ROS and RNS signaling in skeletal muscle: critical signals and therapeutic targets

Abstract

The health of skeletal muscle is promoted by optimal nutrition and activity/exercise through the activation of molecular signaling pathways. Reactive oxygen species (ROS) or reactive nitrogen species (RNS) have been shown to modulate numerous biochemical processes including glucose uptake, gene expression, calcium signaling and contractility. In pathological conditions, ROS/RNS signaling excess or dysfunction contributes to contractile dysfunction and myopathy in skeletal muscle. Here we provide a brief review of ROS/RNS chemistry and discuss concepts of ROS/RNS signaling and its role in physiological and pathophysiological processes within striated muscle.

Introduction

Skeletal muscle is a complex organ system within the body. While we often consider skeletal muscle contraction in support of posture, locomotion or respiration as its main function, skeletal muscle contraction is also important for guarding body cavities (i.e., muscle sphincters), for maintaining body temperature (i.e., shivering thermogenesis), for maintaining glucose control and as a protein reserve. The health of muscle is promoted by optimal nutrition and activity/exercise that activate molecular signaling pathways to promote an increase in the functional and metabolic capacity of the muscle. Conversely, muscle health is negatively impacted by inactivity as well as disease (i.e., diabetes, muscular dystrophies, amyotrophic lateral sclerosis, sarcopenia, critical illness myopathy) often through a dysregulation through similar molecular pathways.

Reactive oxygen species (ROS) or reactive nitrogen species (RNS) have been shown to modulate numerous biochemical processes through the targeted modification of specific protein residues. In striated muscle, contractile activity and/or stretch increases ROS/RNS signaling to modulate a host of biochemical processes including glucose uptake (Chambers, Moylan, Smith, Goodyear, & Reid, 2009), gene expression, calcium signaling and contractility(Chambers, Moylan, & Reid, 2009). In pathological conditions, ROS/RNS signaling excess or dysfunction contributes to contractile dysfunction and myopathy in skeletal muscle.

It is clear that ROS and RNS are generated from numerous tissues and paracrine signaling to skeletal muscle is important in health and disease. In fact, several excellent reviews have been published that consider these pathways (Donoso, Sanchez, Bull, & Hidalgo, 2011; Ferreira & Reid, 2008; Kuster, Hauselmann, Rosc-Schluter, Lorenz, & Pfister, 2010; Lamb & Westerblad, 2011; Powers, Talbert, & Adhihetty, 2011). Emerging research is now revealing pathways by which ROS/RNS are generated within the myocyte at rest and during contraction (i.e., exercise) (Jackson & McArdle, 2011; Powers, Smuder, & Judge, 2012) and novel mechanistic insights into mechanoactivation of ROS signaling have recently been reported (Khairallah et al., 2012; Prosser, Khairallah, Ziman, Ward, & Lederer, 2012). These pathways are being revealed with both in vivo and single cell studies enabled by new methods and reagents. These findings are providing unique insights into both the positive adaptations seen with exercise and the dysfunction seen with inactivity and disease. Here we provide a brief review of ROS/RNS chemistry and discuss concepts of ROS/RNS signaling and its role in physiological and pathophysiological processes within striated muscle.

Molecular Signaling in the context of classical nursing theory

The nurse theorist Mary Levine developed a Conservation Model emphasizing the practical understanding of physiological mechanisms identified in four key principles; Conservation of Energy, Conservation of Structural Integrity, Conservation of Personal Integrity, and Conservation of Social Integrity (Meleis, 2012). In general, these concepts identify attributes that require the nurse to help the client return to a homeostatic balance with respect to each concept. The Conservation Model indicates the nurse acts as an intermediary, helping the client to “adapt” to the life experiences involved with pathophysiological events (Levine, 1996). One of Levine’s assessments to evaluate the biological and physical systems responsible for disease processes was to evaluate the “body movement and positioning” of the client. Understanding the client’s activity with respect to their operational environment (Meleis, 2012) allows nurses to integrate a treatment plan to therapeutically “adapt” an ill client to his/her current condition. Levine’s Model forms a guide for the nurse to help the “ill” client reacquire a new homeostatic balance within their social, personal, and biological milieu. Then as a corollary, one may suggest this model substantiates the need for the modern nurse to consider molecular pathways, such as ROS and RNS and their homeostatic role in physiological signaling and their contribution to disease when the signaling is out of balance.

Reactive Oxygen Signaling and the ROS Paradox

Reactive oxygen species (ROS) are reactive molecules that contain oxygen. ROS are formed as natural byproducts of oxygen metabolism and by enzymatic activity. In both cases, ROS production is highly localized and spatially restricted which makes it a efficient system for cell signaling processes such as glucose transport(Chambers, Moylan, Smith, et al., 2009), eicosanoid (Spiteller, 2010) and cytokine production (Sigala et al., 2011), and mitochondrial biogenesis (Austin, Klimcakova, & St-Pierre, 2011; Kang & Li Ji, 2012). However, during times of environmental stress (e.g., UV or hyperthermia), metabolic stress (e.g., hyperglycemia), or in disease states (e.g., muscular dystrophy, unloaded diaphragm during mechanical ventilation) ROS levels can increase dramatically resulting in aberrant signaling(D. G. Allen, Gervasio, Yeung, & Whitehead, 2010; Davidovich et al., 2013; Faist, Konig, Hoger, & Elmadfa, 2001; Kavazis et al., 2009).

In skeletal muscle, the functional consequences of this biphasic response are well described in experiments where small increases in ROS, as seen with mild exercise, increase muscle force generation. Yet, with excessive fatiguing exercise or with an experimental increase in exogenous ROS, muscle function decreases (Andrade, Reid, & Westerblad, 2001; Ferreira et al., 2012). This biphasic effect of ROS on cell function (Andrade, et al., 2001; Reid, 2001) forms the basis for the ROS “paradox” which acknowledges ROS as necessary for proper signaling pathways yet detrimental to function when in excess (Figure 1).

Figure 1. Model depicting the effects of the cellular oxidation state on muscle force production.

A: basal state from unfatigued muscle. B: unfatigued muscle exposed to reducing agent or antioxidant. C: unfatigued muscle exposed to low-level of ROS. D: Muscle exposed to excess ROS as seen in fatiguing exercise or disease. The dashed vertical lines denote basal redox state (without an additional ROS stimulus) and the optimal redox state (i.e., maximal force). Used with permission (Reid, 2001)

Reactive Oxygen Species

Reactive Oxygen Species (ROS) is a general term encompassing oxygen free radicals; oxygen containing molecules that contain one or more unpaired electrons. (Halliwell & Gutteridge, 2007). It is important to note that not all ROS need to be a radical species. As indicated in Formulas 1 and 2 below, hydrogen peroxide (H₂O₂) is a type of ROS but it is not a “free radical” because it does not contain unpaired electrons.

Superoxide (O₂^·−) is a common ROS molecule within skeletal muscle (Reid, 2001). However, has limited intrinsic reactivity and its negative charge limits its membrane permeability, however O₂^·− has been shown to move through anion channels or aquaporin (Fisher, 2009; Ren, Raucci, Browe, & Baumgarten, 2008). Superoxide’s spontaneous dismutation or enzyme catalyzed dismutation (Formula 1) into the membrane permeable H₂O₂ (Cadenas & Packer, 2002) yields a potent and reactive signaling molecule.

Reactive Oxygen Species Reactions

$${{\mathrm{O}}_2}^{\mathrm{·}-}+{{\mathrm{O}}_2}^{\mathrm{·}-}+2{\mathrm{H}}^{+}\to {\mathrm{H}}_2{\mathrm{O}}_2+{\mathrm{O}}_2$$ Formula 1

$$\mathrm{F}{\mathrm{e}}^{2+}+{\mathrm{H}}_2{\mathrm{O}}_2\to \mathrm{H}{\mathrm{O}}^{-}+\mathrm{O}{\mathrm{H}}^{\mathrm{·}}+\mathrm{F}{\mathrm{e}}^{3+}$$ Formula 2

The formation of H₂O₂ can initiate key redox reactions. H₂O₂ forms the substrate for the production of the extremely reactive hydroxyl radical (OH^·) via a Fenton Reaction with divalent metal cations, but mainly with ferrous iron, Fe²⁺ (Formula 2). The sulfhydryl (–SH) group associated with a protein may initially be oxidized to a sulfenic acid (protein – SOH).The OH^· can then form irreversibly oxidized protein sulfhydryl groups, indicative of protein “damage” (Cadenas & Packer, 2002; Thomas & Mallis, 2001) (Formulas 3 and 4).

Irreversible Protein Oxidation

$$\text{protein}-\mathrm{S}\mathrm{O}\mathrm{H}+({\mathrm{O}}_{\mathrm{x}})\to \text{protein }\mathrm{S}{\mathrm{O}}_2\mathrm{H}\hspace{1em}\text{sulfinic acid}$$ Formula 3

$$\text{protein}-\mathrm{S}{\mathrm{O}}_2\mathrm{H}+({\mathrm{O}}_{\mathrm{x}})\to \text{protein }\mathrm{S}{\mathrm{O}}_3\mathrm{H}\hspace{1em}\text{sulfonic acid}$$ Formula 4

Reactive Nitrogen Species (RNS)

It is important to note other radical and non-radical species have been detected in skeletal muscle, which influences the overall reduction/oxidation (redox) homeostasis within the skeletal myocyte. The nitric oxide (NO^·) molecule forms the parent molecule for reactive nitrogen species. In the presence of O₂^·−, NO^· forms the highly reactive nitrogen intermediate, peroxynitrite, by the reaction in Formula 5.

Peroxynitrite Formation

$$\begin{array}{ccc}\mathrm{N}{\mathrm{O}}^{\mathrm{·}}+\hfill & {{\mathrm{O}}_2}^{\mathrm{·}-}\to \hfill & \mathrm{O}\mathrm{N}\mathrm{O}{\mathrm{O}}^{-}\hfill \\ \text{nitric oxide}\hfill & \text{superoxide}\hfill & \text{peroxynitrite}\hfill \end{array}$$ Formula 5

ROS and RNS can refer to multiple radical and non-radical types of each species. This review primarily focuses on those species indicated below.

ROS/RNS Formation

As outlined above, skeletal muscle generates superoxide and nitric oxide (NO) as the primary ROS/RNS species. Once generated, each lead to the formation of secondary ROS or RNS species with specific signaling roles (Figure 2). They derive from several sources and the amount of each type is increased with contractile activity.

Figure 2. Potential sites for the production of superoxide and nitric oxide in skeletal muscle.

Used with permission (Powers & Jackson, 2008)

Nitric oxide synthase

In skeletal muscle cells, nitric oxide (NO) is generated by neuronal nitric oxide synthase 1 (nNOS;(Ferreira & Reid, 2008)) from the precursor L-arginine. nNOS contains a calmodulin (CaM) binding domain making is Ca²⁺ sensitive and therefore responsive to contractile activity (Supinski & Callahan, 2007). While NO is a potent signaling molecule, it readily reacts with superoxide to form peroxynitrite, which in-turn reacts potently with thiol groups on protein targets. The formation of peroxynitrite also decreases the bioavailability of NO and superoxide, thus modifying the redox balance in the myocytes (Judge, Kass, Thompson, & Wagner, 2011; Kass, Takimoto, Nagayama, & Champion, 2007).

Xanthine Oxidase

In skeletal muscle, xanthine oxidase (XO) exists in two forms whose primary purpose is to catabolize purines into uric acid (Corte & Stirpe, 1972; Harrison, 2002). Under basal activity conditions, XO primarily exists as the enzyme xanthine dehydrogenase (XDH). Increased oxidative stress can oxidize key cysteine amino acids inducing reversible changes of the XDH to XO. Proteolytic enzymes may also cleave the XDH irreversibly to form XO (McNally, Saxena, Cai, Dikalov, & Harrison, 2005). Both enzymes catabolize hypoxanthine to xanthine and then from xanthine to uric acid (Halliwell & Gutteridge, 2007; Harrison, 2002). The primary difference between the two enzymes is that the XDH uses NAD+ as the electron acceptor as opposed to XO that uses O₂. Therefore, generally XO forms the free radical O₂^·− while XDH does not. XO primarily resides on the surface of the endothelial cells lining the skeletal muscle. However, intracellular expression of this enzyme have also been detected (M.-C. Gomez-Cabrera, Domenech, & Viña, 2008; M. C. Gomez-Cabrera et al., 2005; M. C. Gomez-Cabrera, Pallardo, Sastre, Vina, & Garcia-del-Moral, 2003; Harrison, 2002; Houston et al., 1999). It is important to note XO can also form the free radical NO^· under specific hypoxic conditions (Halliwell & Gutteridge, 2007; Harrison, 2002).

Phospholipase A2

Another source for ROS generation in skeletal muscle is from the phospholipase A₂ (PLA₂) enzyme family. These enzymes catalyze the hydrolysis reactions of membrane glycerophospholipids to form arachidonic acid (AA) (Murakami & Kudo, 2002). The enzymatic pathways for cyclooxygenase (COX1, COX2) and the lipoxygenase family then catalyze AA to form eicosanoid inflammatory mediator products such as prostaglandins, leukotrienes, and thromboxanes (Harris, 2007). In skeletal muscle the Ca²⁺ dependent cPLA₂ was detected along the sarcolemma and within the mitochondria while the Ca²⁺ independent constitutively active iPLA₂ isoform was detected within the cytosol.

Nethery and colleagues (1999) used excised rat hemidiaphragm preparations to evaluate cPLA₂ dependent ROS generation in contracting and non-contracting muscle. The cPLA₂ specific inhibitor manoalide significantly decreased the ROS generation during contractile conditions when compared to the untreated control samples (Nethery, Stofan, Callahan, DiMarco, & Supinski, 1999). Furthermore, isolated mitochondrial preparations from the diaphragm muscle also suggested AA interacted with the electron transport chain most likely at Complex I to form ROS, as measured by a H₂O₂ assay (Nethery et al., 2000).

Mitochondria

The mitochondria are a source of free radical generation in muscle. In the process of oxidative phosphorylation, Complexes I–IV and the F₁F₀ ATP synthase protein form the primary quarternary structures to oxidize reducing equivalents. The stepwise reduction transfers electrons to O₂ forming water (H₂O) while establishing a H+ ion gradient between the inner and outer mitochondrial membranes. RS are generated along the electron transport chain (ETC), particularly O₂^·− which spontaneously dismutes to H₂O₂ or the enzyme manganese superoxide dismutase (Mn²⁺SOD) catalyzes the reduction of O₂^·− to H₂O₂ and O₂, Formula 6. The antioxidant Mn²⁺SOD is localized to the mitochondria, while CuZnSOD is located within the cytosol.

Formula 6

This reaction is important because the SOD enzymes remove the O₂^·− from the overall oxidant pool. Initial research estimated approximately 2% of the total oxygen within mitochondrial samples of rat liver, rat heart and pigeon heart generated H₂O₂ (Chance, Sies, & Boveris, 1979). Further insight into mitochondrial oxygen metabolism suggested less RS are generated from the electron transport during oxidative phosphorylation. Using specific Complex inhibitors, St- Pierre and colleagues estimated only 0.1% – 0.2% of the oxygen is consumed for RS formation. The amount and rate of RS generation are important. The O₂^·− is membrane impermeable, but the dismutation product H₂O₂ can form OH^· which acts as a significant oxidant.

NADPH Oxidase

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase or Nox) is a family of multi-protein enzymes first described in phagocytic cells (Hohn & Lehrer, 1975). In phagocytic cells, the Nox enzymes are responsible for generating a large amount of the free radical O₂^·− as a microbicidal mechanism. The active oxidase structure localized to the membrane includes the two membrane proteins, gp91phox and p22phox forming the b₅₅₈ reductase complex (Chowdhury et al., 2005). Three cytosolic proteins, p40phox, p47phox, p67phox, and the small GTP-binding protein Rac form the remaining structure of the Nox enzyme. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the two membrane proteins which results in enzymatic activation and production of O₂^·−. The variants of the catalytic subunit, gp91phox, form the different isoforms: Nox 1–5, and the dual oxidase (Duox) Nox2 homologues, Duox 1 and 2 (Bedard & Krause, 2007; Brown & Griendling, 2009). Continued research of this enzyme family revealed these multiple isoforms are differentially expressed in tissues and do not only perform microbicidal functions. The ROS generated from Nox are key signaling intermediates within inflammatory, angiogenic (Vokurkova, Xu, & Touyz, 2007; Xu & Touyz, 2006), apoptotic, cell proliferation (Sauer, Klimm, Hescheler, & Wartenberg, 2001), and tumorgenic pathways (Arbiser et al.).

The Nox2 and Nox4 isoforms are the predominant Nox enzymes expressed in skeletal muscle. Both have been detected within the transverse tubule system and at the plasma membrane (Hidalgo, Sanchez, Barrientos, & Aracena-Parks, 2006; Javesghani, Magder, Barreiro, Quinn, & Hussain, 2002). It is believed that Nox4 is active at rest while Nox2 may be activated through several mechanisms. For example, Nox2 enzymes have been implicated in ROS formation with depolarization (Espinosa et al., 2006), osmotic stress (Martins, Shkryl, Nowycky, & Shirokova, 2008), as well as the exercise pressor reflex (H. J. Wang, Pan, Wang, Zucker, & Wang, 2009). Additionally, H₂O₂ has been shown to activate the tyrosine kinase Src, in mouse skeletal muscle (Gervasio, Whitehead, Yeung, Phillips, & Allen, 2008). Activation of Src is important because this kinase phosphorylates the cytosolic subunit p47phox. Phosphorylated p47phox translocates to the plasma membrane and complexes with the membrane bound b₅₅₈ subunits. The subunit p67phox binds to the phosphorylated p47phox and is transported concurrently to the plasma membrane. The p40phox and Rac1 subunits then also bind to form a complete Nox2 enzyme. Osmotic stress has been shown to generate ROS, but the physiological importance of this type of mechanical stretch is debatable.

Recent work by our group and others has implicated the Nox pathway as the major source of superoxide ROS in the skeletal muscle cell during repetitive contractions (Michaelson, Shi, Ward, & Rodney, 2010; Sakellariou et al., 2012). ROS production with contraction has been shown to arise from the sarcolemma and t-tubules by activation of transmembrane Nox2 (Espinosa, et al., 2006; Khairallah, et al., 2012; Prosser, Ward, & Lederer, 2011; Sakellariou, et al., 2012). It was further suggested that Nox2 generated superoxide then promoted Nox4 activation within the mitochondria to generate ROS that was released into the cytosol (Whitehead, Streamer, Lusambili, Sachs, & Allen, 2006; Whitehead, Yeung, & Allen, 2006). However, experiments to directly address this hypothesis have not supported that idea (Sakellariou, et al., 2012).

Mechano-transduction activated NOX2 dependent ROS production

Recent investigation into the mechanisms by which exercise or activity regulates ROS/RNS production in the muscle has focused on how the mechanical stress of contraction is converted to a signal that activates ROS (i.e., the mechano-transduction dependent activation of ROS). Experiments and methods used to mechanically manipulate single enzymatically isolated muscle cells were pioneered by several groups (Calaghan, Belus, & White, 2003; Gannier, White, Lacampagne, Garnier, & Le Guennec, 1994; Le Guennec, White, Gannier, Argibay, & Garnier, 1991). These approaches have recently been coupled with high-speed fluorescence microscopy and have begun to reveal new mechanistic insight into the mechano-activation of ROS signaling in skeletal muscle.

The concept of cellular "tensegrity,” proposes that the microtubule (MT) filament network resists mechanical perturbations in cells and in doing so acts as a mechano-transducer (Ingber, 2008; Stamenovic, Mijailovich, Tolic-Norrelykke, Chen, & Wang, 2002; N. Wang, Butler, & Ingber, 1993; N. Wang et al., 2001). The initial hypothesis that the MT network was a mechano-signaling element in heart arose from elegant work in the literature (Calaghan, et al., 2003; Calaghan, Le Guennec, & White, 2004; Iribe et al., 2009). Working with the knowledge that Nox2 is activated by either contraction (i.e, contraction/compression followed by relaxation/stretch), osmotic stress (stretch or compression(Isaeva, Shkryl, & Shirokova, 2005; Martins, et al., 2008; Shkryl et al., 2009; Ullrich, Fanchaouy, Gusev, Shirokova, & Niggli, 2009)), or stretch (Murdoch, Zhang, Cave, & Shah, 2006), our group focused attention on a hypothesis whereby a MT dependent mechano-transduction pathway could translate a mechanical signal to Nox2 to activate ROS. To address this question, we developed new tools and methods (Khairallah, et al., 2012; Prosser, et al., 2011) enabling us to establish a model of stretch activated mechano-transduction in striated muscle. This novel individual muscle fiber device removed the potential confounders of contraction, membrane damaging force (eccentric injury), or non-physiologic experimental stress such as osmotic shock.

Using these new methods coupled with high-speed confocal fluorescence imaging approaches, we revealed that in muscle cells loaded with the ROS indicator dichlorofluorescein (DCF), a brief, acute physiologic stretch elicits a burst of ROS production. As DCF is a non-specific ROS indicator, we used selective inhibitors and genetic approaches to reveal that the microtubule network acted as a mechano-transducer to activate NoX2 dependent ROS generation, a pathway termed X-ROS signaling (Khairallah, et al., 2012; Prosser, et al., 2011). In heart cells, others reported that X-ROS directly or indirectly lead to post-translational modification of the RyR2s, increasing the sensitivity of RyR2s to [Ca²⁺]_i and thus promoting the fidelity of excitation-contraction (EC) coupling. We confirmed many of the features of X-ROS signaling in heart also in skeletal muscle (Khairallah, et al., 2012; Palomero, Pye, Kabayo, & Jackson, 2012), however we demonstrated that the signaling involves a stretch-activated sarcolemmal channel whose opening is enhanced by Nox2-derived ROS. Our ongoing experiments are defining the role of X-ROS in modulating signaling during the contraction cycle. However, we have firmly established that X-ROS is an important pathological component in Duchenne muscular dystrophy, where an increase in microtubule network density and Nox2 expression leads to a detrimental enhancement of X-ROS signaling (Khairallah, et al., 2012).

Targets of ROS/RNS in skeletal muscle

ROS/RNS have been shown to modulate multiple processes in skeletal muscle such as, transcription factor activity, ion transport, apoptosis, metabolism including numerous proteins critical for muscle cell function (R. G. Allen & Tresini, 2000; Barbieri & Sestili, 2012). Within proteins, sulfhydryl groups (−SH) are susceptible to either oxidation or the formation of disulfide bonds. Physiological reduction and oxidation of sulfhydryl moieties take part in the modulation of protein function (Thomas & Mallis, 2001). For example, oxidation, Snitrosylation (Foster, McMahon, & Stamler, 2003; Sun, Xin, Eu, Stamler, & Meissner, 2001), or S-glutathionylation of key cysteine in the RyR1 calcium release channel of skeletal muscle can significantly alter the mean open time and the permeability of the channel (Aracena-Parks et al., 2006; Hamilton & Reid, 2000; Hidalgo, et al., 2006). In addition to the RyR1, the sarcoendoplasmic reticulum calcium ATPase pump (i.e., SERCA pump) (Lehotsky, Kaplan, Murin, & Raeymaekers, 2002; Squier, 2001; Trebak, Ginnan, Singer, & Jourd'heuil, 2010) membrane lipids (Spiteller, 2010), or DNA (Ragu et al., 2007), CaMKinase (Franklin, Rodriguez–Mora, LaHair, & McCubrey, 2006; Pinto, de Sousa, & Sorenson, 2011) and contractile proteins(Ferreira, et al., 2012; Stasko, Hardin, Smith, Moylan, & Reid, 2013) have all been shown to be modified by ROS/RNS.

ROS has also been shown to act as a second messenger in the stretch dependent activation of the nf-KB pathway (Kumar & Boriek, 2003). This pathway, when in excess, promotes degeneration and atrophy of skeletal muscle (Reid & Li, 2001) across multiple disease states (Ferreira, et al., 2012). In addition, several inflammatory disorders/conditions (i.e., rheumatoid arthritis, heart failure, systemic lupus erythematosus) affect skeletal muscle function and new evidence supports cytokine receptor dependent activation and subsequent intracellular second messenger ROS signaling as a contributor in the functional deficits (See Figure 3).

Figure 3. Model of signaling events that mediate inflammation induced skeletal muscle weakness in heart failure.

Model shows hypothetical mechanism by which chronic heart failure could depress specific force of skeletal muscle. Boxes below the sarcolemma (horizontal band) depict intracellular events that regulate TNFR1/oxidant signaling (right edge, red dashed bar) and oxidative inhibition of myofilament function (green hatched bar). Boxes above sarcolemma represent extracellular events that modulate the process (blue bar). TNF, tumor necrosis factor; TNFR1, TNF receptor subtype 1; SMase, sphingomyelinase; PKC, phosphokinase C; ERK, extracellular regulatory kinase; PLA2, phospholipase A2; NO, nitric oxide. Reproduced with permission (Reid & Moylan, 2011)

Finally, ROS has been shown to be a critical modulator in several transcriptional pathways that respond to activity/exercise in muscle. Peroxisome Proliferator-activated Receptor-γ Coactivator-1α (PGC1-alpha) is a transcriptional coactivator shown to activate mitochondria biogenesis(Spiegelman, 2007). As mitochondrial deficits contribute to numerous skeletal muscle diseases (diabetes, amyotrophic lateral sclerosis, Duchene muscular dystrophy) this pathway is thought to represent a novel therapeutic target (Guevel et al., 2011; Hsieh et al., 2011; Santos, Tewari, Goldberg, & Kowluru, 2011). The regulation of the PGC1-a pathway is in fact redox sensitive, which suggests that ROS plays a role in its regulation (Chen et al., 2011; Spiegelman, 2007). The transcriptional regulators histone deacetylase 4 and 5 (HDAC4, 5) are important regulating muscle plasticity and metabolism with activity. Recently, Nox2 dependent ROS has been shown to regulate the nuclear effluxes of the HDAC4, 5 after intense muscle activity(Liu, Hernandez-Ochoa, Randall, & Schneider, 2012). Finally, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that regulates the transcription of over 200 genes responsible for antioxidant defenses in cell and antioxidant enzymes. Nrf2 is kept in the cytoplasm by a cluster of proteins that degrade it quickly. Under oxidative stress, Nrf2 is released from the protein cluster and it travels to the nucleus where it initiates transcription of the antioxidative genes and their proteins(Guerrero-Beltran, Calderon-Oliver, Pedraza-Chaverri, & Chirino, 2012; Mann et al., 2007).

Conclusion

Until recently, ROS/RNS have been considered toxic species resulting in cellular oxidative stress and leading to pathogenesis and premature aging(Amicarelli et al., 1999; Bejma & Ji, 1999; Fielding & Meydani, 1997; Lawler, Cline, Hu, & Coast, 1997; Pansarasa, Bertorelli, Vecchiet, Felzani, & Marzatico, 1999; Reid & Durham, 2002; Vasilaki et al., 2006). We now however understand that ROS/RNS are also important signaling molecules, which regulate physiological processes(Barbieri & Sestili, 2012). This apparent dichotomy in the role of ROS/RNS in skeletal muscle is a focus of much investigation.

It is reasonable to assume that the concentration of ROS/RNS distinguishes between physiological and pathological signaling(Barbieri & Sestili, 2012). In support, generalized ROS scavenging has been shown to be deleterious to acute exercise adaptation in healthy muscle (Petersen et al., 2012; Strobel et al., 2011) showing that a basal level of ROS signaling is important for positive adaptation. This same general ROS reduction strategies are also ineffective as therapeutic strategies in neuromuscular diseases (i.e., idebenone (Buyse et al., 2011), Coenzyme Q₁₀ (Spurney et al., 2011), tocopherol (Arthur, Austin, & Roberts, 1988)) where excess ROS is known to be a disease contributor. In these cases, the source and the species of ROS are likely disease specific and in this case, a targeted approach for therapy is likely needed.

Our understanding of ROS/RNS signaling is moving rapidly. This brief review of ROS/RNS chemistry and concepts of ROS/RNS signaling will hopefully spur the reader to explore this topic further in the reviews and primary literature cited here. We expect that this knowledge will rapidly make its way into clinical practice as novel treatments are developed to target diseases and disorders by ROS/RNS dependent mechanisms.

Table 1.

ROS/RNS

ROS
Species	Effector
O2·−	JNK, base radical for H2O2, OH·, ONOO−
H2O2	nf-κβ, c-Src, Iκβ, TGf-β, RyR1,PKC-α
OH·	c-jun
RNS
NO·	RyR1, guanylate cyclase, albumin, hemoglobin
ONOO−	mitochondrial enzymes (aconitase), RyR1,COX