Mitochondrial Superoxide Flashes Research Articles

Irisin Protects Heart Against Ischemia-Reperfusion Injury Through a SOD2-Dependent Mitochondria Mechanism.

Irisin, a muscle-origin protein derived from the extracellular domain of the fibronectin domain-containing 5 protein (FNDC5), has been shown to modulate mitochondria welfare through paracrine action. Here, we test the hypothesis that irisin contributes to cardioprotection after myocardial infarction by preserving mitochondrial function in cardiomyocytes. Animal model studies show that intravenous administration of exogenous irisin produces dose-dependent protection against ischemia/reperfusion (I/R)-induced injury to the heart as reflected by the improvement of left ventricular ejection fraction and the reduction in serum level of cTnI (n = 15, P < 0.05). I/R-induced apoptosis of cardiomyocytes is reduced after irisin treatment. The irisin-mediated protection has, at least in part, an effect on mitochondrial function because administration of irisin increases irisin staining in the mitochondria of the infarct area. Irisin also reduces I/R-induced oxidative stress as determined by mitochondrial membrane potential evaluation and superoxide FLASH event recording (n = 4, P < 0.05). The interaction between irisin and superoxide dismutase2 (SOD2) plays a key role in the protective process because irisin treatment increases SOD activity (n = 10, P < 0.05) and restores the mitochondria localization of SOD2 in cardiomyocytes (n = 5, P < 0.05). These results demonstrate that irisin plays a protective role against I/R injury to the heart. Targeting the action of irisin in mitochondria presents a novel therapeutic intervention for myocardial infarction.

Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice

BackgroundMutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans.MethodsWe compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.ResultsAbout 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40 ± 1 vs. 86 ± 1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46 ± 3 to 59 ± 2 mN/gr) in CASQ1-null mice.ConclusionsOur findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0035-9) contains supplementary material, which is available to authorized users.

Subsarcolemmal mitochondrial flashes induced by hypochlorite stimulation in cardiac myocytes

Mitochondrial superoxide flash (mitoflash) reflects quantal and bursting superoxide production and concurrent membrane depolarization triggered by transient mitochondrial permeability transition in many types of cells, at the level of single mitochondria. Here we investigate reactive oxygen species (ROS)-mediated modulation of mitoflash activity in cardiac myocytes and report a surprising finding that hypochlorite ions potently and preferentially triggered mitoflashes in the subsarcolemmal mitochondria (SSM), whereas hydrogen peroxide (H2O2) elicited mitoflash activity uniformly among SSM and interfibrillar mitochondria (IFM). The striking SSM mitoflash response to hypochlorite stimulation remained intact in cardiac myocytes from NOX2-deficient mice, excluding local NOX2-mediated ROS as the major player. Furthermore, it occurred concomitantly with SSM Ca2+ accumulation and local Ca2+ and CaMKII signaling played an important modulatory role by altering frequency and unitary properties of SSM mitoflashes. These findings underscore the functional heterogeneity of SSM and IFM and the oxidant-specific responsiveness of mitochondria to ROS, and may bear important ramifications in devising therapeutic strategies for the treatment of oxidative stress-related heart diseases.

Abstract 325: Dissociation of Oxidative Stress from High Fatty Acid-Induced Cardiac Myocyte Death

Metabolic syndrome, featured by obesity and diabetes, is an independent risk factor for cardiovascular disease. The high availability of free fatty acids characteristic of obesity has been proposed to increase fatty acid oxidation, impair mitochondrial function, stimulate reactive oxygen species (ROS) production and eventually lead to oxidative stress, which then would cause myocyte damage and heart dysfunction. However, the role of oxidative stress as the initial cause of obesity-induced heart failure is controversial. We evaluated the time-dependent effect of fatty acids in vitro in adult rat cardiac myocytes (treated with palmitate and oleate, the major saturated and unsaturated fatty acids in the plasma, respectively), and in vivo in mice fed a high-fat diet (fat calories = 60%). Palmitate and oleate dose-dependently stimulated mitochondrial respiration and superoxide flash activity, a respiration-coupled bursting ROS production event in single mitochondria. The flash frequency increased 2-fold (0.1 mM, the physiological level in plasma) and 3-fold (0.3 mM, the plasma level found in obese subjects) after only 2 hr incubation and up to 24 hr, increase that could be blocked by the CPT1 inhibitor etomoxir (100uM). Surprisingly, there was no obvious mitochondrial oxidative stress, although the high level of fatty acids caused mPTP opening and cell death at 48 hr. Further, aconitase activity was normal and NAD/NADH ratio was decreased, indicating a more reduced status rather than oxidative stress. Intriguingly, mitochondrial membrane potential was slightly lowered after only 2 hr of treatment. Finally, the high-fat diet-fed mice developed heart dysfunction after 12 weeks of treatment, but oxidative stress only appeared after 18 weeks. In the light of these results, we hypothesize that high fatty acid supply induced multiple adaptive and mal-adaptive responses in cardiac myocytes. The increased mitochondrial respiration and bursting ROS production is offset by a mild uncoupling and increased reducing equivalents. Therefore, oxidative stress is unlikely an initial cause of myocyte death and heart dysfunction in obesity, but rather a consequence of mitochondrial dysfunction, although the exact cause of this dysfunction remains to be elucidated.

Fission Promotes Respiration and ROS Production in Individual Mitochondria

The balance between fission and fusion essentially participates in mitochondrial biogenesis, transportation and maturation. Whether endogenous fission and fusion processes regulate mitochondrial respiration and ROS production in the heart is not well studied. Cardiac myocytes are excitable cells that featured by dynamic intracellular calcium regulations. Increased intracellular calcium is known to activate dynamin-like protein 1 (DLP1), a key regulator of mitochondrial fission. In this study, we used adenovirus mediated gene transfer of a mitochondrial targeted superoxide indicator and confocal microscopy to investigate the role of fission in mitochondrial respiration and ROS production regulation. We monitored the mitochondrial superoxide flashes (SOFs), which are quantal events of superoxide generation coupled with single mitochondrial respiration, in cultured cardiomyocytes. Acute application of Mdivi-1 (50 µM), a DLP inhibitor suppressed resting SOF activity in rat adult cardiomyocytes and H9C2 cardiac myoblasts. Furthermore, metabolic substrates (20 mM Pyruvate or 20 mM Glucose)-induced SOF events were blocked by Mdivi-1, indicating an essential role of fission in maintaining normal mitochondrial function. Acute stimulation of fission by KCl (50 mM), which induced calcium transients, dramatically stimulated the SOF (SOF frequency from 0.97±0.36 to 2.46±0.34 per 1000 µm2 per 100 s) in H9C2 cells. The effect of KCl is blocked by Mdivi-1. Interestingly, long-term incubation of high glucose (35 mM for 48 hr), which has been shown to induce mitochondrial fragmentation, failed to stimulate SOF activity in H9C2 cells. These results reveal an essential role of mitochondrial fission in regulating physiological functions of individual mitochondria and also highlight that chronically disturbing fission/fusion dynamics may have detrimental effects.

Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of amyloid beta1−42 on neural progenitor cell proliferation

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6–24 hours of exposure to aggregating amyloid β-peptide (Aβ1–42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1–42, during which time global cellular reactive oxygen species increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1–42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1–42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ.

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.

Abstract 224: Mitochondrial Complex I Deficiency Promotes Oxidative Stress During Ischemia Reperfusion Of Cardiac Myocytes

Normal cardiac function relies on highly coordinated intracellular events, such as calcium cycling and contraction, with adequate mitochondrial energy metabolism. However, mitochondrial respiration unavoidably produces reactive oxygen species (ROS) as electrons leak from the electron transport chain (ETC). Complex I of the ETC is believed to be the major site for ROS generation in the mitochondria. However, suppression of Complex I activity by chemical inhibitors leads to oxidative cell damage. In this study, we used a genetic model of Complex I deficiency, in which a key component of Complex I, Ndufs4, was deleted in the heart, to determine the causal role of Complex I in ischemia-reperfusion-induced oxidative stress in adult cardiac myocytes. Germline deletion of Ndufs4 in the heart (Ndufs4H-/-) leads to a ~75% decline of Complex I activity in cardiac mitochondria without obvious disease phenotype in the mice. As predicted, the mitochondrial respiration-coupled superoxide production events, superoxide flashes, were significantly decreased at baseline in the Ndufs4H-/- myocytes. Respiration substrate (pyruvate, 20 mM) failed to stimulate mitochondrial superoxide flash production in Ndufs4H-/- myocytes. This is accompanied by the slightly decreased steady state intracellular and mitochondrial ROS levels determined by the targeted H2O2 indicator, Hyper. The intracellular redox homeostasis is also tilted toward more reduced state, since the NADH/NAD ratio increased 67%. Surprisingly, ischemia reperfusion mimetic treatment of the myocytes caused dramatic increase in mitochondrial ROS production in Ndufs4H-/- groups, which contributed to the elevated overall cellular oxidative status. Overexpression of catalase in the mitochondria prevented these effects. Mechanistically, increased reducing equivalent (NADH) contributed to the dramatic ROS production during ischemia and reperfusion in Ndufs4H-/- myocytes. In summary, mitochondrial Complex I plays a critical role in controlling mitochondrial and cytosolic ROS homeostasis under normal conditions, and compromised Complex I function leads to accumulation of electron donors that paradoxically promote ROS production during ischemia reperfusion.

Imaging ROS signaling in cells and animals

Reactive oxygen species (ROS) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. Understanding such complexity of the ROS signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ROS dynamics at high selectivity, sensitivity, and spatiotemporal resolution. The past decade has witnessed significant progress in ROS imaging at levels of intact cells, whole organs or tissues, and even live organisms. In particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein-based ROS indicators, the use of protein indicator-expressing animal models, and the advent of in vivo imaging technology. Innovative ROS imaging has led to important discoveries in ROS signaling—for example, mitochondrial superoxide flashes as elemental ROS signaling events and hydrogen peroxide transients for wound healing. This review aims at providing an update of the current status in ROS imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions.

Superoxide constitutes a major signal of mitochondrial superoxide flash

AimsMitochondrial flashes detected with an N- and C-terminal circularly-permuted yellow fluorescent protein (cpYFP) have been thought to represent transient and quantal bursts of superoxide production under physiological, stressful and pathophysiological conditions. However, the superoxide nature of the cpYFP-flash has been challenged, considering the pH-sensitivity of cpYFP and the distinctive regulation of the flash versus the basal production of mitochondrial reactive oxygen species (ROS). Thus, the aim of the study is to further determine the origin of mitochondrial flashes. Main methodsWe investigated the origin of the flashes using the widely-used pH-insensitive ROS indicators, mitoSOX, an indicator for superoxide, and 2, 7-dichlorodihydrofluorescein diacetate (DCF), an indicator for H2O2 and other oxidants. Key findingsRobust, quantal, and stochastic mitochondrial flashes were detected with either mitoSOX or DCF in several cell-types and in mitochondria isolated from the heart. Both mitoSOX-flashes and DCF-flashes showed similar incidence and kinetics to those of cpYFP-flashes, and were equally sensitive to mitochondria-targeted antioxidants. Furthermore, they were markedly decreased by inhibitors or an uncoupler of the mitochondrial electron transport chain, as is the case with cpYFP-flashes. The involvement of the mitochondrial permeability transition pore in DCF-flashes was evidenced by the coincidental loss of mitochondrial membrane potential and matrix-enriched rhod-2, as well as by their sensitivity to cyclosporine A. SignificanceThese data indicate that all the three types of mitochondrial flashes stem from the common physiological process of bursting superoxide and ensuing H2O2 production in the matrix of single mitochondrion.

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ

ObjectiveMitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.MethodsChondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.ResultsCultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm2*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.ConclusionsThe superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.

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 Ca(2+) uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca(2+) and ROS. Blocking mitochondrial Ca(2+) transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca(2+) uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca(2+) or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca(2+) 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 Ca(2+) and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca(2+) and ROS signals.

N-Acetylcysteine, a Potent Anti-Oxidant, Rescues the Malignant Hyperthermia and Environmental Heat Stroke Phenotype of Calsequestrin-1 Knockout Mice

Calsequestrin type 1 (CASQ1) is both the major Ca2+ binding protein in the sarcoplasmic reticulum (SR) in skeletal muscle and a direct modulator of type 1 ryanodine receptor (RYR1) Ca2+-release channel activity. Mice lacking CASQ1 (CASQ1-null) exhibit increased susceptibility for Malignant Hyperthermia (MH) and Environmental Heat Stroke (EHS), life-threatening disorders triggered by volatile anaesthetics and high temperature, respectively. Excessive oxidative stress during MH/EHS crises are proposed to result in increased RYR1 nitrosylation, and subsequently, enhanced SR Ca2+ leak that leads to hypercontractures and rhabdomyolysis. Here, we treated CASQ1-null mice for 2 months (from 2-to-4 months of age) with N-acetylcysteine (NAC, a potent anti-oxidant) provided ad libitum in their drinking water (1% w/v). NAC treatment significantly protected CASQ1-null mice from lethal episodes induced by both halothane (2%, 1h at 32°C) and heat challenge (41°C, 1h) with the rate of mortality being 79 vs 25% and 86 vs 30% in control vs NAC-treated mice, respectively. This protection resulted from several factors including: a) reduced oxidative stress measured as both GSH/GSSG ratio and the frequency of mitochondrial superoxide flash activity (P<0.05); b) decreased maximum core temperature during heat challenge (from 42.1 to 40.8°C); and c) reduced number of fibers undergoingrhabdomyolysis (from 37.6 to 11.6 %). Furthermore, in-vitro contracture test (IVCT) showed that the threshold for caffeine-induced contracture is back to normal values. These results support the hypothesis that excessive production of reactive oxygen species plays a critical role in the onset of MH/EHS crises. Therefore, anti-oxidants may be useful for the treatment and prevention of oxidative stress related skeletal muscle disorders.

Mitochondrial Superoxide Production Negatively Regulates Neural Progenitor Proliferation and Cerebral Cortical Development

Although high amounts of reactive oxygen species (ROS) can damage cells, ROS can also play roles as second messengers, regulating diverse cellular processes. Here, we report that embryonic mouse cerebral cortical neural progenitor cells (NPCs) exhibit intermittent spontaneous bursts of mitochondrial superoxide (SO) generation (mitochondrial SO flashes) that require transient opening of membrane permeability transition pores (mPTP). This quantal SO production negatively regulates NPC self-renewal. Mitochondrial SO scavengers and mPTP inhibitors reduce SO flash frequency and enhance NPC proliferation, whereas prolonged mPTP opening and SO generation increase SO flash incidence and decrease NPC proliferation. The inhibition of NPC proliferation by mitochondrial SO involves suppression of extracellular signal-regulated kinases. Moreover, mice lacking SOD2 (SOD2-/- mice) exhibit significantly fewer proliferative NPCs and differentiated neurons in the embryonic cerebral cortex at midgestation compared with wild-type littermates. Cultured SOD2-/- NPCs exhibit a significant increase in SO flash frequency and reduced NPC proliferation. Taken together, our findings suggest that mitochondrial SO flashes negatively regulate NPC self-renewal in the developing cerebral cortex.

Perspectives on: SGP symposium on mitochondrial physiology and medicine: mitochondrial superoxide flashes: from discovery to new controversies.

### Introduction Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated during normal physiological processes are highly reactive with cellular lipids, DNA, and proteins. Superoxide and nitric oxide are the primary ROS and RNS, respectively, produced in cells, and both species

Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease

Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin-D-mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3 ± 2.0 flashes/1000 μm²·100 s before and after 5 tetani) and markedly decreased (to 7.7 ± 1.6 flashes/1000 μm²·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature-dependent increase in mSOF frequency (11.9 ± 0.8 and 19.8 ± 2.6 flashes/1000 μm²·100 s at 23°C and 37°C) was observed in fibers from RYR1(Y522S/WT) mice, a mouse model of malignant hyperthermia and heat-induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress

Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals

The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS). In intact cells, respiratory mitochondria exhibit spontaneous "superoxide flashes", the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT). Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP, and demonstrate their coupling to whole-body glucose metabolism. Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice. In skeletal muscle, imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously. The tMPT/superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation, in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT. Thus, in vivo imaging of tMPT-dependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.

Activity-Dependent Regulation of Mitochondrial Superoxide Flashes in Skeletal Muscle

Reactive oxygen species (ROS) are a class of molecules that oxidize diverse cellular proteins/lipids and are generated mainly in mitochondria by the electron transport chain (ETC). We previously discovered a novel biosensor for superoxide, circularly permutated YFP (cpYFP). Using mitochondrial-targeted cpYFP (mt-cpYFP), localized bursts of superoxide in individual or clusters of mitochondria, termed “superoxide flashes,” are observed in quiescent cells across a wide range of cell types [Wang, W. et al., Cell 134:279-290, 2008]. Here, we examined the properties of superoxide flashes in flexor digitorum brevis muscle fibers from newly generated transgenic mice expressing skeletal muscle-specific mt-cpYFP. A new flash detection and analysis software (“Flash Collector”) was developed to enable automated quantification of flash frequency, amplitude, kinetics, and area. Results demonstrate that skeletal muscle fibers exhibit higher basal mitochondrial superoxide flash frequency, but similar amplitude, kinetics, and area as those observed in cardiac myocytes. Rotenone (5υM) and oligomycin (5υM) reduced flash frequency to ∼20% of control, confirming the ETC dependence of superoxide flash generation. Inhibition of adenine nucleotide translocase by bongkrekic acid (100υM) decreased flash frequency by 50%. Incubation in Ringer's containing 10mM glucose or mitochondrial substrates did not significantly alter flash frequency, but increased flash amplitude and duration by 10-15%. Importantly, superoxide flash activity was enhanced (frequency increased from18.1±1.6 to 22.3±2.0 flashes/100s·100υm2) following five consecutive brief tetani (500ms, 100Hz, 0.2 duty cycle), but was markedly suppressed following a prolonged fatiguing stimulation (40 tetani; frequency reduced from 17.6±2.2 to 7.7±1.6). These results demonstrate skeletal muscle activity regulates mitochondrial superoxide flash production. In adition, muscle-specific mt-cpYFP transgenic mice will be a powerful tool for assessing the physiological role of superoxide flash activity and how this activity is altered and contributes to skeletal muscle aging and disease.

Mitochondrial Superoxide Flashes in Skeletal Muscle are Linked to Metabolism

Reactive Oxygen Species (ROS) constitute important intracellular signalling molecules. Mitochondria are a well known source of ROS production, especially through the electron transport chain. In the present work, the Ca2+ biosensor mt-pericam, kindly provided by Pr Miyawaki (RIKEN, Saitama, Japan) was used as a mitochondria-targeted specific superoxide biosensor, by appropriate choice of the excitation wavelength. Mt-pericam was transfected in vivo into mouse flexor digitorum brevis adult muscles. Fibers were isolated and studied by confocal microscopy. Targeting of the biosensor was specific to the mitochondrial network with little, if any, cytosolic contamination. Fluorescence flashes were detected using excitation at 477 nm which was isosbestic for calcium. Flashes corresponded to superoxide production as shown from simultaneous records of either Mitosox (a mitochondrial superoxide probe) or Rhod-2 fluorescence signals. In addition, the superoxide scavenger Tiron decreased the flash frequency by 85%. On average, flashes have a 20 sec duration, and a F/F0 amplitude of 2. Flashes were recorded in subsarcolemmal, and intermyofibrillar mitochondria. Intermyofibrillar flashes presented three different spatial patterns: longitudinal, transversal or patch-shaped. Flash frequency was increased by application of glucose/pyruvate and decreased by inhibition of the electron transport chain with antimycin A. This is strong evidence for quantal superoxide production being intimately linked to mitochondrial metabolism. Superoxide flashes were also found to cause a decrease of the mitochondrial -potential and -free calcium, as shown by simultaneous measurements of TMRM and Rhod-2/X-Rhod-1, respectively. Together, my results show that superoxide flashes are a physiological phenomenon linked to mitochondrial metabolism that occurs in all subcellular populations of mitochondria in adult skeletal muscle fibers.

In vivo Imaging of Superoxide Flashes in Skeletal Muscle

Emerging evidence suggests that reactive oxygen species (ROS) constitute a class of signaling molecules that regulate diverse cell functions including metabolism, muscle contractility and apoptosis. Recently we have developed and characterized a highly sensitive and reversible superoxide-selective probe, a circularly permuted yellow fluorescent protein (cpYFP), and demonstrated quantal and transient superoxide-producing events (superoxide flashes) within single mitochondria across multiple cell types (Wang et al, Cell, 132, 279).To further understand the physiological significance of flash events, we generated the pan-tissue mt-cpYFP transgenic mice expressing cpYFP in the mitochondria of multiple tissues. In vivo imaging of superoxide signals in gastrocnemius of transgenic mouse under anesthesia revealed mitochondrial superoxide flashes with similar properties (Fig). Further, superoxide flashes were also visualized in isolated skeletal muscle fibers transfected in vivo by electroporation with mt-cpYFP. Our findings support that mitochondrial superoxide flash activity is a physiologically relevant phenomenon that may participate in diverse aspects of cell function and signaling.View Large Image | View Hi-Res Image | Download PowerPoint Slide