Human Skeletal Muscle Biopsies Research Articles

Establishment of Long-Term Myogenic Cultures from Patients with Duchenne Muscular Dystrophy by Retroviral Transduction of a Temperature-Sensitive SV40 Large T Antigen

We have established long-term human myogenic cultures from adult human skeletal muscle biopsies by infecting primary explant cultures with an amphotropic retroviral construct encoding a temperature-sensitive SV40 large T antigen, tsA58-U19. Infected myoblasts expressed the large T antigen and showed greatly enhanced proliferative capacity when cultured at 33°C, compared with noninfected cells. When the infected cultures were incubated at 39°C, the cells withdrew from cycle, aligned, and fused to form multinucleated myotubes which expressed certain antigens that are similarly expressed in nontransduced differentiating muscle cells. Myogenic clones with greatly increased proliferative capacity were generated, for the first time, from biopsies obtained from Duchenne muscular dystrophy patients as well as from normal, dystrophin-positive individuals. Cell lines produced by this approach may prove valuable forin vitrostudies of myogenesis and for investigating the cellular and molecular consequences of inherited muscle diseases.

Increased glutamine:fructose-6-phosphate amidotransferase activity in skeletal muscle of patients with NIDDM.

Overactivity of the hexosamine pathway mediates glucose-induced insulin resistance in rat adipocytes. Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme of this pathway. We determined GFA activity in human skeletal muscle biopsies and rates of insulin-stimulated whole-body, oxidative, and nonoxidative glucose disposal using the euglycemic insulin clamp technique combined with indirect calorimetry (insulin infusion rate (1.5 mU x kg-1 x min-1)) in 12 male patients with NIDDM (age 54 +/- 2 years, BMI 27.5 +/- 0.9 kg/m2, fasting plasma glucose 8.5 +/- 0.6 mmol/l) and 9 matched normal men. GFA activity was detectable in human skeletal muscles and completely inhibited by uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) in all subjects. GFA activity was 46% increased in the NIDDM patients compared with the normal subjects (9.5 +/- 1.3 vs. 6.5 +/- 1.2 pmol, P < 0.05). Whole-body glucose uptake was 58% decreased in patients with NIDDM (20 +/- 3 micromol x kg body wt-1 x min-1) compared with normal subjects (47 +/- 4 micromol x kg body wt-1 x min-1, P < 0.001). This decrease was attributable to decreases in both glucose oxidation (9 +/- 1 vs. 15 +/- 1 micromol x kg-1 x min-1, NIDDM patients vs. control subjects, P < 0.002) and nonoxidative glucose disposal (11 +/- 2 vs. 31 +/- 4 micromol x kg-1 x min-1, P < 0.001). In patients with NIDDM, both HbA1c (r= 0.51, P < 0.05) and BMI (r= -0.57, P < 0.05) correlated with whole-body glucose uptake. HbA1c but not BMI or insulin sensitivity was correlated with basal GFA activity (r = -0.57,P < 0.01) in NIDDM patients and control subjects. We conclude that GFA is found in human skeletal muscle and that all this activity is sensitive to feedback inhibition by UDP-GlcNAc. Chronic hyperglycemia is associated with an increase in skeletal muscle GFA activity, suggesting that increased activity of the hexosamine pathway may contribute to glucose toxicity and insulin resistance in humans.

Serine proteinase inhibitors in human skeletal muscle: expression of beta-amyloid protein precursor and alpha 1-antichymotrypsin in vivo and during myogenesis in vitro.

The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, alpha 1-antichymotrypsin (alpha 1-ACT), and Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-alpha 1-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-beta APP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both alpha 1-ACT and beta APP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, alpha 1-ACT and beta APP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both alpha 1-ACT and beta APP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for alpha 1-ACT and beta APP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation.

Quantification of Carnitine, Acetylcarnitine, and Total Carnitine in Tissues by High-Performance Liquid Chromatography: The Effect of Exercise on Carnitine Homeostasis in Man

A method for the quantitative determination of carnitine, acetylcarnitine, and total carnitine in tissue was developed for application to clinical research and diagnosis. Human skeletal muscle and heart specimens (10–20 mg) were homogenized in 1 ml of water. Aliquots of the resulting homogenates (50 μl) were extracted with 1.0 ml of acetonitrile:methanol (3:1) and the carnitine-related compounds were isolated using columns containing 300 mg of silica gel. Samples were then derivatized with 4′-bromophenacyl trifluoromethanesulfonate for spectrophotometric detection or 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate for fluorescence detection and quantified by high-performance liquid chromatography. Fluorometric detection of 2-(2,3-naphthalimino)ethyl ester derivatives afforded a 500-fold increase in sensitivity when compared to derivatization with 4′-bromophenacyl trifluoromethanesulfonate. This methodology permitted detection of acetylcarnitine in dilute human muscle homogenates at quantities of 790 fmol of acetylcarnitine injected. The method was applied to a series of human skeletal muscle biopsy samples obtained from subjects performing exercise at high work loads. The method permitted quantification of carnitine, acetylcarnitine, and total carnitine (sum of carnitine and all acylcarnitines) and demonstrated the specific redistribution of the carnitine pool from carnitine to acetylcarnitine with exercise above the lactate threshold. This HPLC method is facile, and provides a sensitive and specific approach for use in human biopsy specimens.

Insulin induces translocation of GLUT-4 glucose transporters in human skeletal muscle.

Understanding the molecular mechanisms involved in the regulation of glucose transport into human muscle is necessary to unravel possible defects in glucose uptake associated with insulin resistance in humans. Here we report a strategy to subfractionate human skeletal muscle biopsies (0.5 g) removed from vastus lateralis during a euglycemic insulinemic clamp procedure. A sucrose gradient separated total membranes into five fractions. Fraction 25 (25% sucrose) contained the plasma membrane markers alpha 1- and alpha 2-subunits of the Na(+)-K(+)-adenosinetriphosphatase and the GLUT-5 hexose transporter, recently immunolocalized to the cell surface of human skeletal muscle. The dihydropyridine receptor, a transverse tubule marker, was present exclusively in this fraction. The GLUT-4 glucose transporter was more concentrated in fraction 27.5 (27.5% sucrose) and largely diminished in plasma membrane markers. Open skeletal muscle biopsies were removed before and 30 min after clamping insulin to 550 pM. This increased GLUT-4 protein by 1.61-fold in fraction 25 and lowered it by 50% in fraction 27.5. Thus physiological concentrations of insulin induce translocation of glucose transporters from an internal membrane pool to surface membranes in human skeletal muscle.

An efficient polymerase chain reaction approach for the quantitation of multiple RNAs in human tissue samples.

We describe a PCR quantitation approach that we have set up to study gene expression in human skeletal muscle biopsies. It is characterized by the independent standardization of the individual steps and ignores attempts to control for the efficiency of the PCR. Different RNA extraction/reverse transcription efficiencies are normalized by the addition of an unrelated cRNA. Quantitation is achieved by parallel amplifications of reference samples containing known amounts of PCR products. Precision was achieved by multiple measurements of several samples. Our approach allows for the detection of less than twofold differences (27-88%, depending on the RNA species studied) among samples. A comparison of biopsies from highly trained endurance runners with biopsies from untrained subjects showed that the increased mitochondrial density in the runners' samples is accompanied by a proportional increase in the concentration of the mRNA of cytochrome c oxidase subunit IV.

Expression of major histocompatibility complex antigens in cultures of clonally derived human myoblasts.

We examined class I and class II HLA antigen expression by flow cytometry in clonal cultures derived from normal human skeletal muscle biopsies. Both HLA class I and class II antigens were constitutively expressed in all clones studied. By altering the constituents of the culture medium, we could modulate the expression of HLA class II but not HLA class I antigens. We noted heterogeneity in the expression of HLA class II antigens among different clones; an increased expression was associated with an increased propensity of myoblasts to fuse. These findings indicate that normal aneurally cultured myoblasts may express both HLA class I and class II antigens, that this expression may be modulated by in vitro agents, and that the presence of these antigens may relate to the process of in vitro myoblast fusion.

Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle.

A dystrophin-related protein (DRP) encoded by a gene on chromosome 6 was studied in 14 normal and 79 pathological human skeletal muscle biopsies, as well as in cultured myotubes by light microscopic immunocytochemistry and quantitative immunoblots. In normal muscle immunoreactive DRP was present at the postjunctional surface membrane, at the surface of satellite cells, in the walls of blood vessels, in Schwann cells and in perineurium of intramuscular nerves. All of this produced a weak signal on immunoblots. In Duchenne/Becker dystrophy (DMD/BMD) and in polymyositis (PM) or dermatomyositis (DM) DRP was present throughout the extrajunctional surface membrane of extra- and intrafusal muscle fibers, particularly regenerating ones. This produced a 15-17-fold increase of DRP over normal in DMD/BMD and 4-10-fold increase over normal in PM and DM on immunoblots. In other pathological muscles, DRP localization pattern and quantity was about the same as in normals. Dystrophin-related protein was present in about the same amounts and distribution in normal and DMD cultured myoblasts and myotubes. The molecular stimulus for the marked upregulation of DRP in DMD/BMD and in the inflammatory myopathies is not known. In DMD/BMD the diffuse sarcolemmal DRP may partially compensate for dystrophin deficiency.

A method for quantitative measurement of mitochondrial creatine kinase in human skeletal muscle.

Defects in the mitochondrial energy generating system in patients with a mitochondrial myopathy are known to be localized in various enzyme complexes involved in energy production. Such a defect may exist at the level of mitochondrial creatine kinase (Mi-CK). On that account we have developed a method for measurement of the enzyme activity in human skeletal muscle biopsy material (greater than 10 mg). Interfering creatine kinase isoenzymes are removed by anion exchange and affinity chromatography. The activity of Mi-CK in reference skeletal muscle homogenates amounts to 240 +/- 88 mU/mg protein (30 +/- 8.0 mU/mg wet weight).

Effects of hyperinsulinemia and hyperglycemia on insulin receptor function and glycogen synthase activation in skeletal muscle of normal man

Insulin receptor function, glycogen synthase activity, and activation by phosphatases were studied in biopsies of human skeletal muscle under conditions of hyperglycemia and/or hyperinsulinemia for 150 minutes. Twenty-one healthy volunteers underwent either (A) a hyperinsulinemic, euglycemic clamp (serum insulin, 160.0 ± 7.7 mU/L; plasma glucose, 4.9 ± 0.1 mmol/L; n = 9), (B) a hyperglycemic clamp during normoinsulinemia (serum insulin, 18.1 ± 3.3 mU/L; plasma glucose, 12.9 ± 0.2 mmol/L; n = 6), or (C) a combined hyperinsulinemic, hyperglycemic clamp (serum insulin, 158.3 ± 15.0 mU/L; plasma glucose, 11.4 ± 0.8 mmol/L; n = 6). During all studies, the endogenous insulin secretion was inhibited with somatostatin. Insulin binding and kinase activity of insulin receptors solubilized from vastus lateralis muscle biopsies were unaffected by hyperglycemia and/or hyperinsulinemia. Hyperinsulinemia activated the muscle glycogen synthase with a decrease in the half-maximal activation constant (A 0.5) for glucose-6-phosphate (G6P) from 0.53 ± 0.04 to 0.21 ± 0.02 mmol/L (study A, P < .02) and from 0.53 ± 0.06 to 0.19 ± 0.05 mmol/L (study C, P < .03). In addition, the rate of glycogen synthase activation by phosphatases increased from 0.078 ± 0.017 to 0.134 ± 0.029 U/min/mg protein (study A, P < .03) and from 0.082 ± 0.013 to 0.145 ± 0.033 U/min/mg protein (study C, P = .05). Hyperglycemia during normoinsulinemia did not affect A 0.5 or phosphatase activity. In conclusion, (1) hyperinsulinemia for 2 1 2 hours increases glycogen synthase activity and activation by phosphatases independently on the glycemia; and (2) insulin receptor binding and basal and insulin-stimulated receptor kinase activity are not modified during short-term hyperinsulinemia and/or hyperglycemia.

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle

Single sarcoplasmic reticulum (SR) Ca2+ release channels were reconstituted from normal and malignant hyperthermic (MH) human skeletal muscle biopsies (2-5 g samples). Conduction, gating properties, and myoplasmic Ca2+ dependence of human SR Ca2+ release channels were similar to those in other species (rabbit, pig). The MH diagnostic procedure distinguishes three phenotypes (normal, MH-equivocal, and MH-susceptible) on the basis of muscle contracture sensitivity to caffeine and/or halothane. Single channel studies reveal that human MH muscles (both MH phenotypes) contain SR Ca2+ release channels with abnormally greater caffeine sensitivity. Muscles from MH-equivocal and MH-susceptible patients appear to contain channels with the same abnormality. Further, our data (n = 115, 21 channels, 11 patients) reveals that human MH muscles (both phenotypes) may contain two populations of SR Ca2+ release channels, possibly corresponding to normal and abnormal isoforms. Thus, whole cell phenotypic variation (MH-equivocal vs. MH-susceptible) arises in muscles containing channels with similar caffeine sensitivity suggesting that human MH does not arise from a single defect. These results have important ramifications concerning (a) correlation of functional and genetic MH studies, (b) identification of other, yet to be determined, factors which may influence MH expression, and (c) characterization of normal SR Ca2+ release channel function by exploring genetic channel defects.

Cromakalim (BRL 34915) restores in vitro the membrane potential of depolarized human skeletal muscle fibres.

The purpose of the present study was to analyze the effects of cromakalim (BRL 34915), a potent drug from a new class of drugs characterized as "K+ channel openers", on the electrical activity of human skeletal muscle. Therefore, intracellular recordings were used to measure the effects of cromakalim on the membrane potential and input conductance of fibres from human skeletal muscle biopsies. Cromakalim in a concentration above 1 mumol/l induced an increase in membrane K+ conductance. This effect resulted in a membrane hyperpolarization. The magnitude of this polarization depended on the difference between resting and K+ equilibrium potential. The effect had a rapid onset and was quickly reversible after washing. Fibres from two patients with hyperkalaemic periodic paralysis showed an excessive membrane depolarization during and also after exposure to an slightly elevated extracellular K+ concentration. In the latter situation, cromakalim repolarized the fibres to the normal resting potential. Tolbutamide (1 mmol/l) and Ba2+ (3 mmol/l) strongly antagonized the effect of cromakalim. The data show that cromakalim hyperpolarizes depolarized human skeletal muscle fibres maintained in vitro. The underlying mechanism is probably an activation of otherwise "silent", ATP-regulated K+ channels. Such an effect may be of therapeutic benefit in a situation in which a membrane depolarization causes muscle paralysis.

Developmental regulation of cell-surface glycoproteins in clonal cultures of human skeletal muscle satellite cells.

Pure populations of myogenic cells were obtained by cloning satellite cells from human skeletal muscle biopsies. Cell-surface glycoproteins at various stages of myogenesis were analysed by one- and two-dimensional gel electrophoresis. A total of 14 distinct proteins were detectable at the cell surface, on the basis of their susceptibility to desialation by exogenous neuraminidase or their iodination by exogenous lactoperoxidase. Reproducible changes in lectin binding or iodination of eight of these proteins occurred during myogenesis. Only two of the developmentally regulated proteins were components of the detergent-insoluble extracellular matrix fraction. Developmental regulation of these two proteins was unaffected by growth of cultures in 5-bromo-2'-deoxyuridine to inhibit myogenesis. In contrast, developmental regulation of the other cell-surface proteins was inhibited by growth in 5-bromo-2'-deoxyuridine, suggesting that changes in these proteins are tightly coupled to satellite cell differentiation. These studies represent the first systematic analysis of the surface proteins of pure, clonally derived, primary cultures of normal myogenic cells.

Cromakalim, pinacidil and RP 49356 activate a tolbutamide-sensitive K+ conductance in human skeletal muscle fibres.

S. Quasthoff 1, A. Spulerl, F. Lehmann-Horn 2 and P. Grafe 1 1Deparlment of Physiology, University of Munich 2Department of Neurology, Technical University of Munich, Munich, F.R.G. Introduction Cromakalim (BRL 34915), pinacidil and RP 49356 belong to a new class of drugs characterized as "K+ channel openers" (Weston and Abbott 1987). The present study was concerned with two experimental questions. (a) Do K + channel openers influence electrophysiological parameters of human skeletal muscle? In particular, effects on diseased human muscle would be of interest and may lead to new therapeutic strategies. (b) How do these drugs activate the K+ conductance of skeletal muscle? To answer these questions we performed electrophysiological recordings from human skeletal muscle biopsies maintained in vitro (Iaizzo and Lehmann-Horn 1988; Spuler et al. 1989). Methods Fibre segments (4-6 cm long) from human skeletal muscle biopsies were placed into a perspex chamber (volume 2 ml, flow rate 6-8 ml/min) and superfused at 36~ with a Bretag solution (Bretag 1969). Intracellular -6~,,7 recordings were performed with conventional - microelectrodes. Drugs were applied via the bathing solution. - 801 -90 Results and discussion Resting membrane potentials in fibres from human skeletal muscle biopsies varied between -55 and -85 mV. However, in spite of this wide range in resting potential, an uniform effect was seen when cromakalim, pinacidil, or RP 49356 at a concentration of 100 lxM were applied via the bathing solution. The effect consisted of a membrane hyperpolarization and an increase in membrane conductance. The change in membrane potential was dependent on the difference between resting and K+ equilibrium potential. Only small effects were seen in fibres with resting potentials around -85 mV. Furthermore, current-voltage relationships were determined before and during the action of K+ channel openers. These measurements revealed a reversal potential close to the K+ equilibrium potential. All these observations indicate an increase in membrane K§ conductance. In another series of experiments, the effects of the K+ channel blocker tolbutamide were explored. It was found that tolbutamide strongly antagonized the effects of cromakalim, pinacidil and RP 49356. Fig. 1 illustrates this interaction on three different muscle fibres (upper and lower traces from a patient with myotonic dystrophy, middle trace from a patient with hypokalaemic periodic paralysis). Tolbutamide on its own had no effect on membrane potential or conductance (Spuler et al. 1989).

Protein synthesis in mitochondria isolated from human skeletal muscle: Detection of polymorphism in mitochondrial translation products

The importance of the mitochondrial protein synthesizing system in the development of functional mitochondria, and thus presumably in the pathogenesis of mitochondrial cytopathies has become apparent in recent years. A procedure has been developed to allow the measurement of the protein synthetic activity in mitochondria isolated from human skeletal muscle biopsy materials. The examination of the mitochondrial protein synthesis products revealed two polymorphic variants with the electrophoretic mobilities in SDS-polyacrylamide gel of a 20 22 kDa and a 45 47 kDa protein. Since functional consideration suggests that these variants are most likely to be associated with conservative amino acid substitutions, the observation indicates that it might be possible to electrophoretically detect certain alterations in the mitochondrial translation products in mitochondrial cytopathies due to mutations in the mitochondrial genome.

Hodgkin-Huxley parameters of the sodium channels in human myoballs.

Myoballs were cultured from biopsies of adult human skeletal muscle without the use of antimitotic drugs. The sodium currents flowing during stepwise depolarization of the myoball membrane were investigated with the wholecell recording technique. The temperature range covered 10-37 degrees C. Two types of sodium channel were distinguished by their different sensitivity to tetrodotoxin (TTX). The channel with normal TTX sensitivity seemed identical with the sodium channel in adult muscle, the channel with less TTX sensitivity seemed identical with the juvenile channel found in developing and in denervated muscle. The activation and inactivation parameters of both channel types were quantitatively determined. The activation parameters of the two channel types were identical, but in comparison to the h infinity-curve of the adult sodium channels the h infinity-curve of the juvenile channels was positioned at more negative potentials, had a less steep slope, and when the temperature was decreased, its point of inflection shifted more in negative direction.

Calmodulin-binding profiles for nebulin and dystrophin in human skeletal muscle

Nebulin and dystrophin are two high-molecular-mass skeletal muscle proteins that have both been associated with the defective gene in Duchenne muscular dystrophy, although the function of neither protein is known. Other high-molecular-mass, calmodulin-binding proteins have recently been implicated in regulating calcium release from skeletal muscle. Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin. Dystrophin was absent in a Duchenne muscle biopsy.

Differential effects of halothane on adult and juvenile sodium channels in human muscle

Myoballs were cultured from biopsies of adult human skeletal muscle. Transient currents through the sodium channels were elicited by depolarizing a myoball membrane with the whole-cell patch-clamp technique. The properties of the sodium channels were determined from the Hodgkin-Huxley parameters (INa max, tau m, tau h, h infinity-curve) derived from these transients. Halothane, when applied at 3.4 mmol/l (approximately 15 kPa), blocked about 50% of the current through the adult, TTX-sensitive sodium channels but had little effect on the current through the juvenile, TTX-insensitive sodium channels. At greater than 12 mmol/l, halothane blocked both channel types completely. The time constants of activation and inactivation were decreased in the presence of 3.4 mmol/l halothane but not enough to account for the decrease of the current amplitude. Halothane shifted the h infinity-curves of both channel types toward more negative potentials by an amount that was roughly proportional to its concentration. Myoballs from a man susceptible to malignant hyperthermia (MH) gave the same results as the controls indicating that the halothane effect on the action potential of MH-susceptible muscle are not mediated by a specific effect on the sodium channels.

Automated microanalysis of adenosine phosphates, phosphocreatine, creatine, and lactate in muscle

An automated enzymatic procedure suitable for determination of ATP, ADP, AMP, phosphocreatine, creatine, and lactate in needle biopsies of human skeletal muscle (ca. 30 mg dry wt) using a fast centrifugal analyzer (Multistat III, Instrumentation Laboratory Inc.) is presented. Coefficients of variation ranged from 0.7 to 4.2% for multiple determinations of ATP, ADP, phosphocreatine, and creatine; from 6 to 24% for lactate; and from 9 to 20% for AMP. The procedure should be usable, with appropriate modification, with other tissues and with other fast centrifugal analyzers. Muscle samples are collected into liquid freon, lyophilized, and extracted with 600 μl of 0.65 m perchloric acid. Neutralized supernatants can be stored for up to 3 years at −80°C with no significant deterioration. The procedure takes much less time than similar manual procedures and gives better reproducibility, particularly for ADP and AMP.

Effects of delayed freezing on content of phosphagens in human skeletal muscle biopsy samples.

The concentrations of ATP, phosphocreatine (PCr), creatine, and lactate were determined in muscle biopsy samples frozen immediately or after a delay of 1-6 min. During the delay the samples were exposed to normal air or a gas mixture of 6.5% CO2-93.5% O2. The ATP content was unchanged, but PCr increased significantly from 72 mmol after rapid freezing to 85 mmol X kg dry muscle-1 during the 1st min in air. The lactate concentration increased (2.8 to 5.2 mmol X kg-1). If muscles were made anoxic by circulatory occlusion for 4-6 min before sampling, no increase in PCr was observed. Direct homogenization of fresh tissue in perchloric acid gave the same ATP, PCr, and lactate contents as frozen samples. It is concluded that the ATP and PCr contents in muscle are unaffected by freezing but that the biopsy procedure activates the energy utilization processes resulting in PCr decrease. It is suggested that the muscle PCr content after a 1-min delay in tissue freezing corresponds to the level in resting fresh muscle.