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Biochemical Adaptation in the Skeletal Muscle of Rats Depleted of Creatine with the Substrate Analogue F-Guanidinopropionic Acid

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Biochemical Adaptation in the Skeletal Muscle of Rats Depleted of Creatine with the Substrate Analogue F-Guanidinopropionic Acid Biochem. J. (1985) 232, 125-131 (Printed in Great Britain) 125 Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue f-guanidinopropionic acid Eric A. SHOUBRIDGE,* R. A. John CHALLISS, David J. HAYES and George K. RADDA Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXI 3QU, U.K. 1. Rats were fed on a diet containing 1% ,-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. 2. Maximal activities ofcreatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions ofthe gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. 3. Fast-twitch muscles were smaller in GPA-fed arimals than in controls, but the size ofthe soleus muscle was unchanged. The activities ofaerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. 4. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. 5. The proportion of Type I fibres in the soleus muscle increased from 81 % in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. 6. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. 7. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response. INTRODUCTION known to result in a 90% decrease in [phosphocreatine] and a 50% decrease in [ATP] in skeletal muscle (Fitch et Skeletal muscle is a very adaptable tissue which al., 1974). Although the phosphorylated form of the responds to an altered usage pattern by changing its analogue (PGPA) accumulates in the muscle (Fitch et al., structure to suit the new metabolic demands. The 1975), it cannot substitute for phosphocreatine, because simplest working hypothesis is that there exists an it is a very poor substrate for creatine kinase (Chevli & interplay between structure and function such that Fitch, 1979). Our results indicated that aerobic potential signals which result from altered function feed back to was enhanced in the muscles of GPA-fed animals. Using modify structure. Although a number of stimuli have the same model, Petrofsky & Fitch (1980) showed that the been identified which result in adaptive change, such as isometric twitch characteristics of fast-twitch muscle endurance training (reviewed in Holloszy & Booth, 1976), (plantaris) were unaltered, whereas slow-twitch muscle chronic stimulation (reviewed in Salmons & Henriksson, (soleus) exhibited decreased twitch amplitude, increased 1981), vascular insufficiency (Bylund et al., 1976) and rise and relaxation time and markedly increased iso- altered thyroid state (Fitts et al., 1980), the nature of the metric endurance. The maximum velocity of shortening intracellular signals which trigger the process of wasalsodecreasedinthesoleusmuscle, butwasunchanged adaptation is unknown. in the plantaris muscle (Petrovsky & Fitch, 1980). Taken Using 31P n.m.r., we investigated the metabolic changes together these data suggested that persistent changes in which occur during isometric contraction at 4 Hz in the [phosphocreatine] and/or [ATP] were sufficient to trigger gastrocnemius-plantaris complex (in vivo) in rats which fundamental rearrangements of the system of energy had been depleted ofcreatine by feeding them with a diet metabolism in skeletal muscle. In this study we have containing 1% of the creatine substrate analogue investigated the molecular basis ofthe adaptations which ,f-guanidinopropionic acid (GPA) for 6-10 weeks occur in fast-and slow-twitch skeletal muscles and in (Shoubridge & Radda, 1984). This intervention was cardiac muscle in response to GPA feeding. Abbreviations used: GPA, fl-guanidinopropionic acid; PGPA, its phosphorylated form. *To whom correspondence should be addressed. Present address: Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. Vol. 232 126 E. A. Shoubridge and others MATERIALS AND METHODS buffer, pH 7.4. All activities were expressed per g fresh wt. Chemicals NAD+-isocitrate dehydrogenase and pyruvate de- Enzymes, substrates and cofactors for enzyme and hydrogenase activities were measured in isolated mito- glycogen assays were obtained from Sigma (Poole, chondria. Isocitrate dehydrogenase was measured by the Dorset, U.K.) and Boehringer Mannheim (Lewes, East method of Alp et al. (1976). Pyruvate dehydrogenase was Sussex, U.K.). GPA was synthesized by the method of assayed by the coupling assay of Coore et al. (1971), with Rowley et al. (1971), from ,8-alanine and cyanamide, and modification that [NAD+] was 10 mM. Mitochondria was recrystallized once from hot water. All other were preincubated for 10 min in isolation medium chemicals were of analytical grade. containing 0.1 mM-Ca2+ and 1.0 mM-Mg2+ to fully activate pyruvate dehydrogenase. Activities were ex- Animals pressed per mg of mitochondrial protein. Protein was Male Wistar rats were used in all experiments. The measured by the method of Bradford (1976) with the animals were obtained at 3 weeks of age (50-60 g) and Bio-Rad Laboratories kit. Bovine plasma y-globulin was were given free access to ground laboratory diet or to the used as the standard. same diet containing 1% GPA. Experiments were Glycogen determinations performed 6-10 weeks after feeding was started, but all The gastrocnemius-plantaris muscle complex was experiments used age-paired animals. Animals were killed freeze-clamped in rats under halothane anaesthesia (1%, by cervical dislocation. The following tissues were used in N20/02, 1:1). Glycogen was determined in a HC104 in the enzyme assays: gastrocnemius muscle [whole, or (8% ) extract of these samples by the method of Keppler divided into superficial white, medial and deep red & Decker (1974). portions as in Armstrong & Laughlin (1983)], plantaris muscle, soleus muscle and heart (left ventricle). Mitochondrial isolation Mitochondria were prepared from the gastrocnemius Enzyme assays muscles of control and GPA-fed rats by the trypsin Fresh tissue was used in all assays. For the assays of digestion method of Davies et al. (1981). Each glycogen phosphorylase, hexokinase, 6-phosphofructo- preparation was made from four muscles (GPA-fed) or kinase, 2-oxoglutarate dehydrogenase, lactate dehydro- three muscles (control). The final pellet was resuspended genase, 3-hydroxyacyl-CoA dehydrogenase and creatine in 225 mM-mannitol/75 mM-sucrose/10 mM-Tris/HCl/ kinase, tissues were homogenized with a Polytron (setting 500SM-EDTA, pH 7.4, at a protein concentration of 5, 2 x 20s) in 9 vol. of 50 mM-triethanolamine buffer, pH 5-10 mg/ml. Respiratory activities were measured at 7.4, containing 1 mM-EDTA, 5 mM-MgCI2 and 20 mM- 25 °C as detailed by Morgan-Hughes et al. (1977). 2-mercaptoethanol. The same extraction buffer was used Histochemistry for citrate synthase, except that 2-mercaptoethanol was omitted. All assays were done at 25 'C. Cross-sections (10 ,tm) cut from whole soleus muscles Creatine kinase, lactate dehydrogenase and citrate at -25 °C on a cryostat microtome were stained for synthase activitiesweremeasured inthecrudehomogenate myofibrillar actomyosin ATPase by the method of in the presence of0.1 % Triton X- 100. Creatine kinase was Brooke & Kaiser (1970). The proportions of Type I and assayed by the method of Oscai & Holloszy (1971). Both Type II fibreswere determined ontheentirecross-sectional GPA and PGPA are competitive inhibitors of creatine area. kinase with respect to creatine and phosphocreatine; Presentation of data and statistics however, the inhibition constants are very high, in the range of 50 mm (E. A. Shoubridge, unpublished work). All results are presented as means + S.D. Statistical Since the maximal enzyme activity was assayed in a significance was assessed with a Student's t test. 1:20000 dilution of muscle homogenate, and the concentration of GPA plus PGPA is of the order of RESULTS 30-40 ,umol/g fresh wt, the effects of this inhibition will be negligible. Lactate dehydrogenase was assayed in Body and muscle weight 50 mM-imidazole buffer, pH 7.4, containing 2.0 mm- There was no difference between the body weights of pyruvate and 0.2 mM-NADH. Citrate synthase was control and GPA-fed animals. Control animals weighed assayed by the method of Morgan-Hughes et al. (1977). 312+46 g (n = 25) and GPA-fed animals 295+27 g The crude homogenate was spun
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