Name: Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes (Video) | JoVE Journal
Uploaded: 2021-08-16T14:30:00.000+00:00
Duration: 8 min 4 s
Description: Learn to prepare mitochondria-enriched samples from frozen cardiac tissue using a gentle shredding method. This protocol maximizes yield while reducing live animal use in bioenergetics research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.

<ol><li>Gross, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Measuring+mitochondrial+respiration+in+previously+frozen+biological+samples%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+electron+transfer+cascade+enzyme+activity+assessment+in+cultured+neurons+and+select+brain+regions%5BTitle%5D)+AND+%22Current+Protocols+in+Toxicology%22%5BJournal%5D)">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. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal-engineered+nanomaterial+exposure+disrupts+progeny+cardiac+function+and+bioenergetics%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Maternal-engineered nanomaterial exposure disrupts progeny cardiac function and bioenergetics.</a> American Journal of Physiology-Heart and Circulatory Physiology. 312 (3), 446-458 (2017).</li>
<li>May, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+maternal+aerobic+exercise+during+pregnancy+on+fetal+cardiac+function+and+outflow%5BTitle%5D)+AND+%22American+Journal+of+Obstetrics+%26+Gynecology+MFM%22%5BJournal%5D)">Influence of maternal aerobic exercise during pregnancy on fetal cardiac function and outflow.</a> American Journal of Obstetrics & Gynecology MFM. 2 (2), 100095(2020).</li>
<li>Ehler, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Avoidance+of+malignant+hyperthermia+in+a+porcine+model+for+experimental+open+heart+surgery%5BTitle%5D)+AND+%22Laboratory+Animal+Science%22%5BJournal%5D)">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/pubmed?term=((Effect+of+bovine+serum+albumin+on+mitochondrial+respiration+in+the+brain+and+liver+of+mice+and+rats%5BTitle%5D)+AND+%22Bulletin+of+Experimental+Biology+and+Medicine%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+mitochondrial+respiratory+chain+supercomplexes+using+blue+native+polyacrylamide+gel+electrophoresis+%28BN-PAGE%29%5BTitle%5D)+AND+%22Current+Protocols+in+Mouse+Biology%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22Isolation+of+Functional+Mitochondria+from+Whole+Rat+Heart+Using+a+PBI+Shredder+and+Pressure+Cycling+Technology+%28PCT%29%22">Isolation of Functional Mitochondria from Whole Rat Heart Using a PBI Shredder and Pressure Cycling Technology (PCT)</a>. , South Easton, MA. (2010).</li>
<li>McLaughlin, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+approach+to+quantify+mitochondrial+content+and+intrinsic+bioenergetic+efficiency+across+organs%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Novel approach to quantify mitochondrial content and intrinsic bioenergetic efficiency across organs.</a> Scientific Reports. 10 (1), 17599(2020).</li>
<li>Hom, J., Sheu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+dynamics+of+mitochondria--a+special+emphasis+on+cardiac+muscle+cells%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">Morphological dynamics of mitochondria--a special emphasis on cardiac muscle cells.</a> Journal of Molecular and Cellular Cardiology. 46 (6), 811-820 (2009).</li>
<li>Greggio, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+respiratory+chain+supercomplex+formation+in+response+to+exercise+in+human+skeletal+muscle%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">Enhanced respiratory chain supercomplex formation in response to exercise in human skeletal muscle.</a> Cell Metabolism. 25 (2), 301-311 (2017).</li>
</ol>

The authors declare no conflicts of interest.

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><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 &amp;micro;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

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...

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 ...

measuring-mitochondrial-electron-transfer-complexes-previously-frozen

Research

JoVE Journal

Biochemistry

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

This method exploits the contribution of the mitochondrial permeability transition pore to low-conductance proton leak to determine the voltage threshold for pore opening in neonatal fragile X syndrome mice with increased cardiomyocyte mitochondrial coenzyme Q content compared to wildtype control.

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

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

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.

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Learn to prepare mitochondria-enriched samples from frozen cardiac tissue using a gentle shredding method. This protocol maximizes yield while reducing live animal use in bioenergetics research.

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

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

Explore More Videos