LEARN algorithm: a novel option for predicting non-alcoholic steatohepatitis.

Background: There is an unmet need for accurate non-invasive methods to diagnose non-alcoholic steatohepatitis (NASH). Since impedance-based measurements of body composition are simple, repeatable and have a strong association with non-alcoholic fatty liver disease (NAFLD) severity, we aimed to develop a novel and fully automatic machine learning algorithm, consisting of a deep neural network based on impedance-based measurements of body composition to identify NASH [the bioeLectrical impEdance Analysis foR Nash (LEARN) algorithm]. Methods: A total of 1,259 consecutive subjects with suspected NAFLD were screened from six medical centers across China, of which 766 patients with biopsy-proven NAFLD were included in final analysis. These patients were randomly subdivided into the training and validation groups, in a ratio of 4:1. The LEARN algorithm was developed in the training group to identify NASH, and subsequently, tested in the validation group. Results: The LEARN algorithm utilizing impedance-based measurements of body composition along with age, sex, pre-existing hypertension and diabetes, was able to predict the likelihood of having NASH. This algorithm showed good discriminatory ability for identifying NASH in both the training and validation groups [area under the receiver operating characteristics (AUROC): 0.81, 95% CI: 0.77-0.84 and AUROC: 0.80, 95% CI: 0.73-0.87, respectively]. This algorithm also performed better than serum cytokeratin-18 neoepitope M30 (CK-18 M30) level or other non-invasive NASH scores (including HAIR, ION, NICE) for identifying NASH (P value <0.001). Additionally, the LEARN algorithm performed well in identifying NASH in different patient subgroups, as well as in subjects with partial missing body composition data. Conclusions: The LEARN algorithm, utilizing simple easily obtained measures, provides a fully automated, simple, non-invasive method for identifying NASH.

The Role of Reactive Oxygen Species in ß-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome.

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute ß-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: ß-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to ß-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after ß-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute ß-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

Dietary nitrate improves cardiac contractility via enhanced cellular Ca²âº signaling.

The inorganic anion nitrate (NO3 (-)), which is naturally enriched in certain vegetables (e.g., spinach and beetroot), has emerged as a dietary component that can regulate diverse bodily functions, including blood pressure, mitochondrial efficiency, and skeletal muscle force. It is not known if dietary nitrate improves cardiac contractility. To test this, mice were supplemented for 1-2 weeks with sodium nitrate in the drinking water at a dose similar to a green diet. The hearts from nitrate-treated mice showed increased left ventricular pressure and peak rate of pressure development as measured with the Langendorff heart technique. Cardiomyocytes from hearts of nitrate-treated and control animals were incubated with the fluorescent indicator Fluo-3 to measure cytoplasmic free [Ca(2+)] and fractional shortening. Cardiomyocytes from nitrate-treated mice displayed increased fractional shortening, which was linked to larger Ca(2+) transients. Moreover, nitrate hearts displayed increased protein expression of the L-type Ca(2+) channel/dihydropyridine receptor and peak L-type Ca(2+) channel currents. The nitrate-treated hearts displayed increased concentration of cAMP but unchanged levels of cGMP compared with controls. These findings provide the first evidence that dietary nitrate can affect the expression of important Ca(2+) handling proteins in the heart, resulting in increased cardiomyocyte Ca(2+) signaling and improved left ventricular contractile function. Our observation shows that dietary nitrate impacts cardiac function and adds understanding to inorganic nitrate as a physiological modulator.

Stereoselective Synthesis of Diazabicyclic ß-Lactams through Intramolecular Amination of Unactivated C(sp(3))-H Bonds of Carboxamides by Palladium Catalysis.

An efficient C(sp(3))-H bond activation and intramolecular amination reaction via palladium catalysis at the ß-position of carboxyamides to make ß-lactams was described. The investigation of the substrate scope showed that the current reaction conditions favored activation of the ß-methylene group. Short sequences were developed for preparation of various diazabicyclic ß-lactam compounds with this method as the key step from chiral proline and piperidine derivatives.

Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy.

Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca(2+) uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca(2+) handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.

Effect of the Haoqinqingdan decoction on damp-heat syndrome in rats with influenza viral pneumonia.

OBJECTIVE: To investigate the effect of Chinese medicine prescription-Haoqinqingdan decoction on damp-heat syndrome in rats with influenza viral pneumonia and its influence on the immune function. METHODS: A total of 48 Wistar rats were randomly divided into the normal control group, the damp-heat syndrome model group, the Haoqinqingdan decoction group (high, medium and low dose group) and the ribavirin group. The body temperature and weight of rats in each group were recorded after modeling. After treatment for 6 d, the concentration of T lymphocyte subgroup (CD3(+)CD4(+), CD3(+)CD8(+)) was determined by flow cytometry. The OD value of IFN-γ/IL-4 was detected by double-antibody sandwich ELISA method, and its concentration was acquired through conversion. RESULTS: After modeling, the temperature and weight of rats in each modeling group showed the increasing trend (P<0.01). From the second day of treatment, there was significant difference in the body mass between groups, and the rat weight of the control group was higher than in the modeling group (P<0.05 or 0.01). With the advances of treatment, only the temperature in the medium and high dose Haoqinqingdan decoction groups declined significantly (P<0.05). After treatment, the CD4(+)/CD8(+) ratio of the damp-heat syndrome model group decreased more significantly compared with the control group. Elevated CD3(+) CD8(+) percentages and declined CD4(+)/CD8(+) ratios can be observed in the low dose group and ribavirin group (P<0.05). Moreover, the CD3(+) CD4(+) percentage of ribavirin group was lower than in the control group (P<0.05). After treatment, the IFN-γ and IFN-γ/ IL-4 levels in the peripheral blood of rats in the damp-heat syndrome group were obviously higher than in the control group (P<0.05). CONCLUSIONS: Compared with ribavirin, the high dose Haoqinqingdan decoction can improve the ratio of T lymphocyte subgroup and Th1/Th2 cell balance more effectively.

Impact of Lycium Barbarum Polysaccharide and Danshensu on vascular endothelial growth factor in the process of retinal neovascularization of rabbit.

AIM: To discuss the impact of Lycium Barbarum Polysaccharide (LBP) and Danshensu purified from Traditional Chinese Medicine (TCM) on vascular endothelial growth factor (VEGF) of rabbits with retinal neovascularization. METHODS: Forty rabbits were divided into normal control group, model control group, LBP group and Danshensu group. Animals in the normal control group were fed in the normal oxygen environment. Animals in the other three groups were put into the environment with 70% oxygen for 5 days in order to build the model of oxygen-induced vascular proliferation retinopathy. And then different TCM extract was injected into the abdominal cavities of these annimals. After 7 days, the VEGF content of in the serum of rabbit was measured by double antibody sandwich method. RESULTS: DATA ANALYSIS INDICATED THAT VEGF CONTENT WAS AS FOLLOWS: Danshensu group was lower than model control group (12.92±3.84ng/L vs 19.32±4.15ng/L, P<0.05); LBP group and normal control group were lower than model control group (12.92±3.84ng/L, 9.26±1.61ng/L vs 19.32±4.15ng/L, P<0.01); total blood viscosity, plasma viscosity, cholesterol content, fibrinogen content and triacylglycerol content after peritoneal injection of LBP and Danshensu were obviously lower than before injection. CONCLUSION: TCM extract-LBP and Danshensu can prominently reduce the content of VEGF in the process of vascular proliferative retinopathy of rabbit; can prevent the occurrence of retinal microvascular disease by improving partial oxygen-deficient environment or affecting all kinds of new growth factor.

Impaired mitochondrial respiration and decreased fatigue resistance followed by severe muscle weakness in skeletal muscle of mitochondrial DNA mutator mice.

Mitochondrial dysfunction can drastically impair muscle function, with weakness and exercise intolerance as key symptoms. Here we examine the time course of development of muscle dysfunction in a mouse model of premature ageing induced by defective proofreading function of mitochondrial DNA (mtDNA) polymerase (mtDNA mutator mouse). Isolated fast-twitch muscles and single muscle fibres from young (3-5 months) and end-stage (11 months) mtDNA mutator mice were compared to age-matched control mice. Force and free myoplasmic [Ca(2+)] ([Ca(2+)](i)) were measured under resting conditions and during fatigue induced by repeated tetani. Muscles of young mtDNA mutator mice displayed no weakness in the rested state, but had lower force and [Ca(2+)](i) than control mice during induction of fatigue. Muscles of young mtDNA mutator mice showed decreased activities of citrate synthase and ß-hydroxyacyl-coenzyme A dehydrogenase, reduced expression of cytochrome c oxidase, and decreased expression of triggers of mitochondrial biogenesis (PGC-1α, PPARα, AMPK). Muscles from end-stage mtDNA mutator mice showed weakness under resting conditions with markedly decreased tetanic [Ca(2+)](i), force per cross-sectional area and protein expression of the sarcoplasmic reticulum Ca(2+) pump (SERCA1). In conclusion, fast-twitch muscles of prematurely ageing mtDNA mutator mice display a sequence of deleterious mitochondrial-to-nucleus signalling with an initial decrease in oxidative capacity, which was not counteracted by activation of signalling to increase mitochondrial biogenesis. This was followed by severe muscle weakness in the end stage. These results have implication for normal ageing and suggest that decreased mitochondrial oxidative capacity due to a sedentary lifestyle may predispose towards muscle weakness developing later in life.

Effect of Western medicine therapy assisted by Ginkgo biloba tablet on vascular cognitive impairment of none dementia.

OBJECTIVE: To discuss the clinical effects of Western medicine therapy assisted by Ginkgo biloba tablet (GBT) on patients with vascular cognitive impairment of none dementia (VCIND). METHODS: A total of 80 patients with VCIND were divided into two groups randomly: Conventional treatment group (control group) and combined treatment group. Conventional treatment group was given conventional treatment with anti-platelet aggregation. In this group, 75 mg aspirin was given three times a day for 3 months. While in combined treatment group, 19.2 mg GBT was given three times a day for 3 months together with conventional treatment (anti-platelet aggregation drugs). Montreal cognitive assessment (MoCA) and transcranial Doppler (TCD) were used to observe changes of cognitive ability and cerebral blood flow in VCIND patients before and after treatment in both groups. Then the clinical data were analyzed so as to compare the efficacy in two groups. RESULTS: After 3 month-treatment in combined treatment group, the scores of executive ability, attention, abstract, delayed memory, orientation in the MoCA were significantly increased compared with those before treatment and those in control group after treatment. Besides, blood flow velocity of anterior cerebral artery increased significantly than that before treatment and that in control group after treatment. CONCLUSIONS: GBT tablet can improve the therapeutic efficacy as well improve cognitive ability and cerebral blood flow supply of patients with VCIND.

AICAR reverses ketone body mediated insulin resistance in isolated oxidative muscle.

Recently it was demonstrated that the ketone body ß-hydroxybutyrate (BOH) inhibits insulin-mediated glucose transport in isolated oxidative muscle, which was associated with decreased phosphorylation of Akt/protein kinase B. The purpose of the present study was to determine if activation of AMP-dependent protein kinase by the pharmacological activator AICAR could reverse the insulin resistance induced by BOH. Isolated mouse soleus muscle was incubated in vitro in the absence or presence of 5mM BOH for ∼20 h. Following prolonged incubation, insulin increased 2-deoxyglucose glucose (2-DG) uptake 3-fold, but in the presence of BOH most of the insulin response was lost (only ∼30% remained). Addition of 2mM AICAR during the last 2h of prolonged incubation increased the insulin response in the presence of BOH to ∼80% of the normal insulin effect on 2-DG uptake. The AICAR-mediated reversal of the insulin resistance was not associated with a restoration of the insulin effect on Akt/protein kinase B phosphorylation. However, AICAR enhanced the insulin-induced phosphorylation of the Akt substrate, AS160. In conclusion, these data demonstrate that AICAR reverses the negative effect of BOH on insulin-mediated glucose uptake and this is attributed to activation of a late step in insulin signaling.

Mitochondrial production of reactive oxygen species contributes to the ß-adrenergic stimulation of mouse cardiomycytes.

The sympathetic adrenergic system plays a central role in stress signalling and stress is often associated with increased production of reactive oxygen species (ROS). Furthermore, the sympathetic adrenergic system is intimately involved in the regulation of cardiomyocyte Ca2+ handling and contractility. In this study we hypothesize that endogenously produced ROS contribute to the inotropic mechanism of ß-adrenergic stimulation in mouse cardiomyocytes. Cytoplasmic Ca2+ transients, cell shortening and ROS production were measured in freshly isolated cardiomyocytes using confocal microscopy and fluorescent indicators. As a marker of oxidative stress, malondialdehyde (MDA) modification of proteins was detected with Western blotting. Isoproterenol (ISO), a ß-adrenergic agonist, increased mitochondrial ROS production in cardiomyocytes in a concentration- and cAMP­protein kinase A-dependent but Ca2+-independent manner. Hearts perfused with ISO showed a twofold increase in MDA protein adducts relative to control. ISO increased Ca2+ transient amplitude, contraction and L-type Ca2+ current densities (measured with whole-cell patch-clamp) in cardiomyocytes and these increases were diminished by application of the general antioxidant N-acetylcysteine (NAC) or the mitochondria-targeted antioxidant SS31. In conclusion, increased mitochondrial ROS production plays an integral role in the acute inotropic response of cardiomyocytes to ß-adrenergic stimulation. On the other hand, chronically sustained adrenergic stress is associated with the development of heart failure and cardiac arrhythmias and prolonged increases in ROS may contribute to these defects.

Enhanced cardiomyocyte Ca(2+) cycling precedes terminal AV-block in mitochondrial cardiomyopathy Mterf3 KO mice.

AIMS: Heart disease is commonly associated with altered mitochondrial function and signs of oxidative stress. This study elucidates whether primary cardiac mitochondrial dysfunction causes changes in cardiomyocyte handling of reactive oxygen species (ROS) and Ca(2+). We used a mouse model with a tissue-specific ablation of the recently discovered mtDNA transcription regulator Mterf3 (Mterf3 KO). These mice display a cardiomyopathy with severe respiratory chain dysfunction, cardiac hypertrophy, and shortened lifespan. ROS and Ca(2+) handling were measured using fluorescent indicators and confocal microscopy. RESULTS: Mterf3 KO hearts displayed no signs of increased ROS production or oxidative stress. Surprisingly, Mterf3 KO cardiomyocytes showed enlarged Ca(2+) transient amplitudes, faster sarcoplasmic reticulum (SR) Ca(2+) reuptake, and increased SR Ca(2+) load, resembling increased adrenergic stimulation. Furthermore, spontaneous releases of Ca(2+) were frequent in Mterf3 KO cardiomyocytes. Electrocardiography (measured with telemetry in freely moving mice) showed a terminal state in Mterf3 KO mice with gradually developing bradycardia and atrioventricular block. CONCLUSION: In conclusion, mitochondrial dysfunction induced by Mterf3 KO leads to a cardiomyopathy without signs of oxidative stress but with increased cardiomyocyte Ca(2+) cycling and an arrhythmogenic phenotype. These findings highlight the complex interaction between mitochondrial function, cardiomyocyte contractility, and compensatory mechanisms, such as activation of adrenergic signaling.

Increased fatigue resistance linked to Ca2+-stimulated mitochondrial biogenesis in muscle fibres of cold-acclimated mice.

Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering). Shivering is successively replaced by the recruitment of uncoupling protein-1 (UCP1)-dependent heat production in brown adipose tissue. Interestingly, adaptations observed in skeletal muscles of cold-exposed animals are similar to those observed with endurance training. We hypothesized that increased myoplasmic free [Ca2+] ([Ca2+]i) is important for these adaptations. To test this hypothesis, experiments were performed on flexor digitorum brevis (FDB) muscles, which do not participate in the shivering response, of adult wild-type (WT) and UCP1-ablated (UCP1-KO) mice kept either at room temperature (24°C) or cold-acclimated (4°C) for 4-5 weeks. [Ca2+]i (measured with indo-1) and force were measured under control conditions and during fatigue induced by repeated tetanic stimulation in intact single fibres. The results show no differences between fibres from WT and UCP1-KO mice. However, muscle fibres from cold-acclimated mice showed significant increases in basal [Ca2+]i (∼50%), tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) as compared to fibres from room-temperature mice. Muscles of cold-acclimated mice showed increased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and increased citrate synthase activity (reflecting increased mitochondrial content). Fibres of cold-acclimated mice were more fatigue resistant with higher tetanic [Ca2+]i and less force loss during fatiguing stimulation. In conclusion, cold exposure induces changes in FDB muscles similar to those observed with endurance training and we propose that increased [Ca2+]i is a key factor underlying these adaptations.

{beta}-Hydroxybutyrate inhibits insulin-mediated glucose transport in mouse oxidative muscle.

Blood ketone body levels increase during starvation and untreated diabetes. Here we tested the hypothesis that ketone bodies directly inhibit insulin action in skeletal muscle. We investigated the effect of d,l-beta-hydroxybutyrate (BOH; the major ketone body in vivo) on insulin-mediated glucose uptake (2-deoxyglucose) in isolated mouse soleus (oxidative) and extensor digitorum longus (EDL; glycolytic) muscle. BOH inhibited insulin-mediated glucose uptake in soleus (but not in EDL) muscle in a time- and concentration-dependent manner. Following 19.5 h of exposure to 5 mM BOH, insulin-mediated (20 mU/ml) glucose uptake was inhibited by approximately 90% (substantial inhibition was also observed in 3-O-methylglucose transport). The inhibitory effect of BOH was reproduced with d- but not l-BOH. BOH did not significantly affect hypoxia- or AICAR-mediated (activates AMP-dependent protein kinase) glucose uptake. The BOH effect did not require the presence/utilization of glucose since it was also seen when glucose in the medium was substituted with pyruvate. To determine whether the BOH effect was mediated by oxidative stress, an exogenous antioxidant (1 mM tempol) was used; however, tempol did not reverse the BOH effect on insulin action. BOH did not alter the levels of total tissue GLUT4 protein or insulin-mediated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 but blocked insulin-mediated phosphorylation of protein kinase B by approximately 50%. These data demonstrate that BOH inhibits insulin-mediated glucose transport in oxidative muscle by inhibiting insulin signaling. Thus ketone bodies may be potent diabetogenic agents in vivo.

2-Methoxyoestradiol inhibits glucose transport in rodent skeletal muscle.

2-Methoxyoestradiol (2-ME) is an oestrogen derivative that inhibits superoxide dismutase (which converts superoxide anions to H(2)O(2)). Since reactive oxygen species have been implicated in glucose transport, we determined the effect of 2-ME on glucose transport in skeletal muscle. Experiments were performed on isolated mouse extensor digitorum longus (EDL, glycolytic, fast-twitch) muscle. Glucose uptake was measured using 2-deoxy-d-[1,2-(3)H]glucose. 2-Methoxyoestradiol (50 microm) reduced glucose uptake induced by insulin, contraction and hypoxia by approximately 60%. Exogenous H(2)O(2) activated glucose uptake, and this effect was also blocked by 2-ME, demonstrating that 2-ME was exerting its inhibitory effect on glucose uptake at a site other than superoxide dismutase. When glucose uptake was stimulated by insulin, followed by addition of 2-ME, there was also an attenuation of the effect of insulin (approximately 60%). Moreover, basal glucose uptake was decreased by 2-ME (approximately 50%). In contrast, insulin-mediated translocation of glucose transporter type 4 protein to the plasma membrane was not affected by 2-ME. Similar results were obtained in soleus (oxidative, slow-twitch) muscle. In conclusion, 2-ME appears to decrease glucose transport in skeletal muscle by directly interfering with the function of glucose transport proteins in surface membranes.

Impaired myofibrillar function in the soleus muscle of mice with collagen-induced arthritis.

OBJECTIVE: Progressive muscle weakness is a common feature in patients with rheumatoid arthritis (RA). However, little is known about whether the intrinsic contractile properties of muscle fibers are affected in RA. This study was undertaken to investigate muscle contractility and the myoplasmic free Ca2+ concentration ([Ca2+](i)) in the soleus, a major postural muscle, in mice with collagen-induced arthritis (CIA). METHODS: Muscle contractility and [Ca2+](i) were assessed in whole muscle and intact single-fiber preparations, respectively. The underlying mechanisms of contractile dysfunction were assessed by investigating redox modifications using Western blotting and antibodies against nitric oxide synthase (NOS), superoxide dismutase (SOD), 3-nitrotyrosine (3-NT), carbonyl, malondialdehyde (MDA), and S-nitrosocysteine (SNO-Cys). RESULTS: The tetanic force per cross-sectional area was markedly decreased in the soleus muscle of mice with CIA, and the change was not due to a decrease in the amplitude of [Ca2+](i) transients. The reduction in force production was accompanied by slowing of the twitch contraction and relaxation and a decrease in the maximum shortening velocity. Immunoblot analyses showed a marked increase in neuronal NOS expression but not in inducible or endothelial NOS expression, which, together with the observed decrease in SOD2 expression, favors peroxynitrite formation. These changes were accompanied by increased 3-NT, carbonyl, and MDA adducts content in myofibrillar proteins from the muscles of mice with CIA. Moreover, there was a significant increase in SNO-Cys content in myosin heavy-chain and troponin I myofibrillar proteins from the soleus muscle of mice with CIA. CONCLUSION: These findings show impaired contractile function in the soleus muscle of mice with CIA and suggest that this abnormality is due to peroxynitrite-induced modifications in myofibrillar proteins.

High temperature does not alter fatigability in intact mouse skeletal muscle fibres.

Intense activation of skeletal muscle results in fatigue development, which involves impaired function of the muscle cells resulting in weaker and slower contractions. Intense muscle activity also results in increased heat production and muscle temperature may rise by up to 6 degrees C. Hyperthermia is associated with impaired exercise performance in vivo and recent studies have shown contractile dysfunction and premature fatigue development in easily fatigued muscle fibres stimulated at high temperatures and these defects were attributed to oxidative stress. Here we studied whether fatigue-resistant soleus fibres stimulated at increased temperature show premature fatigue development and whether increasing the level of oxidative stress accelerates fatigue development. Intact single fibres or small bundles of soleus fibres were fatigued by 600 ms tetani given at 2 s intervals at 37 degrees C and 43 degrees C, which is the highest temperature the muscle would experience in vivo. Tetanic force in the unfatigued state was not significantly different at the two temperatures. With 100 fatiguing tetani, force decreased by approximately 15% at both temperatures; the free cytosolic [Ca(2+)] (assessed with indo-1) showed a similar approximately 10% decrease at both temperatures. The oxidative stress during fatigue at 43 degrees C was increased by application of 10 microM hydrogen peroxide or tert-butyl hydroperoxide and this did not cause premature fatigue development. In summary, fatigue-resistant muscle fibres do not display impaired contractility and fatigue resistance at the highest temperature that mammals, including humans, would experience in vivo. Thus, intrinsic defects in fatigue-resistant muscle fibres cannot explain the decreased physical performance at high temperatures.

Upregulation of MHC class I in transgenic mice results in reduced force-generating capacity in slow-twitch muscle.

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment, we used transgenic mice with inducible overexpression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast-twitch) and soleus (slow-twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared with control mice; however, when force was expressed per cross-sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force-generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. Muscle Nerve, 2008.

Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin-mediated glucose uptake in adult skeletal muscle cells.

The involvement of Ca(2+) in the insulin-mediated signaling cascade, resulting in glucose uptake in skeletal muscle, is uncertain. Here, we test the hypothesis that Ca(2+) influx through canonical transient receptor potential 3 (TRPC3) channels modulates insulin-mediated glucose uptake in adult skeletal muscle. Experiments were performed on adult skeletal muscle cells of wild-type (WT) and obese, insulin-resistant ob/ob mice. Application of the diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG) induced a nonselective cation current, which was inhibited by the addition of anti-TRPC3 antibody in the patch pipette and smaller in ob/ob than in WT cells. Knockdown of TRPC3, using a novel technique based on small interfering RNA (siRNA) coupled to functionalized carbon nanotubes, resulted in pronounced (approximately 70%) decreases in OAG-induced Ca(2+) influx and insulin-mediated glucose uptake. TRPC3 and the insulin-sensitive glucose transporter 4 (GLUT4) coimmunoprecipitated, and immunofluorescence staining showed that they were colocalized in the proximity of the transverse tubular system, which is the predominant site of insulin-mediated glucose transport in skeletal muscle. In conclusion, our results indicate that TRPC3 interacts functionally and physically with GLUT4, and Ca(2+) influx through TRPC3 modulates insulin-mediated glucose uptake. Thus, TRPC3 is a potential target for treatment of insulin-resistant conditions.

Nonshivering thermogenesis protects against defective calcium handling in muscle.

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca(2+) leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca(2+) release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca(2+) and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles.