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Domoic acid (DA) is a neurotoxin that is naturally produced by phytoplankton and accumulates in seafood during harmful algal blooms. As the prevalence of DA increases in the marine environment, there is a critical need to identify seafood consumers at risk of DA poisoning. DA exposure was estimated in recreational razor clam ( Siliqua patula ) harvesters to determine if exposures above current regulatory guidelines occur and/or if harvesters are chronically exposed to low levels of DA. Human consumption rates of razor clams were determined by distributing 1523 surveys to recreational razor clam harvesters in spring 2015 and winter 2016, in Washington, USA. These consumption rate data were combined with DA measurements in razor clams, collected by a state monitoring program, to estimate human DA exposure. Approximately 7% of total acute exposures calculated (including the same individuals at different times) exceeded the current regulatory reference dose (0.075 mg DA·kg bodyweight − 1 ·d − 1 ) due to higher than previously reported consumption rates, lower bodyweights, and/or by consumption of clams at the upper range of legal DA levels (maximum 20 mg·kg − 1 wet weight for whole tissue). Three percent of survey respondents were potentially at risk of chronic DA exposure by consuming a minimum of 15 clams per month for at 12 consecutive months. These insights into DA consumption will provide an additional tool for razor clam fishery management. [ABSTRACT FROM AUTHOR]

Summary: To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until ∼5 weeks of age, when ataxic signs began, progressing to death at ∼7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters. [Copyright &y& Elsevier]

Skeletal muscle mitochondrial dysfunction is a key characteristic of aging muscle and contributes to age related diseases such as sarcopenia, frailty, and type 2 diabetes. Mitochondrial oxidative stress has been implicated as a driving factor in these age-related diseases, however whether it is a cause, or a consequence of mitochondrial dysfunction remains to be determined. The development of flexible genetic models is an important tool to test the mechanistic role of mitochondrial oxidative stress on skeletal muscle metabolic dysfunction. We characterize a new model of inducible and reversible mitochondrial redox stress using a tetracycline controlled skeletal muscle specific short hairpin RNA targeted to superoxide dismutase 2 (iSOD2). Methods: iSOD2 KD and control (CON) animals were administered doxycycline for 3- or 12- weeks and followed for up to 24 weeks and mitochondrial respiration and muscle contraction were measured to define the time course of SOD2 KD and muscle functional changes and recovery. Maximum knockdown of SOD2 protein occurred by 6 weeks and recovered by 24 weeks after DOX treatment. Mitochondrial aconitase activity and maximum mitochondrial respiration declined in KD muscle by 12 weeks and recovered by 24 weeks. There were no significant differences in antioxidant or mitochondrial biogenesis genes between groups. Twelve-week KD showed a small, but significant decrease in muscle fatigue resistance. The primary phenotype was reduced metabolic flexibility characterized by impaired pyruvate driven respiration when other substrates are present. The pyruvate dehydrogenase kinase inhibitor dichloroacetate partially restored pyruvate driven respiration, while the thiol reductant DTT did not. We use a model of inducible and reversible skeletal muscle SOD2 knockdown to demonstrate that elevated matrix superoxide reversibly impairs mitochondrial substrate flexibility characterized by impaired pyruvate oxidation. Despite the bioenergetic effect, the limited change in gene expression suggests that the elevated redox stress in this model is confined to the mitochondrial matrix. [Display omitted] • SOD2 knockdown and recovery is achieved by using a shRNA targeted to SOD2 mRNA controlled by a TET-ON System. • SOD2 KD is induced by administering doxycycline (DOX) in the drinking water. • Mitochondrial functional decline and recovery follows the time course of SOD2 protein decline and recovery. • Sustained SOD2 KD drives impaired pyruvate respiration in skeletal muscle mitochondria in the presence of other substrates. [ABSTRACT FROM AUTHOR]

High intensity exercise is a popular mode of exercise to elicit similar or greater adaptive responses compared to traditional moderate intensity continuous exercise. However, the molecular mechanisms underlying these adaptive responses are still unclear. The purpose of this pilot study was to compare high and low intensity contractile stimulus on the Nrf2-mediated redox stress response in mouse skeletal muscle. An intra-animal design was used to control for variations in individual responses to muscle stimulation by comparing a stimulated limb (STIM) to the contralateral unstimulated control limb (CON). High Intensity (HI – 100Hz), Low Intensity (LI – 50Hz), and Naïve Control (NC – Mock stimulation vs CON) groups were used to compare these effects on Nrf2-ARE binding, Keap1 protein, and downstream gene and protein expression of Nrf2 target genes. Muscle stimulation significantly increased Nrf2-ARE binding in LI-STIM compared to LI-CON (p = 0.0098), while Nrf2-ARE binding was elevated in both HI-CON and HI-STIM compared to NC (p = 0.0007). The Nrf2-ARE results were mirrored in the downregulation of Keap1, where Keap1 expression in HI-CON and HI-STIM were both significantly lower than NC (p = 0.008) and decreased in LI-STIM compared to LI-CON (p = 0.015). In addition, stimulation increased NQO1 protein compared to contralateral control regardless of stimulation intensity (p = 0.019), and HO1 protein was significantly higher in high intensity compared to the Naïve control group (p = 0.002). Taken together, these data suggest a systemic redox signaling exerkine is activating Nrf2-ARE binding and is intensity gated, where Nrf2-ARE activation in contralateral control limbs were only seen in the HI group. Other research in exercise induced Nrf2 signaling support the general finding that Nrf2 is activated in peripheral tissues in response to exercise, however the specific exerkine responsible for the systemic signaling effects is not known. Future work should aim to delineate these redox sensitive systemic signaling mechanisms. [Display omitted] • Muscle stimulation decreases Keap1 protein and increases Nrf2-ARE binding. • High intensity muscle stimulation activates Nrf2-ARE binding and decreases Keap1 protein in the contralateral control limb. • NQO1 protein is responsive to muscle stimulation regardless of intensity. • These data suggest an intensity gated redox sensitive exerkine. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction plays an important role in many chronic diseases, including aging. However the role of oxidative stress in this dysfunction remains controversial. We tested whether mitochondrial oxidative stress underlies not only mitochondrial deficits, but also declines in performance of skeletal and cardiac muscle in aged mice. Direct manipulation of mitochondrial H 2 O 2 (mtH 2 O 2 ) production by lifelong overexpression of mitochondrial targeted catalase reduced mitochondrial deficits in aged skeletal muscle and protected against age-related declines in cardiac function. To test whether manipulating mitochondrial oxidative stress late in life could reverse deficits in aged skeletal muscle and heart we used a novel mitochondrial targeted peptide, SS-31, that interacts with cardiolipin on the inner mitochondrial membrane. SS-31 reduces mtH 2 O 2 production in dysfunctional aged mitochondria and improved glutathione redox status while not affecting young healthy mitochondria. Acute (1 hr) and 8 week treatment with SS-31 had similar effects on mitochondrial function and skeletal muscle performance. Both treatments reversed age-related declines in in vivo maximal mitochondrial ATP production and efficiency of oxidative phosphorylation (P/O) while increasing muscle fatigue resistance. Treatment for 8 weeks also reversed cardiac dysfunction, especially diastolic dysfunction. These improvements in skeletal muscle and cardiac function translated to improved treadmill endurance capacity in the aged mice. These results reveal a dynamic relationship between mitochondrial deficits and the redox environment of the cell that affects function in these tissues. To examine the mechanisms underlying this interaction we are examining the effect of manipulating mitochondrial oxidant production on the thiol redox proteome. Aging is associated with increased S-glutathionylation of the thiol proteome in aged skeletal muscle. Treatment with SS-31 partially reverses many of these age-related changes, including proteins involved in E-C coupling, muscle contraction, and protein quality control. These results demonstrate that mitochondrial oxidant production alters redox dependent post-translational modifications throughout the muscle thereby providing a potential mechanism linking mitochondrial oxidative stress to loss of muscle function with age. Furthermore, we demonstrate that reversing this mitochondrial oxidative stress can rapidly improve function highlighting the interaction between mitochondria and cellular redox environment as an attractive target to improve health in the elderly. [ABSTRACT FROM AUTHOR]

In vivo control over metabolism is at the cutting edge of biomedical research. The particulars of mitochondrial function are especially important to understand in vivo to progress metabolic therapies that will be relevant for diseases of aging. Understanding the differences between how mitochondria function in vitro versus in vivo will be a necessary challenge to overcome to achieve mitochondrial medicine. In this article we outline how discoveries in invertebrate models will be informative for understanding the basic biology of mitochondria to streamline translation to mammals and eventually to humans. Further, we highlight examples of how what is known about mitochondria in vitro is translatable to in vivo models and, in some cases, to human diseases. • Mitochondria are complex metabolic hubs that are not fully understood. • Mitochondria in cells, invertebrates, and tissue are important contexts to study. • Achieving mitochondrial medicine will require better understanding in vivo. [ABSTRACT FROM AUTHOR]

High levels of the flame retardant 2,2′,4,4′-tetrabromodiphenyl ether (BDE 47) have been detected in Pacific salmon sampled near urban areas, raising concern over the safety of salmon consumption. However, salmon fillets also contain the antioxidants eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), whose oxidation products induce cellular antioxidant responses. Because oxidative stress is a mechanism of BDE 47 toxicity, we hypothesized that oxidized EPA and DHA can ameliorate the cellular and mitochondrial toxicity of BDE 47. HepG2 cells were treated with a mixture of oxidized EPA and DHA (oxEPA/oxDHA) at a ratio relevant to salmon consumption (1.5/1 oxEPA/oxDHA) followed by exposure to 100 μM BDE 47. Pretreatment with oxEPA/oxDHA for 12 h prior to BDE 47 exposure prevented BDE 47-mediated depletion of glutathione, and increased expression of antioxidant response genes. oxEPA/oxDHA also reduced the level of reactive oxygen species production by BDE 47. The oxEPA/oxDHA antioxidant responses were associated with partial protection against BDE 47-induced loss of viability and also mitochondrial membrane potential. Mitochondrial electron transport system functional analysis revealed extensive inhibition of State 3 respiration and maximum respiratory capacity by BDE 47 were partially reversed by oxEPA/oxDHA. Our findings indicate that the antioxidant effects of oxEPA/oxDHA protect against short exposures to BDE 47, including a protective role of these compounds on maintaining cellular and mitochondrial function. [ABSTRACT FROM AUTHOR]

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly. fx1 [ABSTRACT FROM AUTHOR]

Protein S-glutathionylation (P-SSG) is a reversible redox modification known to alter the activity of transcription factors, kinases, and contractile proteins as well as protect critical cysteine residues from irreversible oxidation. Exercise elicits an acute oxidative stress that is reported to result in beneficial adaptive responses, a process known as redox signaling. We hypothesized that redox signaling will occur through P-SSG of regulatory cysteines following fatiguing muscle contractions. To test this hypothesis, we performed acute fatiguing contractions using an in vivo stimulation protocol on anesthetized CB6F1 (BALB/cBy x C57BL/6) mice. Muscle force production was monitored by a force transducer throughout the 15 minutes of fatiguing stimulations. The right (stimulated) and left (unstimulated) gastrocnemius were collected 60 minutes after the last stimulation and processed for redox proteomics using resin-assisted thiol enrichment and selective reduction. Redox proteomics revealed over 1,500 P-SSG modifications that were significantly increased by fatiguing contractions. Mitochondrial proteins were among the most sensitive to glutathionylation. Ingenuity Pathway Analysis revealed mitochondrial dysfunction, calcium signaling, cytoskeleton signaling, and oxidative stress response in the top ten affected pathways. Critical cysteines on beta-actin, troponin I, ryanodine receptor 1, SERCA1, ATP synthase subunit alpha and NADH dehydrogenase flavoprotein 1 were among those significantly glutathionylated that have previously been reported to alter protein and muscle function. These modifications of redox sensitive proteins implicate rapid regulation of metabolism, excitation/contraction coupling, kinase activity, and contractile protein function, and protection from irreversible oxidation. Together, these data suggest a direct role of ROS in acute exercise signaling and map the redox sensitive proteome in mouse muscle. This study lays the groundwork for future investigation into the redox signaling of altered exercise adaptation associated with chronic conditions, such as sarcopenia, the age-related loss of muscle mass and function. [ABSTRACT FROM AUTHOR]

Muscle redox status regulates diverse aspects of skeletal muscle function, including mitochondrial energetics and contractile performance. In aging skeletal muscle mitochondrial dysfunction contributes to altered redox homeostasis creating a feedforward cycle. Despite this, the role of redox status and its dynamic nature has not been adequately explored as it relates to the decline in skeletal muscle performance with age. To identify mechanisms linking redox status to aged skeletal muscle function we used two long-term treatment strategies to reduce oxidative stress and restore redox homeostasis in aged mice. The first treatment strategy used a short mitochondrial-targeted peptide, elamipretide (SS-31), that our lab has previously shown to acutely improve mitochondrial bioenergetics and muscle fatiguability while also enhancing glutathione redox potential in aged mice. Treating with SS-31 (3 mg/kg) for 8 weeks recapitulates all of the acute improvements while also increasing resting ATP levels and improving treadmill performance compared to both pre-treatment baseline performance and age matched controls. Increased performance was associated with improved redox status as indicated by lower 4-hydroxynonenal-protein adducts and reversal of age-related changes in protein S-glutathionylation. Our second treatment strategy used the powerful antioxidant astaxanthin delivered through the chow of mice at a target dose of 300 mg/kg/day. Astaxanthin treatment paired with exercise training for 8 weeks in aged animals showed a significant improvement in skeletal muscle specific force compared to untreated controls and a trend toward improved treadmill performance. Taken together these results suggest that altering skeletal muscle oxidative stress to restore redox homeostasis is a viable strategy for improving muscle function with age. [ABSTRACT FROM AUTHOR]