Acta Physiol 2014

REVIEW

Characterization of reactive oxygen species in diaphragm L. Zuo,1 T. M. Best,2 W. J. Roberts,1 P. T. Diaz3 and P. D. Wagner4 1 Radiologic Sciences and Respiratory Therapy Division, School of Health and Rehabilitation Sciences, The Ohio State University College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA 2 Division of Sports Medicine, Department of Family Medicine Sports Health and Performance Institute, The Ohio State University, Columbus, OH, USA 3 Division of Pulmonary, Allergy, Critical Care & Sleep Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA 4 Department of Medicine, University of California, San Diego, La Jolla, CA, USA

Received 5 June 2014, revision requested 19 July 2014, revision received 24 September 2014, accepted 16 October 2014 Correspondence: Li Zuo, Molecular Physiology and Rehabilitation Research Lab, School of Health and Rehabilitation Sciences, The Ohio State College of Medicine, The Ohio State University Wexner Medical Center, 453 W. 10th Ave., Columbus, OH 43210, USA. E-mail: [email protected]

Abstract Reactive oxygen species (ROS) exist as natural mediators of metabolism to maintain cellular homeostasis. However, ROS production may significantly increase in response to environmental stressors, resulting in extensive cellular damage. Although several potential sources of increased ROS have been proposed, exact mechanisms of their generation have not been completely elucidated. This is particularly true for diaphragmatic skeletal muscle, the key muscle used for respiration. Several experimental models have focused on detection of ROS generation in rodent diaphragm tissue under stressful conditions, including hypoxia, exercise, and heat, as well as ROS formation in single myofibres. Identification methods include direct detection of ROS with confocal or fluorescent microscopy and indirect detection of ROS through end product analysis. This article explores implications of ROS generation and oxidative stress, and also evaluates potential mechanisms of cellular ROS formation in diaphragmatic skeletal muscle. Keywords confocal, cytochrome c, heat stress, hypoxia, superoxide.

Reactive oxygen species (ROS) play important roles in mediating cellular responses to muscle stress (Barreiro 2013). For instance, skeletal muscle exposure to hypoxia or severe heat stress results in increased levels of ROS, leading to cell dysfunction and/or injury (Zuo et al. 2000, 2003, 2004, Wright et al. 2009). Thus, it is important to develop effective in vitro techniques for identifying ROS production and related mechanisms in muscle tissue. Although exact molecular pathways of ROS generation are complex, underscoring the need to develop the ROS identification in a molecular level, previous research indicates multiple ROS sources in skeletal muscle, including NADPH oxidase (NOX), endothelial xanthine oxidase (XO) and mitochondria (Vina et al. 2000, Jackson 2008, Zuo et al. 2013b) (Fig. 1).

Several experimental models have been used to evaluate both intra- and extracellular ROS generation in skeletal muscle exposed to stressful conditions (Zuo et al. 2000, 2013b). The diaphragm, the major skeletal muscle responsible for respiration, produces excessive levels of ROS under stress (Nethery et al. 2000, Zuo et al. 2000, Zuo & Clanton 2005). For example, patients with chronic obstructive pulmonary disease (COPD) may have increased oxidative stress and skeletal muscle damage due to the stress caused by severe tissue hypoxia (Koechlin et al. 2005, Zuo et al. 2012). Although many previous studies have investigated the role of ROS in skeletal muscle, comprehensive literature focused on the mechanism of ROS formation in diaphragmatic skeletal muscle is highly limited. Unlike other skeletal muscle, the unique fibre composition of

© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12410

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Figure 1 Proposed molecular sources for intra- and extracellular ROS generation in diaphragmatic skeletal muscle, as well as ROS molecular probes including hydroethidine, HFLUOR and Cyt c. ADP, Adenosine Diphosphate; ATP, Adenosine triphosphate; AA, arachidonic acid; Cyt c, Cytochrome c oxidase; DIDS, 4,40 -diisothiocyanatostilbene 2,20 disulfonic acid; DPI, diphenyliodonium; ETYA, 5,8,11,14-eicosatetraenoic acid; HFLUOR, dihydrofluorescein; LOX, lipoxygenase; NOX, NADPH oxidase; PLA2, phospholipase A2; SOD, superoxide dismutase; TTFA, thenoyltrifluoroacetone; XO, xanthine oxidase.

the diaphragm allows it to continually contract. This is necessary given the muscle’s key role in respiration (Ottenheijm et al. 2008); however, the diaphragm may be overworked (Klimathianaki et al. 2011) in chronic cardiopulmonary diseases such as asthma (Durigan et al. 2009), COPD (Ottenheijm et al. 2005), pulmonary fibrosis (Ramos et al. 2008, Borges et al. 2014) and occasionally heart failure (Empinado et al. 2014). Diaphragmatic contractile function can be substantially reduced in critically ill patients with ventilator-induced diaphragm dysfunction (Jubran 2006, Hussain et al. 2010), contributing to persistent respiratory failure. Additional studies have shown an increased ROS level and reduced electron transport chain (ETC) activity in the diaphragmatic mitochondria of rats (Kavazis et al. 2009) and humans (Picard et al. 2012) under mechanical ventilation (MV). This process could be exacerbated in patients at risk for underlying diaphragm dysfunction such as those with COPD or heart failure (Budweiser et al. 2008, Zuo et al. 2012, Empinado et al. 2014). Human patients with severe COPD have been shown to possess elevated levels of superoxide (O_
2 ) in their diaphragmatic mitochondria when compared to controls (Marin-Corral et al. 2009). The resulting oxidized cellular components, such as proteins, are also observed in the diaphragm of cigarette smoke-exposed animals (Barreiro et al. 2010). In response to this alteration of oxidative state, the diaphragm displays intracellular adaptation, including elevation of antioxidant catalase activity that leads to lessen lipid peroxidation, which may be clinically useful to the functional recovery of diaphragm in patients with COPD (Wijnhoven et al. 2

2006, Zuo et al. 2012). Furthermore, increased levels of oxidative stress have been consistently observed in patients suffering from Duchenne muscular dystrophy (DMD) (Rodriguez & Tarnopolsky 2003). Oxidative stress, which can be due to overwhelmed antioxidant defence systems, has been proposed as performing a critical role in the progression of DMD in dystrophin deficient muscles (Kim et al. 2013). Patients diagnosed with DMD suffer from myopathy in the limbs and diaphragm (Kim & Lawler 2012, Kim et al. 2013). As increased levels of oxidative stress are believed to be a key mediator in the progression of DMD, antioxidant therapeutics should be pursued as a method of treating muscle dysfunction, particularly in the diaphragm of these patients (Rando 2002, Lawler 2011). Therefore, research investigating ROS associated pathways in diaphragm has potential clinical relevance. Understanding ROS in rodent diaphragmatic muscle may help elucidate the role of oxidative stress in the human respiratory system and facilitate development of therapeutics targeting ROS-induced muscle injury. Accordingly, this review examines stress-induced ROS formation and potential ROS sources in skeletal muscle, focusing on heat and hypoxic stress in contracting rodent diaphragm muscle (Zuo et al. 2003, Zuo & Clanton 2005).

ROS during exercise and associated heat stress The role of ROS in skeletal muscle is multifaceted. Although low levels of oxygen radicals are produced during normal muscle functioning, formation can substantially increase during repetitive contractions/

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exercise and in response to muscle-related stressors (Reid et al. 1992a, Powers et al. 2011a). As a result, elevated levels of oxygen radicals contribute to oxidative stress and muscle fatigue (Shindoh et al. 1990, Reid et al. 1992a, Mohanraj et al. 1998). During intense exercise, muscles also are exposed to elevated temperatures which may lead to enhanced ROS formation through mitochondrial uncoupling (Brooks et al. 1971, Sachdev & Davies 2008). Consequently, the mitochondrial ETC becomes ‘leaky’ and molecular oxygen reacts with leaked electrons, primarily forming O_
2 (Sachdev & Davies 2008) (Fig. 1). The oxidative stress induced by hyperglycaemia can be relieved with human diaphragmatic breathing as this exercise can enhance antioxidant defences and reduce ROS production (Martarelli et al. 2011). However, molecular pathways downstream of O_
2 generation during heat stress have not been completely defined. Use of confocal microscopy and ROS assays have demonstrated that exposure of resting skeletal muscle to heat stress results in an intracellular ROS burst, detectable with the O_
2 -specific probe hydroethidine (HE), resulting in the production of ethidium (ET) (Zuo et al. 2000). Through the use of the cytochrome c (cyt c) reduction assay, O_
2 can also be detected in the extracellular perfusate, suggesting the possibility of O_
2 release to the extracellular environment in resting skeletal muscle (Close et al. 2005) (Fig. 1). Moreover, it has been shown that when an exogenous O_
2 scavenger is added to the perfusate the level of cyt c reduction diminishes, inferring a decrease in extracellular O_
2 and confirming the existence of ROS (Zuo et al. 2000, 2013a). In addition to O_
2 , H2O2 release from rodent diaphragm during heat stress can be determined using the antioxidant catalase. Previously, research has shown that there is no marked level of H2O2 release in diaphragm muscle during heat stress (Zuo et al. 2000). In this instance, it is likely that intracellular antioxidant defence systems (e.g., endogenous catalase) mitigate release of intracellular H2O2 before its diffusion to the extracellular space (Zuo et al. 2000). Accordingly, the results indicate that skeletal muscle exposed to heat stress shows increased production of intracellular O_
2 and subsequent release (Oosthuizen & Greyling 1999). These findings may help to identify the role of ROS in cell signalling responses to body heat associated with extreme exercise and fatigue. Although the primary type of ROS (O_
2 ) has been identified in response to heat stress, exact cellular sources for ROS production are still unclear. Several sources for both intra- and extracellular ROS in skeletal muscle, including mitochondria, NOX and other oxidative enzymes, will be discussed.

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ROS in hypoxia During hypoxia, rodent striated muscle tissue demonstrates elevated levels of ROS (Park et al. 1991, Zuo & Clanton 2005, Clanton 2007, Chiu et al. 2014). For instance, Vanden Hoek et al. (1997) observed an increase in ET and dichlorofluorescein (DCF) fluorescence during ischaemic conditions, indicating the presence of intracellular ROS, specifically H2O2 and O_
2. A likely explanation for these somewhat paradoxical findings regarding oxygen radical formation in the absence of molecular O2 is intriguing. The formation of the observed intracellular O_
2 can be explicated by low residual levels of intracellular O2 acting as electron acceptors from highly reduced mitochondria (due to lack of O2) (Hess & Manson 1984, Vanden Hoek et al. 1997). In support of this notion, Vanden Hoek et al. used O2 scavengers (dithionite and ascorbate oxidase) during hypoxia to eradicate residual O2. Under these conditions, they observed significant reduction in ET fluorescence during ischaemia, compared with those exposed to ischaemia without O2 scavengers (Vanden Hoek et al. 1997). These results highlight the importance of residual O2 as a key factor for ROS generation during ischaemia in animal myocytes. To assess effects of hypoxia-induced ROS in skeletal muscle, our group exposed resting rat diaphragm to hypoxic conditions and then investigated whether ROS regulate the muscle’s ability to adapt to sudden hypoxia (Zuo & Clanton 2005). Dihydrofluorescein (HFLUOR) was used to evaluate fluorescence and ebselen (a potent scavenger of H2O2) was used to specifically identify whether the ROS signal was attributed to H2O2 (Zuo & Clanton 2005). During hypoxia, ROS-induced fluorescence increased significantly, but could be attenuated through use of ebselen, suggesting H2O2 as a likely mediator for cellular adaptation to hypoxic stress (Zuo & Clanton 2005). Potential implications of these findings are relevant to future clinical studies involving those patients with chronic systemic hypoxic conditions (e.g. COPD or cardiovascular disease) as this in vitro work suggests a specific therapeutic target (Zuo & Clanton 2005, Kirkham & Barnes 2013, Meyer et al. 2013, Robert & Robert 2014). Preconditioning is one such therapy that has been extensively investigated in muscle tissue (Kevin et al. 2003, Matejikova et al. 2009). Results from such research indicate that after exposure to brief periods of ischaemia (or hypoxia), muscle tissue is better able to withstand longer, subsequent intervals of ischaemia (Kevin et al. 2003, Lin et al. 2008, Costa et al. 2013). Because exact mechanisms of this therapeutic technique are unclear, interaction between ROS and ischaemia/hypoxia-related preconditioning resulting in

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muscular adaptation and future resistance to damage from similar stressors is an area of emerging interest. Moreover, understanding the muscle’s dynamic ability to naturally resist ischaemic/hypoxic damage due to ROS through preconditioning may not only enhance therapeutic development for clinical treatment, but also expand the potential role of ROS in other diseases as a potential mechanistic mediator.

Cellular sources and mechanism of ROS Although many sources of ROS in skeletal muscle have been identified (Niess & Simon 2007, Powers et al. 2010, Lamb & Westerblad 2011), it is unclear whether extracellular ROS result primarily from intracellular ROS generation and subsequent extracellular release. Thus, researchers hypothesized that the presence of extracellular O_
2 from resting rodent diaphragm muscle is generated in the mitochondria and released through membrane anion channels (Zuo et al. 2003) (Fig. 1). Utilizing a heat-stressed murine skeletal muscle model, we focused primarily on certain common intracellular sources such as the mitochondrial membrane complexes I, II and III. Anion channel inhibitors including probenecid and 4,40 -diisothiocyanatostilbene 2,20 -disulfonic acid were also applied to prevent potential O_
release during heat stress 2 (Fig. 1), but they had no effects on cyt c reduction assay, which was used to measure extracellular O_
2 levels. It was further determined that the inhibition of mitochondrial complexes I, II and III using rotenone, TTFA (thenoyltrifluoroacetone) and antimycin A respectively, had no effect on cyt c reduction in resting diaphragm (Zuo et al. 2003) (Fig. 1). A related study reached a similar conclusion when experimental results excluded the mitochondrion’s contributions to extracellular O_
2 production (McArdle et al. 2004). This suggests that mitochondrial derived ROS have no effect on extracellular O_
2 levels during normal and heat stress conditions. In addition, exposure to NOX and cell membrane anion channel inhibitors did not decrease observed cyt c reduction, further indicating that other unknown intra- and extracellular sources and molecular mechanisms contribute to increases in cyt c reduction in skeletal muscle during heat stress (Zuo et al. 2003). Correspondingly, previous research has reported several different sources responsible for both intra-and extracellular ROS generation in skeletal muscle (Niess & Simon 2007, Powers et al. 2011b) including XO, heme oxygenase (HO) and arachidonic acid (AA). In the setting of decreased oxygen, reduced ATP levels result in decreased activity of ATP-sensitive Ca2+ pumps (Mohanraj et al. 1998, Gouriou et al. 2013) and build-up of Ca2+, which can indirectly activate 4

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the conversion of xanthine dehydrogenase to XO through the use of proteases (Mohanraj et al. 1998, Ryan et al. 2011, Nanduri et al. 2013) (Fig. 1). While the precise location of xanthine dehydrogenase and XO within skeletal muscle tissue is unclear, it is likely that these enzymes are intracellularly located in both skeletal muscle fibres and their associated endothelial cells (Mohanraj et al. 1998, Ryan et al. 2011). Regardless of their exact location, the resulting high levels of XO mediate breakdown of hypoxanthine, releasing O_
2 in the process (Granger 1988, Chen et al. 2013). Accordingly, the inhibition of XO has been reported to alleviate MV-induced oxidative stress in the exercising diaphragm of rats (Whidden et al. 2009) and decrease extracellular O_
2 levels during isometric contractions (Gomez-Cabrera et al. 2010), further suggesting the role of XO in the generation of extracellular O_
2. Heme oxygenase (HO) is another potential source of ROS generation in diaphragm muscle during hypoxia. HOs are rate-determination enzymes in the degradation process of heme molecules (Barreiro et al. 2002), limiting heme-centred ROS formation. In response to heat stress and hypoxia, the HO inducible isoform HO-1 can be activated transcriptionally, providing beneficiary antioxidant protection during diaphragmatic activity (Barreiro et al. 2002). In addition, AA metabolism can contribute to ROS formation in heat-treated rat diaphragm muscle (Zuo et al. 2004). Generated as a result of phospholipase A2 (PLA2) activation, AA triggers the activity of downstream enzymes such as cyclooxygenase (COX), cytochrome P450 and lipoxygenase (LOX). These enzymes may cause excessive ROS production. For example, during heat stress in resting rat diaphragm, the major source _
of extracellular O_
2 production (O2 release) is AAdependent LOX activity (Zuo et al. 2004) (Fig. 1). Interestingly, LOX inhibition does not have an effect on intracellular ROS generation following resting diaphragm exposure to heat stress (Zuo et al. 2004). The exact reason for this discrepancy is still unclear. However, as LOX is localized to the cell membrane, a potential ability to transfer O_
2 to extracellular space may be an explanation. The inhibition of the upstream AA-producing enzyme, PLA2, also mitigates extracellular O_
2 levels under both normal (37 °C) and elevated temperatures (42 °C). This implies that PLA2 may play a role in regulating O_
2 levels under both normal and heat stress conditions. There may be, therefore, a tight relationship between PLA2 production of AA and LOX activity during heat stress in the diaphragm. Various inhibitors such as DPI (diphenyliodonium) (Zuo et al. 2003, LibikKonieczny et al. 2014), manoalide (Zuo et al. 2004), oxypurinol (Bravard et al. 2011) and ETYA

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(5,8,11,14-eicosatetraenoic acid) (Zuo et al. 2004) have been successfully utilized in studies examining oxidative stress and the mechanism of ROS generation (Fig. 1). Other potential intracellular pathways are likely involved in elevated extracellular O_
2 levels, but remain to be investigated. A recent study by Mangner et al. (2013) reported exercise training limits protein carbonylation in mouse diaphragm injected with the proinflammatory cytokine TNF-a. It also has been shown that TNF-a induces ROS release in muscle through both NOX and XO (Mangner et al. 2013, Zuo et al. 2013a). Consequently, the oxygen radicals produced stimulate the carbonylation of a-actin protein, which is linked to proteasome-induced protein degradation (Mangner et al. 2013). As a result, the muscle force and power decrease, leading to fatigue (Mangner et al. 2013, Zuo et al. 2013a). Interestingly, researchers determined that glutathione peroxidase (GPx, an antioxidant) levels were markedly increased following exercise training. These results indicate that after exercise, GPx may scavenge elevated ROS, thereby attenuating diaphragmatic fatigue through the reduction of protein carbonylation (Mangner et al. 2013). Therapeutic benefits of ROS are again implicated as elucidating redox mechanisms activated during exercise training may provide insight into methods for restoring diaphragmatic contractility.

ROS identification Whole muscle experiments Application of fluorescent probes is currently one of the most effective methods for detecting intracellular ROS in rodent skeletal muscle (Zuo et al. 2000, 2011b, 2013b, Zuo & Clanton 2005). These probes target specific forms of ROS such as hydrogen peroxide (H2O2) or O_
2 . ROS-mediated reactions with the probes can be monitored via fluorescent imaging techniques such as tissue surface fluorometry, as well as confocal/fluorescence microscopy (Zuo & Clanton 2005, Kuznetsov et al. 2011, Xu et al. 2013). Different probes are sensitive to specific ROS, for example, HE is sensitive to O_
2 , while HFLUOR is sensitive to H2O2 (Zuo & Clanton 2002). Maximizing sensitivity of fluorescent probes during experimentation requires understanding of how they function in intracellular environments. The HE probe capitalizes on conversion of HE (a hydrophobic molecule capable of passing the cell membrane) to ET in the presence of ROS (Zuo & Clanton 2002, Xu et al. 2010). This probe is commonly used for detecting intracellular O_
2 because ET fluorescence is markedly stable with no substantial autoxidation, thus reducing potential fluorescent arti-

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facts (Benov et al. 1998, Zuo et al. 2011b). Fluorescence can be visualized through a variety of optical techniques including epifluorescence fluorescent microscopy (Bindokas et al. 1996, Murrant et al. 1999), confocal microscopy (Zuo et al. 2000) and flow cytometry (Hsieh et al. 2013). Previous methods for direct measurement of ROS from intact diaphragm muscle utilized confocal microscopy to perform real-time measurements of ROS levels in the isolated rodent diaphragm tissue, as shown in Fig. 2 (Zuo et al. 2000, Roberts & Zuo 2012). However, certain chemicals (e.g., ferricytochrome c) may oxidize HE, potentially yielding species other than ET (Zuo & Clanton 2002). Moreover in the presence of increased concentrations of O_
2 , HE accelerates the dismutation of O_
, resulting in reduced 2 ET detection (Zuo & Clanton 2002). Tissue overloading of HE can also influence the mitochondrial membrane potential and thus, alter ET fluorescence and probe efficacy (Budd et al. 1997). Under certain conditions, HE detects other specific forms of ROS, such as hydroxyl radicals and peroxynitrite, limiting its application (Zuo et al. 2000, Zuo & Clanton 2002). In addition, researchers have claimed that it may be difficult to determine the intracellular levels of O_
2 using only fluorescence-based microscopic method such as HE assay, as 2-hydroxyethidium (2-OH-E+) can also be formed from the reaction of O_
2 with HE (Zielonka & Kalyanaraman 2010). More studies have shown that 2-OH-E+ is the direct product from the reaction of HE with O_
2 (Zielonka et al. 2006, Fernandes et al. 2007, Meany et al. 2007). As the concentration of 2-OH-E+ from this reaction is usually markedly smaller than ET, alternative methods such as high-performance liquid chromatography (HPLC) should be employed (Kalyanaraman et al. 2014). This would help the understanding of how the HE-O_
2 reaction can be used as an effective intracellular

Figure 2 This schematic demonstrates the confocal setup used for intracellular ROS detection in rodent diaphragm.

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approach for ROS detection (Zielonka & Kalyanaraman 2010). In summary, caution should be taken when HE is used as a major ROS probe. HFLUOR is a commonly used probe to detect intracellular ROS in murine models. After localization of this molecule to the cytosol, it is converted to an oxidized form known as fluorescein (FLUOR) in the presence of H2O2 (Zuo & Clanton 2002, 2005, Zuo et al. 2013b). However, other molecular species such as enzymes (e.g. XO and catalase) and cell signalling molecules (peroxynitrite) may also react with HFLUOR (Zuo & Clanton 2002), rendering the specificity of HFLUOR to H2O2 debatable. Nevertheless, this probe demonstrates several advantages compared with alternative forms such as 20 ,70 -dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR123) (Zuo & Clanton 2002). For instance, HFLUOR has a higher molar fluorescence and a superior intracellular loading efficacy (Zuo & Clanton 2002). It is particularly useful for antioxidant research due to its low reactivity with commonly studied antioxidants [superoxide dismutase (SOD) and catalase] as well as its non-existent interaction with cyt c, as compared to DCFH and DHR123 (Zuo & Clanton 2002, Zuo et al. 2013a). Furthermore, HFLUOR is less sensitive to reactive nitrogen species (Hempel et al. 1999) as well as XO (a popular source of ROS during cellular stress) when compared to other probes (Zuo & Clanton 2002). Several drawbacks are inherent to fluorescent techniques. Limitations are primarily related to standardization of the technique, including tissue loading with the probe, detection system adjustments and amount of external light entering the reaction system. Intracellular molecules (e.g. NADH or FAD) may fluoresce at a similar wavelength as the probe used in ROS detection, potentially affecting fluorescence, creating false positive data, thus masking the signal of interest. Other molecules such as DNA strands may also bind to the oxidized probe and similarly affect the fluorescence signal. In addition, rodent muscle tissue may not effectively retain the probe during the prolonged experiments, resulting in equivocal ROS levels (Zuo & Clanton 2002). Although the benefits of fluorescent labelling are obvious, so are the limitations. Besides direct measurements of intracellular ROS, Nethery et al. (1999) evaluated ROS generation indirectly by measuring ET fluorescence in homogenized rat diaphragm tissue after exercise. Specifically, after diaphragms were isolated from rats and infused with HE, tissues were homogenized, ET extracted, and ET fluorescence measured in the supernatant using a spectrophotofluorometer (Nethery et al. 1999). Although this protocol more accurately quantifies ROS than direct measurements, the longer sample preparation 6

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period may result in false positive results. Alternative methods are therefore necessary to confirm ROS. One such technique frequently employed involves detection of oxidation of glutathione (GSH) to a glutathione disulphide dimer (GSSG) form (Sachdev & Davies 2008, Shortt et al. 2014). As the conversion of GSH to GSSG requires ROS mediation, increases of GSSG in muscle tissue following intense exercise indicate enhanced ROS levels (Lew et al. 1985, Sachdev & Davies 2008). Another technique to assess ROS indirectly entails measurement of lipid peroxidation by-products. Exercise stimulates a radical chain reaction, resulting in the formation of lipid radicals. Oxygen reacts with these radicals, generating lipid peroxyl radical species that react with surrounding fatty acids, ultimately leading to cell and organelle membrane damage (Sachdev & Davies 2008, Fogarty et al. 2011, Pratt et al. 2011). Implicated reactions can be measured by evaluating presence of malondialdehyde (MDA), a by-product of lipid peroxidation. Due to MDA’s unstable nature, however, it is not the most desirable method to evaluate radical-induced damage (Sachdev & Davies 2008). Extracellular ROS measurements supplement intracellular assessments and enhance elucidation of molecular pathways resulting in ROS release in rodent models. One well-established method to detect extracellular O_
2 is the cyt c assay (Reid et al. 1992b, Kolbeck et al. 1997, Zuo et al. 2013a). Cyt c is reduced upon interaction with O_
2 resulting in an increased absorbance peak at 550 nm, readily providing the concentration of reduced cyt c as previously described in exercising diaphragm (Reid et al. 1992b, Kolbeck et al. 1997). Although the cyt c assay is one of the most common methods used to measure ROS release in skeletal muscle (Reid et al. 1992b, Kolbeck et al. 1997, Zuo et al. 2004, Gomez-Cabrera et al. 2010, Lambertucci et al. 2012), ascorbate released from muscle tissue has potential to reduce cyt c, thereby causing background artifacts. To confirm signals are from O_
2 and not chemical interference, the cell impermeable antioxidant enzyme SOD should be used (Zuo et al. 2000). Electron paramagnetic resonance (EPR) employing the spin trap b-phosphorylated nitrone, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) effectively traps and detects O_
2 in solution. By light irradiation to riboflavin and xanthine/XO, Roubaud et al. (1997) demonstrated that EPR with DEPMPO is ~40-fold more sensitive in detecting O_
2 as compared to the cyt c assay. In addition to increased sensitivity, the high specificity for O_
2 using spin trapping with DEPMPO allows for precise detection (Roubaud et al. 1997). Although EPR instrumentation can be finan-

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cially costly and fitting various muscle models into the EPR machine may be difficult, this technique is a potential alternative method for detecting extracellular O_
2 release in skeletal muscle. Together, these methods provide effective techniques to identify and evaluate intra- and extracellular ROS in the muscle tissue.

Single skeletal myofibre experiments Although intracellular detection of ROS in skeletal muscle is generally performed in murine muscle tissue, several limitations arise from this type of model. For instance, the vasculature associated with extracted muscle tissue may interfere with precise determination of ROS sources as endothelial tissue also generates ROS (Golbidi & Laher 2013, Zuo et al. 2013b). In isolated fibres, absence of endothelial tissue nullifies this problem. Furthermore, creating a more homogeneous hypoxic/hyperoxic condition is simplified for single muscle fibres as there is a larger oxygen gradient between intra- and extracellular environments in whole muscle tissue (Zuo et al. 2013b). Monitoring muscle tissue activity under confocal microscopy can similarly be difficult as the high number of contracting fibres result in motion artifact. Previous experiments have been conducted using isolated single myofibres from Xenopus laevis lumbrical muscles (Zuo et al. 2011a, 2013b). Like mammalian muscle, Xenopus muscle fibres exhibit associations between oxidative capacity and fatigue resistance (Westerblad et al. 1991). For this reason, Xenopus fibres may demonstrate similar metabolic responses to ROS-induced cellular stress as compared to the typical mouse or rat skeletal muscle model. Variable PO2 conditions have been shown to impact ROS production in single myofibres. Specifically, higher ROS levels are observed under hypoxic-like conditions as compared to normal PO2 levels (Zuo et al. 2013b). As mentioned previously, these findings likely indicate ROS involvement in cellular adaptation to hypoxia. Single myofibre models allow researchers to measure ROS levels in real time, facilitating studies of ROS generation and signalling in skeletal muscle, especially during muscle contractions. Although single myofibre models circumvent some constraints of whole muscle tissue experiments, other limitations should be noted. For instance, delicate myofibre isolation processes may result in muscle damage. In addition, although easier to create a more homogenous O2 environment in single fibres, intracellular O2 concentrations are still difficult to assess. Finally, single myofibres may respond differently to stimulation and stressors as they have been removed from their normal physiological environment and are no longer part of a whole muscle (Zuo et al. 2000, 2011a, 2013b, Zuo & Clanton 2005). To garner

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a more complete understanding of effects of stressinduced ROS and corresponding redox mechanisms in diaphragmatic skeletal muscle, both whole muscle and single myofibre models should be used.

Conclusion Excessive ROS accumulation can result in substantial cell and tissue damage. For instance, it has been shown that ROS can damage skeletal muscle in the diaphragm and thus lead to muscle contractility and muscle function impairment. Various molecular sources of ROS in skeletal muscle under stressful conditions have been proposed using previously established muscle models. Particularly, diaphragm muscle has been used extensively to measure ROS generation after exposure to hypoxia and heat. Unlike other skeletal muscles, the diaphragm contracts continually to fulfil its role in respiration, and several associated diseases have been implicated by the overproduction of ROS contributed from overworked or dysfunctional diaphragms. Single myofibre models may provide insight into the roles of ROS at the cellular level. Methods including confocal/fluorescent microscopy and specific fluorescent probes have been used successfully for ROS detection in these experiments, although certain limitations may exist. Understanding of ROS may help elucidate different redox pathways involved in skeletal muscle, especially when exposed to stressors. Specifically, patients with increased skeletal muscle strength will have improved respiratory function via the diaphragm. Therefore, this area should be a primary focus of future studies.

Conflict of interest No conflict of interests, financial or otherwise, is declared by the authors. This work was supported by OSU-HRS Fund 013000. We thank Dr. Lan Jiang, Ben Pannell, Chia-Chen Chuang, Anthony Re, Rachael Leahy, Christopher Fortuna, Andrew Graef, and Jiewen Li for their assistance and helpful discussions. Our manuscript is conformed to Persson PB. Good Publication Practice in Physiology 2013 Guidelines for Acta Physiologica. Acta Physiol (Oxf), 2013 Dec; 209(4), 250–3.

References Barreiro, E. 2013. Protein carbonylation and muscle function in COPD and other conditions. Mass Spectrom Rev 33, 219–236. Barreiro, E., Comtois, A.S., Mohammed, S., Lands, L.C. & Hussain, S.N. 2002. Role of heme oxygenases in sepsisinduced diaphragmatic contractile dysfunction and oxidative stress. Am J Physiol Lung Cell Mol Physiol 283, L476–L484.

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