This work is supported by the following grants: NIH/NIGMS T32GM008464 (K.K.G.), Columbia University Irving Medical Center Target of Opportunity Provost award to the Department of Anesthesiology (K.K.G.), Society of Pediatric Anesthesia Young Investigator Research Award (K.K.G.), and NIH/NINDS R01NS112706 (R.J.L.)

Institutional Animal Care and Use Committee of Columbia University Medical Center approval was obtained for all methods described. FXS (Fmr1 KO) (FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J) and control (FVB) (FVB.129P2-Pde6b+ Tyrc-ch/AntJ) mice used as the model systems for this study were commercially acquired (see the Table of Materials). Five to eleven animals were used in each experimental group. Postnatal day 10 (P10) mice were used to model for a time point in human infancy.
1. Mitochondrial isolation from mouse heart
<ol>
	<li>Prepare buffers for mitochondrial isolation and respiration experiments as described in Table 1. Store at 4 &#176;C if made in advance.
	<ol>
		<li>Prepare 15% density gradient medium: dilute commercial density gradient medium to 80% volume/volume (v/v) with density gradient diluent. Further dilute 80% density gradient medium 15% v/v by adding mitochondrial isolation buffer (MI)/bovine serum albumin (BSA).</li>
	</ol>
	</li>
	<li>Isolate mitochondria from fresh, not frozen tissue for these experiments and use on the day of preparation, typically within five hours26. Carry out all isolation steps on ice.
	<ol>
		<li>Decapitate the mouse and excise the heart. Immediately wash 1-2 times in a Petri dish with ice-cold MI/BSA. Cut off the atrium and mince the tissue.</li>
		<li>Transfer the minced heart tissue to a glass-glass homogenizer containing 1 mL of MI/BSA. Homogenize with a looser (A) pestle (10 strokes), then a tighter (B) pestle (10 strokes).</li>
		<li>Centrifuge the homogenate at 1,100 &#215; g for 2 min at 4 &#176;C to remove nuclear and cellular debris.</li>
		<li>Take the supernatant gently without touching any fluffy pellet, and carefully layer it on top of 700 &#956;L of 15% density gradient medium in a centrifuge tube. Centrifuge at 18,500 &#215; g for 15 min at 4 &#176;C.</li>
		<li>Resuspend the pellet in 1 mL of sucrose buffer (SB)/BSA and centrifuged at 10,000 &#215; g for 10 min at 4 &#176;C.</li>
		<li>Completely remove and discard the supernatant. Resuspend the pellet in SB/BSA to a final volume of 55 &#956;L.</li>
		<li>Quantify mitochondrial protein content using a standard assay such as the bicinchoninic acid (BCA) protein assay.</li>
	</ol>
	</li>
</ol>
2. Mitochondrial oxygen (O2) consumption and &#916;&#936;
<ol>
	<li>Oxygen electrode assembly and calibration
	<ol>
		<li>Set up and calibrate the oxygen electrode (see the Table of Materials) according to the manufacturer&#39;s instructions.
		<ol>
			<li>Place a drop of 50% potassium chloride (KCl) electrolyte solution on top of the dome of the electrode disc.</li>
			<li>Place a small piece ~2 cm2 cigarette paper spacer covered with a slightly larger piece of the provided polytetrafluoroethylene (PTFE) membrane over the electrolyte drop.</li>
			<li>Using the applicator tool provided, push the small electrode disc O-ring over the dome of the electrode. 
			NOTE: Ensure that there are no air bubbles and that the membrane is smooth.</li>
		</ol>
		</li>
		<li>Top up the reservoir well with the electrolyte solution.</li>
		<li>Place the larger O-ring in the recess around the electrode disc well.</li>
		<li>Install the disc into the electrode chamber and connect it to the control unit.</li>
		<li>Add 2 mL of air saturated deionized water to the reaction chamber and the PTFE-coated magnet to the chamber.</li>
		<li>Connect the chamber to the rear of the control unit.</li>
		<li>Set the temperature to 37 &#176;C and the stirring speed to 100.</li>
		<li>Allow 10 min for the system temperature to equilibrate before commencing calibration.</li>
		<li>Under the Calibration tab, select Liquid Phase Calibration to perform liquid phase calibration. Confirm that the temperature is set to 37 &#176;C , the stirring speed is 100, and the pressure is set to atmospheric pressure (101.32 kPa).</li>
		<li>Press OK and wait for the signal to plateau.</li>
		<li>When a plateau is reached, press OK.</li>
		<li>Establish zero O2 in the chamber by adding a small amount (~20 mg) of sodium dithionite.</li>
		<li>Press OK and wait for the signal to plateau.</li>
		<li>When a plateau is reached, press Save to accept the calibration.</li>
	</ol>
	</li>
	<li>TPP+-selective electrode assembly
	<ol>
		<li>Fill the TPP+- selective electrode tip with 10 mM TPP solution using the provided syringe and flexible needle, taking care to avoid air bubbles.</li>
		<li>Assemble the TPP+-selective electrode apparatus, including the reference electrode and electrode holder, (see Table of Materials) according to the manufacturer&#39;s instructions.</li>
		<li>Briefly, loosen the electrode holder cap and insert the internal reference electrode into the TPP+&#160;tip. Tighten the cap to secure the tip. Connect the provided cable to the electrode holder and the auxiliary port of the control box.</li>
		<li>Insert the TPP+-selective electrode and reference electrodes into the adapted plunger assembly for ion-selective electrodes. Connect the reference electrode to the reference port of the control box.</li>
		<li>Insert the TPP+-selective and reference electrodes into the adapted plunger assembly for ion-selective electrodes.</li>
	</ol>
	</li>
	<li>Reaction chamber preparation
	<ol>
		<li>Add the reaction mixture to the reaction chamber: 10 mM succinate (complex II substrate), 5 &#956;M rotenone (complex I inhibitor), 80 ng mL&#8722;1 nigericin (to collapse pH gradient across IMM), 2.5 &#956;g mL-1 oligomycin (to induce state 4 respiration), and respiration buffer (RB)/BSA reaction buffer to a final volume of 1 mL. Take care to avoid introducing air bubbles into the chamber.</li>
		<li>Close the chamber with the adapted plunger assembly with the TPP+-selective and the reference electrode in place. Select GO to start recording. Once the chamber is closed, introduce additional reagents directly into the reaction solution using separate microsyringes modified with plastic tubing to adjust the needle length.</li>
	</ol>
	</li>
	<li>TPP+ calibration
	<ol>
		<li>Once a stable voltage signal is obtained, calibrate the TPP+-selective electrode by adding 1 &#956;M increments of a 0.1 mM TPP solution to a final concentration of 3 &#956;M. Observe that there is a logarithmic decline in the TPP+ voltage signal with each addition. 
		NOTE: Calibrate the TPP+-selective electrode at the start of each experiment.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Allow the O2 and TPP+ traces to stabilize, and add 100 &#956;g of freshly prepared cardiomyocyte mitochondria to the reaction chamber to a final concentration of 0.1 mg mL-1 via the reagent addition port in the plunger assembly. Observe the decrease in the O2 levels in the chamber as mitochondria become energized and consume O2 and the abrupt increase in the TPP+ voltage signal as the mitochondria generate a membrane potential and take up TPP+ from the solution.</li>
		<li>Add 2.5 &#956;g mL-1 oligomycin to induce state 4 respiration. 
		NOTE: O2 consumption rates will now represent the rate of proton leak respiration. Oligomycin can be added to the reaction chamber prior to TPP+ as an alternative.</li>
	</ol>
	</li>
	<li>Assessing the open probability of the mPTP
	<ol>
		<li>Observe that &#916;&#936; declines over time during leak respiration. Once the desired &#916;&#936; is reached, add 1 &#956;M CsA (mPTP inhibitor) to the reaction chamber to assess open probability of the mPTP at that specific &#916;&#936;.</li>
		<li>Measure the effect of CsA on O2 consumption and &#916;&#936; before and after CsA addition</li>
		<li>Determine the voltage dependence of mPTP opening by varying the &#916;&#936; at which CsA is added in successive experiments.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

This paper describes a method to assess the open probability of the mPTP. Specifically, the voltage threshold for low-conductance mPTP opening was determined by assessing the effect of CsA inhibition on proton leak over a range of &#916;&#936;s. Using this technique, we could identify differences in voltage gating of the mPTP between FXS mice and FVB controls consistent with their differences in tissue-specific CoQ content. Critical to the success of this methodology is that mitochondria are freshly isolated prior to use...

The mPTP mediates the permeability transition (PT), whereby the IMM becomes abruptly permeable to small molecules and solutes1,2. This striking phenomenon is a distinct departure from the characteristic impermeability of the IMM, which is fundamental for establishing the electrochemical gradient necessary for oxidative phosphorylation3. PT, unlike other mitochondrial transport mechanisms, is a high-conductance, nonspecific, and nonselective process, allowing the passage of a range of molecules up to 1.5 kDa4,5. The mPTP is a voltage-gated channel within the IMM whose opening alters &#916;&#936;, ATP production, calcium homeostasis, reactive oxygen species (ROS) production, and cell viability4.
At the pathologic extreme, uncontrolled and prolonged high-conductance opening of mPTP leads to the collapse of the electrochemical gradient, matrix swelling, depletion of matrix pyridine nucleotides, outer membrane rupture, release of intermembrane proteins (including cytochrome c), and ultimately, cell death4,6. Such pathological mPTP opening has been implicated in cardiac ischemia-reperfusion injury, heart failure, traumatic brain injury, various neurodegenerative diseases, and diabetes1,7. However, low-conductance mPTP opening is physiological in nature and, in contrast to high-conductance opening, does not lead to profound depolarization or mitochondrial swelling4.
Low-conductance opening of the pore restricts permeability to ~300 Da, allows the passage of protons independent of ATP synthesis, and is a potential source of physiological proton leak5. Physiologic mPTP opening causes a controlled decline in &#916;&#936;, increases electron flux through the respiratory transport chain, and results in a short burst or flash of superoxide, contributing to ROS signaling8. Regulation of such transient mPTP opening is important for calcium homeostasis and normal cellular development and maturation4,9,10,11. Transient pore opening in developing neurons, for example, triggers differentiation, while the closure of the mPTP induces maturation in immature cardiomyocytes4,5.
Although the functional significance of the mPTP in health and disease is well established, its precise molecular identity remains debated. Progress on the molecular structure and function of the mPTP has been comprehensively reviewed elsewhere12. Briefly, currently, high- and low- conductance states of the mPTP have been hypothesized to be mediated by distinct entities12. The leading candidates are the F1/F0 ATP synthase (ATP synthase) and adenine nucleotide transporter (ANT) for high- and low-conductance modes, respectively12.
Despite the lack of consensus regarding the exact identity of the pore-forming component of the mPTP, certain key characteristics have been detailed. A well-established feature of the mPTP is that it is regulated by the electrochemical gradient such that depolarization of the IMM leads to pore opening13. Prior work has shown that the redox state of vicinal thiol groups alters the voltage gating of the mPTP, such that oxidation opens the pore at relatively higher &#916;&#936;s, and thiol group reduction results in closed mPTP probability14. However, the identity of the proteinaceous voltage sensor is unknown.
Various small molecules that modulate the open probability of the pore have been identified. For example, the mPTP can be stimulated to open with calcium, inorganic phosphate, fatty acids, and ROS and can be inhibited by adenine nucleotides (particularly ADP), magnesium, protons, and CsA5,12. The mechanisms of action of some of these regulators have been elucidated. Mitochondrial calcium triggers mPTP opening at least in part by binding to the &#946;-subunit of the ATP synthase15. ROS can activate the mPTP by decreasing its affinity for ADP and enhancing its affinity for cyclophilin D (CypD), the best-studied proteinaceous mPTP activator16. The mechanism of activation of the mPTP by inorganic phosphate and fatty acids is less clear. As for endogenous inhibitors, ADP is thought to inhibit the mPTP by binding at the ANT or ATP synthase, while magnesium exerts its inhibitory effect by displacing calcium from its binding site15,17,18,19.
Low pH inhibits mPTP opening by protonating histidine 112 of the regulatory oligomycin sensitivity-conferring protein (OSCP) subunit of the ATP synthase12,20,21. The prototypical pharmacologic inhibitor of the mPTP, CsA, acts by binding CypD and preventing its association with OSCP22,23. Previous work has also shown that a variety of CoQ analogs interact with the mPTP, inhibiting it or activating it24. In recent work, we found evidence of a pathologically open mPTP, excessive proton leak, and inefficient oxidative phosphorylation due to a CoQ deficiency in forebrain mitochondria of newborn FXS mouse pups25.
Closure of the pore with exogenous CoQ blocked the pathologic proton leak and induced morphologic maturity of dendritic spines25. Interestingly, in the same animals, FXS cardiomyocytes had excessive CoQ levels and closed mPTP probability compared to wildtype controls26. Although the cause of these tissue-specific differences in CoQ levels is unknown, the findings underscore the concept that endogenous CoQ is likely a key regulator of the mPTP. However, there is a major gap in our knowledge because the mechanism of CoQ-mediated inhibition of the mPTP remains unknown.
Regulation of the mPTP is a critical determinant of cell signaling and survival4. Thus, detecting mPTP opening within mitochondria is key when considering specific pathophysiological mechanisms. Typically, the threshold for high-conductance pore opening is determined using calcium to trigger the permeability transition. Such calcium loading leads to the collapse of the membrane potential, rapid uncoupling of oxidative phosphorylation, and mitochondrial swelling27,28. We sought to develop a method to detect low-conductance mPTP opening in situ, without inducing it per se.
The approach exploits the role of the mPTP as a proton leak channel. To do so, Clark-Type and TPP+ ion-selective electrodes were employed to simultaneously measure oxygen consumption and membrane potential, respectively, in isolated mitochondria during leak respiration29. 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 in the context of CoQ excess were precisely defined.

<table><tbody><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Adapted plunger assembly for pH or ion-selective electrodes for use with OXYT1</td><td>PP systems</td><td>941039</td><td></td></tr><tr><td>BD Intramedic PE Tubing, PE 50, 0.023 in. 10 ft.</td><td>Fisher Scientific</td><td>14-170-11B</td><td>to modify the length of the hamilton synringe as needed</td></tr><tr><td>Bovine Serum Albumin (BSA). Fatty acid free</td><td>Sigma</td><td>A7030-10G</td><td></td></tr><tr><td>Dri-Ref Reference Electrode, 2 mm</td><td>World Precision Inst. LLC</td><td>DRIREF-2</td><td></td></tr><tr><td>Electrode Holder for KWIK-Tips</td><td>World Precision Inst. LLC</td><td>KWIK-2</td><td>&amp;nbsp;ion selective electrode holder</td></tr><tr><td>Ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid&amp;nbsp; (EGTA)</td><td>Sigma</td><td>324626</td><td></td></tr><tr><td>FVB.129P2-Pde6b&lt;sup&gt;+&lt;/sup&gt; Tyr&lt;sup&gt;c-ch&lt;/sup&gt; Fmr1&lt;sup&gt;tm1Cgr&lt;/sup&gt;/J</td><td>Jackson Laboratory, Bar Harbor, ME</td><td></td><td>FXS mice, &lt;em&gt;Fmr1 &lt;/em&gt;KO&amp;nbsp;</td></tr><tr><td>FVB.129P2-Pde6b&lt;sup&gt;+&lt;/sup&gt; Tyr&lt;sup&gt;c-ch&lt;/sup&gt;/AntJ</td><td>Jackson Laboratory, Bar Harbor, ME</td><td></td><td>FVB mice</td></tr><tr><td>Hamilton 80366 Standard Syringes, 10 uL, Cemented-Needle, 6/pk</td><td>Cole-Parmer</td><td>EW-07938-30</td><td>microsyringe</td></tr><tr><td>Hamilton 80500 Standard Microliter Syringes, 50 uL, Cemented-Needle</td><td>Cole-Parmer</td><td>EW-07938-02</td><td>microsyringe</td></tr><tr><td>Hansatech Instruments Oxytherm+ System (Respiration) Complete</td><td>PP systems</td><td>OXYTHERM+R</td><td>oxygen electrode and software</td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>1374248</td><td></td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M9546-250G</td><td></td></tr><tr><td>P1,P5-diadenosine-5&amp;prime; pentaphosphate pentasodium (AP5A)</td><td>Sigma</td><td>D4022-10MG</td><td></td></tr><tr><td>Percoll</td><td>Sigma</td><td>P1644</td><td>medium for density gradient separation</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>5.43841</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>TPP&lt;sup&gt;+&lt;/sup&gt; Electrode Tips (3)</td><td>World Precision Inst. LLC</td><td>TIPTPP</td><td></td></tr></tbody></table>

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.

Typical O2 consumption and &#916;&#936; curves generated in these experiments are shown (Figure 1A,B). The logarithmic decline in the voltage signal with TPP+ calibration is shown at the start of each experiment. The absence of this logarithmic pattern may suggest a problem with the TPP+ selective electrode. Mitochondria typically generate &#916;&#936; immediately upon addition to respiratory buffer. &#916;&#936; can be interpreted from chang...

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

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 ...

assessment-open-probability-mitochondrial-permeability-transition

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.2K Views.  Columbia University Medical Center. 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. 

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

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Biochemistry

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

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 ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

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 ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...