Name: assessment of open probability of the mitochondrial permeability transition pore in the setting of coenzyme q excess
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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...

<ol>
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A., Hunter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ca2+-induced+membrane+transition+in+mitochondria.+II.+Nature+of+the+Ca2++trigger+site.">The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.</a> Archives of Biochemistry and Biophysics. 195 (2), 460-467 (1979).</li><li>Halestrap, A. P., Connern, C. P., Griffiths, E. J., Kerr, P. 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J., Murphy, E., Liu, J. C., Palmeira, C. M., Moreno, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondrial Bioenergetics: Methods and ProtocolsMethods in Molecular Biology. , 187-196 (2018).</li><li>Carraro, M., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+membrane+permeability+and+the+mitochondrial+permeability+transition.">Measurement of membrane permeability and the mitochondrial permeability transition.</a> Methods in Cell Biology. 155, 369-379 (2020).</li><li>Affourtit, C., Wong, H., Brand, M. D., Palmeira, C. M., Moreno, A. 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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.

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	<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>&#160;ion selective electrode holder</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid&#160; (EGTA)</td>
			<td>Sigma</td>
			<td>324626</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J</td>
			<td>Jackson Laboratory, Bar Harbor, ME</td>
			<td></td>
			<td>FXS mice, Fmr1 KO&#160;</td>
		</tr>
		<tr>
			<td>FVB.129P2-Pde6b+ Tyrc-ch/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 (MgCl2)</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&#8242; 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 (KH2PO4)</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+ 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...

Watch this Scientific Journal Video about Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess at JoVE.com

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 ...

This protocol provides a new way to investigate the low conductance opening of the mitochondrial permeability transition pore. The main advantage of this technique is that it provides an easy way to measure the open probability of the pore as a function of mitochondrial membrane potential. This technique can be readily applied to mitochondria isolated from any organ system and species.Begin with the calibration of the oxygen electrode by placing a drop of 50%potassium chloride electrolyte solution on top of the dome of the electrode disc. Place a small piece of two square centimeter cigarette paper spacer covered with a slightly larger piece of polytetrafluoroethylene membrane over the electrolyte drop. Then use the applicator tool to push the small electrode disc O-ring over the dome of the electrode.Next, top up the reservoir well with the electrolyte solution. Place the larger O-ring in the recess around the electrode disc well. Then install the disc into the electrode chamber, and connect it to the control unit.Add two milliliters of air-saturated deionized water, and the polytetrafluoroethylene-coated magnet to the reaction chamber. When done, connect the chamber to the rear of the control unit. Set the temperature to 37 degrees Celsius, and the stirring speed to 100.Allow the system temperature to equilibrate for 10 minutes before calibration. After ensuring the correct temperature, stirring speed, and pressure, under the Calibration tab, select the Liquid Phase Calibration option to perform liquid phase calibration. Then press OK, and wait for the signal to plateau.Once the plateau is reached, press OK.Then add about 20 milligrams of sodium dithionite to establish zero oxygen in the chamber. Again, press OK, and wait for the signal to plateau before clicking the Save button to accept the calibration. To prepare the tetraphenylphosphonium, or TPP-selective electrode assembly, fill the TPP-selective electrode tip with a 10 millimolar TPP solution using a syringe and a flexible needle.Avoid air bubbles while filling the electrode tip. Loosen the electrode holder cap to insert the internal reference electrode into the TPP tip. When done, assemble the TPP-selective electrode apparatus, including the reference electrode and electrode holder.Tighten the cap to secure the tip. Then connect the cable to the auxiliary port of the control box, and the TPP electrode holder. Insert the TPP-selective and reference electrodes into the adapted plunger assembly for ion-selective electrodes.Connect the reference electrode to the reference port of the control box. Next, to prepare the reaction chamber, add one milliliter of the reaction mixture to the reaction chamber without creating air bubbles. Close the chamber using the adapted plunger assembly with the TPP-selective and reference electrodes in place.Once the chamber is closed, introduce additional reagents directly into the reaction chamber using separate micro-syringes modified with plastic tubing. When the setup is ready, select Go to start recording. Once a stable voltage signal is obtained, calibrate the TPP-selective electrode by adding one micromolar increments of a 0.1 millimolar TPP solution to achieve a final concentration of three micromolar.Observe the logarithmic decline in the TPP voltage signal with each addition. After stabilizing oxygen and TPP traces, add 100 micrograms of freshly prepared cardiomyocyte mitochondria to the reaction chamber via the reagent addition port in the plunger assembly to a final concentration of 0.1 milligrams per milliliter. Observe the decrease in the oxygen levels in the chamber as the mitochondria become energized and consume oxygen.Also, see the abrupt increase in the TPP voltage signal as the mitochondria generates a membrane potential and take up TPP from the solution. Then add 2.5 micrograms per milliliter oligomycin to induce State 4 respiration. To assess the open probability of the mitochondrial permeability transition pore, or mPTP, observe the decline in the membrane potential over the time during leak respiration.Once the desired membrane potential is reached, add one micromolar cyclosporine A as an mPTP inhibitor to the reaction chamber to assess the open probability of the mPTP at that specific membrane potential. Then measure the effect of cyclosporine A on oxygen consumption and membrane potential before and after cyclosporine A addition. In the representative curves of simultaneous oxygen consumption and membrane potential, high membrane potential was set at zero, intermediate at five, and low at 10 millivolts relative to the two micromolar TPP calibration level.The mitochondria at zero millivolts exhibited 100%mPTP closed probability, and at 10 millivolts exhibited 100%open probability. After oligomycin addition, a significant difference in oxygen consumption was not observed, suggesting that adenosine triphosphate, or ATP synthase, contributes minimally to State 2 respiration. Further, the oxygen consumption rate and membrane potential before and after cyclosporine A addition were compared.A decrease in the oxygen consumption rate and an increase in stabilization of membrane potential indicated the closure of an open mPTP. When the mPTP is closed, there was no decrease in oxygen consumption rate, and the membrane potential continued to fall. The results demonstrated similar closed and open mPTP probabilities at high and low membrane potentials in the FVB control and Fmr1 knockout cardiac mitochondria.At the intermediate membrane potential, the Fmr1 knockout cardiac mitochondria demonstrated increased closed mPTP probability compared to the FVB controls. No air bubbles must be introduced into the chamber, as this will lead to unstable oxygen consumption readings, and make interpretation difficult. Following this procedure, calcium loading capacity can be measured.This method assesses the high conductance opening of the pore, and complements the results of our technique.

assessment-open-probability-mitochondrial-permeability-transition

Research

JoVE Journal

Biology

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

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 ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.