We would like to acknowledge Daniel A. Beard and Kalyan C. Vinnakota for their foundational contributions to this protocol. This work was funded by NSF CAREER grant MCB-2237117.

The use and treatment of all vertebrate animals were performed in accordance with approved protocols reviewed and accepted by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University. This protocol was designed using both male and female Hartley albino guinea pigs and Sprague Dawley (SD) rats. For the isolation of cardiac mitochondria from guinea pigs, animals were sacrificed at ages between 4-6 weeks (300-450 g). Cardiac mitochondria from SD rats of both sexes were obtained between the ages o...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Adhering to the methods concisely described in this protocol will ensure the isolation of well-coupled mitochondria from the cardiac tissue of small rodents, in addition to other tissue types and sources. Overall, the process should take a total of 3-3.5 h, during which all animal tissue, samples, and isolates should remain on ice at 4 &#176;C as much as possible to limit degradation. This procedure is robust and can be altered in several ways to better fit experimental goals and models utilized. One modulation that can ...

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.

<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 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Robust Mitochondrial Isolation from Rodent Cardiac Tissue 

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.

Upon completion of mitochondrial isolation, the quality and functionality of the isolates should be tested via quantifying rates of oxygen consumption (JO2) using high-resolution respirometry. To do so, mitochondrial stocks were diluted to 40 mg/mL to allow for working concentrations of 0.1 mg/mL in 2 mL of RB for all respirometry assays using isolated cardiac mitochondria. Respiration was fueled by 5 mM sodium pyruvate and 1 mM L-malate in the presence of 1 mM EGTA, a calcium chelator, and was allowe...

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

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

Mitochondrial Isolation and Quality Assessment in Rodent Cardiac Tissue for Optimizing Respirometry Assays

To isolate mitochondria, place the anesthetized decapitated rat in a prone position on ice. After exposing the heart, cut the aorta approximately four to six millimeters above the aortic root and below the carotid branching points. Cannulate the aorta and perfuse the heart with ice cold cardioplegia solution using a gravity dependent pressure head until the coronary arteries are cleared of blood and the organ appears blanched.Dissect away the atria, cartilaginous valvular tissue and fatty tissues to isolate the ventricular myocardium. Mince the tissue with sharp surgical scissors until the pieces are approximately one cubic millimeter. Transfer the minced tissue into the pre-chilled centrifuge tube, labeled spins one and two containing the protease solution.Add ice cold isolation buffer to a final volume of 25 milliliters. Using a motorized handheld rotor stator homogenizer, disperse the tissue at 18, 000 RPM on ice for 20 to 25 seconds. Centrifuge the homogenized tissue at 8, 000 G for 10 minutes at four degrees Celsius.Discard the supernatant. And gently rinse the pellet with five milliliters of isolation buffer to remove residual protease. After discarding the rinse, add ice cold isolation buffer to a final volume of 25 milliliters.Then vortex to resuspend the pellet. Centrifuge the tissue homogenate at 800 G for 10 minutes at four degrees Celsius. Gently pour the supernatant containing mitochondria into a labeled pre chilled 50 milliliter tube.Now centrifuge the supernatant at 8, 000 G for 10 minutes at four degrees Celsius. Discard the supernatant and retain the mitochondria containing pellet. Using a lint-free wipe, absorb excess supernatant from the inside wall of the tube without disturbing the pellet and place the tube on ice.Add 80 microliters of ice cold isolation buffer to the bottom of the tube and gently resususpend the mitochondria using a micro-pipette. Once resuspended, transfer the mitochondria to a pre-chilled labeled micro centrifuge tube. Determine the mitochondrial protein concentration using the bison kinetic acid protein assay.In a pre-chilled micro centrifuge tube, dilute the mitochondrial stock to the desired working concentration with an isolation buffer. To test the viability and quality of mitochondrial isolates, load 2.3 milliliters of respiration buffer into Oxygraph chambers. Allow the oxygen consumption signal to equilibrate at 37 degrees Celsius for about 10 minutes or until the rate is near zero.Upon equilibration, push down the stoppers and aspirate the excess buffer. Using a 10 microliter Hamilton syringe, add one millimolar EGTA to chelate any residual calcium in the respiration buffer on the Oxygraph. To fuel respiration, add five millimolar sodium pyruvate, and one millimolar al-malate into the buffer.Then add a bolus of diluted mitochondria and allow respiration to occur for five minutes. At the five minute mark, add a bolus of 500 micromolar ADP to initiate state three respiration. To calculate the respiratory control ratio, divide the maximal rate of oxygen consumption during state three by the rate of oxygen consumption just before the addition of ADP in state two.A rapid increase in oxygen consumption was observed immediately after ADP addition indicating successful initiation of oxidative phosphorylation in isolated cardiac mitochondria. Heart mitochondria from guinea pigs, Sprague-Dawley rats and Friend leukemia virus B mice, demonstrated high respiratory control ratios, indicating good mitochondrial isolation quality. Liver and kidney mitochondria from Sprague-Dawley Rats also exhibited acceptable respiratory control ratios supporting successful isolation.HEK293 cells exhibited respiratory control ratio values above the acceptable threshold, confirming the quality of mitochondrial isolation from these cells.

248 Views.  Michigan State University.  Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Summary