			<td>8510-OP</td>
			<td>For buffer (IB)</td>
		</tr>
		<tr>
			<td>Surgical dissection kit</td>
			<td></td>
			<td></td>
			<td>Depends on animal and tissue source</td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td></td>
			<td></td>
			<td>Must cool to 4 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

robust mitochondrial isolation from rodent cardiac tissue 

Mitochondria are subcellular organelles that establish cytoplasmic energetic conditions optimized for specific cell functions. While cellular, tissue, and organism-level studies can provide insights into mitochondrial function, isolating the organelles offers a level of experimental control not possible otherwise. Mitochondrial isolations have been performed since the 1940s, allowing mechanistic studies of metabolism and respiration across a variety of cells and tissues1,2. The historical relevance of mitochondria is also well-documented3. As the main producers of ATP, mitochondria play many key roles that are vital for optimal cellular and organ function4. Within the mitochondrial matrix, substrates are oxidized by the TCA cycle, producing reducing equivalents and mobile electron carriers such as NADH and UQH25,6. Cytochrome C is the third main mobile electron carrier in the mitochondrial biochemical reaction network7. These molecules are then oxidized by the transmembrane complexes of the electron transport system (ETS) embedded in the inner mitochondrial membrane8. Redox reactions of the ETS are coupled with proton translocation from the matrix to the intermembrane space. These processes establish an electrochemical proton gradient that is used to phosphorylate ADP with Pi by F1F0 ATP synthase to produce ATP9,10. The individual processes that occur at each complex can be explored with high-resolution respirometry using Clark-type electrodes or microplate oxygen consumption assays11,12. Additionally, disease models and treatments using isolated mitochondria can determine the impact or importance of mitochondrial function in the progression of certain pathologies. This has proven fruitful in the field of cardiology, where alterations in fuel and substrate delivery have been used to elucidate how mitochondrial dysfunction influences heart failure13,14,15,16. Mitochondria are also known to impact the development of other disease states such as diabetes, cancer, obesity, neurological disorders, and myopathies17,18. Therefore, the use of isolated or purified mitochondria enables mechanistic investigations of oxidative metabolism and ATP production in the source tissue.
There is no shortage of mitochondrial isolation protocols due to their importance in bioenergetic research. Additionally, highly specific methods tailored to subpopulations of mitochondria within tissues and cells can be found19,20. The basic procedural steps are similar between isolation methods, but variations can be made to buffer composition, homogenization steps, and centrifugation spins to improve the amount and quality of mitochondria. Changes to these aspects are based on the metabolic demand of the tissue, overall organ function, mitochondrial density, and other factors. In tissues such as the liver and skeletal muscle, handheld homogenizers are used to preserve mitochondrial integrity and limit damage to the mitochondrial membranes21. However, when isolating from kidneys, some protocols suggest using manually driven homogenization or commercial kits to yield better results22. Although both methods yield functional mitochondria, the quality of the organelles can become compromised by the additional time it takes to complete isolations using these protocols. Centrifugation is also vital to the extraction of mitochondrial protein, as it separates cellular components such as nuclei and other organelles from mitochondria23. During the isolation process, it is debated whether differential or density-based centrifugation should be implemented to obtain purer isolates24. While density centrifugation can separate mitochondria from organelles of similar specific gravity, such as peroxisomes, it is not well-established if mitochondria from these methods better represent in situ organelle function compared to mitochondria isolated using differential centrifugation. In the field of mitochondrial physiology, density-based centrifugation is preferred and can be easily altered to increase isolate purity. Whether changes to g-forces, centrifugation duration, and the number of centrifugation spins are incorporated should be considered before experimentation due to their influence on mitochondrial purification. Furthermore, mitochondrial resuspension, arguably the most crucial step during isolation, differs greatly between studies, with the use of scraping, vortex-based mixing, or homogenization23,25,26. Mechanical resuspension of these types can be too abrasive and impair the membrane integrity of mitochondria. For this reason, gentle washing should be performed to correct this. Despite the plethora of modulations and suggested methodologies, there are fewer comprehensive protocols with high reproducibility and adaptability for rodent models.
The methods described herein outline a detailed, robust, and highly reproducible protocol that yields purified, well-coupled mitochondria from small animal cardiac tissue. As demonstrated, this method can be easily adapted to accommodate the specific needs of each experiment and/or laboratory environment for isolating mitochondria from kidneys, liver, and cultured cells. Further modifications can be made to isolate mitochondria from tissues and other animals not listed here. Buffer recipes used for all isolations are provided and can be modified if needed. Similar to other published protocols, motorized homogenization, and differential centrifugation are implemented; however, adjustments are made to both the shearing time and the force at which the samples are centrifuged to consistently deliver high-quality mitochondrial isolates, depending on the isolation source. Notably, this protocol differs from others by using gentle washing via pipetting to resuspend pelleted mitochondria, which helps preserve mitochondrial membrane integrity and maintain the overall functionality of the organelles. Mitochondrial protein is quantified either by total protein determination or by measuring citrate synthase activity. The utility and broad applicability of this isolation method are further supported by the values of respiratory control ratios (RCR) achieved across various organisms and tissue sources.

<meta charset="utf8"></head><body>作者聚焦：揭示线粒体磷酸钙在心力衰竭和生物能量学中的作用

从啮齿动物心脏组织中稳健地分离线粒体 

Mitochondria involved with many disease pathologies. They are essential organelles that act as rechargeable and renewable biological batteries. They're often referred to as the powerhouse of the cell.They make complex life possible. Our lab is primarily interested in how these biological batteries operate during healthy and disease states to discover effective curatives and develop new therapies. Our lab has identified the presence of calcium phosphate granules embedded in the matrix of mitochondria that disrupt their ability to provide chemical energy for the cell.We have also identified that this effect is sexually dimorphic in nature. Our protocol is a simple and effective way to generate high-quality mitochondria for advanced biomedical research. The protocol consistently produces purified mitochondria with high coupling ratios, one of several measures of quality.Thus, the data we collect are less impacted by isolation artifacts and better reflect their NC2 behavior. Generating data from high-quality mitochondrial isolates enables us to better understand how mitochondria operate under various conditions, and therefore, interrogate possible mechanisms of disease. The calcium phosphate granules we have identified may be causal to heart failure.They cause an impairment in ATP output, which may help explain the bioenergetic abnormality seen in human patients with severe left ventricle hypertrophy and heart failure.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

robust-mitochondrial-isolation-from-rodent-cardiac-tissue

Research

JoVE Journal

Biochemistry

Robust Mitochondrial Isolation from Rodent Cardiac Tissue 

326 次观看.  Michigan State University. 线粒体与许多疾病病理有关。它们是必不可少的细胞器，充当可充电和可再生的生物电池。他们通常被称为细胞的动力源。它们使复杂的生活成为可能。我们的实验室主要对这些生物电池在健康和疾病状态下如何运作感兴趣，以发现有效的治疗方法并开发新的疗法。我们的实验室已经确定线粒体基质中存在嵌入磷酸钙颗粒，这些颗粒会破坏它们...