المؤلفون: Robert J. Bloch, April K. Hartford, George G. Rodney, W. J. Lederer, Andrew P. Ziman, Christopher W. Ward

المصدر: Biophysical Journal. (3):540a-541a

مصطلحات موضوعية: RYR1, Contraction (grammar), Ryanodine receptor, Endoplasmic reticulum, Biophysics, Lumen (anatomy), Skeletal muscle, Biology, chemistry.chemical_compound, medicine.anatomical_structure, Biochemistry, chemistry, cardiovascular system, medicine, Myocyte, Caffeine

الوصف: Excitation-contraction (EC) coupling in skeletal muscle is the process by which an action potential (AP) activates a global increase in [Ca2+]i which then induces contraction. The global increase in [Ca2+]i is mediated by the release of Ca2+ from the lumen of the sarcoplasmic reticulum (SR) through the opening of ryanodine receptors (RyR1). We examined the structure and function of the SR Ca2+ store dynamically, using the low affinity Ca2+ indicator, fluo-5N, which when loaded as an -AM derivative concentrates in the lumen of the SR. Fluo-5N has been used extensively to measure SR Ca2+ and to characterize the Ca2+ stores in cardiac myocytes, with high spatial and temporal resolution (Brochet et al 2005; Wu and Bers 2006). Using dissociated flexor digitorum brevis (FDB) fibers in culture, we studied the dynamics of fluo-5N in the SR and connecting compartments. Fluo-5N accumulates in transverse structures that align with Z-disks, consistent with the location of a major compartment of the SR in adult skeletal muscle. That these structures are primarily SR in nature is indicated by the fact that fluo-5N fluorescence decreases when fibers are exposed to caffeine. FRAP experiments demonstrated similar recovery constants for SR-trapped fluo-5N to those we have previously recorded in rat cardiac myocytes. We will be using fluo-5N to study the organization of the SR, the changes in Ca2+ dynamics in the SR during EC coupling, and the role of different proteins of the SR in regulating SR [Ca2+].

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0312c303c74e73aa57862a2cec6a8abfTest

المؤلفون: Christopher W. Ward, George G. Rodney, Luke Michaelson

المصدر: Biophysical Journal. (3):228a

مصطلحات موضوعية: chemistry.chemical_classification, Reactive oxygen species, Myofilament, NADPH oxidase, animal structures, biology, Biophysics, Skeletal muscle, Stimulation, macromolecular substances, Mitochondrion, musculoskeletal system, RoGFP, Cell biology, medicine.anatomical_structure, chemistry, Biochemistry, biology.protein, medicine, Tetanic stimulation, tissues

الوصف: Skeletal muscles produce low levels of reactive oxygen species (ROS) under resting conditions while during contractile activity the rate of ROS production increases. Low levels of ROS production may act to stimulate adaptive responses in skeletal muscle, while increased ROS dependent oxidation at sarcoplasmic reticulum, myofilaments and other EC coupling components likely lead to decrements in contractile function. Multiple sites exist for ROS production, including mitochondria and NADPH oxidase; however, the contribution of each of these and the factors that regulate the increased production of ROS during contractile activity remains to be determined. Here we use repetitive field stimulation of single FDB myofibers as a model of ROS secondary to repetitive activity. In FDB's loaded with the cytosolic, non-specific ROS probe DCFH, we have imposed intermittent (0.5 Hz) trains (150msec, 0.5msec sq. pulse @, 50Hz) of tetanic stimulation to establish a reliable in vitro model for activity dependent ROS production. In recent studies with this model, we have begun to explore site dependent generation of ROS with a redox sensitive variant of green fluorescent protein (roGFP) that is targeted to the mitochondria (mito-roGFP). Following cDNA electroporation in vivo, expression of mito-roGFP, and FDB isolation, we report evidence of the fidelity and specificity of this probe in mitochondria and the activity dependent mitochondrial redox status during our stimulation paradigm.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::65eff01456dc6fbd1296683aeda5418cTest

المؤلفون: James A. Loehr, Rituraj Pal, Reem Abo-Zahrah, George G. Rodney

المصدر: Biophysical Journal. (2):424a

مصطلحات موضوعية: chemistry.chemical_classification, Reactive oxygen species, NADPH oxidase, medicine.medical_treatment, SOD2, Biophysics, Muscle weakness, Skeletal muscle, Mitochondrion, Biology, medicine.disease_cause, Cell biology, Cytokine, medicine.anatomical_structure, chemistry, Biochemistry, medicine, biology.protein, medicine.symptom, Oxidative stress

الوصف: Increased reactive oxygen species (ROS) are a hallmark of many diseases, such as inflammatory myopathies. Recent evidence suggests elevated cytokine activity increases ROS production resulting in muscle weakness; however, the specific source of ROS production has yet to be fully elucidated. Redox sensitive probes, targeted to NADPH oxidase 2 (Nox2) (p47-roGFP) and the mitochondria (mito-roGFP), were used to assess the sub-cellular site of ROS production in the presence of various cytokines. In addition, we assessed the effect of cytokine induced ROS production on skeletal muscle function. Cytokine stimulation increased p47-roGFP oxidation approximately 15%, but had no effect on mito-roGFP oxidation. Genetic and pharmacological inhibition of Nox2 resulted in decreased Nox2-dependent ROS production while genetic overexpression of SOD2 had no effect on mitochondrial or Nox2-mediated ROS production. Following cytokine administration, skeletal muscle function decreased by 30% and genetic inhibition of Nox2-activity partially rescued muscle function. Genetic inhibition of mitochondrial-ROS provided no protection against decreased muscle function following cytokine stimulation. Collectively, these data indicate that elevated cytokine activity resulted in increased ROS production at specific sub-cellular sites, negatively affecting muscle function. Our data highlight the importance of understanding the source of ROS production in response to physiological and/or pathological stimuli such that targeted therapeutic approaches can be developed to combat the deleterious effects of oxidative stress.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::20139f6504d079fc7a8f356482b18786Test

المؤلفون: Marco Sardiello, Michela Palmieri, George G. Rodney, Shumin Li, Rituraj Pal, James A. Loehr

المصدر: Biophysical Journal. (2):728a

مصطلحات موضوعية: musculoskeletal diseases, congenital, hereditary, and neonatal diseases and abnormalities, mdx mouse, NADPH oxidase, biology, Duchenne muscular dystrophy, Autophagy, Biophysics, Skeletal muscle, medicine.disease, medicine.disease_cause, Cell biology, medicine.anatomical_structure, Biochemistry, medicine, biology.protein, sense organs, PI3K/AKT/mTOR pathway, Oxidative stress, Proto-oncogene tyrosine-protein kinase Src

الوصف: Duchenne muscular dystrophy (DMD) is a fatal degenerative muscle disease, attributed to a defect in the gene that encodes dystrophin. Emerging evidence implicates oxidative stress may impair autophagy in DMD patients and mdx mice, a model of DMD, by activating cytotoxic mediators. However, the specific source of ROS and the mechanism(s) of impaired autophagy have not yet been elucidated in dystrophic muscle. Therefore, understanding the interaction between oxidative stress and defects in autophagy is pivotal as we seek effective therapeutic targets in DMD. Here we demonstrated that nicotinamide adenine dinucleotide phosphatase (NADPH oxidase or Nox2)-induced oxidative stress was linked to impaired autophagy in mdx mice through Nox2-dependent superoxide production, Src kinase activation and further NOX2 activation via p47phox phosphorylation. The defect in autophagy was accompanied by persistent activation of Src kinase, which activated the autophagy repressor mammalian target of rapamycin (mTOR) via PI3K/Akt phosphorylation. Inhibition of Nox2 or Src kinase reduced oxidative stress and partially rescued the defective autophagy in mdx mice. We also have genetically down-regulated Nox2 activity in the mdx mouse to further corroborate that NADPH oxidase was the main source of oxidative stress, which impaired autophagy in DMD. Our data highlights novel pathogenic aspects of DMD and proposes NADPH oxidase as a potential therapeutic target.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::97aa8392d0ca8d49da440468475504eaTest