Superoxide flashes: early mitochondrial signals for oxidative stress-induced apoptosis

Irreversible mitochondrial permeability transition and the resultant cytochrome c release signify the commitment of a cell to apoptotic death. However, the role of transient MPT (tMPT) because of flickering opening of the mitochondrial permeability transition pore remains elusive. Here we show that tMPT and the associated superoxide flashes (i.e. tMPT/superoxide flashes) constitute early mitochondrial signals during oxidative stress-induced apoptosis. Selenite (a ROS-dependent insult) but not staurosporine (a ROS-independent insult) stimulated an early and persistent increase in tMPT/superoxide flash activity prior to mitochondrial fragmentation and a global ROS rise, independently of Bax translocation and cytochrome c release. Selectively targeting tMPT/superoxide flash activity by manipulating cyclophilin D expression or scavenging mitochondrial ROS markedly impacted the progression of selenite-induced apoptosis while exerting little effect on the global ROS response. Furthermore, the tMPT/superoxide flash served as a convergence point for pro- and anti-apoptotic regulation mediated by cyclophilin D and Bcl-2 proteins. These results indicate that tMPT/superoxide flashes act as early mitochondrial signals mediating the apoptotic response during oxidative stress, and provide the first demonstration of highly efficacious local mitochondrial ROS signaling in deciding cell fate.     

线粒体稳定高钙信号诱发超氧炫

线粒体对于细胞钙信号和活性氧信号转导有重要的调控作用．超氧炫是新近发现的单个线粒体超氧阴离子短时程爆发现象,反映了活性氧生成动力学的一种新形式．线粒体钙信号作为重要的细胞功能调控信号,能否及如何调控超氧炫尚待深入研究．本研究对HeLa细胞进行高胞外钙和离子霉素刺激,或用皂苷穿孔细胞质膜后置于高钙细胞内液中,两种方法均显著增加了超氧炫发生的频率．其中,穿孔细胞胞浆高钙诱导的超氧炫依赖于线粒体钙单向转运体,表明超氧炫由线粒体基质内高钙信号所诱发．重要的是,离子霉素诱导的超氧炫发生频率与线粒体稳态钙水平线性相关,而与瞬态线粒体钙无相关性,提示钙离子对超氧炫的调控是一个多步骤、相对缓慢的过程．综上,线粒体基质的稳态高钙是超氧炫的重要调控因子．     

Superoxide Flashes in Mouse Skeletal Muscle Are Produced by Discrete Arrays of Active Mitochondria Operating Coherently

Reactive Oxygen Species (ROS) constitute important intracellular signaling molecules. Mitochondria are admitted sources of ROS, especially of superoxide anions through the electron transport chain. Here the mitochondria-targeted ratiometric pericam (RPmt) was used as a superoxide biosensor, by appropriate choice of the excitation wavelength. RPmt was transfected in vivo into mouse muscles. Confocal imaging of isolated muscle fibers reveals spontaneous flashes of RPmt fluorescence. Flashes correspond to increases in superoxide production, as shown by simultaneous recordings of the fluorescence from MitoSox, a mitochondrial superoxide probe. Flashes occur in all subcellular populations of mitochondria. Spatial analysis of the flashes pattern over time revealed that arrays of mitochondria work as well-defined superoxide-production-units. Increase of superoxide production at the muscle fiber level involves recruitment of supplemental units with no increase in per-unit production. Altogether, these results demonstrate that superoxide flashes in muscle fibers correspond to physiological signals linked to mitochondrial metabolism. They also suggest that superoxide, or one of its derivatives, modulates its own production at the mitochondrial level.     

Superoxide flashes reveal novel properties of mitochondrial reactive oxygen species excitability in cardiomyocytes

Superoxide flash represents quantal and bursting production of mitochondrial reactive oxygen species (ROS) instigated by transient opening of the mitochondrial permeability transition pore (mPTP). Given their critical role in metabolism, ischemia-reperfusion injury, and apoptosis, characterization of flash properties would be valuable to further mechanistic and physiological studies of this newly discovered mitochondrial phenomenon. Here we developed the flash detector FlashSniper based on segmentation of two-dimensional feature maps extracted from time-lapse confocal image stacks, and on the theory for correcting optical distortion of flash-amplitude histograms. Through large-scale analysis of superoxide flashes in cardiomyocytes, we demonstrated uniform mitochondrial ROS excitability among subsarcolemmal and intermyofibrillar mitochondria, and exponential distribution of intervals between consecutive flash events. Flash ignition displayed three different patterns: an abrupt rise from quiescence (44%), a rise with an exponential foot (27%), or a rise occurring after a pedestal precursor (29%), closely resembling action-potential initiation in excitable cells. However, the optical blurring-corrected amplitudes of superoxide flashes were highly variable, as were their durations, indicating stochastic automaticity of single-mitochondrion ROS excitation. Simultaneous measurement of mitochondrial membrane potential revealed that graded, rather than all-or-none, depolarization mirrored the precursor and the primary peak of the flash. We propose that superoxide flash production is a regenerative process dominated by stochastic, autonomous recruitment of a limited number of units (e.g., mPTPs) in single mitochondria.     

Superoxide flashes in single mitochondria

In quiescent cells, mitochondria are the primary source of reactive oxygen species (ROS), which are generated by leakiness of the electron transport chain (ETC). High levels of ROS can trigger cell death, whereas lower levels drive diverse and important cellular functions. We show here by employing a newly developed mitochondrial matrix-targeted superoxide indicator, 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 provide a vital source of superoxide production across many different cell types. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore stimulating superoxide production by the ETC. Furthermore, we observe a flurry of superoxide flash activity during reoxygenation of cardiomyocytes after hypoxia, which is inhibited by the cardioprotective compound adenosine. We propose that superoxide flashes could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.     

Mitochondrial superoxide mediates labile iron level: evidence from Mn-SOD-transgenic mice and heterozygous knockout mice and isolated rat liver mitochondria

Superoxide is the main reactive oxygen species (ROS) generated by aerobic cells primarily in mitochondria. It is also capable of producing other ROS and reactive nitrogen species (RNS). Moreover, superoxide has the potential to release iron from its protein complexes. Unbound or loosely bound cellular iron, known as labile iron, can catalyze the formation of the highly reactive hydroxyl radical. ROS/RNS can cause mitochondrial dysfunction and damage. Manganese superoxide dismutase (Mn-SOD) is the chief ROS-scavenging enzyme and thereby the primary antioxidant involved in protecting mitochondria from oxidative damage. To investigate whether mitochondrial superoxide mediates labile iron in vivo, the levels of labile iron were determined in the tissues of mice overexpressing Mn-SOD and heterozygous Mn-SOD-knockout mice. Furthermore, the effect of increased mitochondrial superoxide generation on labile iron levels was determined in isolated rat liver mitochondria exposed to various electron transport inhibitors. The results clearly showed that increased expression of Mn-SOD significantly lowered the levels of labile iron in heart, liver, kidney, and skeletal muscle, whereas decreased expression of Mn-SOD significantly increased the levels of labile iron in the same organs. In addition, the data showed that peroxidative damage to membrane lipids closely correlated with the levels of labile iron in various tissues and that altering the status of Mn-SOD did not alter the status of other antioxidant systems. Results also showed that increased ROS production in isolated liver mitochondria significantly increased the levels of mitochondrial labile iron. These findings constitute the first evidence suggesting that mitochondrial superoxide is capable of releasing iron from its protein complexes in vivo and that it could also release iron from protein complexes contained within the organelle.     

Energy metabolism in uncoupling protein 3 gene knockout mice

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.     

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Synergistic Triggering of Superoxide Flashes by Mitochondrial Ca2+ Uniport and Basal Reactive Oxygen Species Elevation

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca²⁺ uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca²⁺ and ROS. Blocking mitochondrial Ca²⁺ transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca²⁺ uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca²⁺ or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca²⁺ and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca²⁺ and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca²⁺ and ROS signals.     

Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H(2)O(2) and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H(2)O(2) production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.     

Regulation of superoxide flashes by transient and steady mitochondrial calcium elevations

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling.As a newly discovered universal and fundamental mitochondrial phenomenon,superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria.Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown.Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence,and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response.Quantitatively,the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0?F/F0 of Rhod-2 signal.In contrast,large mitochondrial calcium transients(e.g.,peak△F/F0~2.8,duration~2 min)in the absence of steady-state elevations failed to alter the flash activity.These results indicate that physiological levels of sustained,but not transient,mitochondrial calcium elevation acts as a potent regulator of superoxide flashes,but its mechanism of action likely involves a multi-step,slow-onset process.     

Significance of uncoupling protein 3 in mitochondrial function upon mid- and long-term dietary high-fat exposure

Uncoupling protein 3 (UCP3) may reduce mitochondrial ROS production, and thereby protect against mitochondrial dysfunction in skeletal muscle. UCP3 has been suggested to specifically fulfill this role under high-fat conditions. Here we show that UCP3 knockout mice indeed have elevated mitochondrial ROS production after short-term (8 weeks) high-fat feeding. After 26 weeks of high-fat feeding, UCP3 knockout mice exhibited reduced mitochondrial function as measured ex vivo in isolated mitochondria. In conclusion, these data suggest that UCP3 may have a role in the protection of mitochondria against lipid-induced mitochondrial dysfunction, but only after long-term exposure to high-fat.     

Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Regulate Cortical Neural Progenitor Differentiation

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.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

UCP-mediated energy depletion in skeletal muscle increases glucose transport despite lipid accumulation and mitochondrial dysfunction

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.     

Enhanced lipid-but not carbohydrate-supported mitochondrial respiration in skeletal muscle of PGC-1α overexpressing mice

Skeletal muscle mitochondrial dysfunction has been linked to several disease states as well as the process of aging. A possible factor involved is the peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α), a major player in the regulation of skeletal muscle mitochondrial metabolism. However, it is currently unknown whether PGC-1α, besides stimulating mitochondrial proliferation, also affects the functional capacity per mitochondrion. Therefore, we here tested whether PGC-1α overexpression, besides increasing mitochondrial content, also leads to intrinsic mitochondrial adaptations. Skeletal muscle mitochondria from 10 male, muscle-specific PGC-1α overexpressing mice (PGC-1αTg) and 8 wild-type (WT) mice were isolated. Equal mitochondrial quantities were then analyzed for their oxidative capacity by high-resolution respirometry, fuelled by a carbohydrate-derived (pyruvate) and a lipid (palmitoyl-CoA plus carnitine) substrate. Additionally, mitochondria were tested for reactive oxygen species (superoxide) production and fatty acid (FA)-induced uncoupling. PGC-1αTg mitochondria were characterized by an improved intrinsic mitochondrial fat oxidative capacity as evidenced by pronounced increase in ADP-stimulated respiration (P < 0.001) and maximal uncoupled respiration (P < 0.001) upon palmitoyl-CoA plus carnitine. Interestingly, intrinsic mitochondrial capacity on a carbohydrate-derived substrate tended to be reduced. Furthermore, the sensitivity to FA-induced uncoupling was diminished in PGC-1αTg mitochondria (P = 0.02) and this was accompanied by a blunted reduction in mitochondrial ROS production upon FAs in PGC-1αTg versus WT mitochondria (P = 0.04). Uncoupling protein 3 (UCP3) levels were markedly reduced in PGC-1αTg mitochondria (P < 0.001). Taken together, in addition to stimulating mitochondrial proliferation in skeletal muscle, we show here that overexpression of PGC-1α leads to intrinsic mitochondrial adaptations that seem restricted to fat metabolism.     

Uncoupling protein 3 protects aconitase against inactivation in isolated skeletal muscle mitochondria

Mitochondrial uncoupling proteins only catalyse proton transport when they are activated. Activators include superoxide and reactive alkenals, suggesting new physiological functions for UCP2 and UCP3: their activation by superoxide when protonmotive force is high causes mild uncoupling, which lowers protonmotive force and attenuates superoxide generation by the electron transport chain. This feedback loop acts to prevent excessive mitochondrial superoxide production. Superoxide inactivates aconitase in the mitochondrial matrix, so aconitase activity provides a sensitive measure of the effects of UCPs on matrix superoxide. We find that inhibition of UCP3 in isolated skeletal muscle mitochondria by GDP decreases aconitase activity by 25% after 20 min incubation. The GDP effect is absent in skeletal muscle mitochondria from UCP3 knockout mice, showing that it is mediated by UCP3. Protection of aconitase by UCP3 in the absence of nucleotides does not require added fatty acids. The purine nucleoside diphosphates and triphosphates cause aconitase inactivation, but the monophosphates and CDP do not, consistent with the known nucleotide specificity of UCP3. The IC₅₀ for GDP is about 100 μM. These findings support the proposal that UCP3 attenuates endogenous radical production by the mitochondrial electron transport chain at high protonmotive force.