Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to sham group. Pretreatment with tempol (a SOD mimetic, 50 mg/kg) or co-enzyme Q10 (50 mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via the superoxide over-production and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.
Infarct size; Ischemia-reperfusion injury; Mitochondria; Superoxide; Tourniquet
Mitochondria-mediated apoptosis represents a central process driving age-related muscle loss. However, the temporal relation between mitochondrial apoptotic signaling and sarcopenia as well as the regulation of release of pro-apoptotic factors from the mitochondria has not been elucidated. In this study, we investigated mitochondrial apoptotic signaling in skeletal muscle of rats across a wide age range. We also investigated whether mitochondrial-driven apoptosis was accompanied by changes in the expression of Bcl-2 proteins and components of the mitochondrial permeability transition pore (mPTP). Analyses were performed on gastrocnemius muscle of 8-, 18-, 29- and 37- month-old male Fischer344×Brown Norway rats (9 per group). Muscle weight declined progressively with advancing age, concomitant with increased apoptotic DNA fragmentation. Cytosolic and nuclear levels of apoptosis inducing factor (AIF) and endonuclease G (EndoG) increased in old and senescent animals. In contrast, cytosolic levels of cytochrome c were unchanged with age. Mitochondrial Bcl-2, Bax and Bid increased dramatically in 37-month-old rats, with no changes in the Bax/Bcl-2 ratio in any of the age groups. Finally, expression of cyclophilin D was enhanced at very old age. Our findings indicate that the mitochondrial caspase-independent apoptotic pathway may play a more prominent role in skeletal muscle loss than caspase-mediated apoptosis.
sarcopenia; apoptosis; permeability transition pore; AIF; endonuclease G
To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O2 during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-XL in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-XL, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R.
With the advancement of age, skeletal muscle undergoes a progressive decline in mass, function, and regenerative capacity. Previously, our laboratory has reported an age-reduction in recovery and local induction of IGF-I gene expression with age following tourniquet (TK)-induced skeletal muscle ischemia/reperfusion (I/R). In this study, young (6 mo) and old (24–28 mo) mice were subjected to 2 hours of TK-induced ischemia of the hindlimb followed by 1, 3, 5, or 7 days of reperfusion. Real time-PCR analysis revealed clear age-related reductions and temporal alterations in the expression of IGF-I and individual IGF-I Ea and Eb splice variants. ELISA verified a reduction of IGF-I peptide with age following 7 days recovery from TK. Western blotting showed that the phosphorylation of Akt, mTOR, and FoxO3, all indicators of anabolic activity, were reduced in the muscles of old mice. These data indicate an age-related impairment of IGF-I expression and intracellular signaling does exist following injury, and potentially has a role in the impaired recovery of skeletal muscle with age.
Tourniquet; sarcopenia; muscle regeneration; mTOR; FoxO
Ischemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system.
Ischemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88).
Treatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.
Ablation of mouse occipital cortex induces precisely timed and uniform p53-modulated and Bax-dependent apoptosis of thalamocortical projection neurons in the dorsal lateral geniculate nucleus (LGN) by 7 days postlesion. We tested the hypothesis that this neuronal apoptosis is initiated by oxidative stress and the mitochondrial permeability transition pore (mPTP). Pre-apoptotic LGN neurons accumulate mitochondria, Zn2+ and Ca2+, and generate higher levels of reactive oxygen species (ROS), including superoxide, nitric oxide (NO) and peroxynitrite, than LGN neurons with an intact cortical target. Pre-apoptosis of LGN neurons is associated with increased formation of protein carbonyls, protein nitration, and protein S-nitrosylation. Genetic deletion of nitric oxide synthase 1 (nos1) and inhibition of NOS1 with nitroindazole protected LGN neurons from apoptosis, revealing NO as a mediator. Putative components of the mPTP are expressed in mouse LGN, including the voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (CyPD). Nitration of CyPD and ANT in LGN mitochondria occurs by 2 days after cortical injury. Chemical cross-linking showed that LGN neuron pre-apoptosis is associated with formation of CyPD and VDAC oligomers, consistent with mPTP formation. Mice without CyPD are rescued from neuron apoptosis as are mice treated with the mPTP inhibitors TRO-19622 and TAT-Bcl-XL-BH4. Manipulation of the mPTP markedly attenuated the early pre-apoptotic production of reactive oxygen/nitrogen species in target-deprived neurons. Our results demonstrate in adult mouse brain neurons that the mPTP functions to enhance ROS production and the mPTP and NO trigger apoptosis; thus, the mPTP is a target for neuroprotection in vivo.
Alzheimer’s disease; amyotrophic lateral sclerosis; axotomy; cell death; DNA damage; Parkinson’s disease; target deprivation; traumatic brain injury
In mouse models of familial amyotrophic lateral sclerosis (fALS) motor neurons are especially vulnerable to oxidative stresses in vitro. To determine whether this increased vulnerability also extends to motor nerve terminals in vivo, we assayed the effect of tourniquet-induced ischemia/reperfusion (I/R) injury on motor terminals innervating fast and slow hindlimb muscles in male G93A-SOD1 mice and their wild-type littermates. These mice also expressed yellow fluorescent protein (YFP) in motor neurons. We report that in SOD1-G93A/YFP mice the motor terminals innervating two predominantly fast muscles, extensor digitorum longus (EDL) and plantaris, were more vulnerable to I/R injury than motor terminals innervating the predominantly slow soleus muscle. The mean duration of EDL ischemia required to produce a 50% reduction in endplate innervation in SOD1-G93A/YFP mice was 26 min, compared to 45 min in YFP-only mice. The post-I/R destruction of EDL terminals in SOD1-G93A mice was rapid (< 2 hr) and was not duplicated by cutting the sciatic nerve at the tourniquet site. The increased sensitivity to I/R injury was evident in EDL muscles of SOD1-G93A/YFP mice as young as 31 days, well before the onset of motor neuron death at ~90 days. This early vulnerability to I/R injury may correlate with the finding (confirmed here) that in fALS mice motor nerve terminals innervating fast hindlimb muscles degenerate before those innervating slow muscles, at ages that precede motor neuron death. Early vulnerability of fast motor terminals to I/R injury thus may signal, and possibly contribute to, early events involved in motor neuron death.
Mitochondria serve as a “powerhouse” which provides near 90% of ATP necessary for cell life. However, recent studies provide strong evidence that mitochondria also play a central role in cell death. Mitochondrial permeability transition (mPT) at high conductance in response to oxidative or other cellular stresses is accompanied by pathological and non-specific mPT pore (mPTP) opening in the inner membrane of mitochondria. Mitochondrial PTP can serve as a target to prevent cell death under pathological conditions such as cardiac and brain ischemia/reperfusion injury and diabetes. On the other hand, mPTP can be used as an executioner to specifically induce cell death thus blocking tumorigenesis in cancer diseases. Despite many studies, the molecular identity of the mPTP remains unclear. Cyclophilin D (CyP-D) plays an essential regulatory role in pore opening. This review will discuss direct and indirect mechanisms underlying CyP-D interaction with a target protein of the mPTP complex. Understanding of the mechanisms of mPTP opening will be helpful to further develop new pharmacological agents targeting mitochondria-mediated cell death.
mitochondria; permeability transition pore; cyclophilin D; cell death
Generation of excessive reactive oxygen species (ROS) leads to mitochondrial dysfunction, apoptosis and necrosis in renal ischemia-reperfusion (IR) injury. Previously we showed that lentiviral vector mediated over-expression of superoxide dismutase-1 (SOD1) in proximal tubular epithelial cells (LLC-PK1) reduced cytotoxicity in an in vitro model of IR injury. Here, we examined the effects of SOD1 over-expression on mitochondrial signaling following ATP depletion-recovery (ATP-DR). To examine the role of mitochondrial ROS, a subset of cells were treated with the mitochondrial antioxidant, MitoTEMPO. ATP-DR-mediated increases in mitochondrial calcium ([Ca2+]m), loss of mitochondrial membrane potential (ΔΨm) and increase in mitochondrial permeability transition pore (MPTP) were attenuated by SOD1 and MitoTEMPO (P < 0.01). SOD1 prevented ATP-DR induced mitochondrial Bax translocation, although the release of pro-apoptotic proteins from mitochondria was not prevented by SOD1 alone and required the presence of both SOD1 and MitoTEMPO. SOD1 suppressed the increase in c-jun phosphorylation suggesting that JNK signaling regulates Bax translocation to mitochondria via ROS. ATP-DR-mediated changes in MPTP and mitochondrial signaling increased necrosis and apoptosis, both of which were partially attenuated by SOD1 and MitoTEMPO. These studies show that SOD1 and MitoTEMPO preserve mitochondrial integrity and attenuate ATP-DR mediated necrosis and apoptosis.
ATP depletion; mitochondrial membrane potential; calcium; cytochrome c; Bax; SOD1
The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories—a regimen that increases life span by ~30–40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.
Aging; Oxidative stress; Free radicals; Caloric restriction; Skeletal muscle; Mitochondria; Protein oxidation
Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H2O2 to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging.
Bcl-xL; cytochrome c; hexokinase (HK); mitochon-drial permeability transition pore (mPTP); superoxide; ANT, adenine nucleotide translocase; CHO, Chinese-hamster ovary; CsA, cyclosporine A; DEA, diethylamine NONOate diethylammonium salt; DTT, dithiothreitol; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GMS, 5 mM L-glutamate+2 mM L-malate+5 mM succinate; HK, hexokinase; IMS, intermembrane space; IP, ischaemic pre-conditioning; mPTP, mitochondrial permeability transition pore; OMM, outer mitochondrial membrane; ROS, reactive oxygen species; SOD, superoxide dismutase; VDAC, voltage-dependent anion channel
Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia–reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280 ± 60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.
Reperfusion therapy must be applied as soon as possible to attenuate the ischemic insult of acute myocardial infarction (AMI). However reperfusion is responsible for additional myocardial damage, which likely involves opening of the mitochondrial permeability transition pore (mPTP). In reperfusion injury, mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability. Major mechanisms of mitochondrial dysfunction include the long lasting opening of mPTPs and the oxidative stress resulting from formation of reactive oxygen species (ROS). Several signaling cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning, obtained with brief intermittent ischemia or with pharmacological agents. These pathways converge on a common target, the mitochondria, to preserve their function after ischemia/reperfusion. The present review discusses the role of mitochondria in cardioprotection, especially the involvement of adenosine triphosphate-dependent potassium channels, ROS signaling, and the mPTP. Ischemic postconditioning has emerged as a new way to target the mitochondria, and to drastically reduce lethal reperfusion injury. Several clinical studies using ischemic postconditioning during angioplasty now support its protective effects, and an interesting alternative is pharmacological postconditioning. In fact ischemic postconditioning and the mPTP desensitizer, cyclosporine A, have been shown to induce comparable protection in AMI patients.
Adenosine triphosphate-dependent potassium channels; Cardioprotection; Ischemia-reperfusion injury; Mitochondrial permeability transition pore; Reactive oxygen species
During the aging process, an accumulation of non-heme iron disrupts cellular homeostasis and contributes to the mitochondrial dysfunction typical of various neuromuscular degenerative diseases. Few studies have investigated the effects of iron accumulation on mitochondrial integrity and function in skeletal muscle and liver tissue. Thus, we isolated liver mitochondria (LM), as well as quadriceps-derived subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), from male Fischer 344× Brown Norway rats at 8, 18, 29 and 37 months of age. Non-heme iron content in SSM, IFM and LM was significantly higher with age, reaching a maximum at 37 months of age. The mitochondrial permeability transition pore (mPTP) was more susceptible to the opening in aged mitochondria containing high levels of iron (i.e. SSM and LM) compared to IFM. Furthermore, mitochondrial RNA oxidation increased significantly with age in SSM and LM, but not in IFM. Levels of mitochondrial RNA oxidation in SSM and LM correlated positively with levels of mitochondrial iron, whereas a significant negative correlation was observed between the maximum Ca2+ amounts needed to induce mPTP opening and iron contents in SSM, IFM and LM. Overall, our data suggest that age-dependent accumulation of mitochondrial iron may increase mitochondrial dysfunction and oxidative damage, thereby enhancing the susceptibility to apoptosis.
mitochondrial aging; mitochondrial iron homeostasis; mitochondrial permeability transition pore; mitochondrial RNA; oxidative stress; skeletal muscle subsarcolemmal and interfibrillar mitochondria
Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3-/-Sgcd-/- double targeted mice. Mechanistically, Nol3-/-Sgcd-/- mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause-effect relationships between mSOD1 and mitochrondiopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.
adenine nucleotide translocase; cristae remodeling; mutant SOD1; nitration; porin; ppif; voltage-dependent anion channel
The mitochondrion is the primary source of reactive oxygen species (ROS) in eukaryotic cells. With the aid of a novel mitochondrial matrix-targeted superoxide indicator, here we show that individual mitochondria undergo spontaneous bursts of superoxide generation, termed “superoxide flashes”. Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and reflect elementary events of superoxide production within single mitochondria across a wide diversity of cells. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore (mPTP) and are fueled by electron transfer complexes-dependent superoxide production. While decreased during cardiac hypoxia/anoxia, a flurry of superoxide flash activity contributes to the destructive rebound ROS burst observed during early reoxygenation after anoxia. The discovery of superoxide flashes reveals a novel mechanism for quantal ROS production by individual mitochondria and substantiates the central role of mPTP in oxidative stress related pathology in addition to its well-known role in apoptosis.
Urocortin (Ucn) protects hearts against ischemia and reperfusion injury whether given prior to ischemia or at reperfusion. Here we investigate the roles of protein kinase C, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute Ucn treatment improved hemodynamic recovery during reperfusion after 30 min global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of [3H]-2-deoxyglucose). Ucn pre-treatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that Ucn decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 hr) treatment with Ucn reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells which had been pre-treated with Ucn suggesting that this is the mechanism by which Ucn desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKCε or PKCα translocate to the mitochondria following acute Ucn treatment, inhibition of PKC with chelerythrine eliminated the effect of Ucn on oxidative stress. Our data suggests that acute Ucn treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.
reperfusion; mitochondria; oxygen radicals; peptide hormones
Skeletal muscle contractions increase superoxide anion in skeletal muscle extracellular space. We tested the hypotheses that 1) after an isometric contraction protocol, xanthine oxidase (XO) activity is a source of superoxide anion in the extracellular space of skeletal muscle and 2) the increase in XO-derived extracellular superoxide anion during contractions affects skeletal muscle contractile function. Superoxide anion was monitored in the extracellular space of mouse gastrocnemius muscles by following the reduction of cytochrome c in muscle microdialysates. A 15-min protocol of nondamaging isometric contractions increased the reduction of cytochrome c in microdialysates, indicating an increase in superoxide anion. Mice treated with the XO inhibitor oxypurinol showed a smaller increase in superoxide anions in muscle microdialysates following contractions than in microdialysates from muscles of vehicle-treated mice. Intact extensor digitorum longus (EDL) and soleus muscles from mice were also incubated in vitro with oxypurinol or polyethylene glycol-tagged Cu,Zn-SOD. Oxypurinol decreased the maximum tetanic force produced by EDL and soleus muscles, and polyethylene glycol-tagged Cu,Zn-SOD decreased the maximum force production by the EDL muscles. Neither agent influenced the rate of decline in force production when EDL or soleus muscles were repeatedly electrically stimulated using a 5-min fatiguing protocol (stimulation at 40 Hz for 0.1 s every 5 s). Thus these studies indicate that XO activity contributes to the increased superoxide anion detected within the extracellular space of skeletal muscles during nondamaging contractile activity and that XO-derived superoxide anion or derivatives of this radical have a positive effect on muscle force generation during isometric contractions of mouse skeletal muscles.
contractile function; free radicals; exercise
Reperfusion triggers an oxidative stress. We hypothesized that mild hypoxemia in reperfusion attenuates oxidative brain injury following hypoxia-ischemia (HI). In neonatal HI-mice, the reperfusion was initiated by reoxygenation with room air (RA) followed by the exposure to 100%, 21%, 18%, 15% oxygen for 60 minutes. Systemic oxygen saturation (SaO2), cerebral blood flow (CBF), brain mitochondrial respiration and permeability transition pore (mPTP) opening, markers of oxidative injury, and cerebral infarcts were assessed. Compared with RA-littermates, HI-mice exposed to 18% oxygen exhibited significantly decreased infarct volume, oxidative injury in the brain mitochondria and tissue. This was coupled with improved mitochondrial tolerance to mPTP opening. Oxygen saturation maintained during reperfusion at 85% to 95% was associated (r=0.57) with the best neurologic outcome. Exposure to 100% or 15% oxygen significantly exacerbated brain injury and oxidative stress. Compared with RA-mice, hyperoxia dramatically increased reperfusion CBF, but exposure to 15% oxygen significantly reduced CBF to values observed during the HI-insult. Mild hypoxemia during initial reperfusion alleviates the severity of HI-brain injury by limiting the reperfusion-driven oxidative stress to the mitochondria and mPTP opening. This suggests that at the initial stage of reperfusion, a slightly decreased systemic oxygenation (SaO2 85% to 95%) may be beneficial for infants with birth asphyxia.
brain; hypoxemia; hypoxia-ischemia; neuroprotection; oxidative stress; reperfusion
ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca2+ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F0F1ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca2+-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.
The mitochondrial adenine nucleotide translocators (Ant) are bi-functional proteins that transport ADP and ATP across the mitochondrial inner membrane, and regulate the mitochondrial permeability transition pore (mtPTP) which initiates apoptosis. The mouse has three Ant isoforms: Ant1 expressed in heart, muscle, and brain; Ant2 expressed in all tissues but muscle; and Ant4 expressed primarily in testis. Ant1-deficient mice manifest muscle and heart but not brain pathology. Brain Ant1 is induced by stress, while Ant2 is not. Ant1-deficient mice are resistant to death induced by systemic exposure to the brain excitotoxin, kainic acid (KA), and their hippocampal and cortical neurons are significantly more resistant to neuronal death induced by glutamate, KA, and etoposide‥ The mitochondrial membrane potential of Ant1-deficient brain mitochondria is increased and the mtPTP is more resistance to Ca++ induced permeability transition. Hence, Ant1-deficiency may protect the brain from excitotoxicity by desensitizing the mtPTP and by blocking the pro-apoptotic induction of Ant1 by stress.
Adenine nucleotide translocator; mitochondria; mitochondrial permeability transition; excitotoxicity; apoptosis
Severe myocardial dysfunction and tissue damage resulting from ischemia/reperfusion (I/R) is a common clinical scenario in patients with certain types of heart diseases and therapies such as thrombolysis, percutaneous coronary intervention, coronary artery bypass grafting, and cardiac transplantation. The underlining mechanism of endogenous cardiac protection after I/R injury has been a focus of current research. Growing evidences suggests that soluble receptor for advanced glycation end-products (sRAGE) has a cardioprotective effect; however, its role in I/R injury remains unclear. We hypothesized that exogenous administration of sRAGE during hypoxia/reoxygenation (H/R) induces cardioprotection by inhibiting cardiomyocyte apoptosis via multiple signals, involving mitochondrial membrane potential (MMP), the mitochondrial permeability transition pore (mPTP), mitochondrial cytochrome c, caspase-3, Bcl-2 and Bax. Neonatal rat cardiomyocytes underwent hypoxia for 3-h followed by 2-h reoxygenation or were treated with sRAGE for 10 min before H/R. Compared with H/R alone, sRAGE pretreatment reduced H/R-induced cardiomyocyte apoptosis from 27.9% ± 5.9% to 9.4% ± 0.7% (p < 0.05). In addition, sRAGE treatment significantly inhibited H/R-induced mitochondrial depolarization and mPTP opening, reduced mitochondrial cytochrome c leakage, caspase-3 and caspase-9 activity, and decreased the ratio of Bax to Bcl-2. Therefore, we conclude that the exogenous administration of sRAGE during H/R is involved in cardioprotection by inhibiting apoptosis via the mitochondrial pathway, which, if further confirmed in vivo, may have important clinical implications during H/R.
sRAGE; apoptosis; ischemia; reperfusion; mitochondria
In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca2+ fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation.
Myocardial ischemia-reperfusion injury is a major cause of morbidity and mortality in developed countries. To date, the only treatment of complete ischemia is to restore blood flow; thus the search for new cardioprotective approaches is absolutely necessary to reduce the mortality associated with myocardial ischemia.
Ischemia has long been considered to result in necrotic tissue damage but the reduction in oxygen supply can also lead to apoptosis. Therefore, in the last few years, mitochondria have become the subject of growing interest in myocardial ischemia-reperfusion since they are strongly involved in the regulation of the apoptotic process. Indeed, during ischemia-reperfusion, pathological signals converge in the mitochondria to induce permeabilization of the mitochondrial membrane. Two classes of mechanisms, which are not mutually exclusive, emerged to explain mitochondrial membrane permeabilization. The first occurs via a non-specific channel known as the mitochondrial permeability transition pore (mPTP) in the inner and the outer membranes causing disruption of the impermeability of the inner membrane, and ultimately complete inhibition of mitochondrial function. The second mechanism, involving only the outer membrane, induces the release of cell death effectors. Thus, drugs able to block or to limit mitochondrial membrane permeabilization may be cytoprotective during ischemia-reperfusion. The objective of this review is to examine the pharmacological strategies capable of inhibiting mitochondrial membrane permeabilization induced by myocardial ischemia-reperfusion.
Adenosine Triphosphatases; metabolism; Apoptosis; Mitochondrial Membrane Transport Proteins; antagonists & inhibitors; metabolism; Mitochondrial Membranes; drug effects; metabolism; Myocardial Reperfusion Injury; drug therapy; prevention & control; Permeability; bcl-2 Homologous Antagonist-Killer Protein; metabolism; mitochondria; heart; ischemia-reperfusion; necrosis; apoptosis; mitochondrial membrane permeability.