This work was financially supported by Kansas City University&#39;s intramural grant for Abdulbaki Agbas and Summer Research Fellowship for Daniel Barrera. The authors are thankful for Dr. Jan Talley&#39;s editorial work.

Frozen offspring heart tissues were received from Dr. Sean Newcomer along with the institutional IACUC approval letter. The heart tissues were obtained from a long-term longitudinal study, flash-frozen in liquid nitrogen, and stored at -80 &#176;C for future use. All protocols concerning the processing of offspring heart tissue followed the guidelines of Kansas City University IBC and IACUC committees.
1.&#160;Preparation of buffers and reagents
NOTE: Prepare all samp...

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Analytical Biochemistry. 418 (2), 213-223 (2011).</li><li>Osto, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+respiration+in+previously+frozen+biological+samples.">Measuring mitochondrial respiration in previously frozen biological samples.</a> Current Protocols in Cell Biology. 89 (1), 116 (2020).</li><li>Agbas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+electron+transfer+cascade+enzyme+activity+assessment+in+cultured+neurons+and+select+brain+regions.">Mitochondrial electron transfer cascade enzyme activity assessment in cultured neurons and select brain regions.</a> Current Protocols in Toxicology. 80, 73 (2019).</li><li>Hathaway, Q. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+of+malignant+hyperthermia+in+a+porcine+model+for+experimental+open+heart+surgery.">Avoidance of malignant hyperthermia in a porcine model for experimental open heart surgery.</a> Laboratory Animal Science. 35 (2), 172-175 (1985).</li><li>Panov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bovine+serum+albumin+on+mitochondrial+respiration+in+the+brain+and+liver+of+mice+and+rats.">Effect of bovine serum albumin on mitochondrial respiration in the brain and liver of mice and rats.</a> Bulletin of Experimental Biology and Medicine. 149 (2), 187-190 (2010).</li><li>Jha, P., Wang, X., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+respiratory+chain+supercomplexes+using+blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE).">Analysis of mitochondrial respiratory chain supercomplexes using blue native polyacrylamide gel electrophoresis (BN-PAGE).</a> Current Protocols in Mouse Biology. 6 (1), 1-14 (2016).</li><li>Pressure Biosciences Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolation of Functional Mitochondria from Whole Rat Heart Using a PBI Shredder and Pressure Cycling Technology (PCT). , (2010).</li><li>McLaughlin, K. 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The critical steps for this protocol are indicated here. First, tissue homogenization should be carefully handled so that excessive sheer effects will not be applied during the tissue homogenization process. A tissue shredder should be used, which is a part of pressure cycling technology (PCT) for initial tissue homogenization9. This step will reduce the excessive stroke cycle of glass-on-glass homogenizer (Figure 1B) that may further destroy already fragile mitochond...

The goal of this protocol was to provide detailed steps to obtain mitochondria-enriched preparation from previously frozen heart tissue with a new technology of low energy mechanical disruption of tissue that improves tissue lysis and extraction of mitochondria. With this method, improved extraction efficiency without generating high shear stress or high temperature and short homogenization time (10-12 s) become achievable1.
To obtain mitochondria from archived frozen tissue is a valuable asset to perform both functional2 and biochemical studies3 otherwise not easily repeatable under the exact experimental conditions. A classic Potter-Elvehjem Teflon pestle glass homogenizer or Dounce homogenizer has been used and is still being used in research laboratories to homogenize soft tissues such as liver, kidney, and brain. However, homogenizing hard tissues such as muscle and heart require more homogenization time, enzyme treatment, high-speed homogenization, and extensive user experience. This is disadvantageous for extracting intact organelles such as mitochondria from hard tissue such as muscle and heart. The method described in this protocol is used to obtain high yield mitochondria-enriched preparation to analyze mitochondrial electron transport chain (ETC) protein complexes and their supercomplex formation in heart tissues harvested from offspring born to exercised and sedentary sow, flash-frozen in liquid nitrogen, and stored at -80 &#176;C for future use. This method allows the user to isolate mitochondria enriched preparation from previously frozen archived tissues.
External nanomaterial exposure to pregnant rodents can negatively affect cardiac function and mitochondrial respiration and bioenergetics on progeny during gestation4. Nevertheless, aerobic exercise-induced positive changes in fetal myocyte bioenergetics during pregnancy are yet to be documented. However, emerging studies provide evidence that maternal aerobic exercise during pregnancy has a positive influence on fetal cardiac function5. In order to provide further evidence, an analysis of the longitudinal effects of maternal exercise on offspring cardiac mitochondrial respiratory chain complexes (i.e., Complex I through Complex V) during pregnancy was performed.
This study has significant health relevance since the results may suggest that maternal exercise improves the efficiency of energy production in the cardiac mitochondria of the offspring. In this study, sows (female pig) were used as an animal model for two reasons: (i) cardiac physiology is similar to human6, and (ii) heart tissue harvest from offspring from different time points is feasible under an institutional IACUC approval. The proposed study aims to answer many of the fundamental questions linking maternal exercise and its potential positive effects on the cellular and biochemical makeup of the offspring&#39;s heart tissue. This approach requires gentle yet effective mitochondria isolation techniques from previously frozen cardiac tissue obtained from the lengthy and costly longitudinal studies that addressed the issues of the bioenergetic changes within fetal cardiac myocytes during the pregnancy. The method described in this study allows utilizing large sums of previously frozen archived tissue for mitochondria-enriched preparation for both analytical and functional studies. The study will also help fill the knowledge gap in this field by providing preliminary data, which could lead to future studies determining the effects of maternal exercise on heart health in utero and beyond.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amino caproic acid</td>
			<td>Sigma/Aldrich</td>
			<td>A2504-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Hu Total OxPhos complex kit</td>
			<td>Invitrogen</td>
			<td>458199</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-VDAC antibody</td>
			<td>abcam</td>
			<td>ab15895</td>
			<td>use 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Coomassie G-250</td>
			<td>ThermoSientific</td>
			<td>20279</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie GelCode Blue</td>
			<td>ThermoScientific</td>
			<td>24592</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Cabiochem</td>
			<td>300410</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-Glass pestle homogenizer</td>
			<td>VWR</td>
			<td>KT885451-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Studio</td>
			<td>LICOR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IR-Dye conjugated anti-Rabbit Ab</td>
			<td>LICOR</td>
			<td>LC0725</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell plate reader</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td></td>
		</tr>
		<tr>
			<td>Native molecular weight marker</td>
			<td>ThermoFisher</td>
			<td>BN2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh monofilament</td>
			<td>Small Part Inc</td>
			<td>CMN-74</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>ThermoScientfic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCT Shredder</td>
			<td>Pressure Bioscience Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SEA BLOCK Blocking buffer</td>
			<td>ThermoScienctific</td>
			<td>37527</td>
			<td></td>
		</tr>
		<tr>
			<td>Shredder PULSE Tube</td>
			<td>Pressure Bioscience Inc</td>
			<td>FT500-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Amresco</td>
			<td>M150-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Amresco</td>
			<td>M191-1G</td>
			<td>Requires fresh preparation</td>
		</tr>
	</tbody>
</table>

measuring mitochondrial electron transfer complexes in previously frozen cardiac tissue from the offspring of sow: a model to assess exercise-induced mitochondrial bioenergetics changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Following the protocol, a good yield of mitochondria-enriched protein mixture from heart tissue was prepared. Approximately 15 mg/mL of mitochondria-enriched protein mixture was obtained from an average of 1.2 g frozen heart tissue harvested from the offspring of the sow. Observations indicated that less than 0.5 g of frozen heart tissue did not yield a sufficient amount of mitochondrial-enriched protein mixture to carry out a BN-PAGE assay. The amount of mitochondrial preparation was sufficient to perform (i) a standard...

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Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

This protocol is significant because it allows users to prepare mitochondria samples from frozen tissues collected from previous studies and archived. This protocol also reduces the use of live animals for tissue harvest. This technique allows users to use previously frozen archive tissues to prepare mitochondria enriched samples by employing a gentle tissue shredding method.This method of preparation maximizes the yield of mitochondria preparation. Through reading of the protocol is advisable to ensure the availability of all components and tools especially the fully charged shredder drive and also exercise with some unwanted tissues to get familiar with the steps. Demonstrating the procedure will be Edina Kosa, a Research Associate from my laboratory.To begin with, record the weight of the tube containing the heart tissue. Now weigh the empty tube and subtract it from the previously recorded weight to obtain a net weight of the heart tissue. Put the frozen heart tissue directly into the beaker of ice-cold solution.Stir it for five minutes. Simultaneously using tweezers apply slight pressure to the tissue to get out as much blood as possible. Take the tissue out with forceps and squeeze the heart with a clean filter paper towel to remove blood.Then place the tissue into another beaker of ice-cold solution. After stirring the tissue for five minutes, gently dry the tissue on a paper towel and remove the fat, clotted blood, auricles and fasciae. Pool the ventricular tissue.To shred the tissue samples, place 50 to 300 milligrams of heart tissue into the tissue shredder chamber from the ram side and close the ram side. Then add about 0.2 to 0.8 milliliters of washing buffer to the cap side of the tube. Use the tube tool to cap the shredder chamber with the shredder cap snugly without over tightening it.Place the shredder chamber into the shredder holder unit with the ram side down for engaged fitting. Insert and engage the bit of the fully charged shredder driver with the shredder cap ensuring the bit is snugly locked into place then apply pressure down to shredder driver in shredder chamber until the pressure indicator on the shredder holder reaches a setting of approximately two. Press the trigger and run the shredder driver for about 10 to 12 seconds while maintaining the setting at two.Observe the tube to ensure shredding and passing of all or most of the tissue mass through the lysis disk into the upper shredder chamber and if necessary return the tube to the shredder holder to continue shredding. After shredding the sample lift the shredder chamber out of the holder unit and use the tube tool to remove the shredder cap and place it on a clean surface for later use recommended only within the same sample to avoid cross contamination from other tissue samples. Transfer the shredded heart tissue into the third beaker with 40 milliliters of washing buffer with a stir bar and stirred the tissue for five minutes at a medium speed while on the stir plate.Filter the suspension through a pre-wetted nylon mesh monofilament. After discarding the filtrate wash the shredded tissue on the filter three times with 10 milliliters of washing buffer. Transfer the washed tissue into a 50 milliliter beaker with 20 milliliters of washing buffer placed in the ice bath on the magnetic stir.Add 0.5 milliliter of trypsin solution while constantly stirring for 15 minutes. Approximately halfway through the stirring transfer the tissue suspension into a loosely fitted handheld glass-on-glass homogenizer that can hold 15 milliliters of volume. Homogenize the suspension with four to five gentle strokes without producing froth.Then, place the homogenate into a 50 milliliter beaker and mix on a stirring plate. Next, add 10 milliliters of the isolation buffer containing trypsin inhibitor to the homogenate and incubate while gently stirring. After filtering the suspension again through a nylon filter save the filter in a 50 milliliter conical tube and transfer the particulate fraction left on the filter to the glass-on-glass homogenizer.At 15 to 20 milliliters of the washing buffer and gently homogenize with the hand for 25 to 30 seconds. After combining the homogenate with the filtrate and balancing the tubes, centrifuge and then using a plastic pipette remove the supernatant leaving 0.2 milliliters from the top of the fluffy pellet. Filter the resulting supernatant into a new conical tube using an nylon filter to avoid contamination and centrifuge one more time.After discarding the supernatant wash the pellet two to three times by adding the isolation buffer to the side of the tube to prevent breaking or contaminating the pellet. Discard the fluffy white outer rim layer each time. Add five milliliters of the isolation buffer to each tube and break up the pellet using a pipette with a one milliliter tip or a new transfer pipette Then dilute the suspension with 20 milliliters of additional isolation buffer and centrifuge the suspension at 8, 500 X g for 15 minutes at four degrees Celsius.After discarding the supernatant resuspend the pellet firstly in five milliliters and then in an additional 15 milliliters of the resuspending buffer and centrifuge at 8, 500 X g for 15 minutes at four degrees Celsius. Now discard the supernatant and save the brown pellet containing washed intact mitochondria. Resuspend the mitochondria enriched pellet in 250 microliters of the resuspending buffer including protease inhibitors.Aliquot the suspension in 25 microliters volume, quick freeze in liquid nitrogen and store in a 80 degree Celsius deep freezer for several years years. Following the protocol the mitochondria-enriched protein was prepared from the heart of offspring born to two groups of sows. One was exercised during pregnancy and the other was sedentary.ETC protein profile analysis revealed that the offspring of exercised sows presented relatively reduced levels in some of the ETC complexes. Mitochondrial proteins were resolved in a 3 to 8%gradient BN-PAGE and immunoprobed with antibody cocktails. Both exercised and sedentary groups exhibit multiple supercomplexes.The prominent supercomplex increase observed was at nine months in the exercised group compared to the sedentary group. Mitochondria-enriched proteins obtained from different age groups of 48 hours three months, six months and nine months were analyzed for the Complex I-II activity using a kinetic assay. The exercised group showed an Complex I-II activity as compared to that of the sedentary group.Feasibility of functional studies on mitochondria obtained from frozen tissues and cells is shown by other investigators. The preparation of mitochondria obtained from frozen tissue may be applied for a wide variety of tissues with minimal damage.

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

Abstract