The authors would like to thank Dr. G.C. Shore of McGill University, Dr. A. Strauss of the Washington School of Medicine, and Dr. M.T. Ryan of La Trobe University for the original donations of expression plasmids that were used for this research. This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to D. A. Hood. D. A. Hood is also the holder of a Canada Research Chair in Cell Physiology.

All animals used in these experiments are maintained in the animal care facility at York University. The experiments are conducted in accordance with the Canadian Council on Animal Care guidelines with approval from the York University Animal Care Committee (Permit: 2017-08).
1. Functional isolation of subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle
<ol>
	<li>Reagent preparation:
	<ol>
		<li>Prepare all the buffers and media as mentioned in Table 1.</li>
		<li>Set the buffers to pH 7.4 and store at 4 &#176;C (up to 2 weeks), except nagrase protease.</li>
		<li>Prepare fresh nagarse protease (Table 1) each time.</li>
	</ol>
	</li>
	<li>Tissue removal and mincing 
	NOTE: A flowchart of the steps below for the isolation of mitochondria is provided in Figure 1.
	<ol>
		<li>Fill a glass scintillation vial with ~20 mL of Buffer 1 (Table 1) and place it on ice.</li>
		<li>While the mouse is under anesthetic, harvest skeletal muscles (tibialis anterior, quadriceps, gastrocnemius etc.), approximately 500-1000 mg, and place the muscles in the scintillation vial containing chilled Buffer 1. 
		NOTE: Gaseous isoflurane is used as an anesthetic at 2.5% at a flow rate of 0.4 L of O2/min. A pinch test is performed to ensure that the animal is non-responsive.</li>
		<li>Following tissue collection, euthanize the animal via cervical dislocation.</li>
		<li>Pre-chill a watch glass on ice. Remove any fat or connective tissue from the muscles and mince the tissue on the watch glass until it is a homogenous slurry.</li>
		<li>Place the minced tissue in a pre-chilled 50 mL plastic centrifugation tube (see Table of Materials) and record the exact weight.</li>
		<li>Dilute the minced tissue 10-fold with Buffer 1 + ATP (Table 1).</li>
		<li>Homogenize the muscle sample using an 8 mm twin-blade homogenizer (see Table of Materials) at a power output of 9.8 Hz for 10 s, ensuring no visible chunks of muscle are remaining.</li>
		<li>Centrifuge the samples at 800 x g for 10 min using a high-speed centrifuge (see Table of Materials). 
		NOTE: The purpose of this step is to separate SS and IMF mitochondrial fractions. The supernate contains SS mitochondria, while the pellet contains IMF mitochondria.</li>
	</ol>
	</li>
	<li>SS mitochondrial isolation
	<ol>
		<li>Filter the supernate through a single layer of cheesecloth into another set of 50 mL plastic centrifugation tubes to remove any large debris or contaminants.</li>
		<li>Centrifuge the supernate at 9,000 x g for 10 min at 4 &#176;C using a high-speed centrifuge.</li>
		<li>Discard the supernate and resuspend the pellet in 3.5 mL of Buffer 1 + ATP. 
		NOTE: Resuspend with a P1000 and do this carefully. Mitochondria are fragile organelles. Perform gently and avoid touching the pellet with the pipette tip.</li>
		<li>Centrifuge the sample at 9,000 x g for 10 min at 4 &#176;C using a high-speed centrifuge.</li>
		<li>Discard the supernate unless further processing to isolate a cytosolic fraction devoid of nuclei, mitochondria, and other organelles is desired. Resuspend the pellet carefully in roughly 95 &#181;L of the resuspension medium (Table 1) using a P200 pipette. 
		NOTE: Similar to step 1.3.3, avoid touching the pellet with the pipette tip. The volume of the resuspension medium can be altered depending on the size of the pellet. This is the SS mitochondrial fraction, which can be stored on ice until the IMF fraction is isolated. If desired, the cytosolic fraction, devoid of organelles, can be isolated by centrifuging the supernate at 100,000 x g in an ultracentrifuge (see Table of Materials) and thereafter discarding the pellet.</li>
	</ol>
	</li>
	<li>IMF mitochondrial isolation
	<ol>
		<li>Dilute the pellet from step 1.2.8 by 10-fold in Buffer 1 + ATP. Resuspend using a Teflon pestle by gently mixing the pellet and buffer together until the sample is consistent.</li>
		<li>Homogenize the sample using an 8 mm twin-blade homogenizer (see Table of Materials) at a power output of 9.8 Hz for 10 s.</li>
		<li>Centrifuge the sample at 800 x g for 10 min at 4 &#176;C using a high-speed centrifuge.</li>
		<li>Discard the supernate and dilute the IMF mitochondrial pellet 10-fold using Buffer 2 and resuspend using the Teflon pestle by gently mixing until it is homogeneous.</li>
		<li>Add 25 &#181;L/g of tissue to 10 mg/mL nagarse protease (Table 1) and place the centrifugation tube on its side on ice, gently mixing every minute by rocking the tube side to side. 
		NOTE: The addition of nagarse helps liberate IMF mitochondria by performing limited digestion of the myofibrils.</li>
		<li>After 5 min, add 20 mL of Buffer 2 (Table 1) to dilute the nagarse protease to the point of inactivity and stop the digestion.</li>
		<li>Immediately centrifuge the sample at 5,000 x g for 5 min at 4 &#176;C using a high-speed centrifuge.</li>
		<li>Discard the supernate and dilute the pellet 10-fold using Buffer 2 and resuspend using a Teflon pestle by gently mixing.</li>
		<li>Centrifuge the sample at 800 x g for 15 min at 4 &#176;C to pellet the fibrous material.</li>
		<li>Pour the supernate in a new set of 50 mL plastic centrifuge tubes, careful not to disrupt the pellet, which can be discarded.</li>
		<li>Centrifuge the sample at 9,000 x g for 10 min at 4 &#176;C as in step 1.4.7.</li>
		<li>Discard the supernate, add 3.5 mL of Buffer 2 and gently resuspend the pellet using a P1000 pipette.</li>
		<li>Centrifuge the sample at 9,000 x g for 10 min at 4 &#176;C as in step 1.4.7.</li>
		<li>Discard the supernate and resuspend the pellet gently with roughly 180 &#181;L of resuspension medium using a P200 pipette. 
		&#8203;NOTE: The volume of the resuspension medium can be altered depending on the size of the pellet. This is the IMF mitochondrial fraction, which can be stored on ice until it is to be used in the import assay.</li>
	</ol>
	</li>
</ol>
2. Mitochondrial protein import
<ol>
	<li>In vitro transcription 
	NOTE: These subsequent steps outline how to prepare a radiolabelled protein for import. The import pathway under investigation may dictate the choice of target protein, for example, to evaluate import into the mitochondrial matrix, ornithine carbamyl transferase, OCT, and malate dehydrogenase, MDH, are commonly used, while Tom40 is commonly used as an indication of import into the outer mitochondrial membrane. For the following steps, plasmid DNA encoding the protein of choice is required. The transcription of plasmid DNA can be done prior to the mitochondrial isolation on a separate day, and the mRNA collected from this experiment can be stored and used in future translation and import experiments.
	<ol>
		<li>Linearize the DNA: Combine 40 &#181;L of plasmid DNA (5 &#181;g/&#181;L), 5 &#181;L of 10x enzyme buffer and 5 &#181;L of restriction enzyme and incubate at 37 &#176;C for 30 min. 
		NOTE: The restriction enzymes utilized may vary based on the plasmid, which may require a different enzyme buffer, and experimental conditions DNA can be used in any amount/volume, as the template can be scaled up or down.</li>
		<li>Purify and precipitate the linearized DNA using phenol and ethanol. Carry out all the subsequent steps (2.1.3-2.1.15) in sterile 1.5 mL tubes, and use a tabletop centrifuge (see Table of Materials) for the centrifugation steps.</li>
		<li>Add the appropriate volume of sterile H2O so that the final volume equals 400 &#181;L. Then, add 400 &#181;L of phenol.</li>
		<li>Mix vigorously by inversion for ~10 s, and centrifuge at 17,000 x g for 1 min at 4 &#176;C. Withdraw and save the upper phase.</li>
		<li>Add 400 &#181;L of phenol:chloroform:isoamylalcohol (25:24:1, v:v:v).</li>
		<li>Mix vigorously by inversion for ~10 s, and centrifuge at 17,000 g for 1 min at 4 &#176;C. Withdraw and save the upper phase.</li>
		<li>Add 400 &#181;L of chloroform:isoamylalcohol (24:1, v:v).</li>
		<li>Mix vigorously by inversion for ~10 s, and centrifuge at 17,000 g for 1 min at 4 &#176;C. Withdraw and save the upper phase.</li>
		<li>Add 40 &#181;L of 3M sodium acetate (pH 7.0), and add 1 mL of 95% ethanol.</li>
		<li>Place sterile 1.5 mL tubes in -80 &#176;C freezer on their side for 15 min.</li>
		<li>Centrifuge the samples at 17,000 x g for 10 min at 4 &#176;C.</li>
		<li>Discard the supernate and wash the pellet with 300 &#181;L of 80% ethanol.</li>
		<li>Centrifuge the samples at 17,000 x g for 2 min at 4 &#176;C.</li>
		<li>Discard the supernate and air-dry the pellet. Once the pellet is dry, it should be clear.</li>
		<li>Resuspend the pellet in 20 &#181;L of sterile H2O</li>
		<li>Measure the DNA concentration using a spectrophotometer (see Table of Materials) and ensure that the A260:280 ratio is above 1.8.</li>
		<li>Dilute the DNA to a concentration of 0.8 &#181;g/&#181;L in sterile H2O.</li>
		<li>Transcribe the DNA: Combine plasmid (0.8 &#181;g/&#181;L, 60.8 &#181;L), sterile H2O (8.4 &#181;L), 10 mM NTP (5.2 &#181;L), 10 mM ATP ( 10.0 &#181;L), 1 mM M-7-G (11.6 &#181;L), the mix as mentioned step 2.1.19 (15.6 &#181;L), RNAsin (5.2 &#181;L) and an appropriate RNA polymerase (4.8 &#181;L) to form &#34;one reaction mix&#34; (total volume = 121.6 &#181;L) and incubate for 90 min at the optimum temperature for the polymerase (37 &#176;C for T7 polymerase; 40 &#176;C for SP6 polymerase)</li>
		<li>To prepare the mix add 200 &#181;L of 1M HEPES (pH 7.9), 100 &#181;L of 1 M MgAc2, 500 &#181;L of 4 M KAc, 100 &#181;L of 20 mM spermidine, 100 &#181;L of 0.5 M DTT, and 200 &#181;L of sterile H20. 
		NOTE: The reaction can be scaled up or down in volume as long as the provided proportions of reagents are maintained. The RNA polymerase used is dictated by the bacterial promoter sequence within the plasmid being used.</li>
		<li>Then, purify and precipitate the mRNA using phenol and ethanol as in steps 2.1.2-2.1.15. Bring the volume up to 400 &#181;L with sterile H2O (add 280 &#181;L) and proceed as described in steps 2.1.2-2.1.15. Resuspend the final pellet in 20 &#181;L of sterile H2O.</li>
		<li>Measure the concentration of mRNA using a spectrophotometer, ensure that the A260:280 ratio is ~2.0.</li>
		<li>Dilute the mRNA to 2.8 &#181;g/&#181;L in sterile H2O, and store at -20 &#176;C in 50 &#181;L aliquots.</li>
	</ol>
	</li>
	<li>In vitro translation 
	NOTE: The translation of the desired DNA must be carried out on the same day of the import experiment. This experiment requires the use of radiolabeled amino acids, and therefore the user must have a permit for the use of radioisotopes, and all handling and disposal must be in accordance with the institution&#39;s policies/requirements. Steps involving radioisotope safety measures have been omitted or described briefly, as these will likely differ based on the institution.
	<ol>
		<li>Prepare enough translation reaction mix (Table 2) to supply ~20 &#181;L per sample of mitochondria to be used in the import experiment and include the same volume for a translation lane.</li>
		<li>Incubate the reaction for 30 min at 30 &#176;C (time may vary with mRNA, 25-60 min).</li>
		<li>Record the 35S-methionine use as per the institution&#39;s radioactive safety requirements and dispose of anything that has come into contact with the radioisotope per the institution&#39;s guidelines.</li>
	</ol>
	</li>
	<li>Protein import 
	NOTE: Freshly isolated mitochondria are required for the next steps. Refer to the mitochondrial isolation protocol. Other isolation methods can be used as long as the mitochondria are viable and functional. For the subsequent steps, the mitochondrial samples utilized are resuspended in a resuspension buffer as described above (Table 1). The protein concentration of the mitochondrial sample must also be measured prior to starting the import assay.
	<ol>
		<li>Roughly 15 min following the start of the translation reaction, aliquot 90 &#181;g of mitochondria into sterile 1.5 mL tubes and pre-incubate at 30 &#176;C for ~10 min. 
		NOTE: Aliquot more mitochondria than what will actually be used in the experiment; only 75 &#181;g will be actually used. However, aliquoting in excess is not a requirement.</li>
		<li>In a fresh set of sterile 1.5 mL tubes, combine 75 &#181;g of mitochondria with 18 &#181;L of the translation reaction and incubate at 30 &#176;C for desired time.</li>
		<li>Keep the remaining volume of the translation reaction on ice; this will be utilized later. 
		NOTE: Import is a time-dependent process, so it may be prudent to start with a time-course experiment ranging in incubation times from 1-60 min. A typical reaction time is 30 min.</li>
		<li>During this time, prepare the sucrose cushion by combining 1 g of sucrose, 200 &#181;L of 2.5 M KCl, 10 &#181;L 1 M MgCl2, 100 &#181;L of 1 M HEPES (pH 7.4), and bring the volume to 5 mL using sterile H2O.</li>
		<li>Prepare a new set of sterile 1.5 mL tubes corresponding to each tube in the import reaction. Aliquot 600 &#181;L of the sucrose cushion into each tube and keep on ice until the import reaction is over.</li>
		<li>To terminate the import reaction after the appropriate incubation time, remove the tube from 30 &#176;C, place it on ice and then carefully transfer the import reaction on top of the tube with the sucrose cushion. 
		NOTE: This step needs to be done very slowly to ensure that the mitochondria are not damaged/ruptured. The sucrose cushion helps slow down the migration of the mitochondria and &#34;cushions&#34; its descent to the bottom of the tube during the subsequent centrifugation step.</li>
		<li>Centrifuge the samples + sucrose cushion at 17,000 x g for 15 min at 4 &#176;C.</li>
		<li>Prepare the breaking buffer by combining 25 mL of 2.4 M sorbitol, 2 mL of 1 M HEPES (pH 7.4), and 72 mL of double-distilled H2O.</li>
		<li>Prepare lysis buffer for SDS-PAGE gel electrophoresis: Combine 50 mL of heated glycerol, 11.5 g of SDS, 31.25 mL of 1 M Tris (pH 6.8) and bring the volume to 500 mL with double-distilled H2O.</li>
		<li>For sample preparation, mix 475 &#181;L of lysis buffer with 25 &#181;L of &#946;-mercaptoethanol to be used in a later step. 
		NOTE: Lysis buffer can be made in advance and stored at room temperature; however, the addition of &#946;-mercaptoethanol is to be done fresh.</li>
		<li>Using a P1000 pipette, carefully remove the supernate and do not disturb the pellet. Dispose of the supernate in an appropriate radioactive waste container.</li>
		<li>For import into the outer membrane, perform this using Tom40, resuspend the pellet in 50 &#181;L of freshly prepared 0.1 M sodium carbonate (pH 11.5) and incubate on ice for 30 min.</li>
		<li>Following the incubation, centrifuge the sample 14,000 x g for 5 min at 4 &#176;C. This step is only done for outer membrane import reactions.</li>
		<li>To prepare the samples for SDS-PAGE electrophoresis, add 20 &#181;L of breaking buffer, 20 &#181;L of lysis buffer, and &#946;-mercaptoethanol and resuspend well. Add 5 &#181;L of sample dye.</li>
		<li>Prepare the control/translation lane by combining 3 &#181;L of the remaining translation reaction from step 2.3.3 with 37 &#181;L of lysis buffer and 5 &#181;L of sample dye. Mix well.</li>
		<li>Boil the samples for 5 min at 95 &#176;C and spin down gently at a low speed to avoid pelleting mitochondria.</li>
		<li>Apply the samples to a SDS polyacrylamide gel. 
		NOTE: The gel percentage and subsequent run time will depend on which protein is being imported. For example, OCT is 37 kDa, and its mature processed band is slightly smaller; therefore, a 12% gel allows good separation of these bands.</li>
		<li>Once electrophoresis is complete, remove the gel and place it in double-distilled H2O.</li>
		<li>Boil the gel in 5% TCA in a fume hood for 5 min, continuously stirring/moving the gel to avoid cracking. 
		NOTE: TCA helps solidify the gel for visualization; this can be done in a metal tray over a Bunsen burner. Use enough TCA to generously cover the gel, ~&#189; full.</li>
		<li>Remove the gel and place it back in double-distilled H2O. Agitate it on a rotating plate for 1 min at ~50 rpm.</li>
		<li>Wash the gel in 10 mM Tris on a rotating plate for 5 min at ~50 rpm, again using enough to generously cover the gel, ~&#189; of the container.</li>
		<li>Precipitate the protein by washing the gel in 1 M Salicyclic acid on a rotating plate for 30 min, again using enough Salicyclic acid to generously cover the gel, ~&#189; of the container being used.</li>
		<li>Dehydrate the gel as described in steps 2.3.24-2.3.31. 
		NOTE: This can be done in a number of ways. A gel dryer (see Table of Materials) provides a time-efficient option (described below); however other commercially available methods/kits can be used that may be more cost-effective but more time-consuming. Gel drying kits can be utilized as an alternative that does not require the application of heat or suction but instead allows the gel to air-dry between cellophane sheets to prevent distortion.</li>
		<li>Place a large sheet of blotting paper down on the porous bed of the gel dryer. Place one sheet of paper towel in the area where the gel will be applied.</li>
		<li>Cut a 15 cm x 20 cm piece of blotting paper and place it on the paper towel.</li>
		<li>Scoop out the gel from the container using a second piece of 15 cm x 20 cm blotting paper and lay it down flat on top of the first piece.</li>
		<li>Cut a slightly larger piece of plastic wrap, ~20 cm x 25 cm, and place it down on gel, ensuring no creases or bubbles under the wrap. 
		NOTE: This may take a few attempts, but it is imperative that there are no trapped air pockets.</li>
		<li>Gently lay down the plastic cover of the gel dryer over top of the wrap, again ensuring that there are no bubbles or creases.</li>
		<li>Turn on the pump/vacuum and ensure that the plastic cover has formed a seal. Test the seal by lifting a corner of the plastic cover and wait for it to re-seal.</li>
		<li>Close the gel dryer and run for 90 min. Set the temperature to start at 30 &#176;C, gradually reach 80 &#176;C, and then return to 30 &#176;C at the end of the run.</li>
		<li>Once dehydrated, the gel will solidify and feel paper-thin. Wrap the gel in the plastic wrap used in the drying process.</li>
		<li>Place the dehydrated gel in a film cassette with a phosphorus film (see Table of Materials), with the white side facing the gel. Exposure times vary but can be 24-48 h for visualization of bands.</li>
		<li>Visualize using autoradiography using any suitable imager that is capable of phosphorus imaging.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+ATF5+mediates+a+mammalian+mitochondrial+UPR.">The transcription factor ATF5 mediates a mammalian mitochondrial UPR.</a> Current biology. 26 (15), 2037-2043 (2016).</li><li>Quiros, P. M., 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=Multi-omics+analysis+identifies+ATF4+as+a+key+regulator+of+the+mitochondrial+stress+response+in+mammals.">Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals.</a> The Journal of Cell Biology. 216 (7), 2027-2045 (2017).</li><li>Takahashi, M., Hood, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+import+into+subsarcolemmal+and+intermyofibrillar+skeletal+muscle+mitochondria.+Differential+import+regulation+in+distinct+subcellular+regions.">Protein import into subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Differential import regulation in distinct subcellular regions.</a> The Journal of Biological Chemistry. 271 (44), 27285-27291 (1996).</li><li>Hood, D. A., Memme, J. M., Oliveira, A. N., Triolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+skeletal+muscle+mitochondria+in+health,+exercise,+and+aging.">Maintenance of skeletal muscle mitochondria in health, exercise, and aging.</a> Annual Review of Physiology. 81, (2019).</li><li>Joseph, A., Hood, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrion+plasticity+of+TOM+complex+assembly+in+skeletal+muscle+mitochondria+in+response+to+chronic+contractile+activity.">Mitochondrion plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity.</a> Mitochondrion. 12 (2), 305-312 (2012).</li><li>Singh, K., Hood, D. A. <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+denervation-induced+muscle+disuse+on+mitochondrial+protein+import.">Effect of denervation-induced muscle disuse on mitochondrial protein import.</a> American Journal of Physiology-Cell Physiology. 300 (1), 138-145 (2011).</li><li>Zhang, Y., 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=Altered+mitochondrial+morphology+and+defective+protein+import+reveal+novel+roles+for+Bax+and/or+Bak+in+skeletal+muscle.">Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle.</a> American Journal of Physiology. Cell Physiology. 305 (5), 502-511 (2013).</li><li>Lai, N., Kummitha, C., Rosca, M., Fujioka, H., Tandler, B., Hoppel, C. <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+mitochondrial+subpopulations+from+skeletal+muscle:+optimizing+recovery+and+preserving+integrity.">Isolation of mitochondrial subpopulations from skeletal muscle: optimizing recovery and preserving integrity.</a> Acta Physiologica. 25 (2), 13182 (2019).</li><li>Nargund, A. M., Pellegrino, M. W., Fiorese, C. J., Baker, B. M., Haynes, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+import+efficiency+of+ATFS-1+regulates+mitochondrial+UPR+activation.">Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation.</a> Science. 337 (6094), 587-590 (2012).</li><li>Picard, M., Taivassalo, T., Gouspillou, G., Hepple, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+Isolation,+structure+and+function.">Mitochondria: Isolation, structure and function.</a> Journal of Physiology. 589 (18), 4413-4421 (2011).</li><li>Kras, K. A., Willis, W. T., Barker, N., Czyzyk, T., Langlais, P. R., Katsanos, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsarcolemmal+mitochondria+isolated+with+the+proteolytic+enzyme+nagarse+exhibit+greater+protein+specific+activities+and+functional+coupling.">Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling.</a> Biochemistry and Biophysics Reports. 6, 101-107 (2016).</li><li>S&#225;nchez-Duarte, E., 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=Nicorandil+affects+mitochondrial+respiratory+chain+function+by+increasing+complex+III+activity+and+ROS+production+in+skeletal+muscle+mitochondria.">Nicorandil affects mitochondrial respiratory chain function by increasing complex III activity and ROS production in skeletal muscle mitochondria.</a> Journal of Membrane Biology. 253 (4), 309-318 (2020).</li><li>I&#241;igo, M. R., 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=Estrogen+receptor-&#945;+in+female+skeletal+muscle+is+not+required+for+regulation+of+muscle+insulin+sensitivity+and+mitochondrial+regulation.">Estrogen receptor-&#945; in female skeletal muscle is not required for regulation of muscle insulin sensitivity and mitochondrial regulation.</a> Molecular Metabolism. 34 (2020), 1-15 (2020).</li><li>Newsom, S. A., Stierwalt, H. D., Ehrlicher, S. E., Robinson, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-specific+respiration+of+isolated+skeletal+muscle+mitochondria+after+1+h+of+moderate+cycling+in+sedentary+adults.">Substrate-specific respiration of isolated skeletal muscle mitochondria after 1 h of moderate cycling in sedentary adults.</a> Medicine and Science in Sports and Exercise. 53 (7), 1375-1384 (2021).</li><li>Takahashi, M., Chesley, A., Freyssenet, D., Hood, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+activity-induced+adaptations+in+the+mitochondrial+protein+import+system.">Contractile activity-induced adaptations in the mitochondrial protein import system.</a> The American Journal of Physiology. 274 (5), 1380-1387 (1998).</li><li>Kravic, B., 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=In+mammalian+skeletal+muscle,+phosphorylation+of+TOMM22+by+protein+kinase+CSNK2/CK2+controls+mitophagy.">In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.</a> Autophagy. 8627, 01-65 (2017).</li><li>Opali&#324;ska, M., Meisinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+control+via+the+mitochondrial+protein+import+machinery.">Metabolic control via the mitochondrial protein import machinery.</a> Current Opinion in Cell Biology. 33, 42-48 (2015).</li><li>Gerbeth, 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=Glucose-induced+regulation+of+protein+import+receptor+tom22+by+cytosolic+and+mitochondria-bound+kinases.">Glucose-induced regulation of protein import receptor tom22 by cytosolic and mitochondria-bound kinases.</a> Cell Metabolism. 18 (4), 578-587 (2013).</li></ol>

No conflicts of interest, financial or otherwise, are declared by the authors.

Mitochondria are uniquely dependent on the expression and coordination of both the nuclear and mitochondrial genomes for their synthesis and expansion within cells. However, the nuclear genome encodes the vast majority (99%) of the mitochondrial proteome, and this underscores the importance of the protein import machinery in supporting mitochondrial biogenesis. Import also serves as an important signaling event, as failure to import can promote the initiation of the unfolded protein response and/or mitophagy<sup class="x...

Mitochondria are organelles that exist in complex morphologies in different cell types and are recognized to possess an increasing array of functions that are critical for cellular health. As such, they can no longer be whittled down merely to energy-producing organelles. Mitochondria are key metabolic regulators, determinants of cell fate, and signaling hubs, the functions of which can serve as useful indicators of overall cellular health. In skeletal muscle cells, electron microscopy studies reveal the presence of geographically distinct subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, which exhibit a degree of connectivity1,2,3,4 that is now recognized to be highly dynamic and adaptable to changes in skeletal muscle activity levels, as well as with age and disease. Mitochondrial content and function in muscle can be assessed in numerous ways5,6, and traditional methods of organelle isolation have been applied to better understand the respiratory and enzymatic capacities (Vmax) of mitochondria distinct from the influence of the cellular milieu7,8. In particular, these traditional methods have revealed subtle biochemical distinctions between mitochondria isolated from subsarcolemmal and intermyofibrillar regions, belying possible functional implications for metabolism in these subcellular regions8,9,10,11.
The biogenesis of mitochondria is unique in requiring the contribution of gene products from both nuclear and mitochondrial DNA. However, the vast majority of these are derived from the nucleus since mtDNA transcription only leads to the synthesis of 13 proteins. Since mitochondria normally comprise &#62;1000 proteins involved in diverse metabolic pathways, biogenesis of the organelle requires a tightly regulated means of import and assembly of precursor proteins from the cytosol into the various mitochondrial sub-compartments to maintain proper stoichiometry and function12,13. Nuclear-encoded proteins destined for mitochondria normally carry a mitochondrial targeting sequence (MTS) that targets them to the organelle and facilitates their sub-compartmental localization. Most matrix-bound proteins contain a cleavable N-terminal MTS, while those destined for the outer or inner mitochondrial membrane usually have internal targeting domains14. The import process is carried out by a set of diverse channels that provide multiple avenues for entry into the organelle13. The translocase of the outer membrane (TOM) complex shuttles precursors from the cytosol into the intermembrane space, where they are recognized by the translocase of the inner membrane (TIM) complex. This complex is responsible for importing nuclear-encoded precursors into the matrix, where proteases cleave the N-terminal targeting presequence. Proteins destined for the outer membrane can be directly inserted into this membrane through the TOM complex, while those destined for the inner membrane are inserted by a TIM protein, specifically TIM22. Following their import, proteins are further processed by resident proteases and chaperones and often combine to form larger complexes, such as those found in the electron transport chain.
Mitochondrial protein import itself also serves as a measurement of mitochondrial health, as this process relies on the presence of membrane potential and a source of energy in the form of ATP15. For example, when the membrane potential is dissipated, protein kinase PINK1 cannot be taken up by the organelle, and this leads to phosphorylation signals that trigger the onset of the degradation of the organelle through a pathway called mitophagy16,17. Under similar circumstances, when the import is impeded, the protein ATF5 cannot enter the organelle, and it subsequently translocates to the nucleus, where it serves as a transcription factor for the up-regulation of UPR gene expression18,19. Thus, measuring protein import efficiency can provide comprehensive insight into the health of the organelle, while the gene expression response can be used to indicate the degree of retrograde signaling to the nucleus.
Despite its obvious importance for the biogenesis of mitochondria and for cellular health in general, the import pathway in mammalian mitochondria is remarkably understudied. In this report, we describe the specific steps involved in measuring the import of precursor proteins into skeletal muscle mitochondria and provide data to illustrate the adaptive response of the import system to changes in muscle and disuse, illustrating the contribution of the protein import to the adaptive plasticity of skeletal muscle.

<table><tbody><tr><td>0.2% BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>35S-methionine</td><td>Perkin Elmer</td><td>NEG709A500UC</td><td>Purchase requires a valid radioisotope permit</td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>Blotting paper; Whatman 3MM CHR Paper</td><td>Thermo Fisher</td><td>05-714-5</td><td></td></tr><tr><td>Cassette for film</td><td>Kodak</td><td>Kodak Xomatic</td><td></td></tr><tr><td>Centrifugation Tube</td><td>Thermo Fisher</td><td>3138-0050</td><td></td></tr><tr><td>Chloroform</td><td>Thermo Fisher</td><td>C298-4</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>D9779-5G</td><td></td></tr><tr><td>EDTA</td><td>BioShop</td><td>EDT002</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E4378</td><td></td></tr><tr><td>Gel Dryer</td><td>BioRad</td><td>Model 583</td><td></td></tr><tr><td>Gel Drying Kit</td><td>Sigma or BioRad</td><td>Z377570-1PAK or OW-GDF-10</td><td>Various options are commercially available through many companies, these are just as few examples.</td></tr><tr><td>Glycerol</td><td>Caledon Laboratory Chemicals</td><td>5350-1-40</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>High Speed Centrifuge</td><td>Beckman Coulter</td><td>Avanti J-25 Centrifuge</td><td></td></tr><tr><td>Homogenizer</td><td>IKA</td><td>T25 Digital Ultra Turrex</td><td></td></tr><tr><td>Isoamylalcohol, or 3-methylbutanol</td><td>Sigma</td><td>I9392</td><td></td></tr><tr><td>KAc</td><td>BioShop</td><td>POA301.500</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>M7G</td><td>New England Biolab</td><td>S1404S</td><td>Dilute with 1000ul 20mM HEPES to make 1mM stock</td></tr><tr><td>MgCl</td><td>BioShop</td><td>MAG510</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Thermo Fisher</td><td>M65-500</td><td></td></tr><tr><td>MOPS</td><td>BioShop</td><td>MOP001</td><td></td></tr><tr><td>NaCl</td><td>BioShop</td><td>SOD001</td><td></td></tr><tr><td>NTP</td><td>Thermo Fisher</td><td>R0191</td><td></td></tr><tr><td>OCT Plasmid</td><td>-</td><td>-</td><td>Donated from Dr. G. C. Shore, McGill University, Montreal, Canada</td></tr><tr><td>pGEM4Z/hTom40 Plasmid</td><td>-</td><td>-</td><td>Donated from Dr. M. T. Ryan, La Trobe University, Melbourne, Australia</td></tr><tr><td>pGMDH Plasmid</td><td>-</td><td>-</td><td>Donated from Dr. A. Strauss, Washington University School of Medicine</td></tr><tr><td>Phenol</td><td>Sigma</td><td>P4557</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyalcohol</td><td>Sigma</td><td>P3803</td><td>Can also be made with the ratio provided</td></tr><tr><td>Phosphorus Film</td><td>Fujifilm</td><td>BAS-IP MS 2025</td><td></td></tr><tr><td>Rabbit reticulocyte lysate</td><td>Promega</td><td>L4960</td><td>Avoid freeze-thaw; aliquot lysate upon arrival; amino acids are provided in the kit as well</td></tr><tr><td>RNAsin</td><td>Promega</td><td>N2311</td><td></td></tr><tr><td>Rotor for High Speed Centrifuge</td><td>Beckman Coulter</td><td>JA-25.50</td><td></td></tr><tr><td>SDS</td><td>BioShop</td><td>SDS001.500</td><td>Caution: harmful if ingested or inhaled, wear a mask.</td></tr><tr><td>Sodium acetate</td><td>Bioshop</td><td>SAA 304</td><td></td></tr><tr><td>Sodium Carbonate</td><td>VWR</td><td>BDH9284</td><td></td></tr><tr><td>Sodium salicylate</td><td>Millipore Sigma</td><td>106601</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma</td><td>S6021</td><td></td></tr><tr><td>SP6 RNA Polymerase</td><td>Promega</td><td>P1085</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher</td><td>Nanodrop 2000</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>S-2626</td><td></td></tr><tr><td>Sucrose</td><td>BioShop</td><td>SUC507</td><td></td></tr><tr><td>T7 RNA Polymerase</td><td>Promega</td><td>P2075</td><td></td></tr><tr><td>Tabletop Centrifuge</td><td>Thermo Fisher</td><td>AccuSpin Micro 17</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Thermo Fisher</td><td>A322-500</td><td></td></tr><tr><td>Tris</td><td>BioShop</td><td>TRS001</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M6250-100ML</td><td></td></tr></tbody></table>

measurement of protein import capacity of skeletal muscle mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

We have extensively illustrated that this protocol is a valid assay for determining the rate of import into functional and intact isolated skeletal muscle mitochondria. In comparison to untreated conditions, the import of typical precursor proteins such as malate dehydrogenase (MDH) into the matrix is sensitive to membrane potential because it can be inhibited by valinomycin, a respiratory chain uncoupler (Figure 2A). Import is also impeded when mitochondrial inner and outer...

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Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

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2.6K Views.  York University. Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health. 

Video: Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

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

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

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.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

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.

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 ...

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

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

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.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Biology

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

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

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics.
This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

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.

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

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.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

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

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel.
To do so, oxygen consumption and membrane potential (&#916;&#936;) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP+) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.

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.

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in ...