We have modified the biotin switch assay for protein S-nitrosothiols (SNOs), using resin-assisted capture (SNO-RAC). Compared with existing methodologies, SNO-RAC requires fewer steps, detects high-mass S-nitrosylated proteins more efficiently, and facilitates identification and quantification of S-nitrosylated sites by mass spectrometry. When combined with iTRAQ labeling, SNO-RAC revealed that intracellular proteins may undergo rapid denitrosylation on a global scale. This methodology is readily adapted to analyzing diverse cysteine-based protein modifications, including S-acylation.

Non-technical summary During exercise, skeletal muscle performance depends in great part on the use of aerobic metabolism to supply the energetic demand of contractions. Endurance training increases the muscle aerobic capacity, which is not only associated with enhanced exercise performance, but also with a decreased risk of cardiovascular and metabolic diseases. Recently, it has been shown that regular use of small doses of dark chocolate may result in similar health benefits to exercise training. We show here that mice fed for 15 days with (-)-epicatechin (present in dark chocolate) had improved exercise performance accompanied by: (1) an increased number of capillaries in the hindlimb muscle; and (2) an increased amount of muscle mitochondria as well as signalling for mitochondrial biogenesis. These results suggest that (-)-epicatechin increases the capacity for muscle aerobic metabolism, thereby delaying the onset of fatigue. These findings may have potential application for clinical populations experiencing muscle fatigue. AbstractThe flavanol (-)-epicatechin, a component of cacao (cocoa), has been shown to have multiple health benefits in humans. Using 1-year-old male mice, we examined the effects of 15 days of (-)-epicatechin treatment and regular exercise on: (1) exercise performance, (2) muscle fatigue, (3) capillarity, and (4) mitochondrial biogenesis in mouse hindlimb and heart muscles. Twenty-five male mice (C57BL/6N) were randomized into four groups: (1) water, (2) water-exercise (W-Ex), (3) (-)-epicatechin ((-)-Epi), and (4) (-)-epicatechin-exercise ((-)-Epi-Ex). Animals received 1 mg kg −1 of (-)-epicatechin or water (vehicle) via oral gavage (twice daily). Exercise groups underwent 15 days of treadmill exercise. Significant increases in treadmill performance (∼50%) and enhanced in situ muscle fatigue resistance (∼30%) were observed with (-)-epicatechin. Components of oxidative phosphorylation complexes, mitofilin, porin, nNOS, p-nNOS, and Tfam as well as mitochondrial volume and cristae abundance were significantly higher with (-)-epicatechin treatment for hindlimb and cardiac muscles than exercise alone. In addition, there were significant increases in skeletal muscle capillarity. The combination of (-)-epicatechin and exercise resulted in further increases in oxidative phosphorylation-complex proteins, mitofilin, porin and capillarity than (-)-epicatechin alone. These findings indicate that (-)-epicatechin alone or in combination with exercise induces an integrated response that includes structural and metabolic changes in skeletal and cardiac muscles resulting in greater endurance capacity. These results, therefore, warrant the further evaluation of the underlying mechanism of action of (-)-epicatechin and its potential clinical application as an exercise mimetic.

Physiological sensing of O 2 tension (partial O 2 pressure, pO 2 ) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO 2 to altered calcium release through the ryanodine receptor-Ca 2+ -release channel (RyR1). Reactive oxygen species are generated in proportion to pO 2 by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca 2+ release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O 2 sensor in skeletal muscle, and O 2 -coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O 2 -based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.redox signaling | oxygen sensing | S-nitrosylation S pecialized mammalian sensory cells transduce varying O 2 levels, but the mechanisms of O 2 sensing and O 2 -based signaling have not been elucidated fully. In particular, reversible changes in ion channel activity often are implicated in physiological responses to O 2 , but the molecular bases of these changes are unknown. We previously have described an O 2 -sensing and -signaling mechanism in mammalian skeletal muscle that operates on the ryanodine receptor-Ca 2+ -release channel (RyR1), the principal source of Ca 2+ release from the sarcoplasmic reticulum (SR) (1, 2). At relatively low partial pressure of O 2 (pO 2 ), endogenously generated NO regulates RyR1 activity by S-nitrosylation of a single Cys thiol (1, 3). At higher pO 2 , RyR1 activity is enhanced independently of NO in association with the oxidation of a separate, small set of Cys thiols (1). Oxidation of RyR1 thiols at high pO 2 and reduction following transition from high to low pO 2 are observed in isolated SR vesicles (1). However, the molecular mechanisms within the SR that mediate this O 2 -based redox cycle have not been determined. Here we show that the redox enzyme NADPH oxidase 4 (Nox4) is a constituent of the SR and that hydrogen peroxide (H 2 O 2 ) produced by Nox4 in proportion to pO 2 over a physiological range serves as an essential effector of pO 2 -dependent regulation of RyR1 redox status and function. These data demonstrate physiological regulation by reactive oxygen species (ROS) of RyR1 and suggest insights into the molecular mechanisms of O 2 sensing and O 2 -based signaling in mammalian cells. ResultsRyR1 activity in an SR-enriched subcellular fraction (SR vesicles) was enhanced progressively at pO 2 of 1% O 2 , 5% O 2 , and 20% O 2 (ambient pO 2 ) (Fig. 1A); these levels largely recapitulate the physiological muscle O 2 gradient and extend to oxidative stress (4-7). Production of ROS was enhanced similarly, as assessed ...

Vascular endothelial growth factor (VEGF) is exercise responsive, pro-angiogenic, and expressed in several muscle cell types. We hypothesized that in adult mice, VEGF generated within skeletal myofibers (and not other cells within muscle) is necessary for the angiogenic response to exercise training. This was tested in adult conditional, skeletal myofiber-specific VEGF gene-deleted mice (skmVEGF−/−), with VEGF levels reduced by >80%. After 8 wk of daily treadmill training, speed and endurance were unaltered in skmVEGF−/− mice, but increased by 18% and 99% ( P < 0.01), respectively, in controls trained at identical absolute speed, incline, and duration. In vitro, isolated soleus and extensor digitorum longus contractile function was not impaired in skmVEGF−/− mice. However, training-induced angiogenesis was inhibited in plantaris (wild type, 38%, skmVEGF−/− 18%, P < 0.01), and gastrocnemius (wild type, 43%, P < 0.01; skmVEGF−/−, 7%, not significant). Capillarity was maintained (different from VEGF gene deletion targeted to multiple cell types) in untrained skmVEGF−/− mice. Arteriogenesis (smooth muscle actin+, artery number, and diameter) and remodeling [vimentin+, 5′-bromodeoxycytidine (BrdU)+, and F4/80+ cells] occurred in skmVEGF−/− mice, even in the absence of training. skmVEGF−/− mice also displayed a limited oxidative enzyme [citrate synthase and β-hydroxyacyl CoA dehydrogenase (β-HAD)] training response; β-HAD activity levels were elevated in the untrained state. These data suggest that myofiber expressed VEGF is necessary for training responses in capillarity and oxidative capacity and for improved running speed and endurance.

Sarcopenia is a notable and debilitating age-associated condition. Flavonoids are known for their healthy effects and limited toxicity. The flavanol (−)-epicatechin (Epi) enhances exercise capacity in mice and Epi-rich cocoa improves skeletal muscle structure in heart failure patients. (−)-Epicatechin may thus, hold promise as treatment for sarcopenia. We examined changes in protein levels of molecular modulators of growth and differentiation in young vs. old, human and mouse skeletal muscle. We report the effects of Epi in mice and the results of an initial proof-of-concept trial in humans, where muscle strength and levels of modulators of muscle growth were measured. In mice, myostatin and senescence-associated β-galactosidase levels increase with aging, while those of follistatin and Myf5 decrease. (−)-Epicatechin decreases myostatin and β-galactosidase and increases levels of markers of muscle growth. In humans, myostatin and β-galactosidase increase with aging while follistatin, MyoD and myogenin decrease. Treatment for 7 days with (−)-epicatechin increases hand grip strength and the ratio of plasma follistatin/myostatin. In conclusion, aging has deleterious effects on modulators of muscle growth/differentiation, the consumption of modest amounts of the flavanol (−)-epicatechin can partially reverse these changes. This flavanol warrants its comprehensive evaluation for the treatment of sarcopenia

Background Type 2 diabetes (T2D) and heart failure (HF) are associated with high levels of skeletal muscle (SkM) oxidative stress (OS). Health benefits attributed to flavonoids have been ascribed to antioxidation. However, for flavonoids with similar antioxidant potential, end-biological effects vary widely suggesting other mechanistic venues for reducing OS. Decreases in OS may follow the modulation of key regulatory pathways including antioxidant levels (e.g. glutathione) and enzymes such as mitochondrial superoxide dismutase (SOD2) and catalase. Methods We examined OS-related alterations in SkM in T2D/HF patients (as compared vs. healthy controls) and evaluated the effects of three-month treatment with (−)-epicatechin (Epi) rich cocoa (ERC). To evidence Epi as the mediator of the improved OS profile we examined the effects of pure Epi (vs. water) on SkM OS regulatory systems in a mouse model of insulin resistance and contrasted results vs. normal mice. Results There were severe alterations in OS regulatory systems in T2D/HF SkM as compared with healthy controls. Treatment with ERC induced recovery in glutathione levels and decreases in the nitrotyrosilation and carbonylation of proteins. With treatment, key transcriptional factors translocate into the nucleus leading to increases in SOD2 and catalase protein expression and activity levels. In insulin resistant mice, there were alterations in muscle OS and pure Epi replicated the beneficial effects of ERC found in humans. Conclusions Major perturbations in SkM OS can be reversed with ERC in T2D/HF patients. Epi likely mediates such effects and may provide an effective means to treat conditions associated with tissue OS.

Key points Dietary nitrate supplementation increases plasma nitrite concentration, which provides an oxygen‐independent source of nitric oxide and can delay skeletal muscle fatigue. Nitrate supplementation has been shown to increase myofibre calcium release and force production in mouse skeletal muscle during contractions at a supra‐physiological oxygen tension, but it is unclear whether nitrite exposure can delay fatigue development and improve myofibre calcium handling at a near‐physiological oxygen tension. Single mouse muscle fibres acutely treated with nitrite had a lower force and cytosolic calcium concentration during single non‐fatiguing contractions at a near‐physiological oxygen tension. Nitrite treatment delayed fatigue development during repeated fatiguing isometric contractions at near‐physiological, but not at supra‐physiological, oxygen tension in combination with better maintenance of myofilament calcium sensitivity and sarcoplasmic reticulum calcium pumping. These findings improve understanding of the mechanisms by which increased skeletal muscle nitrite exposure might be ergogenic and imply that this is related to improved calcium handling. Abstract Dietary nitrate (NO3−) supplementation, which increases plasma nitrite (NO2−) concentration, has been reported to attenuate skeletal muscle fatigue development. Sarcoplasmic reticulum (SR) calcium (Ca2+) release is enhanced in isolated single skeletal muscle fibres following NO3− supplementation or NO2− incubation at a supra‐physiological PnormalO2 but it is unclear whether NO2− incubation can alter Ca2+ handling and fatigue development at a near‐physiological PnormalO2. We hypothesised that NO2− treatment would improve Ca2+ handling and delay fatigue at a physiological PnormalO2 in intact single mouse skeletal muscle fibres. Each muscle fibre was perfused with Tyrode solution pre‐equilibrated with either 20% (PnormalO2∼150 Torr) or 2% O2 (PnormalO2 = 15.6 Torr) in the absence and presence of 100 µM NaNO2. At supra‐physiological PnormalO2 (i.e. 20% O2), time to fatigue was lowered by 34% with NaNO2 (control: 257 ± 94 vs. NaNO2: 159 ± 46 s, Cohen's d = 1.63, P < 0.05), but extended by 21% with NaNO2 at 2% O2 (control: 308 ± 217 vs. NaNO2: 368 ± 242 s, d = 1.14, P < 0.01). During the fatiguing contraction protocol completed with NaNO2 at 2% O2, peak cytosolic Ca2+ concentration ([Ca2+]c) was not different (P > 0.05) but [Ca2+]c accumulation between contractions was lower, concomitant with a greater SR Ca2+ pumping rate (P < 0.05) compared to the control condition. These results demonstrate that increased exposure to NO2− blunts fatigue development at near‐physiological, but not at supra‐physiological, PnormalO2 through enhancing SR Ca2+ pumping rate in single skeletal muscle fibres. These findings extend our understanding of the mechanisms by which increased NO2− exposure can mitigate skeletal muscle fatigue development.

There is evidence implicating oxidative stress (OS) as the cause of the deleterious effects of aging. In this study, we evaluated the capacity of the flavanol (-)-epicatechin (Epi) to reduce aging-induced OS and restore mitochondrial biogenesis, as well as, structural and functional endpoints in aged mice. Senile (S; 26-month-old) C57BL/6 male mice were randomly assigned to receive either water (vehicle) or 1mg/kg of Epi via oral gavage (twice daily) for 15 days. Young (Y; 6-month-old) mice were used as controls. In S brain, kidney, heart, and skeletal muscle (compared with Y animals) an increase in OS was observed as evidenced by increased protein-free carbonyls and decreased reduced glutathione levels as well as sirtuin 3, superoxide dismutase 2, catalase, thioredoxin and glutathione peroxidase protein levels. Well-recognized factors (eg, sirtuin 1) that regulate mitochondrial biogenesis and mitochondrial structure- and/or function-related endpoints (eg, mitofilin and citrate synthase) protein levels were also reduced in S organs. In contrast, the aging biomarker senescence-associated β-galactosidase was increased in S compared with Y animals, and Epi administration reduced levels towards those observed in Y animals. Altogether, these data suggest that Epi is capable of shifting the biology of S mice towards that of Y animals.