Different fibre populations distinguished by their calcium transient characteristics in enzymatically dissociated murine flexor digitorum brevis and soleus muscles

Enzymatically dissociated flexor digitorum brevis (FDB) and soleus fibres from mouse were used to compare the kinetics of electrically elicited Ca²⁺ transients of slow and fast skeletal muscle fibres, using the fast Ca²⁺ dye MagFluo4-AM, at 20–22°C. For FDB two Ca²⁺ transient morphologies, types I (MT-I, 11 fibres, 19%) and II (MT-II, 47 fibres, 81%), were found, the kinetic parameters (amplitude, rise time, half width, decay time, and time constants of decay) being statistically different. For soleus (n = 20) only MT-I was found, with characteristics similar to MT-I from FDB. Correlations with histochemically determined mATPase, reduced nicotinamide adenine dinucleotide diaphorase and α-glycerophosphate dehydrogenase activities, as well as immunostaining and myosin heavy chain electrophoretic analysis of both muscles suggest that signals classified as MT-I may correspond to slow type I and fast IIA fibres while those classified as MT-II may correspond to fast IIX/D fibres. The results point to the importance of Ca²⁺ signaling for characterization of muscle fibres, but also to its possible role in determining fibre function.     

Nitric oxide induces metallothionein-I gene expression in mesangial cells

In various forms of injury involving the renal glomerulus, mesangial cells are exposed to potentially toxic concentrations of nitric oxide (NO) caused by activation of the inducible isoform of nitric oxide synthase (NOS). Whether mesangial cells possess systems that can defend against NO mediated oxidative injury is unknown. One putative system is Metallothionein (MT). Metallothioneins constitute a family of cysteine proteins and play a significant role as anti-oxidants. The authors assessed whether NO upregulates MT-I expression in cultured glomerular mesangial cells. Northern blot analysis revealed that steady state MT-I mRNA levels were increased by three different NO donors: sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), and Spermine-NONOate (Sper/NO). The increase in MT-I mRNA levels induced by SNAP-derived NO was attenuated by the antioxidant N-acetylcysteine (NAC), a glutathione (GSH) precursor, which indicates that the mechanism of NO-mediated MT-I expression may involve an oxidative stress response. These observations identify MT-I as a putative antioxidant system in NO-mediated mesangial cell injury.     

Decreased neurofilament density in large myelinated axons of metallothionein-I, II knockout mice

Metallothioneins (MTs) are small proteins, two isoforms (I, II) of which bind metals. Their physiological role has been difficult to establish, but recent reports suggested that they serve an important function in nerve repair and in the protection against oxidative stress in the peripheral nervous system. We previously reported a decreased axon calibre in the large myelinated fibres of the phrenic nerve in the MT-I, II double knock out (MT-I, II KO) mouse model. We propose that this could be due to the effects of oxidative stress on neurofilaments (NFs). In this study, we examined the same subset of large myelinated axons using transmission electron microscopy (TEM). There was a decreased NF density in the axons of MT-I, II KO phrenic nerve (P<0.005). This observation may have novel therapeutic implications in the treatment of amyotrophic lateral sclerosis (ALS), particularly as the terminal phases of the disease involve respiratory insufficiency.     

Metallothionein 1F and 2A overexpression predicts poor outcome of non-small cell lung cancer patients

Metallothioneins (MT) are intracellular, low molecular weight proteins (6–7 kDa) involved in binding of metal ions, scavenging of free radicals, cell proliferation and apoptosis and resistance to certain chemotherapeutics. Four basic families of MT proteins are distinguished: MT-I, MT-II, MT-III, MT-IV, within each of them different isoforms occur. The study aimed at examining the expression level of nine MT isoforms: MT-1A, -1B, -1E, -1F, -1G, -1H, -1X, MT-2A and MT-IV by using real-time PCR and MT-I/II expression by immunohistochemical (IHC) technique in 69 cases of non-small cell lung cancer (NSCLC) and 12 non-malignant lung tissues (NMLT) and to correlate them with patients clinicopathological data and Ki-67 antigen expression. Out of all the analyzed cases, 62 (89.9%) demonstrated an increased MT-I/II expression. MT-1B, 1F, -1G, -1H and MT-1X were significantly up-regulated, whereas MT-1E was significantly down-regulated in NSCLC as compared to NMLT. Only in two cases MT-IV mRNA expression was noted. Significant positive correlations were observed between each particular MT isoform expressions. Higher MT-1F and MT-1A mRNA expression was associated with larger primary tumor size (P = 0.0362 and P < 0.0001, respectively). Moreover, up-regulated MT-1F mRNA expression was associated with higher grade of malignancy of NSCLC (P = 0.0085). Higher MT-1B mRNA expression was associated with squamocellular and adenocarcinoma subtype of NSCLC (P = 0.0358). Univariate analysis showed, that up-regulated MT-1F and MT-2A mRNA predicted poor patients' survival (P = 0.0206 and P = 0.0097, respectively). The levels of MT-1F and MT-2A mRNA could be considered as new markers of poor prognosis of NSCLC patients.     

Effects of dietary extra-virgin olive oil on oxidative stress resulting from exhaustive exercise in rat skeletal muscle: A morphological study

Physical exercise induces oxidative stress through production of reactive oxygen species and can cause damage to muscle tissue. Oxidative stress, resulting from exhaustive exercise is high and improvement of antioxidant defenses of the body may ameliorate damage caused by free radicals. Extra-virgin olive oil is widely considered to possess anti-oxidative properties. The aim of this study was to determine if extra-virgin olive oil improved the adaptive responses in conditions of oxidative stress. Twenty-four 12-week-old male Sprague-Dawley rats were divided in three groups: (1) rats fed with standard chow and not subjected to physical exercise; (2) rats fed with standard chow and subjected to exhaustive exercise; (3) rats fed with a diet rich in oleic acid, the major component of extra-virgin olive oil, and subjected to exhaustive exercise. Exhaustive exercise consisted of forced running in a five-lane 10° inclined treadmill at a speed of 30 m/min for 70–75 min. We studied some biomarkers of oxidative stress and of antioxidant defenses, histology and ultrastructure of the Quadriceps femoris muscle (Rectus femoris). We observed that, in rats of group 3, parameters indicating oxidative stress such as hydroperoxides and thiobarbituric acid-reactive substances decreased, parameters indicating antioxidant defenses of the body such as non-enzymatic antioxidant capacity and Hsp70 expression increased, and R. femoris muscle did not show histological and ultrastructural alterations. Results of this study support the view that extra-virgin olive oil can improve the adaptive response of the body in conditions of oxidative stress.     

Aggravation of 6-hydroxydopamine-induced dopaminergic lesions in metallothionein-I and -II knock-out mouse brain

The effects of two major isoforms of metallothioneins (MTs), MT-I and -II, on dopaminergic neurotoxicity of 6-hydroxydopamine (6-OHDA) were examined using intracerebroventricularly 6-OHDA-injected MT-I, II knock-out (KO) mice. The loss of dopamine neurons in the substantia nigra pars compacta induced by the 6-OHDA injection was significantly aggravated in the MT-I, II KO mice, compared with that in the 6-OHDA-injected wild-type mice. The present results, taken together with the antioxidant properties of MT-I and -II suggest that MT-I and -II exert neuroprotective effects against the dopaminergic neurotoxicity of 6-OHDA at the nigral cell body by scavenging free radicals.     

Exercise and skeletal muscle ageing: cellular and molecular mechanisms

As we age, our skeletal muscle becomes smaller and weaker. In addition, the remaining muscle is more susceptible to damage, particularly following exercise, recovery from damage is severely impaired and muscle is unable to adapt rapidly following sequential periods of exercise. The mechanisms by which skeletal muscle damage occurs are poorly understood and the role that an increased production of free radical species plays in this damage is controversial. However, evidence is emerging which suggests that an increased production of free radicals may act as an activator of the adaptive response in skeletal muscle, resulting in the increased production of antioxidant enzymes and heat shock proteins (HSPs). The increased content of these proteins facilitates rapid remodelling of muscle and provides considerable protection against subsequent periods of damaging exercise. There is considerable evidence that the production of free radicals is modified during the ageing process. The aim of this review is to examine the possible effects of this modification on the ability of muscle cells to respond to stress and the functional effect that this may have on our muscles as we age.     

Exercise, oxidative stress and ageing

Skeletal muscle has the unique ability to increase the rate of oxygen usage during contraction. This has led several workers to suggest that by-products of this increased oxygen consumption, oxygen-derived free radicals, may be primarily responsible for exercise-induced damage to skeletal muscle. However, because of this rapidly changing redox state, skeletal muscle has developed a number of different endogenous mechanisms which adapt rapidly following a period of exercise. These include numerous structural and biochemical changes such as increased muscle activity of antioxidant enzymes and content of stress or heat shock proteins (HSPs). This adaptation is associated with protection against the potentially damaging effects of a second period of exercise. In addition, we have recently demonstrated a significant increase in free radical production during a period of nondamaging exercise, which is rapidly followed by a significant increase in the expression of antioxidant enzymes and HSPs, suggesting that a change in redox state of the muscle may act as signal for adaptation.     

Alterations in the levels of metallothionein and metals in the liver, and unique serum liver enzyme response in metallothionein knock-out mice after burn injury.

OBJECTIVES: Metallothionein (MT) is a small cysteine-rich protein that sequesters and distributes metal ions. Its overexpression stimulates cell proliferation and inhibits apoptosis. We investigated the effects of burn injury on MT expression and metal localization. We also sought to determine roles of MT in the pathophysiologic alterations in the liver after injury. METHODS: Mice (C57BLKS/J, MT-I/II knock-out, KO, and wild-type control mice) were subjected to an 18% burn. Liver tissues harvested after injury were analyzed for the MT expression and the levels of zinc, copper, manganese, and iron. Levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were measured in serum samples from MT-I/MT-II KO mice and controls after injury. RESULTS: Transient induction of MT-I and MT-II mRNAs was observed 3-6 h after injury, while MT-I/MT-II protein peaked on day 1. The induction was localized to hepatocytic nuclei. The intrahepatic levels of zinc, copper, and iron were transiently elevated on day 1, when a downregulation of manganese was evident. Interestingly, only the serum levels of aspartate aminotransferase were significantly augmented in MT-I/MT-II KO mice compared to controls after injury. CONCLUSIONS: These data suggest that MT and metals may participate in the pathogenesis of the liver after burn injury.     

Myopathy-dependent changes in activity of ATPase, SDH and GPDH and NOS expression in the different fibre types of hamster muscles

Proximal (vastus lateralis) and distal (gastrocnemius) muscles of 100-day-old normal and myopathic BIO TO-2 hamsters were analysed to study the effects of myopathy on the different muscle fibre types: SO (slow oxidative), FOG (fast oxidative glycolytic) and FG (fast glycolytic). Cytophotometric measurements of enzyme activities (myofibrillic adenosine triphosphatase, succinate dehydrogenase and glycerol-3-phosphate dehydrogenase), Western blot analysis of nitric oxide synthase (NOS) I, II, III isoforms and NOS II immunohistochemistry were performed. The following alterations were found in myopathic muscle fibres: all fibre types of both proximal and distal myopathic muscles showed decreased myofibrillic adenosine triphosphatase activity indicating depressed contractility. This was associated with depressed oxidative activity of the muscle fibres. A shift to more glycolytic metabolism was observed, mainly in FG fibres of proximal muscle. We found an increased NOS II expression in both myopathic muscle types investigated. It means that increased NO production inhibits force generation in myopathic muscle. NOS II immunoreactivity was found mainly in the cytoplasm of FG fibres. NOS I and NOS III expression was not significantly effected by this form of myopathy. Our findings demonstrate that muscle fibres of proximal and distal skeletal muscles of 100-day-old cardiomyopathic BIO TO-2 hamsters are altered with respect to contractility, metabolism and NOS II expression. FG fibres of the proximal muscle were effected most strongly.     

Effect of exercise on concentrations of free amino acids in pools of type I and type II fibres in human muscle with reduced glycogen stores

A few animal studies have shown that some amino acid concentrations vary between different muscle fibre types. In the present study, amino acid concentrations were measured in separate pools of different fibre types in human skeletal muscle, with reduced glycogen stores, before and after sustained exercise. Five subjects exercised at a submaximal work rate for 60 min and then at a maximal rate for 20 min. Biopsy samples were taken from the vastus lateralis muscle before and after exercise; they were freeze-dried and individual fibres were dissected out. Fragments of these fibres were stained for myosin-adenosine triphosphatase (ATPase) and identified as type I or type II fibres. The concentrations of free amino acids were measured by high performance liquid chromatography (HPLC) in perchloric acid (PCA) extracts containing pools of either type of fibre. After exercise, glycogen was decreased in type I fibres (53%) and in four subjects also in type II fibres. The concentrations of most amino acids were similar in the two fibre types before exercise, but the glutamate, aspartate and arginine levels were 10% higher in type II than in type I fibres. After exercise, the glutamate concentration was decreased by 45% in both fibre types and the branched-chain amino acids (BCAA) were decreased in type II fibres (14%). Exercise caused an increase by 25-30% in tyrosine concentration in both type I and type II fibres. The results show that amino acids can be measured in pools of fibre fragments and suggest that amino acid metabolism play an important role in both type I and type II fibres during exercise.     

Muscle energy metabolism: structural and functional features in different types of porcine striated muscles

Striated muscles exhibit a wide range of metabolic activity levels. Heart and diaphragm are muscles with continuous contractile performance, which requires life-long function. In contrast, skeletal muscles like longissimus muscle can adapt metabolism from resting to different stages of exercise. The aim of this study was to compare the morphological features of these three muscles and the expression of genes that are important for energy metabolism. Therefore, histochemical studies were performed for determination of muscle fibre type composition. Oxidative and glycolytic capacity was assessed by measuring isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities. The mRNA expression of glucose transporter 4 (GLUT 4), growth hormone receptor (GHR) and AMP-activated kinase (AMPK) α₁ and α₂ subunits was studied by semiquantitative Northern blotting. Heart, and to a slightly lesser extent diaphragm were highly oxidative muscles characterised by high expression of oxidative muscle fibres and ICDH activity. Longissimus muscle exhibited the highest percentage of glycolytic fibres and LDH activity. GLUT 4 mRNA was lowest in heart reflecting the dependency of heart muscle on fatty acids as major energy source. Higher expression of GLUT 4 in diaphragm indicated that glucose is an important energy substrate in this oxidative muscle. Highest GLUT 4 expression in longissimus should be essential for the refilling of glycogen stores after exercise. AMPK subunits, which are important stimulators of GLUT 4 protein insertion into the sarcolemma, are also highest expressed in longissimus muscle indicating the strong capacity to adapt energy metabolism to large changes in energy demand. Interestingly, AMPK α₁ subunit expression on protein level is strongly restricted to muscle fibres containing type I myosin in this muscle. GHR mRNA expression was also highest in longissimus muscle indicating that an enhanced effect of growth hormone, which is described to be diabetogenic, could be involved in the lower insulin sensitivity of glycolytic muscles.     

Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle

The molecular events by which eccentric muscle contractions induce muscle damage and remodelling remain largely unknown. We assessed whether eccentric exercise modulates the expression of proteinases (calpains 1, 2 and 3, proteasome, cathepsin B+L), muscle structural proteins (α-sarcoglycan and desmin), and the expression of the heat shock proteins Hsp27 and αB-crystallin. Vastus lateralis muscle biopsies from twelve healthy male volunteers were obtained before, immediately after, and 1 and 14 days after a 30 min downhill treadmill running exercise. Eccentric exercise induced muscle damage as evidenced by the analysis of muscle pain and weakness, creatine kinase serum activity, myoglobinaemia and ultrastructural analysis of muscle biopsies. The calpain 3 mRNA level was decreased immediately after exercise whereas calpain 2 mRNA level was increased at day 1. Both mRNA levels returned to control values by day 14. By contrast, cathepsin B+L and proteasome enzyme activities were increased at day 14. The α-sarcoglycan protein level was decreased immediately after exercise and at day 1, whereas the desmin level peaked at day 14. αB-crystallin and Hsp27 protein levels were increased at days 1 and 14. Our results suggest that the differential expression of calpain 2 and 3 mRNA levels may be important in the process of exercise-induced muscle damage, whereas expression of α-sarcoglycan, desmin, αB-crystallin and Hsp27 may be essentially involved in the subsequent remodelling of myofibrillar structure. This remodelling response may limit the extent of muscle damage upon a subsequent mechanical stress.     

Glycogen and lactate metabolism during low-intensity exercise in man

The influence of high lactate concentration on glycogen metabolism in active type I and inactive type II fibres was investigated. High muscle lactate concentration (26.7 +/- 1.4 mmol kg-1 wet wt) was achieved by three bouts (2 min duration) of bicycle exercise at 112% Vo2 max. Exercise was continued at 40% Vo2 max for 1 h. Serial venous blood samples and biopsies from the vastus lateralis muscle were taken. Over the first 20 min of this low-intensity exercise muscle lactate concentration decreased by 22.9 +/- 0.7 mmol kg-1 wet wt, while glycogen remained unchanged in type I fibres and increased by 20 mmol kg-1 wet wt in type II fibres. During the next 40 min of low-intensity exercise lactate decreased by 1.6 +/- 1.2 mmol kg-1 wet wt, while glycogen concentration decreased by 21 +/- 7 mmol kg-1 wet wt in type I fibres but remained stable in type II fibres. In a second series of experiments, in which lactate was allowed to disappear before the light exercise was started, no changes in glycogen concentration were seen in type II fibres during the 1 h of 40% Vo2 max exercise, while a continuous reduction in glycogen of 28 +/- 8 mmol kg-1 wet wt was found in type I fibres. The results indicate that in the presence of high lactate levels muscle glycogen was resynthesized in inactive type II muscle fibres, while lactate was oxidized in preference to glycogen in type I fibres.     

Identification of non-acetylated metallothioneins induced in rat liver by zinc

In addition to the usual two rat metallothionein (MT) isoforms (MT-I and MT-II), a third isoform of metallothionein (MT-II) is known to be induced by zinc in the liver of rats and mice, and by epidermal growth factors in cultured cells. Although the third isoform has been suggested to be similar and related to MT-II based on its behavior on size-exclusion and ion exchange HPLC columns, further characterization has not been performed. MT-II' was identified in the present study as the unacetylated isoform of MT-II based on mass spectrometric data obtained by matrix assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS) (i.e., MT-II' was 42 Da smaller than MT-II in its molecular mass). Chromatographic properties of MT-II' were consistent with this species being an unacetylated isoform of MT-II arising as a result on lack of acetylation rather than a post-translational deacetylation event (i.e., an isoform of MT-II not co-translationally acetylated, based on the change in its composition relative to MT-II and MT-I after induction of MT expression). Although MT-I' was not separated under the present conditions, the MT-I fraction gave a mass peak corresponding to a species 42 Da smaller than MT-I. This suggested that non-acetylated isoforms of both MT-1 and MT-II were present.