Skeletal Muscle Homogenates Research Articles

Reduction of endothelial NOS and bradykinin-induced extravasation of macromolecules in skeletal muscle of the fructose-fed rat model.

Reduced capillary permeability of the skeletal muscle vascular bed has been suggested to play a role in fructose-fed rats, corroborating a long held view that insulin resistance might partially be explained by the lack of access of insulin and glucose to its target organs, mainly skeletal muscles. The goal of this study was to explore mechanisms underlying this vascular abnormality, and more specifically the role of bradykinin and nitric oxide (NO) on skeletal muscle microcirculation and the extravasation of macromolecules. For that purpose, Sprague-Dawley rats were fed with either a fructose-enriched (F) or a normal chow (N) diet and extravasation of macromolecules was assessed at 4 weeks by measuring in vivo the extravasation of Evans Blue (EB) dye in the quadriceps muscles of both groups after the intravenous injection of the potent vasodilator bradykinin (150 microg/kg). As expected, fructose-fed rats had less extravasation of EB in skeletal muscle in the basal state as compared to controls (F 17.6 +/- 4.4 vs. N 43.6 +/- 6.9 microg/g dry tissue; P<0.01). In response to bradykinin, the EB dye extravasation in skeletal muscle was 89.4% higher in rats fed the normal chow diet compared to the basal state (P<0.03). In contrast, no significant increase in vasopermeability was observed in fructose-fed animals acutely injected with BK (17.6 +/- 4.4 microg/g in the basal state versus 24.6 +/- 3.1 microg/g after the injection of BK; P=NS). To distinguish a functional from an anatomical/structural defect, hematoxylin-eosin sections as well as electron micrographs of skeletal muscle microvessels were examined in both groups of animals: no obvious abnormalities were found. However, in homogenates of skeletal muscles (quadriceps) of fructose-fed rats, there was a marked reduction of NO synthase (NOS) activity (-33.8%; P<0.001) as well as endothelial NOS immunoreactive mass (-23.4%; P<0.04) as compared to control animals. There is unresponsiveness of the skeletal muscle capillary bed to bradykinin in insulin-resistant animals most probably due to a reduction in endothelial NOS (activity and mass). Our results indicate a functional defect possibly involving responsiveness of the precapillary resistance and/or the endothelial barrier to bradykinin in skeletal muscles. Since insulin must cross the endothelial monolayer to reach its target cells on the abluminal side, it is suggested that reduced endothelial NOS and consequent reduced extravasation of macromolecules could exacerbate insulin resistance in skeletal muscles and hypertension in the fructose-fed rat.

Effect of intermittent and continuous hypoxia on ryanodine receptors of rat heart

Intermittent hypoxia (IH) adaptation has been shown to exert beneficial effects on the functions of hearts that had been subjected to insult by ischemia or ischemia/reperfusion. To understand whether calcium release channels/ryanodine receptors (RyRs) were involved, the effects of IH and continuous hypoxia (CH) on [ 3H]ryanodine binding to homogenates of rat hearts were investigated. Similar studies were performed on rat skeletal muscle. The main results on cardiac muscle were as follows: 1) Ischemia for up to 45 min in normal rat hearts had no obvious effect on the equilibrium ryanodine binding constant (K d), while the maximum number of ryanodine binding sites (B max) was affected in a time-dependent manner. B max was significantly increased with 15 min ischemia, which then returned to control levels upon prolonging the ischemia to 30 min. After 45 min ischemia, a small decrease of B max was observed. 2) IH adaptation for up to 28 days did not change B max, but a significant decrease of B max was apparent after longer IH adaptation or after CH exposure. Although B max was not altered by 30 min ischemia, 30 min reperfusion following 30 min ischemia induced an evident decrease of B max. After either IH or CH adaptation, the ischemia/reperfusion- induced decrease of B max was abolished. 3) Several effects on K d of ischemia and ischemia/reperfusion, with and without IH or CH adaptation, were observed. The most distinct and consistent finding was that a clear increase of K d was induced by ischemia or ischemia/reperfusion in CH adapted rats. [ 3H]Ryanodine binding to homogenates of rat skeletal muscle was also affected by IH and CH adaptation. In contrast to that found in cardiac muscle, a decrease of B max in skeletal muscle appeared only after CH adaptation. The physiological significance of these effects is discussed.

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single ∼33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 °C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 °C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.

Fatty acid oxidation by skeletal muscle homogenates from morbidly obese black and white American women

The purpose of this study was to determine if there were differences in the capacity of skeletal muscle from morbidly obese Black and White American women to oxidize fatty acids. The oxidation rates of 14C-palmitate, 14C-palmitoyl-CoA, and 14C-palmitoyl-carnitine were measured in whole homogenates of rectus abdominus from Black and White women who were similar in age and body mass index (BMI). The activities of muscle citrate synthase (CS), β-hydroxy acyl-CoA dehydrogenase (β-HAD), and mitochondrial and microsomal acyl-CoA synthetase (ACS) were measured in the 2 groups. The results showed that the rate of 14C-palmitate oxidation by muscle of Black women was 25% that of Whites (8.7 ± 1.5 v 34.4 ± 6.8 nmol 14CO2 produced/gram tissue wet weight/ hour; P < .05), but the rates of 14C-palmitoyl-CoA and 14C-palmitoyl-carnitine oxidation were not different in the 2 groups. No differences were found in the activities of CS or β-HAD. However, the activities of both mitochondrial and microsomal ACS were lower in the Black women than the Whites (mitochondrial ACS 25.1 ± 3.9 v 36.4 ± 5.0 nmol/mg protein/min; P < .05; microsomal ACS 6.2 ± 0.5 v 8.5 ± 0.5; nmol/mg protein/min; P < .005). The lower rate of palmitate oxidation, and the lack of differences in the rates of palmitoyl-CoA and palmitoyl-carnitine oxidation indicate that there is a defect in the activation of the fatty acid in the muscle of the Black women. This was confirmed by the decrease in mitochondrial ACS activity in the Black women. The decreased fatty acid oxidation by skeletal muscle of obese Black women could result in shunting these fuels from muscle to adipose tissue for storage, which may contribute to the maintenance of obesity in the Black women.

L-2-Hydroxyglutaric acid inhibits mitochondrial creatine kinase activity from cerebellum of developing rats

l-2-Hydroxyglutaric acid (LGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as l-2-hydroxyglutaric aciduria (LHGA). Although this disorder is predominantly characterized by severe neurological findings and pronounced cerebellum atrophy, the neurotoxic mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of LGA, at 0.25–5mM concentrations, on total creatine kinase (tCK) activity from cerebellum, cerebral cortex, cardiac muscle and skeletal muscle homogenates of 30-day-old Wistar rats. CK activity was measured also in the cytosolic (Cy-CK) and mitochondrial (Mi-CK) fractions from cerebellum. We verified that tCK activity was significantly inhibited by LGA in the cerebellum, but not in cerebral cortex, cardiac muscle and skeletal muscle. Furthermore, CK activity from the mitochondrial fraction was inhibited by LGA, whereas that from the cytosolic fraction of cerebellum was not affected by the acid. Kinetic studies revealed that the inhibitory effect of LGA on Mi-CK was non-competitive in relation to phosphocreatine. Finally, we verified that the inhibitory effect of LGA on tCK was fully prevented by pre-incubation of the homogenates with reduced glutathione (GSH), suggesting that this inhibition is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of creatine kinase activity for energy homeostasis, our results suggest that the selective inhibition of this enzyme activity by increased levels of LGA could be possibly related to the cerebellar degeneration characteristically found in patients affected by l-2-hydroxyglutaric aciduria.

Skeletal muscle myosin is the autoantigen for experimental autoimmune myositis

Experimental autoimmune myositis (EAM) is a rodent model for human inflammatory muscle disease (IMD). It can be induced by immunization of rodents with skeletal muscle homogenate and adjuvant. The specific myositogenic autoantigen has not been clearly identified although some evidence points to skeletal muscle myosin. In this report we strengthen this evidence, showing that Lewis rats immunized with purified skeletal muscle myosin develop EAM with the same pattern and severity as EAM induced by whole rabbit skeletal muscle homogenate (WRM). Multiple inflammatory lesions are detected histopathologically in the biceps, quadriceps, and gastrocnemius muscles. Myosin-reactive T cells from animals immunized either with myosin or with WRM have similar patterns of antigen-induced proliferation. The results show that myosin, a component of skeletal muscle, is at least one autoantigen in EAM.

Biochemical and immunohistochemical evidence for a non-muscle myosin at the neuromuscular junction in bovine skeletal muscle.

We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac beta-myosin heavy chain (beta-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac beta-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac beta-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family.

Epinephrine modulates cellular distribution of muscle phosphofructokinase

In this paper, we report evidences that cellular distribution of phosphofructokinase can be affected by epinephrine stimulation in rabbit skeletal muscle homogenates. Through co-sedimentation techniques, we observed that in epinephrine-stimulated tissues, approximately 50% of phosphofructokinase activity is co-located in an actin-enriched fraction, against 29% in control. This phenomenon is companied by a 400% increase in specific phosphofructokinase activity in stimulated homogenates. This effect is reproduced by the β-adrenergic agonist isoprenaline. Here we propose that the modulation of cellular distribution of phosphofructokinase may be one of the mechanisms of control of glycolytic flux in mammalian muscle, by β-adrenergic stimulation.

Detoxification of cytotoxic alpha,beta-unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by liquid chromatography/mass spectrometry in rat skeletal muscle.

Oxidation of polyunsaturated fatty acids containing phospholipids in tissue generates lipid hydroperoxides, which are further degraded to several products, among which unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) play an important role in mediating the pathological effects of oxidative stress. While the reaction of HNE with glutathione (GSH) is a well recognized pathway of detoxification in biological systems, no data are available on HNE interactions with carnosine, a dipeptide (beta-alanyl-L-histidine) present in high concentration in skeletal muscle. The aim of this work was to study the quenching ability of carnosine towards HNE and to characterize the reaction products by electrospray ionization tandem mass spectrometry (ESI-MS/MS), using GSH as a model peptide. GSH incubation with HNE in 1 mM phosphate buffer (pH 7.4) results in the complete disappearance of HNE within 1 h owing to the formation of a Michael adduct, S-(4-hydroxynonanal-3-yl)glutathione. The reaction of HNE with carnosine was studied in different molar ratios and monitored up to 24 h by high-performance liquid chromatography (HPLC) (HNE consumption), MS/MS (infusion) and liquid chromatography mass spectrometry (LC/MS) experiments. Carnosine, although less reactive than GSH, significantly quenched HNE (48.2 +/- 0.9% HNE consumption after 1 h; carnosine:HNE molar ratio 10 : 1). Two reaction products were identified: the Michael adduct, N-(4-hydroxynonanal-3-yl)carnosine involving the imidazolic nitrogen of histidine, and the imine adduct, involving the amino group of the beta-alanine residue. Definitive structure assignment was achieved by chemical reduction with NaBH(4) and multinuclear magnetic resonance experiments. To understand whether carnosine acts as a quencher of unsaturated aldehydes in biological matrices, rat skeletal muscle homogenate was incubated with HNE and the formation of conjugated adducts was determined by LC/MS analysis. Three main products were detected and identified as Michael adducts of HNE with GSH, carnosine and anserine (the N-methylated derivative of carnosine, present in high concentrations in rat muscle). The results indicate that beside GSH, histidine-containing dipeptides could be involved in the detoxification pathway of reactive aldehydes from lipid peroxidation generated in skeletal muscle during physical endurance.

Increased lipogenesis in isolated hepatocytes from cold-acclimated ducklings.

Thermogenic endurance and development of metabolic cold adaptation in birds may critically depend on their ability to synthesize and use fatty acids (FA) as fuel substrates. Hepatic lipogenesis and the capacity to oxidize FA in thermogenic tissues were measured in cold-acclimated (CA) ducklings (Cairina moschata) showing original mechanisms of metabolic cold adaptation in the absence of brown adipose tissue, the specialized thermogenic tissue of rodents. The rate of FA synthesis from [U-(14)C]glucose and from [1-(14)C]acetate, measured in incubated hepatocytes isolated from 5-wk-old thermoneutral (TN; 25 degrees C) or CA (4 degrees C) fed ducklings, was higher than in other species. Hepatic de novo lipogenesis was further increased by cold acclimation with both glucose (+194%) and acetate (+111%) as precursor. Insulin slightly increased (+11-14%) hepatic lipogenesis from both precursors in CA ducklings, whereas glucagon was clearly inhibitory (-29 to -51%). Enhanced de novo lipogenesis was associated with higher (+171%) hepatocyte activity of glucose oxidation and larger capacity (+50 to +100%) of key lipogenic enzymes. The potential for FA oxidation was higher in liver (+61%) and skeletal muscle (+29 to +81%) homogenates from CA than from TN ducklings, suggesting that the higher hepatic lipogenesis may fuel oxidation in thermogenic tissues. Present data underline the high capacity to synthesize lipids from glucose in species like muscovy ducks susceptible to hepatic steatosis. Lipogenic capacity can be further increased in the cold and may represent an important step in the metabolic adaptation to cold of growing ducklings.

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (β-alanyl- l-histidine) acts as a quencher of cytotoxic α,β-unsaturated aldehydes, using 4-hydroxy- trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than l-histidine and N-acetyl-carnosine while β-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that tshe β-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible α,β-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of α,β-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.

Effects of coenzyme Q 10 administration on its tissue concentrations, mitochondrial oxidant generation, and oxidative stress in the rat

Coenzyme Q (CoQ 10) is a component of the mitochondrial electron transport chain and also a constituent of various cellular membranes. It acts as an important in vivo antioxidant, but is also a primary source of O 2 −•/H 2O 2 generation in cells. CoQ has been widely advocated to be a beneficial dietary adjuvant. However, it remains controversial whether oral administration of CoQ can significantly enhance its tissue levels and/or can modulate the level of oxidative stress in vivo. The objective of this study was to determine the effect of dietary CoQ supplementation on its content in various tissues and their mitochondria, and the resultant effect on the in vivo level of oxidative stress. Rats were administered CoQ 10 (150 mg/kg/d) in their diets for 4 and 13 weeks; thereafter, the amounts of CoQ 10 and CoQ 9 were determined by HPLC in the plasma, homogenates of the liver, kidney, heart, skeletal muscle, brain, and mitochondria of these tissues. Administration of CoQ 10 increased plasma and mitochondria levels of CoQ 10 as well as its predominant homologue CoQ 9. Generally, the magnitude of the increases was greater after 13 weeks than 4 weeks. The level of antioxidative defense enzymes in liver and skeletal muscle homogenates and the rate of hydrogen peroxide generation in heart, brain, and skeletal muscle mitochondria were not affected by CoQ supplementation. However, a reductive shift in plasma aminothiol status and a decrease in skeletal muscle mitochondrial protein carbonyls were apparent after 13 weeks of supplementation. Thus, CoQ supplementation resulted in an elevation of CoQ homologues in tissues and their mitochondria, a selective decrease in protein oxidative damage, and an increase in antioxidative potential in the rat.

Compositional Changes in Muscle of Malignant Hyperthermia–Susceptible Pigs Due to Postmortem Alterations in Lipid Metabolism, Lipid Peroxidation and Protein Oxidation

Pigs with mutated calcium release channels are stress-susceptible and are known for increased postmortem metabolism which results in inferior meat quality. The lipid composition, lipid peroxidation, and oxidation of proteins of skeletal muscle homogenates from the longissimus muscle of pigs were investigated. Muscle tissue samples were obtained from malignant hyperthermia-resistant (MHR) and hyperthermia-susceptible (MHS) pigs in a postmortem time course. Differences in the n-3 polyunsaturated fatty acid composition with higher values in the susceptible pigs were detected (MHR versus MHS: 141±5 versus 116±4 μg/g wet wt.; P<0.05). This was accompanied by a dramatic 40% postmortem vitamin E loss in MHS muscles (22 h MHR versus 22 h MHS: 1.66±0.11 versus 1.06±0.16 μg/g wet wt.; P<0.05). Muscle tissue from MHS pigs oxidized at a significantly higher rate in comparison with tissue from MHR animals. This difference was also observed for spontaneous malondialdehyde formation as well as for oxidant-induced peroxidation rate (0.75 h MHR versus 0.75 h MHS: 0.81±0.09 versus 1.33±0.09 nmol/g wet wt.; P<0.05). No difference was observed in protein oxidation level in MHS and MHR animals. It is concluded that the enhanced lipid oxidation rate could contribute to the dramatic changes in meat quality from MHS pigs.

The specificity of the histochemical NADPH diaphorase reaction for nitric oxide synthase-1 in skeletal muscles is increased in the presence of urea

Nitric oxide synthase-1 (NOS-1) can be demonstrated in the sarcolemma region of myofibers in rodent skeletal muscles with the use of NADPH diaphorase histochemistry. Since other, especially intrafibrar enzymes also exhibit NADPH diaphorase activity, we tried to increase the specificity of the histochemical reaction for NOS-1. A qualitative and quantitative analysis was performed on cryostat sections of fast-twitch oxidative myofiber-rich tongue and fast-twitch glycolytic myofibers-rich tibialis anterior muscle derived from C57 mice and NOS-1 deficient knockout mice. All myofibers of both C57 mice and NOS-1 knockout mice contained significant intrafibrar NADPH diaphorase activity which was inhibited to almost background levels when 2 M urea was added to the incubation medium. On the other hand, myofibers of C57 mice but not of NOS-1-deficient knockout mice exhibited NADPH diaphorase activity in their sarcolemma region which was only weakly reduced in the presence of 2 M urea as was demonstrated by image analysis. Quantitative data on the activity of NADPH diaphorase(s) were obtained in situ by photometric analysis of formazan extracted from cryostat sections. The catalytic activity in tongue and tibialis anterior muscle was reduced in presence of 2 M urea to approximately 27% in C57 mice and to 7-17% in NOS-1 knockout mice, respectively. An in vitro NADPH diaphorase assay performed on homogenates of skeletal muscles also revealed an inhibitory effect of 2 M urea in both mouse strains and, additionally, indicated an upregulation of NADPH diaphorase activity in NOS-1 knockout mice. Finally, an immunodepletion analysis demonstrated that NOS-1 comprises 38% of the total NADPH diaphorase activity in tongue and approximately 59% in tibialis anterior muscle in C57 mice. In conclusion, we recommend the addition of 2 M urea to the incubation medium to increase the specificity of the NADPH diaphorase reaction to localise NOS-1 with the use of catalytic histochemistry.

Skeletal muscle oxidative capacity in rats fed high-fat diet.

To investigate whether young rats respond to high-fat feeding through changes in energy efficiency and fuel partitioning at the level of skeletal muscle, to avoid obesity development. In addition, to establish whether the two mitochondrial subpopulations, which exist in skeletal muscle, ie subsarcolemmal and intermyofibrillar, are differently affected by high-fat feeding. Weaning rats were fed a low-fat or a high-fat diet for 15 days. Energy balance and lipid partitioning in the whole animal. State 3 and state 4 oxygen consumption rates in whole skeletal muscle homogenate. State 3 and state 4 oxygen consumption rates, membrane potential and uncoupling effect of palmitate in subsarcolemmal and intermyofibrillar mitochondria from skeletal muscle. Rats fed a high-fat diet showed an increased whole body lipid utilization. Skeletal muscle NAD-linked and lipid oxidative capacity significantly increased at the whole-tissue level, due to an increase in lipid oxidative capacity in subsarcolemmal and intermyofibrillar mitochondria and in NAD-linked activity only in intermyofibrillar ones. In addition, rats fed a high-fat diet showed an increase in the uncoupling effect of palmitate in both the mitochondrial populations. In young rats fed a high-fat diet, skeletal muscle contributes to enhanced whole body lipid oxidation through an increased mitochondrial capacity to use lipids as metabolic fuels, associated with a decrease in energy coupling.

Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs

The objective of this study was to assess the risk of transmission of swine hepatitis E virus (swine HEV) to naı̈ve pigs by inoculation with tissues or feces collected from pigs infected experimentally with swine HEV. Seventy-five, 3-week-old pigs were assigned randomly to 24 groups of 3–4 pigs and inoculated with homogenates of tissues (liver, heart, pancreas, or skeletal muscle) or a suspension of feces from swine HEV-infected pigs collected at 3, 7, 14, 20, 27, or 55 days post inoculation (DPI). Each inoculum was prepared as a 10% suspension (w/v) in PBS buffer and tested by a semi-quantitative RT-PCR for swine HEV RNA and by the swine bioassay. The inoculation route was intravenous for liver, heart and pancreas, and via stomach tube for skeletal muscle and fecal suspension. The liver homogenate inocula and feces collected at 3–7 and 14–20 DPI were positive for swine HEV RNA by RT-PCR. The pigs inoculated with liver homogenates collected at 3–7 and 14–20 DPI developed anti-HEV antibodies and swine HEV RNA was detected in their sera. Pigs inoculated with heart, pancreas, skeletal muscle homogenates or fecal suspensions failed to develop anti-HEV antibodies. These findings suggest that there is a potential risk of transmission of swine HEV via liver tissue from infected pigs in the early stages (3–20 DPI) of infection and the in vitro RT-PCR assay correlates well with the swine bioassay.

Peroxynitrite-induced oxidation of lipids: implications for muscle foods.

Peroxynitrite (ONOO(-)), formed from the nearly diffusion limited reaction between nitric oxide and superoxide, could be an important prooxidant in muscle foods. The objective of this study was to determine whether peroxynitrite caused oxidation of pyrogallol red, liposomes, muscle microsomes, and skeletal muscle homogenate. Oxidation of pyrogallol red, liposomes, and microsomes initiated by peroxynitrite continuously produced by 3-morpholinosydnonimine (SIN-1, 2 mM) was time-dependent and enhanced by CO(2) (1 mM). Reagent peroxynitrite (2 mM) caused concentration-dependent oxidation of pyrogallol red, liposomes, and muscle microsomes that was very rapid with no change after 5 min. Peroxynitrite-induced oxidation was suppressed by CO(2) and low pH. Skeletal muscle homogenate oxidized by reagent peroxynitrite (0.5 mM) exhibited gradual oxidation with time and was suppressed by CO(2), low pH, and metal chelators. These data suggest that peroxynitrite could be an important prooxidant in muscle foods.

Targeted disruption of the nitric oxide synthase 2 gene protects against ischaemia/reperfusion injury to skeletal muscle.

To provide definitive insight into the complicated roles of the nitric oxide synthase (NOS) enzymes in ischaemia/reperfusion (I/R) injury of skeletal muscle, experiments were undertaken in mice with targeted disruption of the inducible NOS (NOS-2 KO) isoform, compared with the wild-type mouse strain. The degree of I/R injury in the NOS-2 KO mice was attenuated relative to that in the wild-type strain. After 70 min of ischaemia (24 h reperfusion), nitroblue tetrazolium (NBT) staining of skeletal muscle showed significant necrosis (40%) in wild-type mice, whilst in NOS-2 KO mice, ischaemia could be prolonged to 90 min before significant necrosis (38%) was apparent. Specific enzyme activities of the mitochondrial respiratory chain enzymes, measured in skeletal muscle homogenates, suggested that direct inhibition of the enzymes is not causal in the I/R injury. Immunohistological examination of skeletal muscle for NOS-2 showed its induction selectively in mast cells. In vitro experiments using bone marrow-derived mast cells showed that NOS-2 induction was associated with increased degranulation of mast cells. These findings suggest that NO generated by induction of NOS-2 has a deleterious effect in I/R injury of skeletal muscle and that NO exerts its damaging effect through factors released by degranulation of mast cells.

Expression of the Na(+)/H(+) exchanger isoform NHE1 in rat skeletal muscle and effect of training.

The expression of the Na(+)/H(+) exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)-muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101-111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle-fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.

Impact of dietary conjugated linoleic acid on the oxidative stability of rat liver microsomes and skeletal muscle homogenates.

Dietary conjugated linoleic acid (CLA; 0-2.0%) increased CLA concentrations in liver microsomes and skeletal muscle homogenates from rats. Dietary CLA decreased oleic and arachadonic acid concentrations in both liver microsomes and skeletal muscle. The presence of CLA in liver microsomes had no impact on linoleic acid, arachadonic acid, and alpha-tocopherol oxidation rates. Dietary CLA (2.0%) also did not alter alpha-tocopherol oxidation rates in liver microsomes or muscle homogenates. Formation of malonaldehyde (MDA) in oxidizing liver microsomes decreased with increasing CLA concentration as determined by measurement of thiobarbituric acid-MDA complexes by HPLC. The ability of CLA to decrease MDA formation without impacting other lipid oxidation markers such as the disappearance of fatty acid and alpha-tocopherol suggests that decreased MDA concentration was the result of CLA's ability to lower polyenoic fatty acids such as arachadonic acid. While CLA does not appear to act as an antioxidant, its ability to decrease polyenoic fatty acid concentrations could decrease the formation of highly cytotoxic lipid oxidation products such as MDA.