In vitro protein degradation measured by differential loss of labeled methionine and 3-methylhistidine: The effect of insulin

A new technique for studying the effect of insulin on protein degradation is reported. The method is based on measuring the parallel release of a reutilizable and a nonreutilizable amino acid from muscle protein. Animals are prelabeled in vivo with [Me-³H]methionine which labels both the nonreutilizable 3-methylhistidine and the reutilizable methionine of tissue protein. The data presented show that insulin has only a trivial effect on the loss of 3-methylhistidine from muscle protein, while it substantially diminishes the efflux of methionine. The analysis of muscle protein confirms the observation that insulin causes the reincorporation of methionine and has a minimum effect on the loss of 3-methylhistidine. This supports the view that the major inhibitory effect of insulin on gluconeogenesis is the diversion of the flow of amino acids away from the gluconeogenesis pathway back toward protein synthesis.     

3-Methylhistidine turnover in the whole body, and the contribution of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat.

The tissue origin of 3-methylhistidine (N tau-methylhistidine) was investigated in adult female rats. The decay of labelling of urinary 3-methylhistidine was compared with the labelling of protein-bound 3-methylhistidine in skeletal muscle and intestine after the injection of [methyl-14C]methionine. The decay curve for urinary 3-methylhistidine was much steeper than that in muscle or intestine, falling to values lower than those in either tissue after 30 days. The lack of decay of labelling in muscle during the first 30 days is shown to result from the persistence of label in the precursor S-adenosylmethionine. The relative labelling of urinary, skeletal-muscle and intestinal 3-methylhistidine cannot be explained in terms of skeletal muscle accounting for a major proportion of urinary 3-methylhistidine. Measurements were also made of the steady-state synthesis rate of protein-bound 3-methylhistidine in intestinal smooth muscle in vivo in adult female rats. This involved measurement of the overall rate of protein synthesis and measurement of the relative rates of synthesis of 3-methylhistidine and of mixed protein. The synthesis rate of 3-methylhistidine was 29.1%/day, compared with the overall rate of 77.1%/day for mixed, non-mucosal intestinal protein. Measurement of the amount of 3-methylhistidine in skeletal muscle (0.632 +/- 0.024 mumol/g) and in the whole body (0.332 +/- 0.013 mumol/g) indicate that, although the muscle pool is 86% of the total, because of its slow turnover rate of 1.1-1.6%/day, it only accounts for 38-52% of the observed excretion. Measurements of the mass of the intestine (9.95 g/250 g body wt.) and protein-bound 3-methylhistidine content (0.160 mumol/g of tissue) indicate a pool size of 1.59 mumol/250 micrograms rat. Thus 463 nmol of the urinary excretion/day would originate from the intestine, 22% of the total. The tissue source of the remaining urinary excretion is not identified, but other non-muscle sources constituting about 10% of the whole-body pool could account for this with turnover rates of only 6%/day, a much lower value than the turnover rate in the intestine.     

Biological activity and the 3-methylhistidine content of actin and myosin

1. The 3-methylhistidine content of myosin varies according to muscle type. It is highest in myosin from white skeletal muscle and lower values are obtained from myosin of red skeletal and smooth muscle. 2. The 3-methylhistidine content of actin was similar in all of the types of muscle from which it was isolated. 3. The 3-methylhistidine of rabbit actin is localized in a single tryptic peptide that was readily modified during fractionation procedures. 4. Photo-oxidation studies indicated that the 3-methylhistidine residues are not essential for adeonsine triphosphatase and actin-combining activities of myosin. 5. During photooxidation G-actin lost completely the ability to polymerize to the F form before all the 3-methylhistidine was destroyed.     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.     

Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells

The rates of [³H]N^τ-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [³H]methionine, were used to measure myofibrillar protein degradation. In fused C₂C₁₂ myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions ± leucine, but in the absence of [³H]-methionine, rates of protein accretion and synthesis over 24–48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types. © 1996 Wiley-Liss, Inc.     

Effect of glucocorticoids on the turnover rate of actin and myosin heavy and light chains on different types of skeletal muscle fibres

The catabolic action of glucocorticoids on the molecular level of the two main muscular proteins, myosin and actin, was found to depend on the type of muscle fibres. The synthesis rate of actin and myosin heavy chain was decreased in all types of muscle fibres, and in myosin light chain only in the slow-twitch red fibres. The turnover rate of actin and myosin heavy chain was also found decreased in all types of muscle fibres. The myosin light chains turned over more rapidly in dexamethasone-treated than in the control rats in all types of muscle fibres except in the case of the slow-twitch red ones as was shown by single and double isotope methods. Dexamethasone treatment enhanced the urinary 3-methylhistidine excretion in rats by 60%.     

Eimeria acervulina infection elevates plasma and muscle 3-methylhistidine levels in chickens

To assess muscle breakdown during avian coccidiosis, the level of the nonmetabolizable amino acid 3-methylhistidine (3MH) was determined in muscle, plasma and excreta from chickens infected with Eimeria acervulina. The changes in 3MH levels during infection were assessed at 1-29 days postinoculation (DPI) in animals given 5 x 10(5) oocysts per bird. The effect of levels of parasitism were evaluated at 8 DPI in birds receiving 5 x 10(3), 5 x 10(4), 5 x 10(5) or 1 x 10(6) oocysts each. The 3MH levels of plasma, muscle, and excreta samples were determined by high-pressure liquid chromatography after derivatization with fluorescamine. Weight gains, breast muscle weight, eviscerated weight, plasma carotenoid levels, dry weight of muscle, and gross lesion scores were also determined. Infected birds had significantly elevated plasma and muscle 3MH at 4 and 8 DPI following a single dose of E. acervulina. The increase in 3MH levels had an inverse relationship with the time course of weight gain and plasma carotenoid levels. Plasma and muscle 3MH levels returned to control values by 15 DPI and remained unchanged from control values through the remainder of the experiment (29 DPI). Breast weight was decreased in infected birds, but the ratio of breast weight to eviscerated body weight was unchanged. Excretion of 3MH decreased relative to controls at 4 and 8 DPI and returned to control levels on 15 DPI. The plasma and muscle levels of 3MH were related to severity of infection; however, levels of excreted 3MH were not. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of E. acervulina infection. The underlying causes for this muscle breakdown was unclear but could involve a physiological response to anorexia and decreased food intake during the acute phase of infection. Levels of excreted 3MH did not increase during infection and this may be the result of decreased excreta output during infection. Plasma and muscle levels of 3MH were correlated with severity of E. acervulina infections but may not be as sensitive an indicator of infection as plasma carotenoid levels or other physiological parameters.     

Isolation of camel brain actin--comparison of its biochemical properties with those of camel skeletal muscle, heart muscle and rabbit skeletal muscle actins

1. Actins were purified from camel brain, skeletal muscle and heart muscle and their properties were compared. 2. Individual actins were homogeneous and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 3. Isoelectric focusing analysis of camel skeletal muscle and heart muscle actin showed a single polypeptide of the alpha-species, while camel brain actin showed two polypeptides of the beta- and gamma-species typical of non-muscle actin. 4. Actins from camel skeletal muscle and heart muscle showed a greater degree of similarity to each other and to rabbit skeletal muscle actin and showed some differences from camel brain actin, as confirmed by amino acid analysis and one-dimensional peptide mapping.     

Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat skeletal muscle fibres

Summary A quantitative histochemical technique was developed for determining the kinetics of the calcium-activated myosin ATPase (Ca²⁺-myosin ATPase) reaction in rat skeletal muscle fibres. Using this technique, the maximum velocity (V_max) and the apparent Michaelis-Menten rate constant for ATP (K_app) of the Ca²⁺-myosin ATPase reaction were measured in type-identified fibres of the rat medial gastrocnemius (MG) muscle. The V_max and the K_app of the Ca²⁺-myosin ATPase reaction were lowest in type I fibres and highest (i.e., approx. two times greater) in type IIb fibres. The K_app in type IIa fibres was similar to that in type I. However, the V_max was 1.5 times greater in type IIa fibres, compared to type I fibres. Evidence is presented to suggest that the type IIb fibre population in the MG does not represent a single myosin isozyme. In addition, the broad range of V_max and K_app values indicates that there is marked heterogeneity in the myosin heavy chain and myosin light chain composition of myosin isozymes among individual fibres.     

Clinical usefulness of urinary 3-methylhistidine excretion in indicating muscle protein breakdown.

Urinary excretion of the post-translationally modified amino-acid 3-methylhistidine, derived from the contractile proteins actin and myosin, was measured in patients with conditions associated with nitrogen loss. The ratio of 3-methylhistidine:creatinine excretion, a measure of the fractional catabolic rate of myofibrillar protein was increased in severe injury, thyrotoxicosis, neoplastic disease, prednisolone administration, and sometimes Duchenne muscular dystrophy. In myxoedema, osteomalacia, and hypothermia the ratio was decreased; and starvation, elective operations, and rheumatoid arthritis had little effect. Provided that the diet is meat free, measurement of urinary 3-methylhistidine may provide useful information on the cause of protein loss.     

Ntau-methylhistidine (3-methylhistidine) and muscle protein turnover: an overview.

Actin and myosin, the contractile proteins of skeletal muscle, are methylated following peptide bond synthesis, with production of Ntau-methylhistidine (3-methylhistidine, 3-MeHis). During intracellular breakdown of these proteins, the 3-MeHis is released and excreted in the urine. Studies on tissue distribution of 3-MeHis and on its qunatitative excretion following administration to rats and to man show that urinary output of this amino acid provides a reliable index of the rate of myofibrillar protein breakdown in the musculature of intact rats and human subjects. Estimates of the fractional rate of muscle protein breakdown based on 3-MeHis data are consistent with rates computed by other techniques. By this technique, it has been shown that the fractional rate of muscle protein breakdown is not significantly different in the elderly as compared with young adults. However, since muscle mass is less in the elderly, it makes a smaller contribution to whole body protein breakdown with aging in humans. Output of 3-MeHis diminishes in growing rats and obese human subjects with protein or energy restriction, though the initial response of myofibrillar protein breakdown in growing rats to protein and protein-energy restriction differs. Measurement of 3-MeHis excretion has also proved useful in exploring the effects of physical and thermal trauma on the rate of muscle useful in exploring the effects of physical and thermal trauma on the rate of muscle protein breakdown.     

Identification of 1- and 3-methylhistidine as biomarkers of skeletal muscle toxicity by nuclear magnetic resonance-based metabolic profiling

Nuclear magnetic resonance (NMR)-based metabolomic profiling identified urinary 1- and 3-methylhistidine (1- and 3-MH) as potential biomarkers of skeletal muscle toxicity in Sprague–Dawley rats following 7 and 14 daily doses of 0.5 or 1 mg/kg cerivastatin. These metabolites were highly correlated to sex-, dose- and time-dependent development of cerivastatin-induced myotoxicity. Subsequently, the distribution and concentration of 1- and 3-MH were quantified in 18 tissues by gas chromatography–mass spectrometry. The methylhistidine isomers were most abundant in skeletal muscle with no fiber or sex differences observed; however, 3-MH was also present in cardiac and smooth muscle. In a second study, rats receiving 14 daily doses of 1 mg/kg cerivastatin (a myotoxic dose) had 6- and 2-fold elevations in 1- and 3-MH in urine and had 11- and 3-fold increases in 1- and 3-MH in serum, respectively. Selectivity of these potential biomarkers was tested by dosing rats with the cardiotoxicant isoproterenol (0.5 mg/kg), and a 2-fold decrease in urinary 1- and 3-MH was observed and attributed to the anabolic effect on skeletal muscle. These findings indicate that 1- and 3-MH may be useful urine and serum biomarkers of drug-induced skeletal muscle toxicity and hypertrophy in the rat, and further investigation into their use and limitations is warranted.     

A rapid,sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 mm sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10³ times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.     

3-Methylhistidine in actin and other muscle proteins

1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7.6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.     

Elimination by formaldehyde of interference with 3-methylhistidine determination: Application of the method to the study of muscle protein degradation

The fluorescent adduct of histidine, but not of 3-methylhistidine, with orthophthalaldehyde is destroyed by reaction with formaldehyde. This is used as the basis for an improved method of detection of 3-methylhistidine in the column effluent of an amino acid analyser. The method is capable of detecting free 3-methylhistidine in physiological fluids in which its analysis by chromatographic methods was previously very difficult due to insufficiently good resolution between histidine and 3-methylhistidine. The method has been used to detect changes of 3-methylhistidine concentration in intramuscular water and in plasma in a variety of circumstances of altered myofibrillar degradation rate.     

A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres

The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.     

Effects of aging and gender on the spatial organization of nuclei in single human skeletal muscle cells

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of the cytoplasm, i.e., the myonuclear domain (MND). We analysed aging‐ and gender‐related effects on myonuclei organization and the MND size in single muscle fibres from six young (21–31 years) and nine old men (72–96 years), and from six young (24–32 years) and nine old women (65–96 years), using a novel image analysis algorithm applied to confocal images. Muscle fibres were classified according to myosin heavy chain (MyHC) isoform expression. Our image analysis algorithm was effective in determining the spatial organization of myonuclei and the distribution of individual MNDs along the single fibre segments. Significant linear relations were observed between MND size and fibre size, irrespective age, gender and MyHC isoform expression. The spatial organization of individual myonuclei, calculated as the distribution of nearest neighbour distances in 3D, and MND size were affected in old age, but changes were dependent on MyHC isoform expression. In type I muscle fibres, average NN‐values were lower and showed an increased variability in old age, reflecting an aggregation of myonuclei in old age. Average MND size did not change in old age, but there was an increased MND size variability. In type IIa fibres, average NN‐values and MND sizes were lower in old age, reflecting the smaller size of these muscle fibres in old age. It is suggested that these changes have a significant impact on protein synthesis and degradation during the aging process.     

Neuromuscular organization of avian flight muscle: architecture of single muscle fibres in muscle units of the pectoralis (pars thoracicus) of pigeon (Columba livia)

The M. pectoralis (pars thoracicus) of pigeons (Columba livia) is comprised of short muscle fibres that do not extend from muscle origin to insertion but overlap ''in-series''. Individual pectoralis motor units are limited in territory to a portion of muscle length and are comprised of either fast twitch, oxidative and glycolytic fibres (FOG) or fast twitch and glycolytic fibres (FG). FOG fibres make up 88 to 90% of the total muscle population and have a mean diameter one-half of that of the relatively large FG fibres. Here we report on the organization of individual fibres identified in six muscle units depleted of glycogen, three comprised of FOG fibres and three comprised of FG fibres. For each motor unit, fibre counts revealed unequal numbers of depleted fibres in different unit cross-sections. We traced individual fibres in one unit comprised of FOG fibres and a second comprised of FG fibres. Six fibres from a FOG unit (total length 15.45 mm) ranged from 10.11 to 11.82 mm in length and averaged (± s.d.) 10.74 ± 0.79 mm. All originated bluntly (en mass) from a fascicle near the proximal end of the muscle unit and all terminated intramuscularly. Five of these ended in a taper and one ended bluntly. Fibres coursed on average for 70% of the muscle unit length. Six fibres from a FG unit (total length 34.76 mm) ranged from 8.97 to 18.38 mm in length and averaged 15.32 ± 3.75 mm. All originated bluntly and terminated intramuscularly; one of these ended in a taper and five ended bluntly. Fibres coursed on average for 44% of the muscle unit length. Because fibres of individual muscle units do not extend the whole muscle unit territory, the effective cross-sectional area changes along the motor unit length. These non-uniformities in the distribution of fibres within a muscle unit emphasize that the functional interactions within and between motor units are complex.     

Morphology and organization of muscle fibres in the thoracic coxal muscle receptor organ and the associated promotor muscle, in a crayfish, Cherax destructor, and mud crab, Scylla serrata

Using confocal laser scanning and conventional light microscopy, the morphology and organization of the muscle fibres in a proprioceptor, the thoracic coxal muscle receptor organ (TCMRO), and the associated 'extrafusal' promotor muscle were investigated in two species of decapod crustacea, the crayfish Cherax destructor and the mud crab Scylla serrata . The diameter of the TCMROs was shown to increase distally, with an increase up to 350% recorded for the crayfish. The tapered shape of the crayfish TCMRO was demonstrated to amplify movements mechanically at the transducer region where the afferent nerves attach. Serial sectioning of the TCMROs, showed that the fibre number increased in the proximal to distal direction from 14 to 30 fibres in the crayfish and from 7 to 20 in the crab. Optical sectioning with the laser scanning confocal microscope revealed that the increase in fibre numbers was the result of muscle fibres branching in the distal third section of the TCMRO. The percentage of muscle tissue in the cross-sectional area in the TCMRO was found to be only 35.2% and 64.6% in the crayfish and crab, respectively. Longitudinal sectioning using laser scanning confocal microscopy revealed the average sarcomere length of the TCMRO muscle fibres of both species to be in the intermediate range for crustacean muscle fibres (4.1 ± 0.1 µm and 4.55 ± 0.34 µm for the crayfish and crab) compared with the long sarcomere muscle fibres in the associated promotor muscles (7.87 ± 0.2 and 10.6 ± 0.6 µm). The distinct morphology of the TCMRO muscle fibres – smaller diameter, intermediate sarcomere length and branching of fibres compared to the larger, long sarcomere promotor fibre muscle fibres – suggest that the TCMRO muscle fibres are specialized in their role of proprioception.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.