Cytochrome c turnover in rat skeletal muscles.

Exercise induces an increase in cytochrome c concentration in skeletal muscle. This adaptation provides an approach to studying the turnover of cytochrome c that avoids the problem of reutilization encountered with isotopic tracers. The half-life of cytochrome c was estimated from the time course of the increase in its concentration to a new, higher, steady state level in response to exercise training, and from the decrease in cytochrome c after cessation of exercise. The half-time of the increase in cytochrome c concentration was approximately 6 days, while the half-time of the decrease was 7 to 8 days in the fast red and slow red types of muscle. The finding that the half-times of the increase and of the decrease in cytochrome c concentration are similar provides evidence that the exercise-induced increase in cytochrome c is due to an increase in its rate of synthesis. These half-times are much shorter than those obtained with isotopic tracers. It had been thought that the heme precursor delta-aminolevulinate is not reutilized. However, the half-time of the decrease in radioactivity of cytochrome c labeled with delta-aminol[14C]levulinate was 45 days, and increased to 60 days in response to exercise, in fast red muscle. The half-time of the decrease in radioactivity of cytochrome c labeled with [(3H)]leucine in gastrocnemius muscle was shorter than with delta-amino[14C]levulinate (18 days compared to 38 days). These results indicate that when delta-amino(14C)levulinate is used to label heme, reutilization is a serious problem in skeletal muscle.     

Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg.kg(-1).day(-1) sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [(14)C]tyrosine (0.1 mM, 0.05 microCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10(-5) M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10(-7), 10(-6), or 10(-5) M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10(-5) M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of beta(2)-adrenoceptors, especially in situations of hormonal or nutritional deficiency.     

Cytochrome c protein-synthesis rates and mRNA contents during atrophy and recovery in skeletal muscle.

It is known that immobilization of the rat hindlimb by plaster casting leads to muscle atrophy and loss of muscle protein. In the present study, immobilization of the rat hindlimb for 6 h resulted in a significant 27% decrease in the absolute rate of cytochrome c synthesis in the red quadriceps muscle, without any change in the relative amount of cytochrome c mRNA. Cytochrome c mRNA in normal red quadriceps muscle was observed to be of four different lengths (1400, 1050, 650 and 580 bases). After 7 days of immobilization, the absolute rate of cytochrome c synthesis remained depressed and cytochrome c mRNA decreased by 40%; each of the cytochrome c mRNAs decreased, with a preferential disappearance of the 1050- and 1400-base lengths. Immobilization was ended on day 7, and the atrophied muscle was allowed to recover. At day 4 of recovery, the absolute rate of cytochrome c synthesis was 92% higher and the amount of cytochrome c mRNA had returned to control values. The abundances of the 1050- and 1400-base cytochrome c mRNAs had increased more than the shorter cytochrome c mRNAs, so that they were higher than control values. It appears that acute decreases in contractile activity of the red quadriceps muscle alter cytochrome c synthesis rates via translational or post-translational mechanisms, whereas chronic periods of modified contractile activity alter its synthesis rate via pre-translational mechanisms.     

Effect of reinnervation on collagen synthesis in rat skeletal muscle.

The effect of reinnervation on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) was studied in gastrocnemius, soleus, and tibialis anterior muscles of rat 19, 26, 40, and 61 days after crush denervation of the sciatic nerve. The GGT activity was elevated in denervated gastrocnemius and soleus muscles and the PH activity in gastrocnemius. Muscular Hyp concentration was increased in denervated tibialis anterior muscle. Both the PH and GGT activities and the Hyp concentration returned to the control level during the reinnervation period (19-61 days from the start of denervation). It seems that denervation atrophy of skeletal muscle is associated with an increased rate of muscular collagen biosynthesis and that during reinnervation collagen synthesis rate decreases despite accelerated muscular growth. The results thus suggest that innervation is a powerful suppressive regulator of muscular collagen biosynthesis.     

Endotoxemia reduces skeletal muscle protein synthesis in neonates

Protein synthesis in skeletal muscle is reduced by as much as 50% as early as 4 h after a septic challenge in adults. However, the effect of sepsis on muscle protein synthesis has not been determined in neonates, a highly anabolic population whose muscle protein synthesis rates are elevated and uniquely sensitive to insulin and amino acid stimulation. Neonatal piglets (n = 10/group) were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 microg. kg(-1). h(-1)]. Plasma amino acid and glucose concentrations were kept at the fed level by infusion of dextrose and a balanced amino acid mixture. Fractional protein synthesis rates were determined by use of a flooding dose of [(3)H]phenylalanine. LPS infusion produced a septic-like state, as indicated by an early and sustained elevation in body temperature, heart rate, and plasma tumor necrosis factor-alpha, interleukin-1, cortisol, and lactate concentrations. Plasma levels of insulin increased, whereas glucose and amino acids decreased, suggesting the absence of insulin resistance. LPS significantly reduced protein synthesis in longissimus dorsi muscle by only 11% and in gastrocnemius by only 15%, but it had no significant effect in masseter and cardiac muscles. LPS increased protein synthesis in the liver (22%), spleen (28%), kidney (53%), jejunum (19%), diaphragm (21%), lung (50%), and skin (13%), but not in the stomach, pancreas, or brain. These findings suggest that, when substrate supply is maintained, skeletal muscle protein synthesis in neonates compared with adults is relatively resistant to the catabolic effects of sepsis.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Hypoxia affects mitochondrial protein expression in rat skeletal muscle

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500?m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.     

Effects of raising muscle glycogen synthesis rate on skeletal muscle ATP turnover rate in type 2 diabetes

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. We hypothesized that any impairment in insulin-stimulated muscle ATP production could merely reflect the lower rates of muscle glucose uptake and glycogen synthesis, rather than cause it. If this is correct, muscle ATP turnover rates in type 2 diabetes could be increased if glycogen synthesis rates were normalized by the mass-action effect of hyperglycemia. Isoglycemic- and hyperglycemic-hyperinsulinemic clamps were performed on type 2 diabetic subjects and matched controls, with muscle ATP turnover and glycogen synthesis rates measured using (31)P- and (13)C-magnetic resonance spectroscopy, respectively. In diabetic subjects, hyperglycemia increased muscle glycogen synthesis rates to the level observed in controls at isoglycemia [from 19 ± 9 to 41 ± 12 μmol·l(-1)·min(-1) (P = 0.012) vs. 40 ± 7 μmol·l(-1)·min(-1) in controls]. This was accompanied by a modest increase in muscle ATP turnover rates (7.1 ± 0.5 vs. 8.6 ± 0.7 μmol·l(-1)·min(-1), P = 0.04). In controls, hyperglycemia brought about a 2.5-fold increase in glycogen synthesis rates (100 ± 24 vs. 40 ± 7 μmol·l(-1)·min(-1), P = 0.028) and a 23% increase in ATP turnover rates (8.1 ± 0.9 vs. 10.0 ± 0.9 μmol·l(-1)·min(-1), P = 0.025) from basal state. Muscle ATP turnover rates correlated positively with glycogen synthesis rates (r(s) = 0.46, P = 0.005). Changing the rate of muscle glucose metabolism in type 2 diabetic subjects alters demand for ATP synthesis at rest. In type 2 diabetes, skeletal muscle ATP turnover rates reflect the rate of glucose uptake and glycogen synthesis, rather than any primary mitochondrial defect.     

Cytochrome c mRNA in skeletal muscles of immobilized limbs

Booth, Frank W., Wei Lou, Marc T. Hamilton, and Zhen Yan.Cytochrome c mRNA in skeletalmuscles of immobilized limbs. J. Appl.Physiol. 81(5): 1941-1945, 1996.Even thoughimmobilization of a slow skeletal muscle in a lengthened positionprevents muscle atrophy, it is unknown whether this treatment wouldprevent a decrease in mitochondrial quantity. We found that, regardless of muscle length in immobilized limbs, the mRNA of a marker for mitochondrial quantity, cytochrome c,decreased. Cytochrome c mRNA permilligram of muscle was 62 and 72% less 1 wk after fixation of thesoleus muscle in shortened and lengthened positions, respectively, thanage-matched controls. Cytochrome cmRNA per milligram wet weight was 36 and 32% less in the tibialisanterior muscle fixed for 1 wk in the shortened and lengthenedpositions, respectively, compared with age-matched controls. Recently,in the 3-untranslated region of cytochromec mRNA a novel RNA-protein interactionthat decreases in chronically stimulated rat skeletal musclewas identified.[Z. Yan, S. Salmons, Y. L. Dang, M. T. Hamilton, and F. W. Booth. Am. J. Physiol. 271 (CellPhysiol. 40): C1157- C1166,1996]. The RNA-protein interaction inthe 3-untranslated region of cytochrome c mRNA in soleus and tibialis anteriormuscles was unaffected by fixation in either shortened or lengthenedposition. We conclude that, whereas lengthening muscle during limbfixation abates the loss of total muscle protein, the percentagedecrease in cytochrome c mRNA isproportionally greater than total protein. This suggests that thedesign of countermeasures to muscle atrophy should include differentexercises to maintain total protein and mitochondria.

     

Cortisol binding in rat skeletal muscle.

Studies of the reversible binding of [3H]cortisol by rat gastrocnemius muscle cytoplasm in vitro reveal specific binding in the 27,000 times g supernatant fraction at 0 degrees. The [3H]cortisol-binding molecule had an apparant Kd value of 1.7 times 10-7 M and the number of binding sites was 0.99 pmol per mg of cytosol protein. Only a single class of [3H]cortisol-binding sites could be detected, whose protein nature was suggested by its susceptibility to nagarse. The [3H]cortisol-protein complex sedimented at similar to 4 S in a 5 to 20% sucrose gradient either in the presence or absence of 0.3 M KCl. Binding increased more than 2-fold in adrenalectomized rats and was markedly reduced in the muscle of rats pretreated with cortisol. In contrast to the binding of [3H]dexamethasone and [3H]triamcinolone acetonide to receptor proteins in muscle, no correlation was found between the ability of various steroids to complete wtth [3H]cortisol binding and their glucocorticoid potency: [3H]cortisol binding was inhibited by a 1000-fold higher concentration of unlabeled cortisol and progesterone but not by dexamethasone or triamcinolone acetonide. It is therefore suggested that the [3H]cortisol-binding reaction is not directly involved in the biological effects of all potent glucocorticoids in skeletal muscle. The [3H]cortisol-binding protein in muscle cytosol could not be unequivocally distinguished from rat plasma corticosteroid-binding globulin, because both had similar steroid specificity and temperature stability, were not markedly affected by--SH reagents, and displayed similar sedimentation properties.     

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.