Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function.

Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.

Nonmelanoma Skin Cancer in Patients Older Than Age 85 Years Presenting for Mohs Surgery: A Prospective, Multicenter Cohort Study.

Importance: It has been suggested that Mohs surgery for skin cancer among individuals with limited life expectancy may be associated with needless risk and discomfort, along with increased health care costs. Objective: To investigate patient- and tumor-specific indications considered by clinicians for treatment of nonmelanoma skin cancer in older individuals. Design, Setting, and Participants: This multicenter, prospective cohort study was conducted using data from US private practice and academic centers. Included patients were those older than age 85 years presenting for skin cancer surgery and referred for Mohs surgery, with reference groups of those younger than age 85 years receiving Mohs surgery and those older than age 85 years not receiving Mohs surgery. Data were analyzed from November 2018 through January 2019. Exposures: Mohs surgery for nonmelanoma skin cancer. Main Outcomes and Measures: Reason for treatment selection. Results: Among 1181 patients older than age 85 years referred for Mohs surgery (724 [61.9%] men among 1169 patients with sex data; 681 individuals aged >85 to 88 years [57.9%] among 1176 patients with age data) treated at 22 sites, 1078 patients (91.3%) were treated by Mohs surgery, and 103 patients (8.7%) received alternate treatment. Patients receiving Mohs surgery were more likely to have tumors on the face (738 patients [68.5%] vs 26 patients [25.2%]; P < .001) and nearly 4-fold more likely to have high functional status (614 patients [57.0%] vs 16 patients [15.5%]; P < .001). Of 15 distinct reasons provided by surgeons for opting to proceed with Mohs surgery, the most common were patient desire for treatment with a high cure rate (712 patients [66.0%]), good or excellent patient functional status for age (614 patients [57.0%]), and high risk associated with the tumor based on histology (433 patients [40.2%]). Conclusions and Relevance: This study found that older patients who received Mohs surgery often had high functional status, high-risk tumors, and tumors located on the face. These findings suggest that timely surgical treatment may be appropriate in older patients given that their tumors may be aggressive, painful, disfiguring, and anxiety provoking.

Enhanced small neutral but not branched chain amino acid transport after epigenetic sodium coupled neutral amino acid transporter-2 (SNAT2) cDNA expression in myoblasts.

BACKGROUND: Skeletal muscle mass and function are partly maintained by the supply of amino acids, altered amino acid transport is an important cause of frailty that can lead to decreased independence with increasing age and slow trauma recovery. The system-A sodium coupled neutral amino acid transporter (SNAT)-2 coded by gene family SLC38A2 generates a 506 amino acid 56 kDa protein that is an important transporter of amino acids in skeletal muscle. Ageing is associated with a decrease in expression of SNAT2 transporters. METHODS: In this study, we used the C2C12 cell line, using myoblast cells and cells differentiated into myotubes. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis. RESULTS: In control myoblasts and myotubes, we found significantly decreased expression of SNAT2 (6.5× decrease, n = 4 per group, P < 0.05) in myotubes than found in myoblasts. After transfection with a SNAT2-eGFP cDNA plasmid, C2C12 myoblasts significantly increased perinuclear punctate SNAT2-eGFP expression that persisted and was more cytoplasmic after differentiation into myotubes. Interestingly, transfected cells were significantly more responsive to the hormone 5α-dihydrotestosterone (DHT, 4.5 nM, by 1.6×, n = 3 per group, P < 0.04). Starvation significantly enhanced the amino acid C14 -MeAIB transport (1.7×, n = 3 per group, P < 0.05) indicating increased function of SNAT2. Inhibiting SNAT2 with high concentrations of MeAIB (3.3 or 5 mM) significantly reduced C14 -Isoleucine transport by L-type amino acid transporter (LAT2, 52.8% and 77%, respectively, n = 3 per group, P < 0.05). However, there was no increase in the LAT2 transport of C14 -isoleucine detectable in SNAT2-eGFP transfected cells after DHT (4.5 nM) exposure. This indicated that small amino acid availability was not rate limiting to LAT2 function in myoblasts. CONCLUSIONS: Overall, these data show that transfection of SNAT2-eGFP expression enhanced its function following starvation and treatment with physiological levels of DHT. Enhanced SNAT2 expression in muscle cells offers a viable epigenetic target in pathological conditions associated with altered amino acid transport.

Non-canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus.

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.

Oximetry-Guided normoxic resuscitation following canine cardiac arrest reduces cerebellar Purkinje neuronal damage.

BACKGROUND: Animal studies indicate that maintaining physiologic O2 levels (normoxia) immediately after restoration of spontaneous circulation (ROSC) from cardiac arrest (CA) results in less hippocampal neuronal death compared to animals ventilated with 100% O2. This study tested the hypothesis that beneficial effects of avoiding hyperoxia following CA are apparent in the cerebellum and therefore not limited to one brain region. METHODS: Adult beagles were anesthetized and mechanically ventilated. Ventricular fibrillation CA was induced by electrical myocardial stimulation and cessation of ventilation. Ten min later, dogs were ventilated with 100% O2 and resuscitated using 3 min of open chest CPR followed by defibrillation. Dogs were ventilated for 1 h with either 100% O2 or with O2 titrated rapidly to maintain hemoglobin O2 saturation at 94-96%. FiO2 was adjusted in both groups between one and 24 h post-arrest to maintain normoxic PaO2 of 80-120 mm Hg. Following 24 h critical care, dogs were euthanized and cerebellum analyzed for histochemical measures of neuronal damage and inflammation. RESULTS AND CONCLUSIONS: Hyperoxic resuscitation increased the number of injured Purkinje cells by 278% and the number of activated microglia/macrophages by 18% compared to normoxic resuscitation. These results indicate that normoxic resuscitation promotes favorable histopathologic outcomes in the cerebellum (in addition to hippocampus) following CA/ROSC. These findings emphasize the importance of avoiding unnecessary hyperoxia following CA/ROSC.

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP-/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.

Global carbon dioxide removal rates from forest landscape restoration activities.

BACKGROUND: Forest landscape restoration (FLR) has been adopted by governments and practitioners across the globe to mitigate and adapt to climate change and restore ecological functions across degraded landscapes. However, the extent to which these activities capture CO2 with associated climate mitigation impacts are poorly known, especially in geographies where data on biomass growth of restored forests are limited or do not exist. To fill this gap, we developed biomass accumulation rates for a set of FLR activities (natural regeneration, planted forests and woodlots, agroforestry, and mangrove restoration) across the globe and global CO2 removal rates with corresponding confidence intervals, grouped by FLR activity and region/climate. RESULTS: Planted forests and woodlots were found to have the highest CO2 removal rates, ranging from 4.5 to 40.7 t CO2 ha-1 year-1 during the first 20 years of growth. Mangrove tree restoration was the second most efficient FLR at removing CO2, with growth rates up to 23.1 t CO2 ha-1 year-1 the first 20 years post restoration. Natural regeneration removal rates were 9.1-18.8 t CO2 ha-1 year-1 during the first 20 years of forest regeneration, followed by agroforestry, the FLR category with the lowest and regionally broad removal rates (10.8-15.6 t CO2 ha-1 year-1). Biomass growth data was most abundant and widely distributed across the world for planted forests and natural regeneration, representing 45% and 32% of all the data points assessed, respectively. Agroforestry studies, were only found in Africa, Asia, and the Latin America and Caribbean regions. CONCLUSION: This study represents the most comprehensive review of published literature on tree growth and CO2 removals to date, which we operationalized by constructing removal rates for specific FLR activities across the globe. These rates can easily be applied by practitioners and decision-makers seeking to better understand the positive climate mitigation impacts of existing or planned FLR actions, or by countries making restoration pledges under the Bonn Challenge Commitments or fulfilling Nationally Determined Contributions to the UNFCCC, thereby helping boost FLR efforts world-wide.

Greenhouse gas emissions from tropical forest degradation: an underestimated source.

BACKGROUND: The degradation of forests in developing countries, particularly those within tropical and subtropical latitudes, is perceived to be an important contributor to global greenhouse gas emissions. However, the impacts of forest degradation are understudied and poorly understood, largely because international emission reduction programs have focused on deforestation, which is easier to detect and thus more readily monitored. To better understand and seize opportunities for addressing climate change it will be essential to improve knowledge of greenhouse gas emissions from forest degradation. RESULTS: Here we provide a consistent estimation of forest degradation emissions between 2005 and 2010 across 74 developing countries covering 2.2 billion hectares of forests. We estimated annual emissions of 2.1 billion tons of carbon dioxide, of which 53% were derived from timber harvest, 30% from woodfuel harvest and 17% from forest fire. These percentages differed by region: timber harvest was as high as 69% in South and Central America and just 31% in Africa; woodfuel harvest was 35% in Asia, and just 10% in South and Central America; and fire ranged from 33% in Africa to only 5% in Asia. Of the total emissions from deforestation and forest degradation, forest degradation accounted for 25%. In 28 of the 74 countries, emissions from forest degradation exceeded those from deforestation. CONCLUSIONS: The results of this study clearly demonstrate the importance of accounting greenhouse gases from forest degradation by human activities. The scale of emissions presented indicates that the exclusion of forest degradation from national and international GHG accounting is distorting. This work helps identify where emissions are likely significant, but policy developments are needed to guide when and how accounting should be undertaken. Furthermore, ongoing research is needed to create and enhance cost-effective accounting approaches.

Mitochondrial ROS regulate oxidative damage and mitophagy but not age-related muscle fiber atrophy.

Age-related loss of skeletal muscle mass and function is a major contributor to morbidity and has a profound effect on the quality of life of older people. The potential role of age-dependent mitochondrial dysfunction and cumulative oxidative stress as the underlying cause of muscle aging remains a controversial topic. Here we show that the pharmacological attenuation of age-related mitochondrial redox changes in muscle with SS31 is associated with some improvements in oxidative damage and mitophagy in muscles of old mice. However, this treatment failed to rescue the age-related muscle fiber atrophy associated with muscle atrophy and weakness. Collectively, these data imply that the muscle mitochondrial redox environment is not a key regulator of muscle fiber atrophy during sarcopenia but may play a key role in the decline of mitochondrial organelle integrity that occurs with muscle aging.

Long-term administration of the mitochondria-targeted antioxidant mitoquinone mesylate fails to attenuate age-related oxidative damage or rescue the loss of muscle mass and function associated with aging of skeletal muscle.

Age-related skeletal muscle dysfunction is the underlying cause of morbidity that affects up to half the population aged 80 and over. Considerable evidence indicates that oxidative damage and mitochondrial dysfunction contribute to the sarcopenic phenotype that occurs with aging. To examine this, we administered the mitochondria-targeted antioxidant mitoquinone mesylate {[10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenylphosphonium; 100 µM} to wild-type C57BL/6 mice for 15 wk (from 24 to 28 mo of age) and investigated the effects on age-related loss of muscle mass and function, changes in redox homeostasis, and mitochondrial organelle integrity and function. We found that mitoquinone mesylate treatment failed to prevent age-dependent loss of skeletal muscle mass associated with myofiber atrophy or alter a variety of in situ and ex vivo muscle function analyses, including maximum isometric tetanic force, decline in force after a tetanic fatiguing protocol, and single-fiber-specific force. We also found evidence that long-term mitoquinone mesylate administration did not reduce mitochondrial reactive oxygen species or induce significant changes in muscle redox homeostasis, as assessed by changes in 4-hydroxynonenal protein adducts, protein carbonyl content, protein nitration, and DNA damage determined by the content of 8-hydroxydeoxyguanosine. Mitochondrial membrane potential, abundance, and respiration assessed in permeabilized myofibers were not significantly altered in response to mitoquinone mesylate treatment. Collectively, these findings demonstrate that long-term mitochondria-targeted mitoquinone mesylate administration failed to attenuate age-related oxidative damage in skeletal muscle of old mice or provide any protective effect in the context of muscle aging.-Sakellariou, G. K., Pearson, T., Lightfoot, A. P., Nye, G. A., Wells, N., Giakoumaki, I. I., Griffiths, R. D., McArdle, A., Jackson, M. J. Long-term administration of the mitochondria-targeted antioxidant mitoquinone mesylate fails to attenuate age-related oxidative damage or rescue the loss of muscle mass and function associated with aging of skeletal muscle.

The effect of lengthening contractions on neuromuscular junction structure in adult and old mice.

Skeletal muscles of old mice demonstrate a profound inability to regenerate fully following damage. Such a failure could be catastrophic to older individuals where muscle loss is already evident. Degeneration and regeneration of muscle fibres following contraction-induced injury in adult and old mice are well characterised, but little is known about the accompanying changes in motor neurons and neuromuscular junctions (NMJs) following this form of injury although defective re-innervation of muscle following contraction-induced damage has been proposed to play a role in sarcopenia. This study visualised and quantified structural changes to motor neurons and NMJs in Extensor digitorum longus (EDL) muscles of adult and old Thy1-YFP transgenic mice during regeneration following contraction-induced muscle damage. Data demonstrated that the damaging contraction protocol resulted in substantial initial disruption to NMJs in muscles of adult mice, which was reversed entirely within 28 days following damage. In contrast, in quiescent muscles of old mice, ∼15 % of muscle fibres were denervated and ∼80 % of NMJs showed disruption. This proportion of denervated and partially denervated fibres remained unchanged following recovery from contraction-induced damage in muscles of old mice although ∼25 % of muscle fibres were completely lost by 28 days post-contractions. Thus, in old mice, the failure to restore full muscle force generation that occurs following damage does not appear to be due to any further deficit in the percentage of disrupted NMJs, but appears to be due, at least in part, to the complete loss of muscle fibres following damage.

Manipulation of environmental oxygen modifies reactive oxygen and nitrogen species generation during myogenesis.

Regulated changes in reactive oxygen and nitrogen species (RONS) activities are important in maintaining the normal sequence and development of myogenesis. Both excessive formation and reduction in RONS have been shown to affect muscle differentiation in a negative way. Cultured cells are typically grown in 20% O2 but this is not an appropriate physiological concentration for a number of cell types, including skeletal muscle. The aim was to examine the generation of RONS in cultured skeletal muscle cells under a physiological oxygen concentration condition (6% O2) and determine the effect on muscle myogenesis. Primary mouse satellite cells were grown in 20% or 6% O2 environments and RONS activity was measured at different stages of myogenesis by real-time fluorescent microscopy using fluorescent probes with different specificities i.e. dihydroethidium (DHE), 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and 5-(and-6)-chloromethyl-2',7' -dichlorodihydrofluorescein diacetate (CM-DCFH-DA). Data demonstrate that satellite cell proliferation increased when cells were grown in 6% O2 compared with 20% O2. Myoblasts grown in 20% O2 showed an increase in DCF fluorescence and DHE oxidation compared with myoblasts grown at 6% O2. Myotubes grown in 20% O2 also showed an increase in DCF and DAF-FM fluorescence and DHE oxidation compared with myotubes grown in 6% O2. The catalase and MnSOD contents were also increased in myoblasts and myotubes that were maintained in 20% O2 compared with myoblasts and myotubes grown in 6% O2. These data indicate that intracellular RONS activities in myoblasts and myotubes at rest are influenced by changes in environmental oxygen concentration and that the increased ROS may influence myogenesis in a negative manner.

Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres.

KEY POINTS: Many studies have previously suggested the existence of stress hormone receptors on the cell membrane of many cell types, including skeletal muscle fibres; however, the exact localisation of these receptors and how they signal to the rest of the cell is poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the physiological functions of these receptors in mouse skeletal muscle cells. We found that the receptors were present throughout muscle development and that, in adult muscle fibres, they were localised in the extracellular matrix, satellite cells (muscle stem cells) and close to mitochondria. We also found that they signalled to the rest of the cell by activating enzymes called mitogen-activated protein kinases. From these results we suggest that, at physiological concentrations, stress hormones may be important in skeletal muscle differentiation, repair and regeneration. ABSTRACT: A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway.

Family centered brief intensive treatment: a pilot study of an outpatient treatment for acute suicidal ideation.

Family Centered Brief Intensive Treatment (FC BIT), a hospital diversion treatment program for individuals with acute suicidal ideation, was developed to treat suicidal clients and their families. Individuals who met criteria for hospitalization were treated as outpatients using FC BIT (n = 19) or an intensive outpatient treatment without the family component (IOP; n = 24). Clients receiving FC BIT identified family members or supportive others to participate in therapy. FC BIT clients had significantly greater improvement at the end of treatment compared to IOP clients on measures of depression, hopelessness, and suicidality. Further research is needed to test the efficacy of FC BIT.

Skeletal muscle contractions induce acute changes in cytosolic superoxide, but slower responses in mitochondrial superoxide and cellular hydrogen peroxide.

Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

In vitro susceptibility of thioredoxins and glutathione to redox modification and aging-related changes in skeletal muscle.

Thioredoxins (Trx's) regulate redox signaling and are localized to various cellular compartments. Specific redox-regulated pathways for adaptation of skeletal muscle to contractions are attenuated during aging, but little is known about the roles of Trx's in regulating these pathways. This study investigated the susceptibility of Trx1 and Trx2 in skeletal muscle to oxidation and reduction in vitro and the effects of aging and contractions on Trx1, Trx2, and thioredoxin reductase (TrxR) 1 and 2 contents and nuclear and cytosolic Trx1 and mitochondrial Trx2 redox potentials in vivo. The proportions of cytosolic and nuclear Trx1 and mitochondrial Trx2 in the oxidized or reduced forms were analyzed using redox Western blotting. In myotubes, the mean redox potentials were nuclear Trx1, -251 mV; cytosolic Trx1, -242mV; mitochondrial Trx2, -346mV, data supporting the occurrence of differing redox potentials between cell compartments. Exogenous treatment of myoblasts and myotubes with hydrogen peroxide or dithiothreitol modified glutathione redox status and nuclear and cytosolic Trx1, but mitochondrial Trx2 was unchanged. Tibialis anterior muscles from young and old mice were exposed to isometric muscle contractions in vivo. Aging increased muscle contents of Trx1, Trx2, and TrxR2, but neither aging nor endogenous ROS generated during contractions modified Trx redox potentials, although oxidation of glutathione and other thiols occurred. We conclude that glutathione redox couples in skeletal muscle are more susceptible to oxidation than Trx and that Trx proteins are upregulated during aging, but do not appear to modulate redox-regulated adaptations to contractions that fail during aging.

A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue.

BACKGROUND: We describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component) based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing. FINDINGS: The described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of "house-keeper" marker proteins specific for each cellular compartment. CONCLUSION: This low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at -80°C for future analysis and/or additional processing at a later date.

Vasculopathy related to cocaine adulterated with levamisole: A review of the literature.

BACKGROUND: Recently, there have been numerous case reports and series describing patients presenting with cutaneous vasculopathy that has been linked to the levamisole frequently found in cocaine. OBJECTIVE: The purpose of this study was to review all published case reports and series of patients reported with cutaneous vasculopathic findings of lemavisole induced vasculopathy (LIV) associated with cocaine use. METHODS: A review of PubMed was performed searching the keywords: levamisole, cocaine, in combination with vasculitis, and vasculopathy. Twenty-two case reports and series were available with sufficient data on reported patients to be included. Four patients from the authors' clinical experience are included as well. RESULTS: A number of common clinical and pathological findings are reviewed, including lower extremity (46/55 patients, 84%) and ear involvement (40/55 patients, 73%), and positive anti-neutrophil cytoplasmic antibodies (ANCA) findings (p-ANCA 42/48 patients, 88%; anti human neutrophil elastase 11/11 patients, 100%). Similar numbers of patients were treated with systemic corticosteroids as were treated conservatively; there was comparable improvement on follow up. CONCLUSIONS: There are a number of clinical and laboratory findings that are commonly found in patients with LIV. There is currently insufficient data to recommend treatment with systemic corticosteroids in patients with this condition.

The age-related failure of adaptive responses to contractile activity in skeletal muscle is mimicked in young mice by deletion of Cu,Zn superoxide dismutase.

In muscle, aging is associated with a failure of adaptive responses to contractile activity, and this is hypothesized to play an important role in age-related loss of muscle mass and function. Mice lacking the Cu,Zn superoxide dismutase (Cu,ZnSOD, SOD1) show an accelerated, age-related loss of muscle mass and function. This work determined whether adult mice lacking Cu,ZnSOD (Sod1(-/-) mice) show a premature failure of adaptive responses to contractions in a similar manner to old wild-type (WT) mice. Adult Sod1(-/-) mice (6-8 months of age) had a ∼30% reduction in gastrocnemius muscle mass compared with age-matched WT mice. This lower muscle mass was associated with an activation of DNA binding by NFκB and AP-1 at rest. Measurements of the activity of reactive oxygen species (ROS) in single fibres from the muscles of Sod1(-/-) mice at rest indicated an elevation in activity compared with fibres from WT mice. Following 15 min of isometric contractions, muscle fibres from WT mice showed an increase in the intracellular ROS activities and activation of NFκB and AP-1, but no changes in either ROS activity or NFκB and AP-1 activation were seen in the muscles of Sod1(-/-) mice following contractions. This pattern of changes mimics that seen in the muscles of old WT mice, suggesting that the attenuated responses to contractile activity seen in old mice result from chronic exposure to increased oxidant activity. Data support the use of the Sod1(-/-) mouse model to evaluate potential mechanisms that contribute to the loss of muscle mass and function in the elderly.

Measurement of vasoactive metabolites (hydroxyeicosatetraenoic and epoxyeicosatrienoic acids) in uterine tissues of normal and compromised human pregnancy.

BACKGROUND: Preeclampsia and intrauterine growth restriction define two disorders of a multifactorial etiology that compromise maternal and fetal well being as well as cardiovascular health in later life. Many of the overt symptoms of preeclampsia are attributable to the systemic endothelial dysfunction observed in the uteroplacental and systemic circulation, leading to a generalized vasoconstriction, hypertension and inadequate placental perfusion. Mounting evidence implicates nonprostanoid eicosanoids, epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) in the control of vascular function and dysfunction. OBJECTIVE: To determine whether levels of EETs and HETEs are altered in preeclampsia and intrauterine growth restriction compared with normal term pregnancy. METHODS: An analytical liquid chromatography-tandem mass spectrometry profiling method was utilized in order to analyze differential levels of EETs and HETEs in intrauterine tissues of term nonlaboring, laboring and preeclamptic women as well as women with a growth-restricted pregnancy. RESULTS: Placentae of preeclamptic women contained significantly (P < 0.05) larger amounts of 5-HETE, 12-HETE and 15-HETE known to possess either vasoconstrictive or proinflammatory actions. Laboring tissues were characterized by significantly higher (P < 0.05) EET levels in the amnion compared with the other clinical groups. EET and HETE levels in preeclampsia and intrauterine growth restriction were positively correlated (P < 0.05), whereas in normal and laboring pregnancies, EETs and HETEs were negatively correlated. CONCLUSION: Increased production of 5-HETE, 12-HETE and 15-HETE metabolites in preeclamptic placentae indicates an important role for this family of eicosanoids in the cause of this disease.