We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91 phox membrane subunit and the cytoplasmic regulatory p47 phox subunit co-localized in transverse tubules of adult mice fibers with the ␣ 1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91 phox and p22 phox , which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47 phox and p67 phox . NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [ 3 H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca 2؉ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.The NADPH oxidases (NOX) 3 are flavoprotein enzymes that use NADPH as electron donor to mediate the univalent reduction of molecular oxygen to superoxide anion (1), a free radical that by spontaneous or enzymatically catalyzed dismutation is readily converted into H 2 O 2 . The phagocytic NOX isoform (NOX2) was first discovered as a pivotal component of the neutrophil respiratory burst (2, 3). The functional NOX2 enzyme is composed of two integral plasma membrane subunits, gp91 phox and p22phox , which make up cytochrome b 558 , plus three cytosolic regulatory subunits: p40 phox , p47 phox , and p67 phox (2, 4). A variety of tissues, including endothelial cells (5), smooth muscle cells (6), neurons (7-9), and astrocytes (7, 10), possess nonphagocytic NOX homologues (11,12). Several reports indicate that NOX2 and its homologues have a central role in the generation of reactive oxygen species (ROS) in response to diverse physiological extracellular stimuli (13-17). Moreover, membrane depolarization stimulates NOX activity in phagocytes (18) and endothelial cells (19,20). NOX stimulation is also apparent following agonist-induced stimulation of N-methyl-D-aspartate receptors in hippocampal neurons (21). Some N...

Triclosan (TCS), a high-production-volume chemical used as a bactericide in personal care products, is a priority pollutant of growing concern to human and environmental health. TCS is capable of altering the activity of type 1 ryanodine receptor (RyR1), but its potential to influence physiological excitation–contraction coupling (ECC) and muscle function has not been investigated. Here, we report that TCS impairs ECC of both cardiac and skeletal muscle in vitro and in vivo. TCS acutely depresses hemodynamics and grip strength in mice at doses ≥12.5 mg/kg i.p., and a concentration ≥0.52 μM in water compromises swimming performance in larval fathead minnow. In isolated ventricular cardiomyocytes, skeletal myotubes, and adult flexor digitorum brevis fibers TCS depresses electrically evoked ECC within ∼10–20 min. In myotubes, nanomolar to low micromolar TCS initially potentiates electrically evoked Ca 2+ transients followed by complete failure of ECC, independent of Ca 2+ store depletion or block of RyR1 channels. TCS also completely blocks excitation-coupled Ca 2+ entry. Voltage clamp experiments showed that TCS partially inhibits L-type Ca 2+ currents of cardiac and skeletal muscle, and [ 3 H]PN200 binding to skeletal membranes is noncompetitively inhibited by TCS in the same concentration range that enhances [ 3 H]ryanodine binding. TCS potently impairs orthograde and retrograde signaling between L-type Ca 2+ and RyR channels in skeletal muscle, and L-type Ca 2+ entry in cardiac muscle, revealing a mechanism by which TCS weakens cardiac and skeletal muscle contractility in a manner that may negatively impact muscle health, especially in susceptible populations.

ABSTRACT3 H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic [Ca 2ϩ ] rest . R163C channels are inherently more active than WT channels, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the Ca 2ϩ release unit of adult fibers.

Malignant hyperthermia (MH) and central core disease in humans have been associated with mutations in the skeletal ryanodine receptor (RyR1). Heterozygous mice expressing the human MH/central core disease RyR1 R163C mutation exhibit MH when exposed to halothane or heat stress. Considering that many MH symptoms resemble those that could ensue from a mitochondrial dysfunction (e.g. metabolic acidosis and hyperthermia) and that MH-susceptible mice or humans have a higher than normal cytoplasmic Ca 2؉ concentration at rest, we evaluated the role of mitochondria in skeletal muscle from R163C compared with wild type mice under basal (untriggered) conditions. R163C skeletal muscle exhibited a significant increase in matrix Ca 2؉ , increased reactive oxygen species production, lower expression of mitochondrial proteins, and higher mtDNA copy number. These changes, in conjunction with lower myoglobin and glycogen contents, Myh4 and GAPDH transcript levels, GAPDH activity, and lower glucose utilization suggested a switch to a compromised bioenergetic state characterized by both low oxidative phosphorylation and glycolysis. The shift in bioenergetic state was accompanied by a dysregulation of Ca 2؉ -responsive signaling pathways regulated by calcineurin and ERK1/2. Chronically elevated resting Ca 2؉ in R163C skeletal muscle elicited the maintenance of a fast-twitch fiber program and the development of insulin resistance-like phenotype as part of a metabolic adaptation to the R163C RyR1 mutation.

Based on these combined results, we propose (i) that BDNF-induced RyR2-mediated Ca release and ROS generation via NOS/NOX2 are strictly required for the dendritic spine remodeling and the RyR2 upregulation induced by BDNF, and (ii) that RyR2 channel expression is crucial for spatial memory processes. Antioxid. Redox Signal. 29, 1125-1146.