The interactive effect of sustained sleep restriction and resistance exercise on skeletal muscle transcriptomics in young females.

Both sleep loss and exercise regulate gene expression in skeletal muscle, yet little is known about how the interaction of these stressors affects the transcriptome. The aim of this study was to investigate the effect of nine nights of sleep restriction (SR), with repeated resistance exercise (REx) sessions, on the skeletal muscle transcriptome of young, trained females. Ten healthy females aged 18-35 yr old undertook a randomized cross-over study of nine nights of SR (5 h time in bed) and normal sleep (NS; ≥7 h time in bed) with a minimum 6-wk washout. Participants completed four REx sessions per condition (days 3, 5, 7, and 9). Muscle biopsies were collected both pre- and post-REx on days 3 and 9. Gene and protein expression were assessed by RNA sequencing and Western blot, respectively. Three or nine nights of SR had no effect on the muscle transcriptome independently of exercise. However, close to 3,000 transcripts were differentially regulated (false discovery rate < 0.05) 48 h after the completion of three resistance exercise sessions in both NS and SR conditions. Only 39% of downregulated genes and 18% of upregulated genes were common between both conditions, indicating a moderating effect of SR on the response to exercise. SR and REx interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.NEW & NOTEWORTHY This study investigated the effect of nine nights of sleep restriction, with repeated resistance exercise sessions, on the skeletal muscle transcriptome of young, trained females. Sleep restriction and resistance exercise interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.

A gene therapy targeting medium-chain acyl-CoA dehydrogenase (MCAD) did not protect against diabetes-induced cardiac pathology.

Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. KEY MESSAGES: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications.

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signaling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and the activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long noncoding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1,000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signaling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response to a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signaling, which could have important implications for cardiac function.NEW & NOTEWORTHY Calcium is essential for signaling, excitation contraction, and energy homeostasis in the heart. Despite this, molecular regulators of these processes are not completely understood. We report that knockdown of lncRNA Tug1 alters the calcium handling transcriptome and increases mitochondrial calcium uniporter expression via a mechanism involving CaMKII. As overexpression of MCU is known to be protective against pathological cardiac remodeling, targeting Tug1 may be a potential strategy for treating cardiovascular disease.

Long non-coding RNA Tug1 modulates mitochondrial and myogenic responses to exercise in skeletal muscle.

BACKGROUND: Mitochondria have an essential role in regulating metabolism and integrate environmental and physiological signals to affect processes such as cellular bioenergetics and response to stress. In the metabolically active skeletal muscle, mitochondrial biogenesis is one important component contributing to a broad set of mitochondrial adaptations occurring in response to signals, which converge on the biogenesis transcriptional regulator peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α), and is central to the beneficial effects of exercise in skeletal muscle. We investigated the role of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1), which interacts with PGC-1α in regulating transcriptional responses to exercise in skeletal muscle. RESULTS: In human skeletal muscle, TUG1 gene expression was upregulated post-exercise and was also positively correlated with the increase in PGC-1α gene expression (PPARGC1A). Tug1 knockdown (KD) in differentiating mouse myotubes led to decreased Ppargc1a gene expression, impaired mitochondrial respiration and morphology, and enhanced myosin heavy chain slow isoform protein expression. In response to a Ca2+-mediated stimulus, Tug1 KD prevented an increase in Ppargc1a expression. RNA sequencing revealed that Tug1 KD impacted mitochondrial Ca2+ transport genes and several downstream PGC-1α targets. Finally, Tug1 KD modulated the expression of ~300 genes that were upregulated in response to an in vitro model of exercise in myotubes, including genes involved in regulating myogenesis. CONCLUSIONS: We found that TUG1 is upregulated in human skeletal muscle after a single session of exercise, and mechanistically, Tug1 regulates transcriptional networks associated with mitochondrial calcium handling, muscle differentiation and myogenesis. These data demonstrate that lncRNA Tug1 exerts regulation over fundamental aspects of skeletal muscle biology and response to exercise stimuli.

The mitochondrial profile in women with polycystic ovary syndrome: impact of exercise.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting pre-menopausal women and involves metabolic dysregulation. Despite the high prevalence of insulin resistance, the existence of mitochondrial dysregulation and its role in the pathogenesis of PCOS is not clear. Exercise is recommended as the first-line therapy for women with PCOS. In particular, high-intensity interval training (HIIT) is known to improve metabolic health and enhance mitochondrial characteristics. In this narrative review, the existing knowledge of mitochondrial characteristics in skeletal muscle and adipose tissue of women with PCOS and the effect of exercise interventions in ameliorating metabolic and mitochondrial health in these women are discussed. Even though the evidence on mitochondrial dysfunction in PCOS is limited, some studies point to aberrant mitochondrial functions mostly in skeletal muscle, while there is very little research in adipose tissue. Although most exercise intervention studies in PCOS report improvements in metabolic health, they show diverse and inconclusive findings in relation to mitochondrial characteristics. A limitation of the current study is the lack of comprehensive mitochondrial analyses and the diversity in exercise modalities, with only one study investigating the impact of HIIT alone. Therefore, further comprehensive large-scale exercise intervention studies are required to understand the association between metabolic dysfunction and aberrant mitochondrial profile, and the molecular mechanisms underlying the exercise-induced metabolic adaptations in women with PCOS.

Sensitivity to behavioral stress impacts disease pathogenesis in dystrophin-deficient mice.

Mutation to the gene encoding dystrophin can cause Duchenne muscular dystrophy (DMD) and increase the sensitivity to stress in vertebrate species, including the mdx mouse model of DMD. Behavioral stressors can exacerbate some dystrophinopathy phenotypes of mdx skeletal muscle and cause hypotension-induced death. However, we have discovered that a subpopulation of mdx mice present with a wildtype-like response to mild (forced downhill treadmill exercise) and moderate (scruff restraint) behavioral stressors. These "stress-resistant" mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal and renin-angiotensin-aldosterone pathways following behavioral stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to "stress-sensitive" mdx mice. Stress-resistant mdx mice also presented with a less severe striated muscle histopathology and greater exercise and skeletal muscle oxidative capacity at rest. Most interestingly, female mdx mice were more physically active following behavioral stressors compared to male mdx mice; a response abolished after ovariectomy and rescued with estradiol. We demonstrate that the response to behavioral stress greatly impacts disease severity in mdx mice suggesting the management of stress in patients with DMD be considered as a therapeutic approach to ameliorate disease progression.

Reactive oxygen species in exercise and insulin resistance: Working towards personalized antioxidant treatment.

Reactive oxygen species (ROS) are well known for their role in insulin resistance and the development of cardiometabolic disease including type 2 diabetes mellitus (T2D). Conversely, evidence supports the notion that ROS are a necessary component for glucose cell transport and adaptation to physiological stress including exercise and muscle contraction. Although genetic rodent models and cell culture studies indicate antioxidant treatment to be an effective strategy for targeting ROS to promote health, human findings are largely inconsistent. In this review we discuss human research that has investigated antioxidant treatment and glycemic control in the context of health (healthy individuals and during exercise) and disease (insulin resistance and T2D). We have identified key factors that are likely to influence the effectiveness of antioxidant treatment: 1) the context of treatment including whether oxidative distress or eustress is present (e.g., hyperglycemia/lipidaemia or during exercise and muscle contraction); 2) whether specific endogenous antioxidant deficiencies are identified (redox screening); 3) whether antioxidant treatment is specifically designed to target and restore identified deficiencies (antioxidant specificity); 4) and the bioavailability and bioactivity of the antioxidant which are influenced by treatment dose, duration, and method of administration. The majority of human research has failed to account for these factors, limiting their ability to robustly test the effectiveness of antioxidants for health promotion and disease prevention. We propose that a modern "redox screening" and "personalized antioxidant treatment" approach is required to robustly explore redox regulation of human physiology and to elicit more effective antioxidant treatment in humans.

Antioxidant supplements and endurance exercise: Current evidence and mechanistic insights.

Antioxidant supplements are commonly consumed by endurance athletes to minimize exercise-induced oxidative stress, with the intention of enhancing recovery and improving performance. There are numerous commercially available nutritional supplements that are targeted to athletes and health enthusiasts that allegedly possess antioxidant properties. However, most of these compounds are poorly investigated with respect to their in vivo redox activity and efficacy in humans. Therefore, this review will firstly provide a background to endurance exercise-related redox signalling and the subsequent adaptations in skeletal muscle and vascular function. The review will then discuss commonly available compounds with purported antioxidant effects for use by athletes. N-acetyl cysteine may be of benefit over the days prior to an endurance event; while chronic intake of combined 1000 mg vitamin C + vitamin E is not recommended during periods of heavy training associated with adaptations in skeletal muscle. Melatonin, vitamin E and α-lipoic acid appear effective at decreasing markers of exercise-induced oxidative stress. However, evidence on their effects on endurance performance are either lacking or not supportive. Catechins, anthocyanins, coenzyme Q10 and vitamin C may improve vascular function, however, evidence is either limited to specific sub-populations and/or does not translate to improved performance. Finally, additional research should clarify the potential benefits of curcumin in improving muscle recovery post intensive exercise; and the potential hampering effects of astaxanthin, selenium and vitamin A on skeletal muscle adaptations to endurance training. Overall, we highlight the lack of supportive evidence for most antioxidant compounds to recommend to athletes.

Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance.

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called protonmotive force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria-targeted light-activated proton pump that we name mitochondria-ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose-dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy-sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well-characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.

Effects of repeated local heat therapy on skeletal muscle structure and function in humans.

The purpose of the present study was to examine the effects of repeated exposure to local heat therapy (HT) on skeletal muscle function, myofiber morphology, capillarization, and mitochondrial content in humans. Twelve young adults (23.6 ± 4.8 yr, body mass index 24.9 ± 3.0 kg/m2) had one randomly selected thigh treated with HT (garment perfused with water at ~52°C) for 8 consecutive weeks (90 min, 5 days/wk) while the opposite thigh served as a control. Biopsies were obtained from the vastus lateralis muscle before and after 4 and 8 wk of treatment. Knee extensor strength and fatigue resistance were also assessed using isokinetic dynamometry. The changes in peak isokinetic torque were higher (P = 0.007) in the thigh exposed to HT than in the control thigh at weeks 4 (control 4.2 ± 13.1 Nm vs. HT 9.1 ± 16.1 Nm) and 8 (control 1.8 ± 9.7 Nm vs. HT 7.8 ± 10.2 Nm). Exposure to HT averted a temporal decline in capillarization around type II fibers (P < 0.05), but had no effect on capillarization indexes in type I fibers. The content of endothelial nitric oxide synthase was ~18% and 35% higher in the thigh exposed to HT at 4 and 8 wk, respectively (P = 0.003). Similarly, HT increased the content of small heat shock proteins HSPB5 (P = 0.007) and HSPB1 (P = 0.009). There were no differences between thighs for the changes in fiber cross-sectional area and mitochondrial content. These results indicate that exposure to local HT for 8 wk promotes a proangiogenic environment and enhances muscle strength but does not affect mitochondrial content in humans.NEW & NOTEWORTHY We demonstrate that repeated application of heat therapy to the thigh with a garment perfused with warm water enhances the strength of knee extensors and influences muscle capillarization in parallel with increases in the content of endothelial nitric oxide synthase and small heat shock proteins. This practical method of passive heat stress may be a feasible tool to treat conditions associated with capillary rarefaction and muscle weakness.

Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2•-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2•--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2•- quantum yield (ΦO2•-) of SuperNova was determined to be 1.5 × 10-3, KillerRed was 0.97 × 10-3, and mCherry 1.2 × 10-3. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 µM O2•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield (Φ1O2) for SuperNova to be 22.0 × 10-3, KillerRed 7.6 × 10-3, and mCherry 5.7 × 10-3. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.

Cold water immersion attenuates anabolic signaling and skeletal muscle fiber hypertrophy, but not strength gain, following whole-body resistance training.

We determined the effects of cold water immersion (CWI) on long-term adaptations and post-exercise molecular responses in skeletal muscle before and after resistance training. Sixteen men (22.9 ± 4.6 y; 85.1 ± 17.9 kg; mean ± SD) performed resistance training (3 day/wk) for 7 wk, with each session followed by either CWI [15 min at 10°C, CWI (COLD) group, n = 8] or passive recovery (15 min at 23°C, control group, n = 8). Exercise performance [one-repetition maximum (1-RM) leg press and bench press, countermovement jump, squat jump, and ballistic push-up], body composition (dual X-ray absorptiometry), and post-exercise (i.e., +1 and +48 h) molecular responses were assessed before and after training. Improvements in 1-RM leg press were similar between groups [130 ± 69 kg, pooled effect size (ES): 1.53 ± 90% confidence interval (CI) 0.49], whereas increases in type II muscle fiber cross-sectional area were attenuated with CWI (-1,959 ± 1,675 µM2 ; ES: -1.37 ± 0.99). Post-exercise mechanistic target of rapamycin complex 1 signaling (rps6 phosphorylation) was blunted for COLD at post-training (POST) +1 h (-0.4-fold, ES: -0.69 ± 0.86) and POST +48 h (-0.2-fold, ES: -1.33 ± 0.82), whereas basal protein degradation markers (FOX-O1 protein content) were increased (1.3-fold, ES: 2.17 ± 2.22). Training-induced increases in heat shock protein (HSP) 27 protein content were attenuated for COLD (-0.8-fold, ES: -0.94 ± 0.82), which also reduced total HSP72 protein content (-0.7-fold, ES: -0.79 ± 0.57). CWI blunted resistance training-induced muscle fiber hypertrophy, but not maximal strength, potentially via reduced skeletal muscle protein anabolism and increased catabolism. Post-exercise CWI should therefore be avoided if muscle hypertrophy is desired.NEW & NOTEWORTHY This study adds to existing evidence that post-exercise cold water immersion attenuates muscle fiber growth with resistance training, which is potentially mediated by attenuated post-exercise increases in markers of skeletal muscle anabolism coupled with increased catabolism and suggests that blunted muscle fiber growth with cold water immersion does not necessarily translate to impaired strength development.

Epigenetic Reprogramming of Immune Cells in Women With PCOS Impact Genes Controlling Reproductive Function.

CONTEXT: Polycystic ovary syndrome (PCOS) is a chronic disease affecting reproductive function and whole-body metabolism. Although the etiology is unclear, emerging evidence indicates that the epigenetics may be a contributing factor. OBJECTIVE: To determine the role of global and genome-wide epigenetic modifications in specific immune cells in PCOS compared with controls and whether these could be related to clinical features of PCOS. DESIGN: Cross-sectional study. PARTICIPANTS: Women with (n = 17) or without PCOS (n = 17). SETTING: Recruited from the general community. MAIN OUTCOME MEASURES: Isolated peripheral blood mononuclear cells were analyzed using multicolor flow cytometry methods to determine global DNA methylation levels in a cell-specific fashion. Transcriptomic and genome-wide DNA methylation analyses were performed on T helper cells using RNA sequencing and reduced representation bisulfite sequencing. RESULTS: Women with PCOS had lower global DNA methylation in monocytes (P = 0.006) and in T helper (P = 0.004), T cytotoxic (P = 0.004), and B cells (P = 0.03). Specific genome-wide DNA methylation analysis of T helper cells from women with PCOS identified 5581 differentially methylated CpG sites. Functional gene ontology enrichment analysis showed that genes located at the proximity of differentially methylated CpG sites belong to pathways related to reproductive function and immune cell function. However, these genes were not altered at the transcriptomic level. CONCLUSIONS: It was shown that PCOS is associated with global and gene-specific DNA methylation remodeling in a cell type-specific manner. Further investigation is warranted to determine whether epigenetic reprogramming of immune cells is important in determining the different phenotypes of PCOS.

High-intensity interval training in chronic kidney disease: A randomized pilot study.

INTRODUCTION: High-intensity interval training (HIIT) increases mitochondrial biogenesis and cardiorespiratory fitness in chronic disease populations, however has not been studied in people with chronic kidney disease (CKD). The aim of this study was to compare the feasibility, safety, and efficacy of HIIT with moderate-intensity continuous training (MICT) in people with CKD. METHODS: Fourteen individuals with stage 3-4 CKD were randomized to 3 supervised sessions/wk for 12 weeks, of HIIT (n = 9, 4 × 4 minute intervals, 80%-95% peak heart rate [PHR]) or MICT (n = 5, 40 minutes, 65% PHR). Feasibility was assessed via session attendance and adherence to the exercise intensity. Safety was examined by adverse event reporting. Efficacy was determined from changes in cardiorespiratory fitness (VO2 peak), exercise capacity (METs), and markers of mitochondrial biogenesis (PGC1α protein levels), muscle protein catabolism (MuRF1), and muscle protein synthesis (p-P70S6k Thr389 ). RESULTS: Participants completed a similar number of sessions in each group (HIIT = 33.0[7.0] vs MICT = 33.5[3.3] sessions), and participants adhered to the target heart rates. There were no adverse events attributable to exercise training. There was a significant time effect for exercise capacity (HIIT = +0.8 ± 1.2; MICT = +1.3 ± 1.6 METs; P = 0.01) and muscle protein synthesis (HIIT = +0.6 ± 1.1; MICT = +1.4 ± 1.7 au; P = 0.04). However, there were no significant (P > 0.05) group × time effects for any outcomes. CONCLUSION: This pilot study demonstrated that HIIT is a feasible and safe option for people with CKD, and there were similar benefits of HIIT and MICT on exercise capacity and skeletal muscle protein synthesis. These data support a larger trial to further evaluate the effectiveness of HIIT.

Mitochondrial Reactive Oxygen Species Generated at the Complex-II Matrix or Intermembrane Space Microdomain Have Distinct Effects on Redox Signaling and Stress Sensitivity in Caenorhabditis elegans.

Aims: How mitochondrial reactive oxygen species (ROS) impact physiological function may depend on the quantity of ROS generated or removed, and the subcellular microdomain in which this occurs. However, pharmacological tools currently available to alter ROS production in vivo lack precise spatial and temporal control. Results: We used CRISPR/Cas9 to fuse the light-sensitive ROS-generating protein, SuperNova to the C-terminus of mitochondrial complex II succinate dehydrogenase subunits B (SDHB-1::SuperNova) and C (SDHC-1::SuperNova) in Caenorhabditis elegans to localize SuperNova to the matrix-side of the inner mitochondrial membrane, and to the intermembrane space (IMS), respectively. The presence of the SuperNova protein did not impact complex II activity, mitochondrial respiration, or C. elegans development rate under dark conditions. ROS production by SuperNova protein in vitro in the form of superoxide (O2˙-) was both specific and proportional to total light irradiance in the 540-590 nm spectra, and was unaffected by varying the buffer pH to resemble the mitochondrial matrix or IMS environments. We then determined using SuperNova whether stoichiometric ROS generation in the mitochondrial matrix or IMS had distinct effects on redox signaling in vivo. Phosphorylation of PMK-1 (a p38 MAPK homolog) and transcriptional activity of SKN-1 (an Nrf2 homolog) were each dependent on both the site and duration of ROS production, with matrix-generated ROS having more prominent effects. Furthermore, matrix- but not IMS-generated ROS attenuated susceptibility to simulated ischemia reperfusion injury in C. elegans. Innovation and Conclusion: Overall, these data demonstrate that the physiological output of ROS depends on the microdomain in which it is produced. Antioxid. Redox Signal. 31, 594-607.

Acute HIIE elicits similar changes in human skeletal muscle mitochondrial H2O2 release, respiration, and cell signaling as endurance exercise even with less work.

It remains unclear whether high-intensity interval exercise (HIIE) elicits distinct molecular responses to traditional endurance exercise relative to the total work performed. We aimed to investigate the influence of exercise intensity on acute perturbations to skeletal muscle mitochondrial function (respiration and reactive oxygen species) and metabolic and redox signaling responses. In a randomized, repeated measures crossover design, eight recreationally active individuals (24 ± 5 yr; V̇o2peak: 48 ± 11 ml·kg-1·min-1) undertook continuous moderate-intensity [CMIE: 30 min, 50% peak power output (PPO)], high-intensity interval (HIIE: 5 × 4 min, 75% PPO, work matched to CMIE), and low-volume sprint interval (SIE: 4 × 30 s) exercise, ≥7 days apart. Each session included muscle biopsies at baseline, immediately, and 3 h postexercise for high-resolution mitochondrial respirometry ( Jo2) and H2O2 emission ( Jh2o2) and gene and protein expression analysis. Immediately postexercise and irrespective of protocol, Jo2 increased during complex I + II leak/state 4 respiration but Jh2o2 decreased ( P < 0.05). AMP-activated protein kinase and acetyl co-A carboxylase phosphorylation increased ~1.5 and 2.5-fold respectively, while thioredoxin-reductase-1 protein abundance was ~35% lower after CMIE vs. SIE ( P < 0.05). At 3 h postexercise, regardless of protocol, Jo2 was lower during both ADP-stimulated state 3 OXPHOS and uncoupled respiration ( P < 0.05) but Jh2o2 trended higher ( P < 0.08) and PPARGC1A mRNA increased ~13-fold, and peroxiredoxin-1 protein decreased ~35%. In conclusion, intermittent exercise performed at high intensities has similar dynamic effects on muscle mitochondrial function compared with endurance exercise, irrespective of whether total workload is matched. This suggests exercise prescription can accommodate individual preferences while generating comparable molecular signals known to promote beneficial metabolic adaptations.

Use the Protonmotive Force: Mitochondrial Uncoupling and Reactive Oxygen Species.

Mitochondrial respiration results in an electrochemical proton gradient, or protonmotive force (pmf), across the mitochondrial inner membrane. The pmf is a form of potential energy consisting of charge (∆ψm) and chemical (∆pH) components, that together drive ATP production. In a process called uncoupling, proton leak into the mitochondrial matrix independent of ATP production dissipates the pmf and energy is lost as heat. Other events can directly dissipate the pmf independent of ATP production as well, such as chemical exposure or mechanisms involving regulated mitochondrial membrane electrolyte transport. Uncoupling has defined roles in metabolic plasticity and can be linked through signal transduction to physiologic events. In the latter case, the pmf impacts mitochondrial reactive oxygen species (ROS) production. Although capable of molecular damage, ROS also have signaling properties that depend on the timing, location, and quantity of their production. In this review, we provide a general overview of mitochondrial ROS production, mechanisms of uncoupling, and how these work in tandem to affect physiology and pathologies, including obesity, cardiovascular disease, and immunity. Overall, we highlight that isolated bioenergetic models-mitochondria and cells-only partially recapitulate the complex link between the pmf and ROS signaling that occurs in vivo.

Light-induced oxidant production by fluorescent proteins.

Oxidants play an important role in the cell and are involved in many redox processes. Oxidant concentrations are maintained through coordinated production and removal systems. The dysregulation of oxidant homeostasis is a hallmark of many disease pathologies. The local oxidant microdomain is crucial for the initiation of many redox signaling events; however, methods to control oxidant product are limited. Some fluorescent proteins, including GFP, TagRFP, KillerRed, miniSOG, and their derivatives, generate oxidants in response to light. These genetically-encoded photosensitizers produce singlet oxygen and superoxide upon illumination and offer spatial and temporal control over oxidant production. In this review, we will examine the photosensitization properties of fluorescent proteins and their application to redox biology. Emerging concepts of selective oxidant species production via photosensitization and the impact of light on biological systems are discussed.

High dietary fat intake increases fat oxidation and reduces skeletal muscle mitochondrial respiration in trained humans.

High-fat, low-carbohydrate (CHO) diets increase whole-body rates of fat oxidation and down-regulate CHO metabolism. We measured substrate utilization and skeletal muscle mitochondrial respiration to determine whether these adaptations are driven by high fat or low CHO availability. In a randomized crossover design, 8 male cyclists consumed 5 d of a high-CHO diet [>70% energy intake (EI)], followed by 5 d of either an isoenergetic high-fat (HFAT; >65% EI) or high-protein diet (HPRO; >65% EI) with CHO intake clamped at <20% EI. During the intervention, participants undertook daily exercise training. On d 6, participants consumed a high-CHO diet before performing 100 min of submaximal steady-state cycling plus an ∼30-min time trial. After 5 d of HFAT, skeletal muscle mitochondrial respiration supported by octanoylcarnitine and pyruvate, as well as uncoupled respiration, was decreased at rest, and rates of whole-body fat oxidation were higher during exercise compared with HPRO. After 1 d of high-CHO diet intake, mitochondrial respiration returned to baseline values in HFAT, whereas rates of substrate oxidation returned toward baseline in both conditions. These findings demonstrate that high dietary fat intake, rather than low-CHO intake, contributes to reductions in mitochondrial respiration and increases in whole-body rates of fat oxidation after a consuming a high-fat, low-CHO diet.-Leckey, J. J., Hoffman, N. J., Parr, E. B., Devlin, B. L., Trewin, A. J., Stepto, N. K., Morton, J. P., Burke, L. M., Hawley, J. A. High dietary fat intake increases fat oxidation and reduces skeletal muscle mitochondrial respiration in trained humans.

Exercise and Mitochondrial Dynamics: Keeping in Shape with ROS and AMPK.

Exercise is a robust stimulus for mitochondrial adaptations in skeletal muscle which consequently plays a central role in enhancing metabolic health. Despite this, the precise molecular events that underpin these beneficial effects remain elusive. In this review, we discuss molecular signals generated during exercise leading to altered mitochondrial morphology and dynamics. In particular, we focus on the interdependence between reactive oxygen species (ROS) and redox homeostasis, the sensing of cellular bioenergetic status via 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), and the regulation of mitochondrial fission and fusion. Precisely how exercise regulates the network of these responses and their effects on mitochondrial dynamics is not fully understood at present. We highlight the limitations that exist with the techniques currently available, and discuss novel molecular tools to potentially advance the fields of redox biology and mitochondrial bioenergetics. Ultimately, a greater understanding of these processes may lead to novel mitochondria-targeted therapeutic strategies to augment or mimic exercise in order to attenuate or reverse pathophysiology.