Articles in PresS. Am J Physiol Endocrinol Metab (June 2, 2015). doi:10.1152/ajpendo.00001.2015
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Contractile function and energy metabolism of skeletal muscle in
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rats with secondary carnitine deficiency
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Running title: Carnitine Deficiency & Skeletal Muscle
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Paul A. Roberts1,2*, Jamal Bouitbir1,2*, Annalisa Bonifacio1,2, François Singh1,2, Priska
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Kaufmann1,2, Albert Urwyler2,3 & Stephan Krähenbühl1,2
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contributed equally to this work
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Division of Clinical Pharmacology & Toxicology, 2Department of Biomedicine, 3Department
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of Anesthesia, University Hospital Basel, Switzerland
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Address for correspondence
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Stephan Krähenbühl, MD, PhD
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Division of Clinical Pharmacology & Toxicology
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University Hospital Basel
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4031 Basel
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Switzerland
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Phone: +41 61 265 4715
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Fax: +41 61 265 4560
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E-mail: [email protected]
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Copyright © 2015 by the American Physiological Society.
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Summary
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The consequences of carnitine depletion upon metabolic and contractile characteristics of
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skeletal muscle remains largely unexplored. We therefore investigated the effect of N-
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trimethyl-hydrazine-3-propionate (THP) administration, a carnitine analogue inhibiting
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carnitine biosynthesis and renal reabsorption of carnitine, on skeletal muscle function and
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energy metabolism. Male Sprague Dawley rats were fed a standard rat chow in the absence
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(CON; n=8) or presence of THP (n=8) for 3 weeks. Following treatment, rats were fasted for
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24h prior to excision of their soleus and EDL muscles for biochemical characterization at rest
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and following 5min of contraction in vitro. THP treatment reduced the carnitine pool by ~80%
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in both soleus and EDL muscles compared to CON. Carnitine depletion was associated with
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a 30% decrease soleus muscle weight, whilst contractile function (expressed per g muscle),
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free coenzyme A and water content remained unaltered from CON. Muscle fiber distribution
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and fiber area remained unaffected whereas markers of apoptosis were increased in soleus
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muscle of THP-treated rats. In EDL muscle, carnitine depletion was associated with reduced
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free coenzyme A availability (-25%, p<0.05), impaired peak tension development (-44%,
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p<0.05) and increased glycogen hydrolysis (52%, p<0.05) during muscle contraction, whilst
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PDC activation, muscle weight and water content remained unaltered from CON. In
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conclusion, myopathy associated with carnitine deficiency can have different causes. While
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muscle atrophy, most likely due to increased apoptosis, is predominant in muscle composed
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predominantly of type I fibers (soleus), disturbance of energy metabolism appears to be the
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major cause in muscle composed of type II fibers (EDL).
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Keywords:
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carbohydrate metabolism; muscle atrophy; apoptosis
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N-trimethyl-hydrazine-3-propionate; secondary carnitine deficiency;
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Introduction
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Carnitine is a naturally occurring compound that is found in all mammalian tissues. L-
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carnitine, the biologically effective isomer, plays a key role within several cellular energy
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producing pathways (9). For instance, carnitine is essential for the transport of long-chain
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fatty acids across the inner mitochondrial membrane towards their oxidative fate inside the
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mitochondrial matrix (15), is important for the removal of potentially toxic acyl-CoAs from the
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mitochondria by forming acylcarnitines (3, 6) and serves as a temporal acetyl group buffer in
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the oxidation of carbohydrates during periods of augmented pathway flux (14, 31).
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Fundamental to our understanding of the role of carnitine within the body is the ability to
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manipulate the size of the tissue carnitine pool and to investigate the consequences of such
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upon cellular, tissue and whole-body functions, at rest and in response to external stresses
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such as muscular contraction. Although recent studies have shown that the skeletal muscle
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carnitine content can be increased in the presence of high circulating insulin concentrations
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by approximately 20% in humans (39, 40, 46), it appears to be more difficult to increase the
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skeletal muscle carnitine content in rodents (26). On the other hand, well characterized
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models of carnitine deficiency are available in rodents (21, 37). In rodents, it is therefore
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easier to assess the effect of carnitine deficiency on skeletal muscle function and energy
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metabolism.
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In humans and in animals, carnitine deficiency can be divided into two forms, primary and
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secondary. Primary carnitine deficiency is associated with mutations in OCTN2, a sodium-
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dependent, high affinity carnitine carrier with a high expression in the renal proximal tubules
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(29, 42). Lack of OCTN2 activity is associated with systemic carnitine deficiency both in
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humans (29) and in mice (21). Secondary carnitine deficiency is associated with diseases
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such as organic acidurias (35), hemodialysis (13, 19, 44), drugs such as valproate or
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pivmecillinam (7), or reduced dietary intake (24). Patients with primary carnitine deficiency
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typically present in early childhood with recurrent episodes of hypoketotic hypoglycemia and
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possibly coma (43). Patients with secondary carnitine deficiency typically present later in life
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and myopathy as well as cardiomyopathy are leading symptoms (19, 35, 43).
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We have previously reported on a rat model with secondary carnitine deficiency induced by
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treatment with the carnitine analogue N-trimethyl-hydrazine-3-propionate (THP) (37). Two to
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three weeks of THP treatment has been shown to reduce the carnitine content of heart, liver,
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plasma and skeletal muscle (quadriceps femoris) by 72-83% (37), to cause liver steatosis
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and peroxisomal proliferation (38) and impaired myocardial function (47). To date, the
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characterization of this model of carnitine deficiency has focused predominantly upon THP’s
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effect upon liver, heart and kidney, whereas skeletal muscle metabolism and function have
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received little attention. Taking into account that myopathy is a leading symptom of
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secondary carnitine deficiency, rats treated with THP appear to be a good animal model to
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learn more about this disease. It is for instance currently not known whether the observed up
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to 80% reduction in the skeletal muscle carnitine pool in THP-treated rats is sufficient to
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induce alterations in energy metabolism and a decrease in muscle function.
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The present study wishes to address this point by examining the consequences of carnitine
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deficiency induced by THP treatment upon the composition of coenzyme A and carnitine
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pools, tissue water content and substrates of energy metabolism at rest and in response to
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short-term contraction in rat skeletal muscle. To further our understanding, we examined the
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above in two functionally and biochemically distinct muscle groups, namely soleus (SOL;
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84% slow-twitch fibers) (1) and extensor digitorum longus (EDL; 98% fast-twitch fibers) (2),
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where the role of the carnitine pool varies from predominantly facilitating fat translocation
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(SOL) to preserving the free coenzyme A content for sustained carbohydrate oxidation
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(EDL).
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Methods
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Animal handling
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All in vivo procedures were performed in full accordance with the ‘Ethical Principles and
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Guidelines for Scientific Experiments on Animals’ of the Swiss Academy of Medical Sciences
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and following Cantonal approval from the Veterinary Department of Basel, Switzerland.
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Sixteen male Sprague Dawley rats, weighing ~200g, were obtained from the Süddeutsche
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Versuchstierfarm (Tuttlingen, Germany) and were acclimatized to the laboratory one week
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prior to the start of the study. The animals were housed in a temperature (20-22° C) and light
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(12h light – 12h dark cycle) controlled animal facility.
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Study design
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Two different types of experiments were performed. In the first experiment (experimental
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group 1), THP-treated (n=8) and control rats (n=8) were used to study the strength of soleus
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and EDL muscle as well as for biochemical characterization. In the second experiment
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(experimental group 2), THP-treated (n=5) and control rats (n=5) were used for studying
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muscle histology, mechanisms of cell death and mRNA expression.
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Following a week of habituation, animals were randomly assigned to one the treatments
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groups and received a standard powdered rat chow (Kliba Futter 3433, Basel, Switzerland)
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ad libitum. Treatment with THP was performed as described earlier (20mg THP 100g-1 body
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weight x day-1 for 21 days) (37). THP was added to the rat chow, which was obtained
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grinded. Based upon the findings of our previous study, rats were fed a standard rat chow
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(carnitine content 16.9 nmol g-1; Kliba Futter, Basel, Switzerland), as the effect of carnitine-
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deficient food upon carnitine homeostasis is minor in rats treated with THP. Water was
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provided ad libitum throughout the study. The THP was commercially prepared by
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ReseaChem GmbH (Burgdorf, Switzerland) as described previously (37). In the first
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experimental group, food consumption and changes in body weight were examined daily.
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Following 21 days of treatment, animals were starved for 24 h prior to the induction of
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anesthesia with 160 mg kg-1 i.p. ketamine (Ketalar, Park-Davis, Zurich, Switzerland). Once
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adequate anesthesia was established, the animal’s soleus and EDL muscles were carefully
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isolated (total time <5 min), tendon-to-tendon, from both the left and right hind-limbs and
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processed as described below.
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Muscle strength and biochemistry (experimental group 1)
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For experimental group 1, one soleus and one EDL muscle were snap-frozen in liquid
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nitrogen for future biochemical analysis of basal metabolite values. The distal and proximal
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tendons of the remaining soleus and EDL muscles were tied with nylon suture (Surgilon,
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grade 3-0) prior to their removal from the animal and were immediately placed in continually
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gassed (95% oxygen – 5% carbon dioxide) Krebs buffer (135mM NaCl, 5mM KCl, 1mM
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Na2H2PO4, 15mM NaHCO3, 11mM glucose, 1mM MgSO4, 2.5mM CaCl2; pH 7.4; 25oC). In an
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attempt to maintain the in vivo appearance (fiber alignment and 3D architecture), the
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muscles were loaded with ~1.2g upon the distal tendon whilst in Krebs suspension prior to
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the assessment of their in vitro contractile properties.
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Contractile function of isolated muscles (experimental group 1)
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Muscles were mounted within the electrical stimulatory apparatus following their excision
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from the animal (<10 min). The contractile properties of the isolated muscles were
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individually assessed, with the EDL muscle always the first to be studied. Muscles were
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vertically mounted in the water-tight chamber of the electrical stimulatory apparatus
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(manufactured in-house), by the anchoring of the distal tendon to a fixed hook at the base of
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the bath and the attachment of the proximal tendon, by thread, to the isometric force
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transducer (Grass FT03, Quincy, Medfield, MA, USA). The force transducer was mounted
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upon the micropositioner and was calibrated over the range of 0.25-1.00 g prior to each
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electrical stimulation protocol. Following muscle connection, the chamber of the apparatus
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was filled with 20 ml Krebs buffer and continually gassed with a mixture of 96% O2 and 4%
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CO2 and maintained at 37.5oC (Julabo 5B, JD Instruments, Houston, TX, USA) for the
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duration of the muscle stimulation protocol. Optimal muscle length (length producing maximal
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twitch tension) was determined by varying the muscle length in increments of 1mm with the
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micropositioner and by performing a single ‘twitch’ stimulus. Once optimal muscle length was
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determined, all subsequent measurements were made at this setting and the muscle was
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allowed to equilibrate for 5 min prior to the commencement of the contractile, electrical
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stimulation protocol. Muscular contraction was electrically induced by two platinum-plate
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electrodes located at the proximal and distal origins of the muscle. Muscles were stimulated
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for 5 min (1 s train interval, 333 ms train duration, 25 ms pulse interval, 1 ms pulse duration,
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130 V), in accordance with the work of Brass et al. (8). Contractile function was recorded
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using a 1-channel chart recorder (LKB Bromma 2210, Bromma, Sweden). Following the
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assessment of the EDL muscle, the stimulatory protocol was repeated for the soleus muscle
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which had already been mounted within a second electrical stimulatory apparatus containing
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Krebs buffer gassed with a mixture of 96% O2 and 4% CO2 at 37.5oC. Immediately following
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the contraction protocol (<1 s), muscles were snap frozen in liquid nitrogen for subsequent
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biochemical determinations.
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Biochemical analysis of skeletal muscle (experimental group 1)
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All muscle samples, basal and post-contraction, were divided into two equal portions under
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liquid nitrogen. Subsequently, one portion was freeze-dried, dissected free from visible blood
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and connective tissue and powdered. Total muscle water content of samples was determined
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by weighing the samples before and after freeze-drying. Freeze-dried samples were then
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extracted in 0.5 M perchloric acid containing 1 mmol EDTA, with the resulting supernatant
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neutralized with 2.2 M KHCO3 and used for the spectrophotometric determination of ATP,
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phosphocreatine, creatine and muscle lactate (18). The extract was also used for the
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radioenzymatic determination of free carnitine, acetylcarnitine and total acid soluble carnitine
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(5, 10) as well as for free CoA (CoASH) and acetyl-CoA (10). Finally, the extract was
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analyzed fluorimetrically for the determination of the tricarboxylic acid cycle intermediate
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citrate (25). Freeze-dried muscle powder was also used for the spectrophotometric
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determination of muscle glycogen (18). The remaining portion of frozen, wet muscle was
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used to assess the activation status of the pyruvate dehydrogenase complex (11).
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Muscle histology, TUNEL assay and mRNA and protein expression (experimental group 2)
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For experimental group 2, both EDL and soleus muscles were isolated, snap frozen and
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stored at -80° C. Histology was performed using cryosections as described previously (26).
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Frozen sections were stained with hematoxylin/eosin and with NADH-tetrazolium in order to
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differentiate type I from type II muscle fibers and photographs were captured using an
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Olympus BX61 microscope (Olympus, Hamburg, Germany). The fiber area was determined
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by randomly selecting muscle fibers with a distinct cell membrane and excluding elongated
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fibers indicating an oblique section. We employed Image J (version 1.41) software to
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measure muscle fibers within at least 4 muscle cross-sections of each muscle and each rat.
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Mechanisms of cell death were studied by quantifying the expression of cleaved caspase 3,
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the Bax/Bcl2 mRNA ratio and by a TUNEL assay. Cleaved caspase 3 was quantified by
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Western blotting according to Bonifacio et al. (4). Isolation of mRNA from frozen skeletal
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muscle, synthesis of cDNA and real time quantitative PCR for Bax and Bcl2 and for GLUT-4
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and hexokinase were performed as described previously (4, 41).
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TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) staining of
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myonuclei positive for DNA strand breaks was performed using a commercially available
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fluorescence detection kit (Life Technologies, Zug, Switzerland). Cross sections (10 μm) of
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muscles cut with a cryostat microtome were fixed with 4% paraformaldehyde for 15 min and
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permeabilized with 2 mg/mL proteinase K. The TUNEL reaction mixture containing terminal
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deoxynucleotidyltransferase (TdT) and fluorescein-labeled dUTP was added to the sections
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in portions of 50 μL and then incubated for 60 min at 37°C in a humidified chamber in the
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dark. After incubation, the sections were rinsed three times in PBS for 1 min each. Following
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embedding with ProLong diamond antifade mountant with DAPI (Life Technologies, Zug,
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Switzerland), the sections were investigated with a fluorescence microscope (Olympus
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BX61, Germany; 40X objective).
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Calculations & statistics
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All data are reported as means ± SEM. Comparisons between treatments were carried out
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using two-way analysis of variance (ANOVA) with repeated-measures. When a significant F-
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value was obtained (P<0.05) an LSD post-hoc test was used to locate any differences (SPSS
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Base 12.0).
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Results
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Animal Data
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There were no significant differences in body mass between the treatment groups during the
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21 days of chow feeding in the presence and absence of THP; with each group linearly
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gaining ~90g during the course of the study. Daily food consumption was not different
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between treatment groups during the 21 days of treatment (CON = 38.7±0.2 vs. THP =
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38.3±0.3 g per day). The activity of creatine kinase in plasma was numerically higher in THP-
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treated rats without reaching statistical significance (CON = 79±7 vs. THP = 105±12 U/L,
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p=0.098).
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Muscle mass, water content, histology and apoptosis
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No differences in muscle water content existed between CON and THP groups following 21
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days of treatment in either the EDL (CON = 76.9±0.3 vs. THP = 76.5±0.2 % water) or soleus
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muscles (CON = 75.4±0.2 vs. THP = 75.3±0.1 % water).
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The absolute muscle weight of tendon-to-tendon excised soleus muscle was reduced by
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30% in THP-treated animals when compared to CON (CON = 187±19 vs. THP = 132±15 mg
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wet muscle, P<0.05). This muscle atrophy was maintained when soleus weight was
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expressed relative to the animals body weight (CON = 0.47±0.05 vs. THP = 0.34±0.03 mg
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wet muscle g-1 body mass, P<0.05). In contrast, no significant reduction in absolute weight
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(CON = 141±4 vs. THP = 124±15 mg wet muscle) or muscle weight relative to body weight
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(CON = 0.35±0.01 vs. THP = 0.32±0.03 mg wet muscle g-1 body mass) was observed for
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EDL muscle of THP-treated compared CON rats.
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There was no significant change in muscle fiber type and fiber area in soleus muscle
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(Figures 1A and B). In contrast, in EDL muscle, the fiber area was 25% larger for THP-
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treated compared to control rats regarding type I fibers and 27% larger regarding type II
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fibers (Figures 1C and D). Due to a large variability mainly in THP-treated rats, these
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differences did not reach statistical significance (p=0.199 for type 1 and 0.123 for type II
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fibers). On sections stained with hematoxylin/eosin, no necrotic areas could be detected in
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both soleus and EDL muscle (data not shown).
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In order to find out the mechanism for soleus muscle atrophy in THP-treated rats, we
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assessed markers of apoptosis. As shown in figure 2A and B, expression of cleaved caspase
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3 was significantly higher in soleus muscle from THP-treated to CON rats, suggesting
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increased myocyte apoptosis in soleus of THP-treated rats. This was not the case for EDL
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muscle. Similarly, TUNEL positive nuclei were only detected in soleus muscle from THP-
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treated rats, but not in soleus from control rats or in EDL muscle (Figure 2C). In addition, the
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ratio of the mRNA expression of Bax/Bcl2 was increased in soleus from THP-treated
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compared to control rats, but not in EDL muscle (Figure 2D).
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Skeletal muscle contractile properties
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Peak isometric force generated was reduced by 44% in the EDL muscle following THP-
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treatment when compared to CON and the force remained significantly lower throughout
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contraction (figure 3 and table 1). Despite the difference in peak force generation, no
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difference in the rate of fatigue development existed between the two treatment groups, as
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reflected by similar slopes of the decline in the force development curve (figure 3 and table
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1). Similar to peak isometric force generation, the entire isometric force generated, as
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reflected by the area under the force development curve (AUC0-300sec), was reduced by 48%
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in EDL muscles from THP-treated compared to control rats (table 1).
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Although peak isometric force and entire force generation (AUC0-300sec) of soleus muscle
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were reduced by 17% and 20%, respectively, in THP-treated compared to CON rats, this
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reduction did not reach statistical significance (figure 3 and table 1). Similar to EDL muscle,
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the development of muscle fatigue (as reflected by the slope of the force development
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decline) was not different for soleus muscle obtained from THP-treated or CON rats.
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Skeletal muscle carnitine pool
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Treatment with THP reduced all carnitine fractions determined (free carnitine, acetylcarnitine,
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total acid soluble carnitine) by 70-85% compared to CON in both EDL and soleus muscles,
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both at rest and following muscle contraction (table 2). The individual carnitine fractions
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determined (free carnitine, acetylcarnitine, total acid soluble carnitine) did not change with
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muscle contraction in both soleus and EDL muscle for both treatment groups (table 2).
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The acetylcarnitine to free carnitine ratio was increased significantly by ~95% in EDL muscle
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and by ~125% in soleus muscle following THP treatment when compared to CON rats at rest
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and also following contraction (table 2). Muscle contraction did not change this ratio both in
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the CON and in the THP group in both muscle types investigated.
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Skeletal muscle coenzyme A pool
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The free CoA (CoASH) content was decreased by ~12% in soleus and by ~25% in EDL
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muscle in THP-treated compared to CON rats, reaching statistical significance only for EDL
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muscle, both at rest and after muscle contraction (table 3). Regarding acetyl-CoA, there were
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no significant differences between THP-treated and CON rats for both soleus and EDL
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muscle. Similar to carnitine, muscle contraction was not associated with significant changes
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in the CoASH and acetyl-CoA pools in both muscles studied and in THP-treated as well as
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CON rats.
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The acetyl-CoA to CoASH ratio was increased in THP-treated as compared to CON rats,
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reaching statistical significance for EDL muscle both at rest (46% increase) and after muscle
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contraction (53% increase). Muscle contraction was associated with slight increases in the
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acetyl-CoA to CoASH ratio in EDL and soleus muscle in both THP-treated and CON rats,
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reaching statistical significance only for EDL muscle of THP-treated rats.
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Muscle metabolites at rest & during contraction
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No differences in the ATP content existed between THP-treated and CON rats at rest or
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during contraction in either EDL or soleus muscle (table 4). During contraction, the ATP
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content had a tendency to decrease compared to resting conditions, reaching statistical
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significance in EDL muscle from THP-treated rats (40% decrease).
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Similar to the ATP content, the skeletal muscle creatine, phosphocreatine and total creatine
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contents were not different between THP-treated and CON rats for both EDL and soleus
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muscle under resting conditions and during contraction. Skeletal muscle contraction was
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associated with a significant decrease in the phosphocreatine content in both muscle types
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and both treatment groups studied (decrease by ~70% irrespective of treatment and muscle
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type), whereas the creatine content was not affected. The total creatine content decreased
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by 10 to 20% with muscle contraction; this decrease reached statistical significance for
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soleus muscle in both treatment groups.
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The resting skeletal muscle lactate content was not affected by treatment with THP in both
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muscle types studied. During muscle contraction, the lactate content showed a tendency to
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increase in both treatment groups and both muscles studied; this increase reached statistical
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significance for EDL muscle in CON rats (54% increase).
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The glycogen content was not significantly different between treatment groups in EDL and
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soleus muscles both under resting conditions and after muscle contraction. The only
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exception was a 52% decrease in EDL muscle of THP-treated compared to control rats after
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muscle contraction.
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The muscle citrate content at resting conditions was not statistically different between the
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treatment groups in soleus, but was increased by 108% in THP-treated compared to CON
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rats in EDL muscle.
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Active form of the pyruvate dehydrogenase complex (PDCa)
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At rest, the PDCa activity was increased in both muscles types investigated in THP-treated
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compared to control rats by ~50%, but this increase did not reach statistical significance
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(figure 4). Electrical stimulation was associated with an increase in the PDCa activity by 50 to
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100% in muscles from THP-treated and by approximately 200% in muscles from control rats.
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This increase was significant for all conditions studied, except for soleus muscle of THP-
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treated rats. After electrical stimulation, there were no significant differences in PDCa activity
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between muscles from THP-treated and control rats.
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mRNA expression of GLUT-4 and hexokinase
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mRNA expression of GLUT-4 was decreased by 25% in soleus and increased by 54% EDL
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muscle in THP-treated compared to CON rats. These differences did not reach statistical
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significance. mRNA expression of hexokinase was increased by 24% in soleus and by 67%
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in EDL muscle of THP-treated compared to CON rats, reaching statistical significance
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(p<0.05) for EDL.
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Discussion
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This is the first study to examine the consequences of carnitine depletion upon the metabolic
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and contractile characteristics of predominantly slow- and fast-twitch skeletal muscles from
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otherwise healthy rats. Twenty-one days of treatment with THP, a competitive inhibitor of
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carnitine biosynthesis and renal reabsorption (37), reduced the total-carnitine pool to the
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same extent in both soleus and EDL muscles from that measured in control animals by
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~80%, to a level comparable to that reported in JVS mice (20) and in human sufferers of
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primary carnitine-deficiency (36). The THP-induced decrease of the muscle carnitine pool
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was associated with a marked atrophy in soleus muscle, whilst contractile function, total-
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coenzyme A and water content were not altered significantly compared to control muscle. In
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comparison, carnitine depletion was associated with impaired peak tension development,
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ATP homeostasis, a reduction in free-coenzyme A availability and increased glycogen
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hydrolysis during contraction in EDL muscle, whilst muscle weight and water content were
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not significantly altered from control muscle.
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Skeletal muscle atrophy
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Taking into account that the body weight increased by 21% during the observation period but
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that soleus muscle in THP-treated rats was 30% lower than in control rats, it can be assumed
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the decrease in soleus muscle weight in THP-treated rats cannot be explained only by
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impaired muscle growth but that muscle atrophy has at least to contribute.
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Muscle atrophy refers to the wasting or loss of muscular tissue that occurs as a result of
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disuse, damage to the nerves that supply the muscle, from a disease of the muscle itself or
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from toxic effects on the muscle (22). In the present study, we observed a significant 30%
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reduction in the mass of the soleus muscle in response to THP treatment, whereas the effect
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on EDL did not reach statistical significance (minus 14% in THP treated rats, p = 0.292). As
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the soleus muscle is composed of more than 80% slow-twitch fibers and the fiber
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composition remained unchanged in THP-treated compared to control rats (figure 1), our
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findings imply a predominant type I fiber loss in response to carnitine-depletion. Given that
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THP treatment results in no alteration in rodent overnight activity (P. Kaufmann, unpublished
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observation), it would appear that the atrophy reported herein was not the result of disuse,
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but resulted from an abnormality within the muscle itself. It is noteworthy that a type I atrophy
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has been reported in an 18 year old female with a secondary carnitine deficiency (16).
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Our mechanistic studies are compatible with increased apoptosis of type I muscle fibers as
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the mechanism for soleus muscle atrophy (figure 2). Apoptosis is indeed one of the major
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determinants of skeletal muscle atrophy (23, 45). In vitro studies have demonstrated that L-
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carnitine can inhibit apoptosis at several levels (12, 28). For example, L-carnitine can
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interfere with Fas-ligand mediated apoptosis, thereby reducing acid sphingomyelinase
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activity and the ability of ceramide to stimulate the pro-apoptotic Bax/Bad complexes (12,
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27). L-carnitine can also impair opening of the mitochondrial membrane permeability
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transition pore, which normally leads to apoptosis (28, 30). Clearly, low skeletal muscle
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carnitine stores are likely to promote rather than suppress apoptosis. Further studies are
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required, however, to understand the predominant effect of the decreased carnitine muscle
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stores on type I vs. type II muscle fibers. This could be attributable to the higher
15
389
mitochondrial content and higher CPT1 levels in type I compared to type II muscle fibers. In
390
this context, it is worth noting that 24 weeks of L-carnitine administration has been shown to
391
have a selective trophic effect upon type I muscle fibers in man (17).
392
In comparison to soleus, EDL muscle of THP treated rats was not atrophic, but showed a
393
non-significant approximately 25% increase of the area of type I and II fibers, compatible with
394
fiber hypertrophy. A possible explanation for these findings could be that low skeletal muscle
395
carnitine content is associated with muscle fiber loss. Certain muscles (such as EDL) can
396
compensate this loss with fiber hypertrophy while this is not possible for other muscles (such
397
as soleus). The reason for the assumed difference in the adaption to fiber loss is currently
398
not known and deserves further investigation. In total, THP-treated rats can preserve their
399
muscle mass as suggested by maintained body weight and urinary excretion of creatinine
400
compared to control rats (37).
401
402
Contractile function
403
At present, very little is known regarding carnitine’s effect upon the contractile properties of
404
skeletal muscle; especially with regard to peak tension development and towards the
405
maintenance of contractile function. Work by Brass et al. (8) examining the contractile
406
properties of soleus and EDL muscle strips in vitro documented an improved maintenance of
407
contractile function throughout 5 minutes of electrical stimulation when soleus was incubated
408
in a media rich in carnitine. However, no carnitine-mediated improvements in contractile
409
function were observed in EDL muscle in this study.
410
Since we wanted to be comparable with existing data, we used the protocol of Brass et al. (8)
411
for skeletal muscle stimulation. Using this protocol, an intense muscle contraction is induced,
412
for which carbohydrates are the main energy source (32). Based on these considerations
413
and the previous findings of Brass et al. (8), we expected carnitine depletion to impair peak
414
tension and to increase fatigability of the musculature mainly by impairing the ability to buffer
415
excess acetyl-CoA generated through carbohydrate oxidation.
16
416
However, peak tension and the decline in contractile function during 5 min of electrical
417
stimulation of soleus muscle in vitro were not significantly impaired in THP-treated compared
418
to control rats. The absence of THP-induced impairment in function of the soleus muscle
419
could possibly be explained by the intensity of the stimulation. Though we observed the
420
expected decrease in the phosphocreatine content in THP-treated and control muscle in
421
response to contraction, there was no increase in the acetylcarnitine content or in the
422
acetylcarnitine to carnitine ratio. It therefore appears that acetyl-CoA delivery through the
423
carbohydrate pathways did not exceed the demands of the TCA cycle for oxidative
424
substrates. Therefore, carnitine’s ability to buffer excess acetyl-CoA may not have been
425
sufficiently tested using the current intensity of contraction.
426
In contrast to soleus, peak isometric tension production was reduced in EDL muscle in
427
response to THP pre-treatment, although the maintenance of peak tension was not different
428
from control muscle. As will be discussed below, a likely explanation for this finding is the
429
reduced availability of free CoASH to stimulate oxidative ATP synthesis in response to
430
treatment with THP.
431
432
Effect of carnitine deficiency on skeletal muscle energy metabolism
433
Similarly to soleus muscle, treatment with THP reduced the carnitine content in EDL muscle
434
by approximately 80%. However, in contrast to soleus, the acetyl-CoA to free CoASH ratio
435
was significantly increased and the concentration of free CoASH, a key substrate for the
436
PDC reaction, was significantly lowered when compared to CON muscle. Despite these
437
differences in free CoASH availability in EDL muscle, no difference in PDC activation existed
438
between THP-treated and control rats. These findings indicate that, despite a similar degree
439
of PDC activation, acetyl-CoA and NADH delivery via this enzyme complex (i.e. flux) was
440
reduced in EDL muscle from THP-treated vs. CON rats.
441
The decline in free-CoASH concentration in EDL vs. soleus muscle following 21 days of THP
442
is a surprising observation that warrants further investigation. One explanation is the
443
increased acetyl-CoA to free-CoASH ratio in response to the decline in tissue carnitine
17
444
content and a concomitant reduced ability of carnitine acyltransferase to buffer excess acetyl
445
groups to maintain free-CoASH. An additional complication of an elevated acetyl-CoA to
446
free-CoASH ratio may be its effect upon pantothenate kinase, the master regulator of
447
coenzyme A biosynthesis in mammalian cells (33). Evidence is emerging that acetyl-CoA
448
and free-CoASH differentially regulate pantothenate kinase; with acetyl-CoA potently
449
inhibiting activity, leading to a reduction in total-CoASH content and with free-CoASH
450
stimulating coenzyme A biosynthesis (34).
451
Interestingly, the citrate content in EDL muscle was increased in THP-treated compared to
452
control rats under resting conditions. Increased formation of citrate by the Krebs cycle is
453
unlikely taking into account the impaired flux across the PDC. Impaired activity of the Krebs
454
cycle distal to the formation of citrate is also unlikely regarding the maintained ATP and
455
phosphocreatine content without a significant increase in the lactate content. Am more likely
456
possibility is a decreased flux across the cytosolic citrate lyase reaction, which uses free
457
CoASH as a substrate to produce acetyl-CoA and oxaloacetate from citrate.
458
Another important observation in EDL muscle is the decrease in the glycogen content in
459
response to muscle contraction in THP-treated compared to control rats. This could result
460
from impaired transport and/or phosphorylation of glucose as well as impaired glycogen
461
synthesis. Taking into account the increased mRNA expression of GLUT-4 and hexokinase
462
and the maintained cellular ATP concentrations in resting EDL of THP-treated rats, impaired
463
transport and phosphorylation of glucose are both unlikely. Glycogen synthesis was not
464
assessed directly, but maintained glycogen stores under resting conditions in EDL of THP-
465
treated rats suggest a normal activity of glycogen synthesis. A more likely explanation for the
466
reduced glycogen content in post-contraction EDL muscle of THP-treated rats is therefore
467
increased glycogen consumption. As mentioned above, the flux across the PDC reaction,
468
which is at the same time an important source of substrates for the Krebs cycle, was
469
decreased in EDL of THP-treated rats. Under resting conditions, this decrease was not large
470
enough to impair oxidative metabolism of glucose. During muscle contraction, however,
471
Krebs cycle activity may have been too limited in EDL from THP-treated rats to maintain the
18
472
cellular ATP stores and glycogenolysis/glycolysis may have been necessary for ATP
473
generation. The lactate content after contraction was numerically increased in EDL muscle
474
from THP-treated compared to control rats (70±7 vs. 49±15 mmol/kg dry muscle, p=0.225),
475
supporting this explanation.
476
Impaired flux across PDC and citrate lyase and, as a consequence, increased cellular citrate
477
and reduced glycogen stores may explain the observed decrease in maximal contraction in
478
EDL of THP-treated rats. In comparison, the findings in the soleus muscle concerning the
479
effect of the loss of carnitine on the CoA pool go into the same direction as observed in the
480
EDL muscle, but are less accentuated. Accordingly, citrate did not accumulate and glycogen
481
stores were maintained during contraction in soleus from THP-treated rats and soleus
482
muscle contraction was not significantly impaired.
483
It is also possible reduced contractile function of EDL is not a consequence of changes in
484
energy metabolism, but may be related to the observed morphological changes which may
485
affect muscle contraction directly or via impairing excitation-contraction coupling.
486
In conclusion, we present novel data demonstrating that a THP-induced carnitine-deficiency,
487
in otherwise healthy skeletal muscle, results in a predominant loss of soleus (type I) muscle
488
fibers, whilst the biochemical and functional properties of the soleus muscle remained largely
489
normal. Conversely, despite the preservation of EDL muscle mass, the biochemical and
490
functional properties of this predominantly type II fibred muscle were impaired, with an
491
increased reliance upon energy production from non-oxygen dependent routes when
492
compared to control muscle. Our findings suggest that myopathy associated with carnitine
493
deficiency can have different reasons, namely muscle atrophy in muscles composed
494
predominantly of type I fibers and impaired mitochondrial energy production in muscles
495
composed predominantly of type II fibres.
496
497
498
499
19
500
Financial support
501
The study supported by a grant from Lonza AG, Basel, Switzerland and by a grant of the
502
Swiss National Science Foundation to SK (310000-112483).
503
504
505
Acknowledgements
506
We wish to express our appreciation to Lonza Group, Basel, Switzerland; especially towards
507
Miss Ulla Held & Dr Paula Gaynor, for supporting this research. The authors would also like
508
to thank Dr Michael Török for his kind assistance whilst commencing the animal studies.
509
20
510
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511
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651
Figure legends
652
653
Figure 1
654
Muscle fiber composition and fiber area. Muscle fibers were stained with NADH-tetrazolium
655
using cryosections as described in Methods. Type I fibers are dark, type II fibers bright. The
656
fiber area was quantified using an Olympus BX61 microscope equipped with Image J
657
(version 1.41) software. A. Soleus muscle sections. B. Quantification of type I fiber area in
658
soleus muscle. C. Extensor digitorum longus (EDL) muscle sections. D. Quantification of
659
type I and type II fiber area in EDL muscle. CON: control rats (n=5), THP: N-trimethyl-
660
hydrazine-3-propionate treated rats (n=5). Results are expressed as means ± SEM. There
661
were no statistically significant differences in fiber area between control and THP-treated
662
animals.
663
664
Figure 2
665
Markers of apoptosis in extensor digitorum longus (EDL) and soleus muscle. Apoptosis was
666
investigated by quantifying cleaved caspase 3, TUNEL staining and determination of the
667
Bax/Bcl2 mRNA ratio. A: Western blot for cleaved caspase 3. GAPDH protein expression
668
was used to correct for different protein loading. B: Quantification of the Western blots (n=3
669
independent experiments). C: TUNEL staining of EDL and soleus muscle sections. The
670
arrows show TUNEL positive nuclei. D: Ratio of the Bax/Bcl2 mRNA expression in EDL and
671
soleus muscle. CON: control rats (n=5), THP: N-trimethyl-hydrazine-3-propionate treated rats
672
(n=5). Results are expressed as means ± SEM. *p<0.05 vs. control rats.
673
674
Figure 3
675
Muscle force and fatigue development. Isometric tension production and fatigue development
676
were investigated in extensor digitorum longus and soleus muscles excised tendon-to-tendon
677
from control (CON, n=8) and N-trimethyl-hydrazine-3-propionate treated (THP, n=8) rats.
678
Electrical stimulation was performed for 5 minutes and force generation was recorded as
25
679
described in Methods. Results are expressed as the percentage of the maximal force
680
generated by control muscles (see Table 1 for absolute values and statistical analysis) and
681
are given as means ± SEM.
682
683
Figure 4
684
Pyruvate Dehydrogenase Complex activation (PDCa) in extensor digitorum longus and
685
soleus muscles. Muscles were studied at rest and following 5 min of electrically induced
686
contraction from control (CON, n=8) and N-trimethyl–hydrazine-3-propionate (THP, n=8)
687
treated rats. The reaction was carried out at 37°C and the results are expressed as means ±
688
SEM. *p<0.05 vs. resting conditions within the same group. There were no significant
689
differences between THP-treated and control rats.
690
26
691
Table 1
692
Skeletal muscle force generation. Isometric tension production and fatigue development
693
were determined in extensor digitorum longus and soleus muscles excised tendon-to-tendon
694
from control (CON) and N-trimethyl-hydrazine-3-propionate treated (THP) rats as described
695
in Methods. The muscles were incubated at 37°C and electrical stimulation was performed
696
for 5 minutes. Results are expressed as means ± SEM. aDifferent from respective CON
697
(P<0.05).
698
EDL
Soleus
CON (n=8)
THP (n=8)
CON (n=8)
THP (n=8)
9.65 ± 0.81
5.36 ± 1.00a
3.80 ± 0.33
3.14 ± 0.61
40 ± 13
55 ± 17
35 ± 4
50 ± 17
2520 ± 170
1320 ± 270a
960 ± 110
770 ± 160
-11.1 ± 1.8
-8.98 ± 2.36
-5.91 ± 0.73
-5.11 ± 1.10
-1
Forcemax (N x g wet
weight)
Tmax (sec)
AUC0-300sec (N x g
-1
wet weight x sec)
Slope (N x g-1 wet
weight x sec-1 x 10-3)
699
700
27
701
Table 2
702
Skeletal muscle carnitine (Cn) content. The carnitine fractions were determined using a
703
radioenzymatic assay in freeze-dried muscle as described in Methods. The rats investigated
704
were either untreated (control rats, CON) or treated with N-trimethyl-hydrazine-3-propionate
705
(THP) for 3 weeks. Muscles (EDL and soleus) were studied at rest or during contraction.
706
Results are expressed as means ± SEM with units of mmol kg-1 dry muscle. ap<0.05 vs. the
707
respective control group. There were no significant differences between contraction and rest
708
within the same treatment groups.
709
CON (n=8)
Rest
THP (n=8)
Contraction
Rest
Contraction
Extensor digitorum longus
Free carnitine (Cn)
3.54±0.25
3.10±0.22
0.61±0.10a
0.41±0.06 a
Acetylcarnitine
2.79±0.40
2.96±0.57
0.91±0.16a
0.68±0.07a
Total acid soluble
6.53±0.58
6.15±0.71
1.59±0.26a
1.19±0.13a
0.78±0.09
0.93±0.16
1.50±0.14a
1.81±0.17a
Free carnitine (Cn)
2.93±0.48
2.78±0.25
0.45±0.11a
0.40±0.07a
Acetylcarnitine
2.87±0.64
2.86±0.40
0.77±0.11a
0.77±0.16a
Total acid soluble
5.95±1.07
5.84±0.56
1.29±0.22a
1.27±0.21a
0.98±0.12
1.03±0.14
2.23±0.45a
2.22±0.51a
carnitine
Acetyl-Cn/free Cn
Soleus
carnitine
Acetyl-Cn/free Cn
710
711
28
712
Table 3
713
Skeletal muscle coenzyme A (CoA) content. The coenzyme A fractions were determined
714
using a radioenzymatic method in freeze-dried muscle as described in Methods. The rats
715
investigated were either untreated (control rats, CON) or treated with N-trimethyl-hydrazine-
716
3-propionate (THP) for 3 weeks. Muscles (extensor digitorum longus and soleus) were
717
studied at rest or during contraction. Results are expressed as means ± SEM with units of
718
µmol kg-1 dry muscle. ap<0.05 vs. the respective control group; bp<0.05 vs. respective value
719
at rest.
720
CON (n=8)
Rest
THP (n=8)
Contraction
Rest
Contraction
Extensor digitorum longus
Free CoA (CoASH)
110±7
109±8
81±6a
77±8a
Acetyl-CoA
14±2
17±1
16±2
18±2
0.13±0.01
0.15±0.01
0.19±0.01a
0.23±0.01a,b
117±8
115±9
103±8
102±9
8±2
10±2
7±3
9±2
0.068±0.008
0.087±0.011
0.068±0.007
0.088±0.009
Acetyl-CoA/CoASH
Soleus
Free CoA (CoASH)
Acetyl-CoA
Acetyl-CoA/CoASH
721
722
29
723
Table 4
724
Skeletal muscle metabolite content. Metabolite concentrations were determined using
725
enzymatic methods as described in Methods. The rats investigated were either untreated
726
(control rats, CON) or treated with N-trimethyl-hydrazine-3-propionate (THP) for 3 weeks.
727
Muscles (extensor digitorum longus and soleus) were studied at rest or during contraction.
728
All values are expressed as means ± SEM. ATP, phosphocreatine, creatine, total creatine,
729
lactate and citrate are expressed as mmol x kg-1 dry muscle. Glycogen is expressed as mmol
730
glycosyl units x kg-1 dry muscle. ap<0.05 vs. control; bp<0.05 vs. resting conditions within the
731
same group. n/a: not available.
732
CON (n=8)
Rest
THP (n=8)
Contraction
Rest
Contraction
Extensor digitorum longus
ATP
29.0 ± 3.8
21.8 ± 3.6
31.5 ± 4.2
18.7 ± 4.0b
Phosphocreatine
33.3 ± 5.5
11.6 ± 2.5b
39.5 ± 9.0
9.0 ± 1.9b
Creatine
151 ± 6
157 ± 10
144 ± 11
157 ± 11
Total creatine
184 ± 6
169 ± 10
183 ± 7
166 ± 10
Lactate
41 ± 7
63 ± 6b
49 ± 15
70 ± 7
Glycogen
47 ± 5
50 ± 4
46 ± 10
22 ± 8a,b
Citrate
0.25 ± 0.04
n/a
0.52 ± 0.06a
n/a
24.8 ± 4.2
21.7 ± 1.9
25.1 ± 2.8
21.3 ± 1.6
16.7 ± 3.7
5.6 ± 0.8b
Soleus
ATP
733
b
Phosphocreatine
19.3 ± 5.1
4.2 ± 3.3
Creatine
142 ± 7
131 ± 4
139 ± 5
129 ± 5
Total creatine
161 ± 9
135 ± 6b
155 ± 4
134 ± 5b
Lactate
46 ± 9
50 ± 5
46 ± 10
48 ± 3
Glycogen
51 ± 6
50 ± 5
39 ± 10
44 ± 8
Citrate
0.29 ± 0.07
n/a
0.35 ± 0.06
n/a
Fig. 1
A
B
Soleus
Type I
Fiber area ( m2)
4000
3000
2000
1000
100 μm
100 μm
0
Control
CON
D
Extensor digitorum longus
THP
Type I
Type II
3000
6000
100 μm
Fiber area ( m2)
5000
Fiber area ( m2)
C
THP
2000
1000
THP
3000
2000
1000
100 μm
0
Control
4000
0
CON
THP
CON
THP
Fig. 2
A
C
EDL
CON
THP
EDL
THP
CON
cleaved
caspase 3
GAPDH
30 μm
Soleus
CON
THP
cleaved
caspase 3
30 μm
Soleus
THP
CON
GAPDH
30 μm
3.0
3.0
2.5
2.5
Densitometric Units
Densitometric Units
*
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.0
THP
D
EDL
Soleus
2.0
2.0
0.5
CON
30 μm
Soleus
CON
THP
Bax/Bcl-2 mRNA expression
EDL
Bax/Bcl-2 mRNA expression
B
1.5
1.0
0.5
0.0
*
1.5
1.0
0.5
0.0
CON
THP
CON
THP
Fig. 3
Soleus
120
CON
THP
110
100
90
80
70
60
50
40
30
20
10
0
0
50
100
150
200
Time (seconds)
250
300
Force (relative to maximal force of CON)
Force (relative to maximal force of CON)
Extensor digitorum longus
120
CON
THP
110
100
90
80
70
60
50
40
30
20
10
0
0
50
100
150
200
Time (seconds)
250
300
Fig. 4
1.4
1.2
*
1.0
*
0.8
0.6
0.4
0.2
0.0
CON
Rest
THP
CON
THP
Contraction
Soleus
PDCa (mmol x min-1 x kg-1 wet weight)
PDCa (mmol x min-1 x kg-1 wet weight)
Extensor digitorum longus
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
CON
Rest
THP
CON
THP
Contraction