Changes In Mitochondrial Volume Research Articles

A Scoping Review Investigating the "Gene-Dosage Theory" of Mitochondrial DNA in the Healthy Skeletal Muscle.

This review provides an overview of the evidence regarding mtDNA and valid biomarkers for assessing mitochondrial adaptions. Mitochondria are small organelles that exist in almost all cells throughout the human body. As the only organelle, mitochondria contain their own DNA, mitochondrial DNA (mtDNA). mtDNA-encoded polypeptides are subunits of the enzyme complexes in the electron transport chain (ETC) that are responsible for production of ATP to the cells. mtDNA is frequently used as a biomarker for mitochondrial content, since changes in mitochondrial volume are thought to induce similar changes in mtDNA. However, some exercise studies have challenged this "gene-dosage theory", and have indicated that changes in mitochondrial content can adapt without changes in mtDNA. Thus, the aim of this scoping review was to summarize the studies that used mtDNA as a biomarker for mitochondrial adaptions and address the question as to whether changes in mitochondrial content, induce changes in mtDNA in response to aerobic exercise in the healthy skeletal muscle. The literature was searched in PubMed and Embase. Eligibility criteria included: interventional study design, aerobic exercise, mtDNA measurements reported pre- and postintervention for the healthy skeletal muscle and English language. Overall, 1585 studies were identified. Nine studies were included for analysis. Eight out of the nine studies showed proof of increased oxidative capacity, six found improvements in mitochondrial volume, content and/or improved mitochondrial enzyme activity and seven studies did not find evidence of change in mtDNA copy number. In conclusion, the findings imply that mitochondrial adaptions, as a response to aerobic exercise, can occur without a change in mtDNA copy number.

Short communication: Acute hypoxia does not alter mitochondrial abundance in naked mole-rats

Hypoxia poses a significant energetic challenge and most species exhibit metabolic remodelling when exposed to prolonged hypoxia. One component of this remodelling is mitochondrial biogenesis/mitophagy, which alter mitochondrial abundance and helps to adjust metabolic throughput to match changes in energy demands in hypoxia. However, how acute hypoxia impacts mitochondrial abundance in hypoxia-tolerant species is poorly understood. To help address this gap, we exposed hypoxia-tolerant naked mole-rats to 3 h of normoxia or acute hypoxia (5% O2) and measured changes in mitochondrial abundance using two well-established markers: citrate synthase (CS) enzyme activity and mitochondrial DNA (mtDNA) abundance. We found that neither marker changed with hypoxia in brain, liver, or kidney, suggesting that mitochondrial biogenesis is not initiated during acute hypoxia in these tissues. Conversely in skeletal muscle, the ratio of CS activity to total protein decreased 50% with hypoxia. However, this change was likely driven by an increase in soluble protein density in hypoxia because CS activity was unchanged relative to wet tissue weight and the mtDNA copy number was unchanged. To confirm this, we examined skeletal muscle mitochondria using transmission electron microscopy and found no change in mitochondrial volume density. Taken together with previous studies of mitochondrial respiratory function, our present findings suggest that naked mole-rats primarily rely on tissue-specific functional remodelling of metabolic pathways and mitochondrial respiratory throughput, and not physical changes in mitochondrial number or volume, to adjust to short-term hypoxic exposure.

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨm). Chemiosmosis requires ion exchangers to remove Na+, K+, Ca2+, PO43−, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K+, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H2O by osmosis. The effects of K+ uptake through ligand-specific mK+ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K+ influx is likely its effects on H+ cycling and bioenergetics facilitated by mitochondrial (m) K+/H+ exchange (mKHE), though the kinetics and consequences of K+ efflux by KHE are not well described. We hypothesized that a major role of K+ influx/efflux is stimulation of respiration via the influx of H+ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK+ cycle to capture changes in mitochondrial volume, pHm, ΔΨm, and respiration that would reflect a role for H+ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K+ buffer at pH 6.9, or in a 140 mM Cs+ buffer at pH 7.6 or 6.9 with added 10 mM K+, minimal Ca2+ and free of Na+. O2 content was measured by a Clark electrode, and pHm, ΔΨm, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpHm = pHin–pHext) at pHext 6.9> > 7.6, so that ΔΨm was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ ionophore valinomycin depolarized ΔΨm. We postulate that K+ efflux-induced H+ influx via KHE causes an inward H+ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpHm, the smaller component of the overall proton motive force, ΔμH+. Thus ΔpHm establishes and maintains the ΔΨm required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K+ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.

Biophysical properties of a novel mitochondrial large ubiquitous non-selective amiloride sensitive-LUNA-current

Inner mitochondrial membrane (IMM) ion channels and transporters account for communication of the matrix with the intermembrane space (IMS) and the cytosol. Transport of solutes and ions is keep under strict regulation mainly because small changes in solute concentrations could generate changes in mitochondrial volume or membrane potential (ΔΨm), interrupting ATP synthesis and leading to mitochondrial damage. The list of recently discovered mitochondrial ion channels has been growing in the past decades.

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

Glucocorticoids regulate mitochondrial fatty acid oxidation in fetal cardiomyocytes

The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesized that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon a glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24h later. Instead, at E17.5, fatty acid oxidation genes were downregulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also downregulated in fetal hearts at E17.5, 24h after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were downregulated in heart. These data suggest that endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by downregulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. KEY POINTS: Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acid oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice downregulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) downregulates the glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by downregulating the ability to respond to glucocorticoids.

Abstract P334: Biophysical Properties Of A Novel Mitochondrial Large Ubiquitous Non-selective Amiloride Sensitive (luna) Current

Inner mitochondrial membrane (IMM) ion channels and transporters account for communication of the matrix with the intermembrane space (IMS) and the cytosol. Transport of solutes and ions is keep under strict regulation mainly because small changes in solute concentrations could generate changes in mitochondrial volume or membrane potential (ΔΨ m ), interrupting ATP synthesis and leading to mitochondrial damage. The list of recently discovered mitochondrial ion channels has been growing in the past decades. In this work, using the patch-clamp technique we observed the activity of a novel mitochondrial current, named here LUNA current, in mitoplasts (IMM striped of outer membrane) from mouse liver, spleen, brain and heart, as well as established cell lines. LUNA is a novel non-selective cation current (K + >Na + >NMDG + >H + ) active at depolarized membrane potentials. The basal activity of whole-mitoplast LUNA currents from wild type mice hearts changed from 445±106 pA/pF to 1232±287 pA/pF upon chelation of external divalent cations (Ca 2+ and Mg 2+ ). Moreover, the activity of LUNA is independent of the mitochondrial Ca 2+ uniporter and of the non-selective reactive oxygen species modulator channel (ROMO1). In the heart, the activity of LUNA was enhanced in both the Tfam -KO mice, which have impaired electron transport chain (ETC) activity and are a model for mitochondrial cardiomyopathies, and mice with cardiac pressure overload due to transverse aortic constriction (TAC) compared to sham-operated hearts (729±197; n=7 vs 283±137 pA/pF). LUNA current is reversibly inhibited by amiloride with no sensitivity to the vast majority of common K + , Na + and Ca 2+ channels and ETC inhibitors. The molecular identity of mitochondrial LUNA current remains to be determined.

Mitochondrial division inhibitor 1 (mdivi-1) increases oxidative capacity and contractile stress generated by engineered skeletal muscle.

In skeletal muscle fibers, mitochondria are densely packed adjacent to myofibrils because adenosine triphosphate (ATP) is needed to fuel sarcomere shortening. However, despite this close physical and biochemical relationship, the effects of mitochondrial dynamics on skeletal muscle contractility are poorly understood. In this study, we analyzed the effects of Mitochondrial Division Inhibitor 1 (mdivi-1), an inhibitor of mitochondrial fission, on the structure and function of both mitochondria and myofibrils in skeletal muscle tissues engineered on micromolded gelatin hydrogels. Treatment with mdivi-1 did not alter myotube morphology, but did increase the mitochondrial turbidity and oxidative capacity, consistent with reduced mitochondrial fission. Mdivi-1 also significantly increased basal, twitch, and tetanus stresses, as measured using the Muscular Thin Film (MTF) assay. Finally, mdivi-1 increased sarcomere length, potentially due to mdivi-1-induced changes in mitochondrial volume and compression of myofibrils. Together, these results suggest that mdivi-1 increases contractile stress generation, which may be caused by an increase in maximal respiration and/or sarcomere length due to increased volume of individual mitochondria. These data reinforce that mitochondria have both biochemical and biomechanical roles in skeletal muscle and that mitochondrial dynamics can be manipulated to alter muscle contractility.

Real time mitochondrial dimension measurements.

Mitochondrial volume is correlated with cell function and internal cell processes. Changes in mitochondrial volume were associated with advanced states of cardiac disease. Thus, measurements of mitochondrial dimension deformations are important to the understanding of cell function and its deterioration. Existing methods either allow measurements of the volume of isolated mitochondria, which are an inferior model to that of isolated cells, or they allow short time measurements that are toxic to the cells. Recent studies have discovered that mitochondrial deformation along a given cell axis can be measured by using the Fourier transformation on the variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows. However, this method was used only offline and in a line scan mode, making it impossible to measure both axes. We designed an open source program in LabVIEW to take advantage of the transmitted light diffraction technique and quantify mitochondrial two dimension (2D) deformation in cardiomyocytes, in situ in real time for long periods (more than several seconds). We validated the program on synthetic and on experimental images from rabbit and rat ventricular myocytes. The program can analyze offline and real time simultaneous 2D mitochondrial deformation dynamics as well as also sarcomere length dynamics. Moreover, the program can accurately analyze images acquired from different cameras. Quantification of mitochondrial 2D deformations is a powerful tool for exploring cell biophysics and bioenergetics mechanisms and will lay the foundation for a future clinical tool for quantifying mitochondrial volume changes associated with different cardiac diseases.

Parvalbumin alters mitochondrial dynamics and affects cell morphology

The Ca2+-binding protein parvalbumin (PV) and mitochondria play important roles in Ca2+ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.

Effects of Temperature and Hypoxia‐Reoxygenation on Mitochondrial Oxidative Capacity and Volume, Oncorhynchus mykiss

We investigated the effects of temperature and hypoxia‐reoxygenation (H‐RO) on mitochondrial bioenergetics and volume regulation. Oxygen consumption rates in isolated rainbow trout liver mitochondria were measured at 5, 13 and 25 oC before and after exposure to varying durations (5, 15 and 30 min) of hypoxia and reoxygenation. Subsequently, exposure to 15 min of H‐RO at 13 oC was used to probe the involvement of mitochondrial potassium channels (mitoKATP) in H‐RO‐induced mitochondrial volume changes. Our results showed that the stimulatory effect of temperature on state 3 respiration was reduced by H‐RO. In contrast, H‐RO acted cooperatively with temperature to stimulate state 4 respiration. Interestingly, while H‐RO reduced the thermal sensitivity of state 3 at the low (5‐13 oC) temperature range, it increased that of state 4 at both the low and high (13‐25 oC) ranges. These disparate effects of H‐RO on both the rates of state 3 and 4 respiration and their thermal sensitivities markedly reduced the mitochondrial coupling efficiency. We also found that diazoxide, a mitoKATP opener, protected mitochondria against H‐RO‐induced mitochondrial swelling. Overall, our findings indicate that the effects of H‐RO on mitochondrial oxidative capacity are exacerbated by temperature and that mitoKATP mediate H‐RO‐induced mitochondrial volume changes.

Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss)

We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0–100μM) at three (5, 13 and 25°C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25°C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q10 values for state 3 respiration increased at low temperature (5–13°C) while those for state 4 increased at high temperature (13–25°C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and intensifying the effects. Lastly, using specific modulators of mitochondrial ion channels, we demonstrated that the mitochondrial volume changes were associated with Cd uptake via the mitochondrial calcium uniporter (MCU) without significant contribution of the permeability transition pore and/or potassium channels. Overall, it appears that high temperature exacerbates Cd-induced mitochondrial dysfunction and volume changes in part by increasing metal uptake through the MCU.

Excess Ca2+ and succinate‐induced mitochondrial H+ permeability (572.2)

Mitochondrial permeability transition pore (mPTP) opening is a key event in ischemia and reperfusion injury. Several proteins were suggested to constitute mPTP, but the molecular nature of mPTP remains elusive. An increased probability of mPTP opening when using mitochondrial complex I substrates led to a suggestion that complex I might constitute part of the pore. In this study, we entertained the idea that complex I, under certain conditions that lead to excess reactive oxygen species generation, may increase permeability across mitochondrial membranes that is not the typical mPTP. Guinea pig heart mitochondria were suspended in experimental buffer +/‐ excess Ca2+ and supplemented with either pyruvate or succinate. Excess Ca2+ combined with succinate induced mitochondrial permeability that was mainly due to H+ leak as evidenced by matrix acidification without a change in mitochondrial volume. This was associated with impaired Ca2+ uptake. Adding rotenone restored/prevented matrix acidification (H+ leak) and improved Ca2+ uptake in a time dependent manner. We propose that under conditions similar to those that occur during and after extended ischemia i.e., excess Ca2+ with high succinate, mitochondrial complex I or another site may become permeable to H+. The mechanism for this H+ permeability requires further investigation.Grant Funding Source: Supported by grants from the National Institutes of Health (R01HL095122,A.K.S.Camara/R.K.Dash)

The role of the ATP-sensitive potassium channel in the activation of the K+ cycle in rat liver mitochondria

It is known that the mitochondrial ATP-sensitive potassium channel (mitoKATP) plays a key role in protecting myocardium during ischemia. We have suggested that the mechanism of this protection is associated with the potassium cycle in mitochondria. In this paper, for the first time, a direct proof was obtained of the existence of a cycle of potassium ions in rat liver mitochondria that are associated with the functioning of mitoKATP. Activation of the cycle was recorded by optical density changes of mitochondrial suspension in the form of two or three swelling-contraction waves of the organelles. Using activators and inhibitors of mitoKATP we showed that a significant role in the potassium cycle belongs to the channel. It was found that in vitro sildenafil has a direct effect on mitoKATP, being its activator. The results obtained indicate that the cardioprotective effect of sildenafil observed previously is associated with the activation of mitoKATP. In order to study the structure and volume changes of mitochondria in various stages of the cycle in the presence of potassium channel modulators, the electron microscopy studies of mitochondria preparations were carried out. A correlation between the optical density decrease of mitochondrial suspension and the swelling of mitochondria was revealed. The data obtained in this study suggest participation of mitoKATP in the protection of tissues from hypoxic damage.

Age-dependent changes in mitochondrial morphology and volume are not predictors of lifespan

Mitochondrial dysfunction is a hallmark of skeletal muscle degeneration during aging. One mechanism through which mitochondrial dysfunction can be caused is through changes in mitochondrial morphology. To determine the role of mitochondrial morphology changes in age-dependent mitochondrial dysfunction, we studied mitochondrial morphology in body wall muscles of the nematodeC. elegans. We found that in this tissue, animals display a tubular mitochondrial network, which fragments with increasing age. This fragmentation is accompanied by a decrease in mitochondrial volume. Mitochondrial fragmentation and volume loss occur faster under conditions that shorten lifespan and occur slower under conditions that increase lifespan. However, neither mitochondrial morphology nor mitochondrial volume of five- and seven-day old wild-type animals can be used to predict individual lifespan. Our results indicate that while mitochondria in body wall muscles undergo age-dependent fragmentation and a loss in volume, these changes are not the cause of aging but rather a consequence of the aging process.

Effects of all-trans-retinoic acid on the permeability transition and bioenergetic functions of rat liver mitochondria in combination with endoxifen

AimsThe clinical utilization of the combinations of all-trans-retinoic acid (RA) with antiestrogens, which present synergism of action in breast cancer, has been limited by RA adverse effects, including hepatotoxicity, which may be related with mitochondrial damage. This work evaluated the effects of RA alone and in combination with the antiestrogen endoxifen (EDX) on liver mitochondria. Main methodsMitochondrial permeability transition (MPT) was assessed by using Calcium Green-5N fluorescence and a tetraphenylphosphonium selective electrode. Oxidative stress was evaluated by oxygen consumption and thiobarbituric acid method. Mitochondrial bioenergetic was monitored by measuring oxygen consumption and mitochondrial membrane potential (ΔΨ). Osmotic volume changes of mitochondria were followed at 540nm. Key findingsEDX prevents the MPT induced by RA, allowing mitochondria pre-incubated with RA to accumulate Ca2+ and inhibiting the depolarization of ΔΨ. RA above 10nmol/mg protein depresses the phosphorylation capacity of mitochondria, as shown by the increase in the time required for ADP phosphorylation as well as by the decrease in state 3 respiration. At 20nmol/mg protein, RA decreases the ΔΨ and increases the state 4 respiration, suggesting that high concentrations of RA permeabilize the membrane to protons, possibly due to a proton leak through the Fo fraction of complex V. Moreover, the effects of RA on mitochondrial bioenergetics are not changed by EDX. SignificanceRA-induced hepatotoxicity may be related with induction of MPT and alterations in bioenergetic parameters; the combination with EDX, which reduces mitochondrial dysfunction and synergistically potentiates the anticancer activity, may provide a safer therapeutic strategy.

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.

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 established(1), accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca(2+) homeostasis(2), bioenergetics and redox signaling( 3). mtPTP opening is triggered by matrix Ca(2+) 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 acids(4). In vitro, mtPTP opening can be achieved by increasing Ca(2+) concentration inside the mitochondrial matrix through exogenous additions of Ca(2+) (calcium retention capacity). When Ca(2+) levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca(2+) 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 Ca(2+) handling (uptake and release, as measured by Ca(2+) concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca(2+) 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 Ca(2+), 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.

35 Protective action of synthetic diarylamines on mitochondrial permeability transition pore

Mitochondria can undergo a generalised increase of the permeability of the inner membrane, called mitochondrial permeability transition (MPT), due to the formation of non-specific pores (MPTP), which is an early...

The Activity of the Plant Mitochondrial Inner Membrane Anion Channel (PIMAC) of Maize Populations Divergently Selected for Cold Tolerance Level is Differentially Dependent on the Growth Temperature of Seedlings

The activity of the plant inner membrane mitochondrial anion channel (PIMAC) is involved in metabolite shuttles and mitochondrial volume changes and could have a role in plant temperature tolerance. Our objectives were to investigate (i) the occurrence and (ii) the temperature dependence of anion fluxes through PIMAC in mitochondria isolated from seedlings of three maize populations differing in terms of cold tolerance; and (iii) the relationships between the PIMAC activity kinetics and the level of cold tolerance. Populations were the source population (C0) and two populations divergently selected for high (C4H) and low (C4L) cold tolerance. Such divergently selected populations are expected to share most of their genes, with the main exception of those genes controlling cold tolerance. Arrhenius plots of PIMAC chloride fluxes showed a linear temperature dependence when seedlings were grown at 25 or 14°C, whereas a non-linear temperature dependence was found when seedlings were grown at 5°C, with or without acclimation at 14°C. The activation energy and other thermodynamic parameters of PIMAC activity varied depending on temperature treatments during seedling growth. When seedlings were grown at 14 and 5°C with acclimation, PIMAC activity of the C4H population increased, while that of C4L declined, as compared with the activities of seedlings grown at 25°C. These symmetric responses indicate that PIMAC activity changes are associated with genetically determined differences in the cold tolerance level of the investigated populations. We conclude that anion fluxes by PIMAC depend upon changes on growth temperature and are differentially related to the tolerance level of the tested populations.