Valerie P. Wright

Redox modulation of global phosphatase activity and protein phosphorylation in intact skeletal muscle

Skeletal muscles produce transient reactive oxygen species (ROS) in response to intense stimulation, disuse atrophy, heat stress, hypoxia, osmotic stress, stretch and cell receptor activation. The physiological significance is not well understood. Protein phosphatases (PPases) are known to be highly sensitive to oxidants and could contribute to many different signalling responses in muscle. We tested whether broad categories of PPases are inhibited by levels of acute oxidant exposure that do not result in loss of contractile function or gross oxidative stress. We also tested if this exposure results in elevated levels of global protein phosphorylation. Rat diaphragm muscles were treated with either 2,3‐dimethoxy‐1‐naphthoquinone (DMNQ; 1, 10, 100 μm; a mitochondrial O2•−/H2O2 generator) or exogenous H2O2 (5, 50, 500 μm) for 30 min. Supernatants were assayed for serine/threonine PPase (Ser/Thr‐PPase) or protein tyrosine PPase (PTP) activities. With the exception of 500 μm H2O2, no other oxidant exposures significantly elevated protein carbonyl formation, nor did they alter the magnitude of twitch force. DMNQ significantly decreased all categories of PPase activity at 10 and 100 μm and reduced PTP at 1 μm. Similar reductions in Ser/Thr‐PPase activity were seen in response to 50 and 500 μm H2O2 and PTP at 500 μm H2O2. ROS treatments resulted a dose‐dependent increase in the phosphorylation states of many proteins. The data are consistent with the concept that PPases, within intact skeletal muscles, are highly sensitive to acute changes in ROS activity and that localized ROS play a critical role in lowering the barriers for effective phosphorylation events to occur in muscle cells, thus increasing the probability for cell signalling responses to proceed.

Dithiothreitol improves recovery from in vitro diaphragm fatigue.

There is increasing evidence that reactive oxygen species are produced during strenuous skeletal muscle work and that they contribute to the development of muscle fatigue. Although the precise cellular mechanisms underlying such a phenomenon remain obscure, it has been hypothesized that endogenously produced reactive oxygen species may down-regulate force production during fatigue by oxidizing critical sulfhydryl groups on important contractile proteins. To test this hypothesis, we fatigued rat diaphragm strips in vitro for 4 min at 20 Hz stimulation and a duty cycle of 0.33. Following fatigue, the tissue baths were drained and randomly replaced with either physiologic saline or physiologic saline containing the disulfide reducing agent, dithiothreitol (DTT) at varying doses (0.1-5.0 mM). Force-frequency characteristics were then measured over a 90-min recovery period. At the 0.5 and 1.0 mM doses, DTT treatment was associated with significantly greater force production in the recovery period. DTTs effects were observed at most frequencies tested, but appeared more prominent at the higher frequencies. The beneficial effects of DTT were not evident at the 0.1 or 5.0 mM doses and appeared to be specific for fatigued muscle. These recovery-enhancing effects of a potent disulfide reducing agent suggest that important contractile proteins may be oxidized during fatigue; such changes may be readily reversible.

Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro.

We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC‐derived microvesicles (MVs) were responsible for monocyte suppression.

Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition

Mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. This study identified the critical temperature, resulting in a loss of contractile function in isolated diaphragm (thermal tolerance), and then tested the hypotheses 1) that increased reactive oxygen species (ROS) production contributes to the loss of contractile function at this temperature, and 2) eicosanoid metabolism plays an important role in preservation of contractile function in hyperthermia. Contractile function and passive force were measured in rat diaphragm bundles during and after 30 min of exposure to 40, 41, 42 or 43 degrees C. Between 40 and 42 degrees C, there were no effects of hyperthermia, but at 43 degrees C, a significant loss of active force and an increase in passive force were observed. Inhibition of ROS with the antioxidants, Tiron or Trolox, did not inhibit the loss of contractile force at 43 degrees C. Furthermore, treatment with dithiothreitol, a thiol (-SH) reducing agent, did not reverse the effects of hyperthermia. A variety of global lipoxygenase (LOX) inhibitors further depressed force during 43 degrees C and caused a significant loss of thermal tolerance at 42 degrees C. Cyclooxygenase (COX) inhibitors also caused a loss of thermal tolerance at 42 degrees C. Blockage of phospholipase with phospholipase A(2) inhibitors, bromoenol lactone or arachidonyltrifluoromethyl ketone failed to significantly prevent the loss of force at 43 degrees C. Overall, these data suggest that ROS do not play an apparent role in the loss of contractile function during severe hyperthermia in diaphragm. However, functional LOX and COX enzyme activities appear to be necessary for maintaining normal force production in hyperthermia.

Adipocyte DIO2 Expression Increases in Human Obesity but Is Not Related to Systemic Insulin Sensitivity

Deiodinase type II (D2), encoded by DIO2, catalyzes the conversion of T4 to bioactive T3. T3 not only stimulates adaptive thermogenesis but also affects adipose tissue (AT) lipid accumulation, mitochondrial function, inflammation, and potentially systemic metabolism. Although better defined in brown AT, the precise role of DIO2 expression in white AT remains largely unknown, with data derived only from whole fat. Therefore, the purpose of this study was to determine whether subcutaneous (SAT) and visceral (VAT) adipocyte-specific gene expression of DIO2 differs between obese and lean patients and whether these differences relate to alterations in mitochondrial function, fatty acid flux, inflammatory cytokines/adipokines, and ultimately insulin sensitivity. Accordingly, adipocytes of 73 obese and 21 lean subjects were isolated and subjected to gene expression analyses. Our results demonstrate that obese compared to lean human individuals have increased adipocyte-specific DIO2 expression in both SAT and VAT. Although higher DIO2 was strongly related to reduced fatty acid synthesis/oxidation and mitochondrial function, we found no relationship to proinflammatory cytokines or insulin resistance and no difference based on diabetic status. Our results suggest that adipocyte-derived DIO2 may play a role in weight maintenance but is likely not a major contributor to obesity-related insulin resistance.