Role Of Mitochondrial Permeability Transition Research Articles

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.

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.

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.

Abstract 854: The Role of Mitochondrial Permeability Transition in the Regulation of ETC Supercomplexes Assembly by OPA1

Optic atrophy type 1 (OPA1), a dynamin-related GTPase is the inner mitochondrial membrane (IMM) protein, which, in addition to mitochondrial fusion, participates in maintaining the structural integrity of the IMM. The function and topography of the IMM is also regulated by changes in the matrix volume. Opening of the mitochondrial permeability transition pores (PTP) is the leading cause of mitochondrial matrix swelling. However, the cause-and-effect relationship between OPA1 proteolytic cleavage and PTP induction remains unknown. Here, we elucidated the role of PTP in OPA1-mediated regulation of electron transport chain (ETC) supercomplexes (SCs) assembly. First, intact mitochondria isolated from the rat heart were used to analyze the effect of PTP induction on the cleavage of long (L)-OPA1. Calcium-induced mitochondrial swelling increased L-OPA1 cleavage (66%, P&lt;0.001) associated with accumulation of short (S)-OPA1. Inhibition of swelling by sanglifehrin A (a PTP inhibitor) prevented L-OPA cleavage whereas tert-butyl hydroperoxide (TBH) had no effect on L-OPA levels. Second, OPA1 knockdown (KD) by siRNA in HEK cells resulted in a 74% decrease (P&lt;0.001) in OPA1 protein expression but did not change the proportion of L-OPA1 to total OPA. Mitochondria isolated from OPA1 KD cells showed reduced calcium-stimulated swelling with no changes in protein levels of cyclophilin D, a major PTP regulator. OPA1 KD cells demonstrated a 20% increase (P&lt;0.05) in calcium-induced ROS generation. Analysis of individual ETC complexes revealed significant reduction of protein levels of the complexes I, III, and IV in OPA1 KD cells. Likewise, activities of complexes I and IV were reduced (48% and 37%, respectively, P&lt;0.01 for both). OPA1 KD cells contained 38% (P&lt;0.05) less ATP whereas the citrate synthase activity reflecting mitochondria mass was not affected. Blue native PAGE analysis demonstrated a 35% decrease (P&lt;0.01) of high molecular weight proteins (&gt;100 kDa) and a 24% decrease (P&lt;0.05) of the respirasome, the main SC, levels in OPA1 KD mitochondria. We conclude that mitochondrial swelling by PTP opening stimulates OPA1 cleavage, and reduced OPA1 levels result in cristae malformation including reduced respirasome levels and the ETC complexes activity.

Sex Differences in Ischemia/Reperfusion Injury: The Role of Mitochondrial Permeability Transition.

Brain and heart ischemia are among the leading causes of death and disability in both men and women, but there are significant sex differences in the incidence and severity of these diseases. Ca2+ dysregulation in response to ischemia/reperfusion injury (I/RI) is a well-recognized pathogenic mechanism leading to the death of affected cells. Excess intracellular Ca2+ causes mitochondrial matrix Ca2+ overload that can result in mitochondrial permeability transition (MPT), which can have severe consequences for mitochondrial function and trigger cell death. Recent findings indicate that estrogens and their related receptors are involved in the regulation of MPT, suggesting that sex differences in I/RI could be linked to estrogen-dependent modulation of mitochondrial Ca2+. Here, we review the evidence supporting sex differences in I/RI and the role of estrogen and estrogen receptors in producing these differences, the involvement of mitochondrial Ca2+ overload in disease pathogenesis, and the estrogen-dependent modulation of MPT that may contribute to sex differences.

Cyclosporin A protects against Lead neurotoxicity through inhibiting mitochondrial permeability transition pore opening in nerve cells

Mitochondria play a key role in the process of lead (Pb)-induced impairment in nervous system. To further clarify the underlying mechanism of Pb neurotoxicity, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and cyclophilin D (CyPD), a component of MPT pore (MPTP), in Pb-induced mitochondrial apoptosis in nerve cells. In SH-SY5Y and PC12 cells, Cyclosporin A (CSA), a special inhibitor of CyPD, could alleviate cell death, lactate dehydrogenase (LDH) leakage and adenosine 5 triphosphate (ATP) decrease caused by PbAc. In the following experiments, we found PbAc increased the protein level of CyPD and induced MPT pore (MPTP) opening. When cells were pretreated with CSA to inhibit MPTP opening, the Pb-induced impairment of mitochondrial morphology (swelling and rupture) and the loss of mitochondria were attenuated. In addition, CSA obviously ameliorated the Pb-induced damage of mitochondrial function, such as reactive oxygen species (ROS) boost and mitochondrial membrane potential (MMP) collapse, as well as the release of cytochrome C (Cyto C) and apoptosis-inducing factor (AIF) from mitochondria. These beneficial effects could finally result in cell survival under Pb-exposure conditions. Furthermore, scavenging ROS also significantly abrogated MPTP opening and attenuated Pb neurotoxicity. Therefore, we found that MPT played an important role in Pb-induced mitochondrial damage and, ultimately, cell death. Our results provided a potential strategy for inhibiting PbAc neurotoxicity. However, due to the high Pb concentrations used in this study further investigations at Pb concentrations closer to human exposure are needed to verify the results.

Alleviation of Lead-Induced Apoptosis by Puerarin via Inhibiting Mitochondrial Permeability Transition Pore Opening in Primary Cultures of Rat Proximal Tubular Cells.

Previous study has demonstrated that mitochondrial-dependent apoptotic pathway is involved in the nephroprotective effect of puerarin (PU) against lead-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells. To further clarify how PU exerts its antiapoptotic effects, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and subsequent apoptotic events in the process of PU against Pb-induced cytotoxicity in rPT cells. The results showed that Pb-mediated mitochondrial permeability transition pore (MPTP) opening together with mitochondrial cytochrome c release, activations of caspase-9 and caspase-3, and subsequent poly-ADP-ribose polymerase (PARP) cleavage can be effectively blocked by the addition of PU. Simultaneously, upregulation and downregulation of Bcl-2 and Bax with increased Bcl-2/Bax ratio due to PU administration further alleviated Pb-induced mitochondrial apoptosis. Moreover, PU can reverse Pb-induced ATP depletion by restoring mitochondrial fragmentation to affect ATP production and by regulating expression levels of ANT-1 and ANT-2 to improve ATP transport. In summary, PU produced a significant protection against Pb-induced mitochondrial apoptosis in rPT cells by inhibiting MPTP opening to ameliorate the mitochondrial dysfunction.

Mitochondrial permeability transition and its regulatory components are implicated in apoptosis of primary cultures of rat proximal tubular cells exposed to lead.

Previous studies have already demonstrated that mitochondria play a key role in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells. To further clarify the underlying mechanism of Pb-induced mitochondrial apoptosis, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and its regulatory components in Pb-induced apoptosis in rPT cells. Mitochondrial permeability transition pore (MPTP) opening together with disruption of mitochondrial ultrastructure, translocation of cytochrome c from mitochondria to cytoplasm and subsequent caspase-3 activation were observed in this study, suggesting that MPT is involved in Pb-induced apoptosis in rPT cells. Simultaneously, Pb-induced caspase-3 activation and apoptosis can be significantly inhibited by three MPTP inhibitors (CsA, DIDS, BA), which target different regulatory components of MPTP (Cyp-D, VDAC, ANT), respectively, demonstrating that Cyp-D, VDAC and ANT participate in MPTP regulation during lead exposure. Moreover, decreased ATP levels and increased ADP/ATP ratio induced by lead treatment can be significantly reversed by BA, indicating that Pb-mediated ANT dysfunction resulted in ATP depletion. In addition, up-regulation of VDAC-1, ANT-1 together with down-regulation of Cyp-D, VDAC-2 and ANT-2 at both the levels of transcription and translation were revealed in rPT cells under lead exposure conditions. In conclusion, Pb-mediated mitochondrial apoptosis in rPT cells is dependent on MPTP opening. Different expression levels in each isoform of three regulatory components contribute to alteration in their functions, which may promote the MPTP opening.

Hypoxic preconditioning-induced mitochondrial protection is not disrupted in a cell model of mtDNA T8993G mutation-induced F1F0-ATP synthase defect: the role of mitochondrial permeability transition

Transient opening of the mitochondrial permeability transition pore plays a crucial role in hypoxic preconditioning-induced protection. Recently, the cyclophilin-D component of the mitochondrial permeability transition pore has been shown to interact with and regulate the F1F0-ATP synthase. However, the precise role of the F1F0-ATP synthase and the interaction between cyclophilin-D and F1F0-ATP synthase in the mitochondrial permeability transition pore and hypoxic preconditioning remain uncertain. Here we found that a 1-h hypoxic preconditioning delayed apoptosis and improved cell survival after stimulation with various apoptotic inducers including H2O2, ionomycin, and arachidonic acid in mitochondrial DNA T8993G mutation (NARP) osteosarcoma 143B cybrids, an F1F0-ATP synthase defect cell model. This hypoxic preconditioning protected NARP cybrid cells against focal laser irradiation-induced oxidative stress by suppressing reactive oxygen species formation and preventing the depletion of cardiolipin. Furthermore, the protective functions of transient opening of the mitochondrial permeability transition pore in both NARP cybrids and wild-type 143B cells can be augmented by hypoxic preconditioning. Disruption of the interaction between cyclophilin-D and F1F0-ATP synthase by cyclosporin A attenuated the mitochondrial protection induced by hypoxic preconditioning in both NARP cybrids and wild-type 143B cells. Our results demonstrate that the interaction between cyclophilin-D and F1F0-ATP synthase is important in the hypoxic preconditioning-induced cell protection. This finding improves our understanding of the mechanism of mitochondrial permeability transition pore opening in cells in response to hypoxic preconditioning, and will be helpful in further developing new pharmacological agents targeting hypoxia–reoxygenation injury and mitochondria-mediated cell death

Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein

Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established; however, the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca2+ were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase 3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein.

The role of mitochondrial permeability transition in reperfusion-induced cardiomyocyte death depends on the duration of ischemia

Mitochondrial permeability transition (MPT) is critical in cardiomyocyte death during reperfusion but it is not the only mechanism responsible for cell injury. The objectives of the study is to investigate the role of the duration of myocardial ischemia on mitochondrial integrity and cardiomyocyte death. Mitochondrial membrane potential (ΔΨm, JC-1) and MPT (calcein) were studied in cardiomyocytes from wild-type and cyclophilin D (CyD) KO mice refractory to MPT, submitted to simulated ischemia and 10min reperfusion. Reperfusion after 15min simulated ischemia induced a rapid recovery of ΔΨm, extreme cell shortening (contracture) and mitochondrial calcein release, and CyD ablation did not affect these changes or cell death. However, when reperfusion was performed after 25min simulated ischemia, CyD ablation improved ΔΨm recovery and reduced calcein release and cell death (57.8±4.9% vs. 77.3±4.8%, P<0.01). In a Langendorff system, CyD ablation increased infarct size after 30min of ischemia (61.3±6.4% vs. 45.3±4.0%, P=0.02) but reduced it when ischemia was prolonged to 60min (52.8±8.1% vs. 87.6±3.7%, P<0.01). NMR spectroscopy in rat hearts showed a rapid recovery of phosphocreatine after 30min ischemia followed by a marked decay associated with contracture and LDH release, that were preventable with contractile blockade but not with cyclosporine A. In contrast, after 50min ischemia, phosphocreatine recovery was impaired even with contractile blockade (65.2±4% at 2min), and cyclosporine A reduced contracture, LDH release and infarct size (52.1±4.2% vs. 82.8±3.6%, P<0.01). In conclusion, the duration of ischemia critically determines the importance of MPT on reperfusion injury. Mechanisms other than MPT may play an important role in cell death after less severe ischemia.

Complement 1q-binding protein inhibits the mitochondrial permeability transition pore and protects against oxidative stress-induced death

Opening of the MPT (mitochondrial permeability transition) pore is a critical event in mitochondrial-mediated cell death. However, with the exception of CypD (cyclophilin D), the exact molecular composition of the MPT pore remains uncertain. C1qbp (complement 1q-binding protein) has recently been hypothesized to be an essential component of the MPT pore complex. To investigate whether C1qbp indeed plays a critical role in MPT and cell death, we conducted both gain-of-function and loss-of-function experiments in MEFs (mouse embryonic fibroblasts). We first confirmed that C1qbp is a soluble protein that localizes to the mitochondrial matrix in mouse cells and tissues. Similarly, overexpression of C1qbp in MEFs using an adenovirus resulted in its exclusive localization to mitochondria. To our surprise, increased C1qbp protein levels actually suppressed H2O2-induced MPT and cell death. Antithetically, knockdown of endogenous C1qbp with siRNA (small interfering RNA) sensitized the MEFs to H2O2-induced MPT and cell death. Moreover, we found that C1qbp could directly bind to CypD. Therefore C1qbp appears to act as an endogenous inhibitor of the MPT pore, most likely through binding to CypD, and thus protects cells against oxidative stress.

Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines

Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

C1qbp localizes to mitochondria and protects against oxidative stress‐induced mitochondrial dysfunction and cell death

Opening of the mitochondrial permeability transition (MPT) pore is thought to be a critical event in mitochondrial‐mediated cell death, resulting in dissipation of mitochondrial potential, inhibition of ATP synthesis, and mitochondrial swelling and rupture. However, with the exception of cyclophilin‐D (CypD), the exact molecular composition of the mitochondrial pore remains uncertain. To this end we conducted proteomic analyses of CypD‐containing complexes isolated from liver mitochondria. One protein we identified was complement component 1q binding protein (C1qbp), which has been proposed to play a role in many cellular functions including mitochondrial homeostasis. To investigate whether C1qbp plays a role in MPT and cell death, we conducted both gain‐ and loss‐of function experiments in mouse embryonic fibroblasts (MEFs). Overexpression of C1qbp in MEFs using an adenovirus resulted in its exclusive localization to mitochondria. To our surprise, increased C1qbp protein levels actually suppressed hydrogen peroxide‐induced MPT and cell death. Antithetically, knockdown of endogenous C1qbp with siRNA sensitized the MEFs to oxidative stress‐induced MPT and cell death. Therefore, C1qbp, a novel CypD binding protein, appears to act as an endogenous inhibitor of the MPT pore and protects cells against oxidative stress.

Abstract P81: Role of Mitochondrial Permeability Transition Pore (mPTP) in the Transition from Reversible to Irreversible Myocardial Ischemia and Reperfusion

Introduction . The aim of this study was to evaluate the role of mitochondrial permeability transition in the transition from reversible to irreversible injury following increasing times of ischemia- reperfusion. Methods . Rat hearts (n=72) were perfused according to Langendorff technique, arrested and submitted to global warm ischemia during 0 (sham), 10, 20, 30, 40 or 60 minutes at 37°C, and 60 min of reperfusion. Infarct size was evaluated by TTC staining and CK and LDH release. Functional recovery was assessed by rate-pressure product (RPP). Mitochondria were isolated to assess Ca 2+ resistance capacity (CRC) of the MPTP. Results . a transition from reversible to irreversible ischemia occurred between 20 and 30 minutes of warm ischemia. Hearts ischemic for less than 20 minutes exhibited better functionnal recovery with RPP averaging 31264±1341 and 28578±2176 mmHg/beats/min, respectively. In contrast, hearts that underwent 30, 40 and 60 min of ischemia displayed dramatic RPP decrease, averaging 12183±1895, 7411±578 and 4916±698 mmHg/min, respectively (p&lt;0.001 vs 20 minutes ischemia). CK and LDH release and TTC staining confirmed absence of infarction with ischemia &lt; 20 min (Panel A). In line with this, mean CRC was significantly reduced when ischemia lasted more than 20 minutes (p&lt;0.001, Panel B). Conclusion . This study shows a parallel evolution between the alteration of mitochondrial calcium retention capacity of the appearance of irreversible damage in the reperfused heart.

Having a swell time – mitochondrial morphology and plant cell death programmes

Cell death is a vital process in multi-cellular eukaryotes. Rather than being a contradiction in terms, this statement highlights the importance of limited and localized cell killing to the health and normal development of complex organisms. The main focus of this article is the role of mitochondrial morphological changes during cell death programmes, and the conserved role of mitochondrial permeability transition (increased permeability of either the outer or inner membrane) as an early mechanistic event preceding cell death in both plant and non-plant eukaryotes. A second focus of this article is a review of the terminology and fundamental paradigms underpinning cell death research. Because of the importance of the process of cell death, there has been an enormous quantity of research performed to try to understand the underlying biological mechanisms. One result of such a large and varied research effort, and a result that is perhaps particularly evident to investigators coming into the field anew, is that some of the basic tenets of cell death research appear to have become confused. In this short article, I make an attempt to clarify the subject, focussing on the role of mitochondria, and the difficulties in comprehensibility arising from the sometimes-erroneous, or at least unnecessarily confusing use of specific terminology; there are several key terms in the cell death literature that appear interchangeable when they are not, or are interchanged when they should not be.

Mechanisms of Chloroform-Induced Hepatotoxicity: Oxidative Stress and Mitochondrial Permeability Transition in Freshly Isolated Mouse Hepatocytes

The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform (12 mM) with hepatocytes resulted in cell death (alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3–5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.

Evaluation of Mitochondrial Function in Isolated Rat Hepatocytes and Mitochondria during Oxidative Stress

The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.

The mitochondrial permeability transition pore as a target of cardioprotective signaling

it is has been over two decades since Murry and colleagues ([15][1]) published their seminal study describing ischemic preconditioning, a phenomenon whereby brief antecedent periods of ischemia-reperfusion profoundly protect the heart against a subsequent lethal period of ischemia and reperfusion.

Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca 2+, AAI caused mitochondrial swelling, leakage of Ca 2+, membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid.