Induction Of Mitochondrial Permeability Transition Research Articles

Arsenic-Induced Injury of Mouse Hepatocytes through Lysosome and Mitochondria: An In Vitro Study.

Background and Aims The cellular mechanism of liver injury related to arsenic toxicity is ill defined. It is thought that oxidative stress and mitochondrial dysfunction may play some role in arsenic-induced liver damage. In this study, we evaluated subcellular events within the primary cultured mouse hepatocytes when exposed to inorganic arsenic. Methods Primary cultured mouse hepatocytes were treated with 10 μM arsenic for different time periods. Reactive oxygen species (ROS) formation, functional changes of the lysosome and mitochondria, and mode of hepatocytes death were studied by laser confocal microscopy, fluorescence spectroscopy, and flow cytometry. Expression of proapoptotic member of the BCL-2 family of genes BAX and antiapoptotic BCL-2 mRNA expression were studied by real-time PCR. Cytochrome c expression was studied by Western blotting. Results Fluorescence spectroscopy as well as flow cytometric analysis revealed that arsenic-induced formation of ROS was time dependent. Confocal microscopy showed initiation of ROS formation from periphery of the hepatocytes at 30 min of arsenic exposure that progressed to central part of the hepatocytes at 3 h of arsenic exposure. The ROS formation was found to be NADPH oxidase (NOX) dependent. This low level of intracellular ROS induced lysosomal membrane permeabilization (LMP) and subsequently released cathepsin B to the cytosol. The LMP further increased intracellular ROS which in turn triggered induction of mitochondrial permeability transition (MPT). Pretreatment of hepatocytes with LMP inhibitor bafilomycin A (BafA) significantly decreased, and LMP inducer chloroquine (ChQ) significantly increased the production of ROS suggesting that LMP preceded enhanced ROS generation in response to arsenic. MPT was accompanied with increase in BAX : BCL2 mRNA ratio resulting in upregulation of caspase 3 and increased hepatocyte apoptosis. Conclusion Although arsenic-related oxidative liver injury is well established, neither the site of origin of ROS nor the early sequence of events in arsenic toxicity due to ROS is known. We believe that our study provides evidences elucidating the early sequence of events that culminates in the death of the mouse hepatocytes during arsenic exposure.

A review on the mitochondrial toxicity of “ecstasy” (3,4-methylenedioxymethamphetamine, MDMA)

3,4-Methylenedioxymethamphetamine (MDMA or “ecstasy”) is a drug of abuse used by millions worldwide. MDMA human abuse and dependence is well described, but addictive properties are not always consistent among studies. This amphetamine is a substrate type releaser, binding to monoamine transporters, leading to a pronounced release of serotonin and noradrenaline and to a minor extent dopamine. The toxicity of MDMA is well studied at the pre-clinical level, with neurotoxicity and hepatotoxicity being particularly described.In this review, we describe the most relevant MDMA effects at the mitochondrial level found in in vitro and in vivo models, these later conducted in mice and rats. Most of these reports focus on the mitochondria of brain or liver. In in vitro models, MDMA causes depletion of ATP levels and inhibition of mitochondrial complex I and III, loss in mitochondrial membrane potential (ΔΨm) and induction of mitochondrial permeability transition. The involvement of mitochondria in the apoptotic cell death evoked by MDMA has also been shown, such as the release of cytochrome c. Additionally, MDMA or its metabolites impaired mitochondrial trafficking and increased the fragmentation of axonal mitochondria. In animal studies, MDMA decreased mitochondrial complex I activity and decreased ATP levels. Moreover, MDMA-evoked oxidative stress has been shown to cause deletion on mitochondrial DNA and impairment in mitochondrial protein synthesis.Although the concentrations and doses used in some studies do not always correlate to the human scenario, the mitochondrial abnormalities evoked by MDMA are well described and are in part responsible for its mechanism of toxicity.

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers.

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.

Metformin induces apoptosis via uterus mitochondrial permeability transition pore opening and protects against estradiol benzoate-induced uterine defect and associated pathophysiological disorder in female Wistar rats

BackgroundSome antitumor or anticancer agents have been shown to execute cell death by induction of mitochondrial permeability transition (mPT) pore opening in order to elicit their chemotherapeutic effect. Therefore, this study investigated the effect of metformin on cell death via rat uterus mPT pore and estradiol benzoate-induced uterine defect and associated pathophysiological disorder in female rat. Mitochondria were isolated using differential centrifugation. The mPT pore opening, cytochrome c release and mitochondrial ATPase activity were determined spectrophotometrically. Caspases 9 and 3 activities, MDA and estradiol levels and SOD, GSH activities, were determined using ELISA technique. Histological and histochemical assessments of the uterine section were carried out using standard methods.ResultsMetformin at concentrations 10–90 μg/mL, showed no significant effect on mPT pore opening, mATPase activity and release of cytochrome c. However, oral administration of metformin caused mPT pore opening, enhancement of mATPase activity and activation of caspases 9 and 3 significantly at 300 and 400 mg/kg. Metformin protected against estradiol benzoate (EB)-induced uterine defect and other associated pathophysiological disorder. It also improved the antioxidant defense system. The histological evaluation revealed the protective effect of metformin on the cellular architecture of the uterus while the histochemical examination showed severe hyperplasia in the uterine section of EB-treated rats, remarkably reversed by metformin co-treatment.ConclusionThis study suggests that metformin at high doses induces apoptosis via rat uterus mPT pore opening and protects against EB-induced uterine defect (hyperplasia) and associated pathophysiological disorder.

Tobacco‐Smoke Induced Muscle Atrophy Depends Upon Aryl Hydrocarbon Receptor Activity‐Mediated Activation of Mitochondrial Permeability Transition

INTRODUCTION Chronic smokers exhibit skeletal muscle atrophy and mitochondrial impairments, which exacerbate the impact of normal aging and can increase risk of death. Tobacco smoke (TS) exposure drives these adverse effects by unknown mechanisms. TS toxins are degraded by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor which upregulates xenobiotic enzymes. Notably, chronic AHR activation phenocopies changes seen with mitochondrial permeability transition (MPT), a well-established cytotoxic mechanism in ischemia-reperfusion injury. Despite this, the role of chronic AHR activation in TS-induced skeletal muscle impact, and the potential involvement of MPT, has not been considered. Therefore, we designed experiments to establish: (1) the impact of TS exposure on MPT and its reliance on AHR activation, and (2) the necessity of AHR activation and MPT in TS-induced muscle atrophy. METHODS MPT is associated with the release of apoptosis inducing factor (AIF), which can translocate to the nucleus thereby providing a read-out of MPT occurrence. Thus, to evaluate the effect of TS on MPT occurrence, we exposed C2C12 myotubes to TS or vehicle control (DMSO) for 24 h, with and without the MPT inhibitor cyclosporin A (CsA; 1µM) and quantified nuclear:AIF co-localization. To evaluate if AHR antagonism could prevent MPT in TS-exposed cells, myotubes were co-treated with CH223191 (an AHR antagonist; 1µM) and nuclear:AIF co-localization was measured. Next, to establish the dependence of TS-induced muscle atrophy on AHR activation and AHR activity-mediated MPT, we exposed isolated single mouse FDB myofibers to TS, TS + CH223191 (1µM), or TS + isoxazole 63 (an MPT inhibitor; 0.5µM). Muscle atrophy was determined as the change in myofiber diameter from 0 to 24 h by measuring diameter at 10 points along the length of each fiber. RESULTS Nuclear:AIF co-localization was greatest in TS-exposed C2C12’s (Mander's Coefficient in TS: 0.49±0.03) compared with all other treatments (DMSO: 0.39±0.02; CsA: 0.40±0.03; CH223191: 0.40±0.02, p=0.001 for all), consistent with TS causing MPT in skeletal muscle in a manner that depends upon AHR activation. TS reduced single FDB fiber diameter over 24 hrs (0 h: 26.3±6.6 vs. 24 h: 22.1±4.8 µm, p=0.002). In contrast, fibers treated with TS + isoxazole 63 (0 h: 27.5±5.5 vs. 24 h: 25.4±5.4 µm, p=0.085) or TS + CH223191 (0 h: 28.0±5.6 vs. 24 h: 26.7±5.8 µm, p=0.302) did not atrophy significantly over 24 h, suggesting AHR-activity induced MPT is necessary for TS-induced muscle atrophy. DISCUSSION Our results show that TS increases nuclear:AIF co-localization in myotubes, consistent with TS causing MPT in skeletal muscle. Further, this effect was dependent upon AHR activation, linking chronic AHR activity with induction of MPT by TS exposure. Our results also show that AHR antagonism or MPT inhibition can prevent TS-induced atrophy in single myofibers, implicating AHR-mediated MPT in the mechanism by which TS causes muscle atrophy.

Mitochondrial Permeability Transition Induces Skeletal Muscle Atrophy in Single Living Myofibers

Introduction In advanced age (≥75 y), skeletal muscle atrophy leads to physical frailty, impaired mobility, and increased mortality. Mitochondrial dysfunction has been linked to aging skeletal muscle atrophy, although the specific mechanisms are unclear. Mitochondrial permeability transition (MPT) has been implicated in aging biology, which involves formation of a pore across the inner mitochondrial membrane that causes dissipation of mitochondrial membrane potential and cytoplasmic release of reactive oxygen species (ROS) and mitochondrial proteins. Although we find mitochondria are sensitized to MPT in aging skeletal muscle, the role of MPT in aging muscle biology has not been explored. To gain insight to the impact of MPT in aging muscle, we recently examined the cross-sectional area (CSA) of muscle fibers with myonuclei that contained apoptosis inducing factor (AIF), a protein released from mitochondria during MPT. On average, fibers containing AIF-positive myonuclei had smaller CSA, suggesting a link between MPT and muscle atrophy in advanced age. To more directly test the involvement of MPT in muscle atrophy, we used cultured mouse skeletal muscle fibers to determine if acute chemical induction of MPT would cause fiber atrophy that would be prevented by MPT inhibition. We also hypothesized that treatment with an MPT inhibitor would prevent atrophy in a model of muscle fiber ‘disuse’ where isolated single mouse myofibers were maintained in culture conditions for 5 d. Methods Single fibers were isolated from mouse flexor digitorum brevis (FDB) muscles, plated, and treated with DMSO (vehicle control), doxorubicin (DOX; 1µM), a chemotherapy agent known to induce MPT, or DOX plus the MPT inhibitor cyclosporine A (CsA; 1 µM). Fibers were imaged at 20x upon plating and after 24h treatment. Myofiber diameter was assessed in ImageJ (average of 10 diameter measurements per fiber). Individual fibers were matched pre- to post-treatment for determination of percent change in myofiber diameter. Previous studies have established that maintaining single isolated muscle fibers in culture conditions is associated with fiber atrophy and can therefore be used as a model of disuse/denervation atrophy. On this basis, we isolated single mouse FDB myofibers and treated them with vehicle (DMSO) or 1 µM CsA every 24h under standard culture conditions for 5 d. Myofiber diameter was determined at baseline and day 5, as described above. Results Whereas 24h treatment of myofibers with DOX induced significant atrophy compared to baseline (percent change in myofiber diameter; DOX: -16.64 ± 7.78%, P<0.05), the addition of CsA prevented atrophy over this brief period (DOX+CsA: 0.22 ± 15.38%, NS). Similarly, in a model of muscle disuse, MPT inhibition using CsA prevented myofiber atrophy (baseline: 38.11 ± 5.87 µm vs. day 5: 36.49 ± 7.66 µm, NS) compared with DMSO (baseline: 35.65 ± 7.30 µm vs. day 5: 31.15 ± 7.01 µm, P<0.05). Discussion Consistent with our hypotheses, both inducing MPT using DOX and 5 days of muscle disuse led to atrophy in single living myofibers that was prevented by MPT inhibition. Therefore, our results identify MPT as a novel mechanism of skeletal muscle atrophy and a mechanism that appears relevant to causing the atrophy seen in aging skeletal muscle.

The Regulation of Non-Specific Membrane Permeability Transition in Yeast Mitochondria under Oxidative Stress

In this study, the mechanism of non-specific membrane permeability (yPTP) in the Endomyces magnusii yeast mitochondria under oxidative stress due to blocking the key antioxidant enzymes has been investigated. We used monitoring the membrane potential at the cellular (potential-dependent staining) and mitochondrial levels and mitochondria ultra-structural images with transmission electron microscopy (TEM) to demonstrate the mitochondrial permeability transition induction due to the pore opening. Analysis of the yPTP opening upon respiring different substrates showed that NAD(P)H completely blocked the development of the yPTP. The yPTP opening was inhibited by 5–20 mM Pi, 5 mM Mg2+, adenine nucleotides (AN), 5 mM GSH, the inhibitor of the Pi transporter (PiC), 100 μM mersalyl, the blockers of the adenine nucleotide transporter (ANT) carboxyatractyloside (CATR), and bongkrekic acid (BA). We concluded that the non-specific membrane permeability pore opens in the E. magnusii mitochondria under oxidative stress, and the ANT and PiC are involved in its formation. The crucial role of the Ca2+ ions in the process has not been confirmed. We showed that the Ca2+ ions affected the yPTP both with and without the Ca2+ ionophore ETH129 application insignificantly. This phenomenon in the E. magnusii yeast unites both mitochondrial unselective channel (ScMUC) features in the Saccharomyces cerevisiae mitochondria and the classical membrane pore in the mammalian ones (mPTP).

Modulatory effect of methanol extract of Annona muricata stem bark on mitochondrial membrane permeability transition pore in normal rat liver and monosodium glutamate-induced uterine hyperplasia

Uterine fibroids are benign tumors that develop in many women of reproductive age. Surgery is the main approach to treatment while other options are also associated with adverse effects. Studies have shown that certain bioactive agents present in medicinal plants elicit their anti-tumor activity by induction of mitochondrial permeability transition (mPT) opening. This research therefore aimed at investigating the effect of methanol extract of Annona muricata (MEAM) on mPT pore opening in normal and monosodium glutamate-induced uterine hyperplasia using female Wistar rats. Mitochondria, isolated from rat liver were exposed to different concentrations (20, 60, 100, 140 and 180μg/mL) of MEAM. The mPT pore opening, cytochrome c release, mitochondrial ATPase (mATPase) activity and the percentage lipid peroxidation were assessed spectrophotometrically. Histological effects of MEAM on the liver, brain and uterus of normal and MSG-treated rats were investigated. The in vitro results showed a significant induction of mPT pore opening by 2.4, 4.2 and 6.4 folds, release of cytochrome c and enhancement of mATPase activity at 100,140 and 180μg/mL, respectively. However, oral administration of MEAM did not induce mPT pore opening, neither any significant release of cytochrome c nor enhancement of mATPase activity at all the dosages used. However, histological assay revealed the presence of MSG-induced cellular damage and uterine hyperplasia which was ameliorated by MEAM co-administration. These findings suggest that MEAM contains phytochemicals that can ameliorate MSG-induced damage and uterine hyperplasia in rats; however, the mechanism might not be via upregulation of mitochondrial-mediated apoptosis.

Recent Advances in the Pathophysiology of Fatty Acid Oxidation Defects: Secondary Alterations of Bioenergetics and Mitochondrial Calcium Homeostasis Caused by the Accumulating Fatty Acids.

Deficiencies of medium-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein, isolated long-chain 3-hydroxyacyl-CoA dehydrogenase, and very long-chain acyl-CoA dehydrogenase activities are considered the most frequent fatty acid oxidation defects (FAOD). They are biochemically characterized by the accumulation of medium-chain, long-chain hydroxyl, and long-chain fatty acids and derivatives, respectively, in tissues and biological fluids of the affected patients. Clinical manifestations commonly include hypoglycemia, cardiomyopathy, and recurrent rhabdomyolysis. Although the pathogenesis of these diseases is still poorly understood, energy deprivation secondary to blockage of fatty acid degradation seems to play an important role. However, recent evidence indicates that the predominant fatty acids accumulating in these disorders disrupt mitochondrial functions and are involved in their pathophysiology, possibly explaining the lactic acidosis, mitochondrial morphological alterations, and altered mitochondrial biochemical parameters found in tissues and cultured fibroblasts from some affected patients and also in animal models of these diseases. In this review, we will update the present knowledge on disturbances of mitochondrial bioenergetics, calcium homeostasis, uncoupling of oxidative phosphorylation, and mitochondrial permeability transition induction provoked by the major fatty acids accumulating in prevalent FAOD. It is emphasized that further in vivo studies carried out in tissues from affected patients and from animal genetic models of these disorders are necessary to confirm the present evidence mostly achieved from in vitro experiments.

Methyl palmitate reversed estradiol benzoate-induced endometrial hyperplasia in female rats

Early detection and treatment of endometrial hyperplasia (EH) is mandatory for endometrial cancer prevention. Several bioactive agents of plant origin have been shown to elicit their chemotherapeutic effect against tumors and cancer via induction of mitochondrial permeability transition(mPT) pore opening. This research was therefore aimed at evaluating the potential chemopreventive effect of methyl palmitate (MP), on estradiol benzoate(EB)-induced EH, looking at the mitochondrial-mediated pathway and other possible mechanisms of action. Mitochondria were isolated using differential centrifugation. The mPT pore, mitochondrial ATPase (mATPase) activity, lipid peroxidation and cytochrome c release were determined by standard methods using spectrophotometer. Uterine interleukin 1b, MDA levels and SOD, GSH activities, were determined using commercially available kits. The uterine histological and immunohistochemical assessment of estrogen receptor (ERα), IL-1b and caspas-3 were carried out. The fibroblast cell count density was determined using histomorphometry. At all the concentrations of MP used, there was no significant induction of mPT pore opening, neither any enhancement of mATPase activity nor release of cytochrome c when compared to the control. Similar pattern of results were recorded for the in vivo study. However, there was marked increase in the uterine MDA and interleukin 1b levels, with concurrent decrease in SOD and GSH activities, in the EB-treated group, which was significantly reversed by MP co-administration. Endometrial Hyperplasia observed in the EB-treated group was ameliorated by MP co-administration. The immunoexpression of ERα and IL-1b in the EB-treated group was reversed by MP co-administration. This study suggests anti-inflammatory, antioxidant and anti-proliferative potential of MP against EB-induced EH.

Monosodium Glutamate Induces Cytotoxicity in Rat Liver via Mitochondrial Permeability Transition Pore Opening.

Monosodium glutamate (MSG) is a major food additive used as a flavor enhancer. A lot of controversies have been generated over the use of MSG. The present study therefore investigated whether MSG would induce cytotoxicity via the induction of mitochondrial permeability transition (mPT) pore opening. 36 male albino rats were used for this study. The rats were equally divided into six groups: group I is the control while group II, III, IV, V, and VI were orally treated with MSG (25, 50, 100, 200, and 400 mg/kg) daily for 28 days. The opening of the pore, cytochrome c release, mitochondrial ATPase activity, mitochondrial lipid peroxidation and hepatic DNA fragmentation were determined spectrophotometrically. Histological assessment of prostate and brain was carried out. The results show that MSG at concentrations ≤30 µg/ml did not induce mPT pore opening while higher concentrations caused significant induction of pore opening. Also, at lower doses (25 and 50 mg/kg), MSG did not cause any significant induction of mPT pore opening while at higher doses, there were significant induction of pore opening. Similar trend of results was recorded for cytochrome c release, mitochondrial ATPase activity and lipid peroxidation. The histological results show that at low doses (25 and 50 mg/kg), no significant lesion was observed while higher doses caused benign prostatic hyperplasia (BPH) in the prostate and necrotic damage in the brain. MSG administration at low dose is tolerable while high doses induce cytotoxicity via mPT pore opening.

Physiopathology of the Permeability Transition Pore: Molecular Mechanisms in Human Pathology.

Mitochondrial permeability transition (MPT) is the sudden loss in the permeability of the inner mitochondrial membrane (IMM) to low-molecular-weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate outer-mitochondrial-membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade and caspase-independent cell-death mechanisms. The induction of MPT is mostly dependent on mitochondrial reactive oxygen species (ROS) and Ca2+, but is also dependent on the metabolic stage of the affected cell and signaling events. Therefore, since its discovery in the late 1970s, the role of MPT in human pathology has been heavily investigated. Here, we summarize the most significant findings corroborating a role for MPT in the etiology of a spectrum of human diseases, including diseases characterized by acute or chronic loss of adult cells and those characterized by neoplastic initiation.

P0017ESTIMATION OF KIDNEY MITOCHONDRIA TOLERANCE VIA FLUORESCENCE MICROSCOPY

Abstract Background and Aims The functioning of mitochondria is a key parameter that determines the normal activity of kidney cells and triggers life/death transition in pathological conditions. The main pathological event in the mitochondria is the opening of a permeability transition pore, observed in renal ischemia and other acute nephrological pathologies. The development of non-specific mitochondrial permeability leads to a burst of ROS generation and the release of proapoptotic factors. Assessment of mitochondrial resistance to induction of permeability transition is an important characteristic for the analysis of kidney tolerance to damaging factors, such as ischemia. In this study, we developed a method for evaluating mitochondrial permeability transition in renal tubular cell culture using fluorescence microscopy. Method The primary renal tubular epithelial cells were loaded with a fluorescent probe, TMRE, which accumulates in the mitochondria depending on the transmembrane potential. Cells in the culture were analyzed on a fluorescent microscope for 180 seconds under constant exposure with exciting light and the video was recorded at a frequency of 1 frame/sec. To analyze the images, a Python algorithm was created that allocated the individual mitochondria in the image and analyzed the shape, size, and dynamics of the TMRE fluorescence of each mitochondrion. In total, more than 10^5 mitochondria were analyzed in each experiment. Results The initial state of mitochondrial transmembrane potential was evaluated by the intensity of TMRE fluorescence in the first frame in each series when phototoxicity of the dye was not manifested yet. Population analysis revealed that the total distribution of mitochondria by TMRE fluorescence intensity was a single-modal. In this case, the histogram of the distribution had a wide arm of mitochondria with greater TMRE fluorescence (that means high transmembrane potential). This distribution suggests that renal cells have mitochondria with high and low TMRE fluorescence values. This can be interpreted as the presence of mitochondria in cells with higher transmembrane potential values. Analysis of the dynamics of TMRE fluorescence intensity revealed that some mitochondria after a certain time demonstrated a sharp drop in the fluorescence intensity, which we interpret as the opening of the mitochondrial pore (Fig.1). However, this drop did not occur in all kidney cells mitochondria, but only in about 15% of the cells. A small number of mitochondria had several repeated falls/rises in TMRE fluorescence. This can be interpreted as the closing and subsequent opening of the permeability transition pores. Analysis of a subpopulation of mitochondria with low TMRE values revealed that low-energized mitochondria have a later pore opening time. It is important to note that mitochondria with higher TMRE fluorescence are more susceptible to photodynamic effects when the probe is excited by light. Conclusion Renal epithelial cells happen to be very heterogeneous in terms of mitochondrial potential and the time of the induction of mitochondrial permeability transition. Correlations were found between the value of the mitochondrial potential (TMRE accumulation) and the cell's resistance to induction of non-specific mitochondrial pore.

Mitochondrial Permeability Transition Induces Atrophy in C2C12 Myotubes

INTRODUCTIONIn advanced age (&gt;75 yrs), muscle atrophy can lead to physical frailty, impaired mobility, and increased mortality. Impairment and functional decline of mitochondria within skeletal muscle are seen with aging, including a sensitization to mitochondrial permeability transition (MPT). MPT is an established pathological mechanism causing ischemic organ damage and has been observed in aged motoneurons and skeletal muscle. However, the role of MPT in age‐related muscle atrophy has not been determined. To better understand how MPT may induce skeletal muscle atrophy, we determined the impact of inducing MPT on myotube size in a cell culture model and the impact of modulating MPT on classical atrophy signaling transcripts. We hypothesized that induction of MPT in myotubes would cause atrophy, and MPT would modulate atrophy‐related signaling pathways.METHODSTo determine if MPT can induce myotube atrophy, C2C12 myotubes were treated with doxorubicin (Dox; 1 μM), a known inducer of MPT, or DMSO (vehicle, 1 μM) on day 4 post‐differentiation for 12, 24, and 48 h. At each time point, cells were fixed/permeabilized in icecold methanol/acetone (1:1), blocked, and incubated with a primary antibody against sarcomeric myosin (MF‐20). Cells were incubated with a fluorescent secondary antibody and imaged at 20X on an inverted fluorescent microscope. Images (n=4 per well, 5 wells/treatment) were analyzed to quantify MF20+ area (myotube area) using a custom written program in CellProfiler.To assess signaling mechanisms downstream of MPT, C2C12 myotubes were treated with Dox (1μM), Dox + an inhibitor of MPT (bongkrekic acid [BA]; DOX+BA, each 1 μM), or DMSO (vehicle;1 μM) for 12 h on day 4 post‐differentiation (n=3 biological replicates; n=4 technical replicates per treatment). At 12 h post‐treatment, cells were collected in TRIzol and their RNA isolated for RT‐PCR analysis of transcriptional regulators of muscle atrophy (FOXO1, NfKB1, Gadd45a, ATF4), an autophagy‐regulating gene (p62/SQSTM), and E3 ubiquitin ligases (Mafbx, MuRF). The comparative threshold cycle (CT) method was used to calculate fold changes in expression with 18s as an endogenous control. Relative fold changes in expression are presented as 2−ΔΔCT and normalized to DMSO‐treated samples.Two‐tailed Student’s t‐tests were used to assess differences between DOX vs. DMSO, and DOX vs. DOX+BA, respectively. Significance was accepted at P&lt;0.05.RESULTSDOX treatment significantly reduced myotube area by 24 h post‐treatment, and this persisted through 48 h (P&lt;0.05). Inhibiting MPT with BA attenuated mRNA levels of transcription factors FOXO1 and ATF4, Gadd45a, the atrogene MuRF1, and the autophagy regulating gene p62 at 12 h post‐treatment (P&lt;0.05 for all). There was no difference between DOX vs. DOX+BA for MAFbx (P=0.076) or NfKB1 (0.692) expression.DISCUSSIONConsistent with our hypothesis, induction of MPT with DOX caused myotube atrophy. Additionally, inhibiting MPT in the presence of DOX attenuated mRNA levels of classical atrophy signaling pathway components, including FOXO1, ATF4, Gadd45α, MuRF1 and p62. Thus, our results suggest that MPT is a cause of atrophy in aging skeletal muscle.Support or Funding InformationSB is supported by a T32 from the National Institute of Child Health and Human Development Grant T32HD043730.

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2−1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.

Ketamine treatment affects hippocampal but not cortical mitochondrial function in prepubertal rats.

Previous reports have shown that ketamine triggered apoptosis in immature developing brain involving mitochondrial-mediated pathways. However, no data for ketamine effects on hippocampal and cortical mitochondrial function are available in prepubertal rats. Twenty-one-day-old Sprague-Dawley rats received ketamine (40mg/kg i.p.) for 3days and were killed 24hr after thelast injection. Hippocampal mitochondria from ketamine-treated rats showed decreased malate-glutamate state 4 and 3 respiratory rates and an inhibition in complex I and IV activities. Hippocampal mitochondrial membrane depolarization and mitochondrial permeability transition induction were observed. This was not reflected in an increment of H2 O2 production probably due to increased Mn-SOD and catalase activities, 24hr after treatment. Interestingly, increased H2 O2 production rates and cardiolipin oxidation were found in hippocampal mitochondria shortly after ketamine treatment (45min). Unlike the hippocampus, ketamine did not affect mitochondrial parameters in the brain cortex, being the area less vulnerable to suffer ketamine-induced oxidative damage. Results provide evidences that exposure of prepubertal rats to ketamine leads to an induction of mitochondrial ROS generation at early stages of treatment that was normalized by the triggering of antioxidant systems. Although hippocampal mitochondria from prepubertal rats were capable of responding to the oxidative stress, they remain partially dysfunctional.

Aged garlic extract and its constituent, S-allyl-L-cysteine, induce the apoptosis of neuroblastoma cancer cells due to mitochondrial membrane depolarization.

Aged garlic extract (AGE) has been demonstrated to have therapeutic properties in tumors; however its mechanisms of action have not yet been fully elucidated. A previous study revealed that AGE exerts an anti-proliferative effect on a panel of both sensitive [wild-type (WT)] and multidrug-resistant (MDR) human cancer cells. Following treatment of the cells with AGE, cytofluorimetric analysis revealed the occurrence of dose-dependent mitochondrial membrane depolarization (MMD). In this study, in order to further clarify the mechanisms of action of AGE, the effects of AGE on mitochondria isolated from rat liver mitochondria (RLM) were also examined. AGE induced an effect on the components of the electrochemical gradient (ΔµH+), mitochondrial membrane potential (ΔΨm) and mitochondrial electrochemical gradient (ΔpHm). The mitochondrial membrane dysfunctions of RLM induced by AGE, namely the decrease in both membrane potential and chemical gradient were associated with a higher oxidation of both the endogenous glutathione and pyridine nucleotide content. To confirm the anti-proliferative effects of AGE, experiments were performed on the human neuroblastoma (NB) cancer cells, SJ-N-KP and the MYCN-amplified IMR5 cells, using its derivative S-allyl-L-cysteine (SAC), with the aim of providing evidence of the anticancer activity of this compound and its possible molecular mechanism as regards the induction of cytotoxicity. Following treatment of the cells with SAC at 20 mM, cell viability was determined by MTT assay and apoptosis was detected by flow cytometry, using Annexin V-FITC labeling. The percentages of cells undergoing apoptosis was found to be 48.0% in the SJ-N-KP and 50.1% in the IMR5 cells. By cytofluorimetric analysis, it was suggested that the target of SAC are the mitochondria. Mitochondrial activity was examined by labeling the cells with the probe, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanine iodide (JC-1). Following treatment with SAC at 50 mM, both NB cell lines exhibited a marked increase in MMD. On the whole, the findings of this study indicate that both natural products, AGE and SAC, cause cytotoxicity to tumor cells via the induction of mitochondrial permeability transition (MPT).

Involvement of the biogenic active amine agmatine in mitochondrial membrane permeabilization and release of pro-apoptotic factors.

Agmatine (AGM) produces a dual effect on the mitochondrial permeability transition (MPT) mechanism in rat liver mitochondria: at low concentrations, it induces the phenomenon, at high ones, inhibits it. The prevention at high concentrations is evidenced by the significant inhibition of mitochondrial swelling induced by Ca2+ and phosphate; in this condition, AGM both prevents the release of Apoptosis Inducing Factor (AIF) and enhances the release of other pro-apoptotic factors, such as cytochrome c (cyt c) and Smac/DIABLO. As these factors are released without MPT induction, the involvement of mitochondrial outer membrane permeabilization (MOMP) could be hypothesized. Cyclosporin A (CsA), a powerful inhibitor of MPT, enhanced the AGM-mediated inhibition of swelling, and surprisingly, prevented the release of cyt c and Smac/DIABLO. In the presence of Ca2+, AGM also activated the Bcl-2 family protein Bax, a key factor in inducing MOMP, which is inactivated by CsA. Together with the voltage-dependent anion channel (VDAC), Bax forms channels in the outer membrane further supporting the involvement of MOMP in the release of pro-apoptotic factors. In view of the fact that VDAC was inactivated by ruthenium red, which in turn inhibited the release of cyt c, it can be hypothesized that, on the one hand, AGM inhibits MPT induction and, on the other, it selectively permeabilizes the outer membrane via MOMP induction.

Disturbance of mitochondrial functions associated with permeability transition pore opening induced by cis-5-tetradecenoic and myristic acids in liver of adolescent rats

Patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency commonly present liver dysfunction whose pathogenesis is poorly known. We demonstrate here that major metabolites accumulating in this disorder, namely cis-5-tetradecenoic acid (Cis-5) and myristic acid (Myr), markedly impair mitochondrial respiration, decreasing ATP production in liver mitochondrial preparations from adolescent rats. Other parameters of mitochondrial homeostasis such as membrane potential (ΔΨm) and Ca2+retention capacity were strongly compromised by these fatty acids, involving induction of mitochondrial permeability transition. The present data indicate that disruption of mitochondrial bioenergetics and Ca2+homeostasis may contribute to the liver dysfunction of VLCAD deficient patients.

Impairment of mitochondrial bioenergetics and permeability transition induction caused by major long-chain fatty acids accumulating in VLCAD deficiency in skeletal muscle as potential pathomechanisms of myopathy

cis-5-Tetradecenoic (cis-5) and myristic (Myr) acids predominantly accumulate in patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. They commonly manifest myopathy with muscular pain and rhabdomyolysis, whose underlying mechanisms are poorly known. Thus, in the present study we investigated the effects of cis-5 and Myr on mitochondrial bioenergetics and Ca2+ homeostasis in rat skeletal muscle. cis-5 and Myr decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration, especially when mitochondria were supported by NADH-linked as compared to FADH2-linked substrates. In contrast, these fatty acids increased resting respiration (state 4). Similar effects were observed in skeletal muscle fibers therefore validating the data obtained with isolated mitochondria. Furthermore, cis-5 and Myr markedly decreased mitochondrial membrane potential and Ca2+ retention capacity that were avoided by cyclosporin A plus ADP and ruthenium red, indicating that cis-5 and Myr induce mitochondrial permeability transition (MPT). Finally, docosanoic acid did not disturb mitochondrial homeostasis, indicating selective effects for Myr and cis-5. Taken together, our findings indicate that major long-chain fatty acids accumulating in VLCAD deficiency behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and MPT inducers. It is presumed that these pathomechanisms contribute to the muscular symptoms and rhabdomyolysis observed in patients affected by VLCAD deficiency.