Inhibitor Of Mitochondrial Permeability Transition Research Articles

Disturbance of mitochondrial functions caused by N-acetylglutamate and N-acetylmethionine in brain of adolescent rats: Potential relevance in aminoacylase 1 deficiency

Aminoacylase 1 (ACY1) deficiency is a rare genetic disorder that affects the breakdown of short-chain aliphatic N-acetylated amino acids, leading to the accumulation of these amino acid derivatives in the urine of patients. Some of the affected individuals have presented with heterogeneous neurological symptoms such as psychomotor delay, seizures, and intellectual disability. Considering that the pathological mechanisms of brain damage in this disorder remain mostly unknown, here we investigated whether major metabolites accumulating in ACY1 deficiency, namely N-acetylglutamate (NAG) and N-acetylmethionine (NAM), could be toxic to the brain by examining their in vitro effects on important mitochondrial properties. We assessed the effects of NAG and NAM on membrane potential, swelling, reducing equivalents, and Ca2+ retention capacity in purified mitochondrial preparations obtained from the brain of adolescent rats. NAG and NAM decreased mitochondrial membrane potential, reducing equivalents, and calcium retention capacity, and induced swelling in Ca2+-loaded brain mitochondria supported by glutamate plus malate. Notably, these changes were completely prevented by the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP and by ruthenium red, implying the participation of MPT and Ca2+ in these effects. Our findings suggest that NAG- and NAM-induced disruption of mitochondrial functions involving MPT may represent relevant mechanisms of neuropathology in ACY1 deficiency.

The effects of pretreatment with Cyclosporin A and Docetaxel before vitrification of porcine immature oocytes on subsequent embryo development

In the present study, we attempted to improve the developmental competence of vitrified immature porcine oocytes by the preservation of mitochondrial properties using Cyclosporin A (CsA, inhibitor of mitochondrial membrane permeability transition) and Docetaxel (stabilizer of microtubules, hence mitochondrial distribution). In Experiment 1, Mitotracker red staining revealed reduced mitochondrial activity (MA) in vitrified/warmed oocytes at 0 and 22 h of in vitro maturation (IVM) compared with fresh ones. However, by at 46 h of IVM, MA levels in vitrified oocytes were similar to those in fresh control. Treatment of oocytes with CsA or Docetaxel improved MA at 0 h and 22 h of IVM compared with non-treated vitrified oocytes. However, there were no significant differences among groups in percentages of survival, maturation and embryo development after subsequent IVM and parthenogenetic activation. Nevertheless, a pretreatment with a combination of 10 µg/mL CsA and 0.05 µM Docetaxel improved the blastocyst formation of vitrified oocytes compared with non-treatment counterparts (11.2 ± 1.6% vs 5.9 ± 1.6%, P < 0.05). In conclusion, vitrification reduced mitochondrial activity in GV-stage oocytes during 0–22 h of IVM; however, it was normalized by 46 h IVM. Docetaxel or CsA pretreatment alone did not improve development competence of vitrified oocytes. However, pretreatment with a combination of CsA and Docetaxel could improve blastocyst formation rates.

Enhanced resistance to Ca2+-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

Antioxidant Potential and Inhibition of Mitochondrial Permeability Transition Pore by Myricetin Reduces Aluminium Phosphide-Induced Cytotoxicity and Mitochondrial Impairments.

Oxidative stress and mitochondrial dysfunction are involved in the mechanisms of cardiac toxicity induced by aluminum phosphide (AlP). AlP-induced cardiotoxicity leads to cardiomyocyte death, cardiomyopathy, cardiac dysfunction, and eventually severe heart failure and death. Importantly, protecting cardiomyocytes from death resulting from AlP is vital for improving survival. It has been reported that flavonoids such as myricetin (Myr) act as modifiers of mitochondrial function and prevent mitochondrial damage resulting from many insults and subsequent cell dysfunction. In this study, the ameliorative effect of Myr, as an important antioxidant and mitochondrial protective agent, was investigated in cardiomyocytes and mitochondria isolated from rat heart against AlP-induced toxicity, oxidative stress, and mitochondrial dysfunction. Treatment of AlP (20 μg/ml) significantly increased cytotoxicity; reduced glutathione (GSH) depletion, cellular reactive oxygen species (ROS) formation, malondialdehyde (MDA) level, ATP depletion, caspase-3 activation, mitochondrial membrane potential (ΔΨm) collapse, and lysosomal dysfunction; and decreased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in intact cardiomyocytes. Also, treatment of AlP (20 μg/ml) significantly increased mitochondrial dysfunction and swelling in isolated mitochondria. Myr (80 µM) appeared to ameliorate AlP-induced cytotoxicity in isolated cardiomyocytes; significantly lessened the AlP-stimulated intracellular ROS and MDA production and depletion of GSH; and increased the activities of SOD, CAT, and GSH-Px. Furthermore, Myr (40 and 80 µM) lowered AlP-induced lysosomal/mitochondrial dysfunction, ATP depletion, and caspase-3 activation. In the light of these findings, we concluded that Myr through antioxidant potential and inhibition of mitochondrial permeability transition (MPT) pore exerted an ameliorative role in AlP-induced toxicity in isolated cardiomyocytes and mitochondria, and it would be valuable to examine its in vivo effects.

Gallic acid inhibits celecoxib-induced mitochondrial permeability transition and reduces its toxicity in isolated cardiomyocytes and mitochondria.

Mitochondria are the main target organelles through which drugs and chemicals exert their toxic effect on cardiomyocytes. The mitochondria-related mechanisms of celecoxib-induced cardiotoxicity have been extensively studied. Accumulated evidence shows natural molecules targeting mitochondria have proven to be effective in preventing cardiotoxicity. In the present study, we examined the ameliorative effect of gallic acid (GA) against celecoxib-induced cellular and mitochondrial toxicity in isolated cardiomyocytes and mitochondria. The isolated cardiomyocytes and mitochondria were divided into various group, namely, control, celecoxib, celecoxib + GA (10, 50, and 100µM). Several cellular and mitochondrial parameters such as cell viability, lipid peroxidation, succinate dehydrogenase (SDH) activity, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse, and mitochondrial swelling were assessed in isolated cardiomyocytes and mitochondria. Our results showed that administration of celecoxib (16µg/ml) induced cytotoxicity and mitochondrial dysfunction at 6h and 1h, respectively, which is associated with lipid peroxidation intact cardiomyocytes, mitochondrial ROS formation, MMP collapse, and mitochondrial swelling. The cardiomyocytes and mitochondria treated with celecoxib + GA (10, 50, and 100µM) significantly and dose-dependently restore the altered levels of cellular and mitochondrial parameters. We concluded that GA through antioxidant potential and inhibition of mitochondrial permeability transition (MPT) pore exerted ameliorative role in celecoxib-induced toxicity in isolated cardiomyocytes and mitochondria. The data of the current study suggested that GA supplementation may reduce celecoxib-induced cellular and mitochondrial toxicity during exposure and may provide a potential prophylactic and defensive candidate for coxibs-induced mitochondrial dysfunction, oxidative stress, and cardiotoxicity.

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.

Alisporivir Improves Mitochondrial Function in Skeletal Muscle of mdx Mice but Suppresses Mitochondrial Dynamics and Biogenesis.

Mitigation of calcium-dependent destruction of skeletal muscle mitochondria is considered as a promising adjunctive therapy in Duchenne muscular dystrophy (DMD). In this work, we study the effect of intraperitoneal administration of a non-immunosuppressive inhibitor of calcium-dependent mitochondrial permeability transition (MPT) pore alisporivir on the state of skeletal muscles and the functioning of mitochondria in dystrophin-deficient mdx mice. We show that treatment with alisporivir reduces inflammation and improves muscle function in mdx mice. These effects of alisporivir were associated with an improvement in the ultrastructure of mitochondria, normalization of respiration and oxidative phosphorylation, and a decrease in lipid peroxidation, due to suppression of MPT pore opening and an improvement in calcium homeostasis. The action of alisporivir was associated with suppression of the activity of cyclophilin D and a decrease in its expression in skeletal muscles. This was observed in both mdx mice and wild-type animals. At the same time, alisporivir suppressed mitochondrial biogenesis, assessed by the expression of Ppargc1a, and altered the dynamics of organelles, inhibiting both DRP1-mediated fission and MFN2-associated fusion of mitochondria. The article discusses the effects of alisporivir administration and cyclophilin D inhibition on mitochondrial reprogramming and networking in DMD and the consequences of this therapy on skeletal muscle health.

Peptide Based Therapy for Neurological Disorders.

Peptides are small molecules composed of amino acids linked together by peptide bonds. The targeted action of these peptides along with their magnificent ability to reach locations in the body that are complicated to access, is being considered of tremendous potential in disease modifying therapies. Synthetic as well as natural peptides like Carnosine are currently under research for the treatment of Neurodegenerative Disorders (NDDs). Peptide based vaccines are currently under immense research for diseases like dementia. Toxicity of peptide-based drugs towards eukaryotic cells due to their increased haemolytic activity is of major concern and this is being tackled by introducing modifications into the peptide structure. Some crucial peptide inhibitors currently in use for neurodegenerative disorders include Aβ (16-20) KLVFF for Alzheimer's disease, NAPVSIPQ (NAP) for Parkinson's disease, towards eukaryotic cells Vasoactive Intestinal Peptides (VIP) for Huntington's disease, Polyglutamine Binding Peptide-1(QBP1) for Dentatorubral- pallidoluysian atrophy (DRPLA). Certain peptides are involved in inhibition of Mitochondrial Permeability Transition (MPT) that plays a prominent part in the materialization of neurodegenerative diseases, one such example of peptides being Ba-V which is obtained from Bothrops atrox snake venom. New therapeutic peptides are being identified using bioinformatics tools like High Throughput Screening (HTS). These tools are being used to explore the selectivity, stability, extent of immune response and toxic side effects of peptides. Apart from neurodegenerative diseases, the potential of bioactive peptides is also being tested against cancer, diabetes and microbes. This review focuses on the recent advances in peptide therapeutics and novel peptides discovered for the treatment of NDs.

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.

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.

The cyclophilin inhibitor NIM-811 increases muscle cell survival with hypoxia in vitro and improves gait performance following ischemia\u2013reperfusion in vivo

Acute ischemia–reperfusion injury in skeletal muscle is a significant clinical concern in the trauma setting. The mitochondrial permeability transition inhibitor NIM-811 has previously been shown to reduce ischemic injury in the liver and kidney. The effects of this treatment on skeletal muscle are, however, not well understood. We first used an in vitro model of muscle cell ischemia in which primary human skeletal myoblasts were exposed to hypoxic conditions (1% O2 and 5% CO2) for 6 h. Cells were treated with NIM-811 (0–20 µM). MTS assay was used to quantify cell survival and LDH assay to quantify cytotoxicity 2 h after treatment. Results indicate that NIM-811 treatment of ischemic myotubes significantly increased cell survival and decreased LDH in a dose-dependent manner. We then examined NIM-811 effects in vivo using orthodontic rubber bands (ORBs) for 90 min of single hindlimb ischemia. Mice received vehicle or NIM-811 (10 mg/kg BW) 10 min before reperfusion and 3 h later. Ischemia and reperfusion were monitored using laser speckle imaging. In vivo data demonstrate that mice treated with NIM-811 showed increased gait speed and improved Tarlov scores compared to vehicle-treated mice. The ischemic limbs of female mice treated with NIM-811 showed significantly lower levels of MCP-1, IL-23, IL-6, and IL-1α compared to limbs of vehicle-treated mice. Similarly, male mice treated with NIM-811 showed significantly lower levels of MCP-1 and IL-1a. These findings are clinically relevant as MCP-1, IL-23, IL-6, and IL-1α are all pro-inflammatory factors that are thought to contribute directly to tissue damage after ischemic injury. Results from the in vitro and in vivo experiments suggest that NIM-811 and possibly other mitochondrial permeability transition inhibitors may be effective for improving skeletal muscle salvage and survival after ischemia–reperfusion injury.

The mitochondrial permeability transition phenomenon elucidated by cryo-EM reveals the genuine impact of calcium overload on mitochondrial structure and function

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves cristae despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

Mitochondria as a promising target for developing novel agents for treating Alzheimer's disease.

The mitochondria-targeting drugs can be conventionally divided into the following groups: those compensating for the energy deficit involved in neurodegeneration, including stimulants of mitochondrial bioenergetics and activators of mitochondrial biogenesis; and neuroprotectors, that are compounds increasing the resistance of mitochondria to opening of mitochondrial permeability transition (MPT) pores. Although compensating for the energy deficit and inhibition of MPT are obvious targets for drugs used in the very early stages of Alzheimer-like pathology, but their use as the monotherapy for patients with severe symptoms is unlikely to be sufficiently effective. It would be optimal to combine targets that would provide the cognitive-stimulating, the neuroprotective effects and the ability to affect specific disease-forming mechanisms. In the design of such drugs, assessment of their potential mitochondrial-targeted effects is of particular importance. The possibility of targeted drug design for simultaneous action on mitochondrial and neurotransmitter's receptors targets is, in particularly, based on the known interplay of various cellular pathways and the presence of common structural components. Of particular interest is directed search for multitarget drugs that would act simultaneously on mitochondrial calcium-dependent functions, the targets (receptors, enzymes, etc.) facilitating neurotransmission, and the molecular targets related to the action of so-called disease-modifying factors, in particular, the formation and overcoming of the toxicity of β-amyloid or hyperphosphorylated tau protein. The examples of such approaches realized on the level of preclinical and clinical trials are presented below.

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.

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.

Betulin attenuated liver damage by prevention of hepatic mitochondrial dysfunction in rats with alcoholic steatohepatitis.

Betulin, a pentacyclic triterpene, possesses antioxidant, anti-inflammatory and hepatoprotective properties. The aim of this study was to evaluate the impact of liver mitochondria in hepatoprotection of betulin using a rat model of alcoholic steatohepatitis induced by ethanol administration (4g/kg, intragastric) for 8weeks. The treatment with betulin (50 and 100mg/kg b.w., intragastric) during this period attenuated the histological signs of steatohepatitis and lowered the serum and liver triglyceride contents, as well as the serum activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Betulin (100mg/kg) decreased the liver/body weight ratio and inhibited the increase in the serum levels of TNFα, IL-1β, TGFβ, and hyaluronic acid, demonstrating hepatoprotective, anti-inflammatory, and antifibrotic potential. Betulin also inhibited the formation of superoxide anions in mitochondria and the end-products of lipid peroxidation in liver tissue, the amount of which was significantly increased in ethanol-treated rats. The disturbances in mitochondrial respiration, uncoupling of oxidative phosphorylation and decreasing of mitochondrial complex I, II, and IV activities in rats with steatohepatitis, were reverted by betulin administration. The increased susceptibility of mitochondria to Ca2+-induced permeability transition pore formation in the hepatitis group was improved in rats treated with betulin. In conclusion, betulin, having antioxidant properties, exerts a beneficial effect in the rat model of alcoholic steatohepatitis via prevention of liver mitochondria dysfunction, which may be attributed to the inhibition of mitochondrial permeability transition.

Inhibition of mitochondrial permeability transition by deletion of the ANT family and CypD.

The mitochondrial permeability transition pore (MPTP) has resisted molecular identification. The original model of the MPTP that proposed the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component was challenged when mitochondria from Ant1/2 double null mouse liver still had MPTP activity. Because mice express three Ant genes, we reinvestigated whether the ANTs comprise the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPTP formation, and when also given cyclosporine A (CsA), the MPTP was completely inhibited. Moreover, liver mitochondria from mice with quadruple deletion of Ant1, Ant2, Ant4, and Ppif (cyclophilin D, target of CsA) lacked Ca2+-induced MPTP formation. Inner-membrane patch clamping in mitochondria from Ant1, Ant2, and Ant4 triple null mouse embryonic fibroblasts showed a loss of MPTP activity. Our findings suggest a model for the MPTP consisting of two distinct molecular components: The ANTs and an unknown species requiring CypD.

Mondia whitei, an African Spice Inhibits Mitochondrial Permeability Transition in Rat Liver.

Mondia whitei is an African traditional spice with aphrodisiac properties. Inhibition of mitochondrial permeability transition (mPT) pore is an important cytoprotective process essential for cell survival. In this study, the effects of methanol extract (ME), dichloromethane (DCMF), ethyl acetate (EF), and methanol fractions (MF) of Mondia whitei on mPT, mitochondrial ATPase, lipid peroxidation, testosterone hormone (TH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were investigated; sperm analyses were also carried out. Male experimental rats were treated intraperitoneally with 25, 50, and 100 mg/kg bw of ME, DCMF, EF, and MF of Mondia whitei for two weeks. The positive and the negative controls received sildenafil citrate and the vehicle, respectively. The results showed that mPT was inhibited by MF at the highest dose. The ME, DCMF, and MF did not enhance ATPase activity. The levels of TH, FSH, and LH varied linearly with the drug dose only in EF. Malondialdehyde levels in the treated groups were significantly higher than the normal control. There were no significant defects in sperm produced by the animals in all the treated groups relative to the control. This study showed that the extract and fractions of Mondia whitei have cytoprotective effects and may prevent mitochondrial-mediated cell death.

Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step.

A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60–80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨm)-sensitive dye, TMRM, was used as the time of MPT (TMPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (TSP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (TSP-TMPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (TSP -TMPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5–20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H2O2 (200–800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.

Metabolite accumulation in VLCAD deficiency markedly disrupts mitochondrial bioenergetics and Ca2+ homeostasis in the heart.

We studied the effects of the major long-chain fatty acids accumulating in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, namely cis-5-tetradecenoic acid (Cis-5) and myristic acid (Myr), on important mitochondrial functions in isolated mitochondria from cardiac fibers and cardiomyocytes of juvenile rats. Cis-5 and Myr at pathological concentrations markedly reduced mitochondrial membrane potential (ΔΨm ), matrix NAD(P)H pool, Ca2+ retention capacity, ADP- (state 3) and carbonyl cyanide 3-chlorophenyl hydrazine-stimulated (uncoupled) respiration, and ATP generation. By contrast, these fatty acids increased resting (state 4) respiration (uncoupling effect) with the involvement of the adenine nucleotide translocator because carboxyatractyloside significantly attenuated the increased state 4 respiration provoked by Cis-5 and Myr. Furthermore, the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP, as well as the Ca2+ uptake blocker ruthenium red, fully prevented the Cis-5- and Myr-induced decrease in ΔΨm in Ca2+ -loaded mitochondria, suggesting, respectively, the induction of MPT pore opening and the contribution of Ca2+ toward these effects. The findings of the present study indicate that the major long-chain fatty acids that accumulate in VLCAD deficiency disrupt mitochondrial bioenergetics and Ca2+ homeostasis, acting as uncouplers and metabolic inhibitors of oxidative phosphorylation, as well as inducers of MPT pore opening, in the heart at pathological relevant concentrations. It is therefore presumed that a disturbance of bioenergetics and Ca2+ homeostasis may contribute to the cardiac manifestations observed in VLCAD deficiency.