Neuromuscular Disorders 12 (2002) 247–257 www.elsevier.

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A quantitative study of bioenergetics in skeletal muscle lacking utrophin and dystrophin
M.A. Cole a, J.A. Rafael b,1, D.J. Taylor a, R. Lodi a,2, K.E. Davies b, P. Styles a,*
a

MRC Biochemical and Clinical Magnetic Resonance Unit, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK b MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, UK Received 2 April 2001; received in revised form 27 July 2001; accepted 6 August 2001

Abstract Muscle energetics and function were investigated in the hindlimb of mice lacking dystrophin (mdx), utrophin and dystrophin (utr-dys) and controls (C57Bl/10) using 31P and 1H magnetic resonance techniques, electrical nerve stimulation and direct biochemical analysis. At rest, [adenosine triphosphate] and [total creatine] were lowest in utr-dys, while [inorganic phosphate] was elevated. Calculated [adenosine diphosphate] was 3-fold higher in mdx and 5-fold higher in utr-dys than in controls, consistent with an increased adenosine triphosphate requirement for ion pump activity. During stimulation, force production was low only in utr-dys, and this was reflected in the bioenergetic changes. Initial recovery rates of [phosphocreatine] and [adenosine diphosphate] after stimulation were rapid in all groups, indicative of normal mitochondrial adenosine triphosphate production in utr-dys and mdx. Recovery of pH was slow in utr-dys. The data indicate that the severe abnormalities which are present in the absence of utrophin and dystrophin leave basic muscle energetics intact and appear confined to processes involving the sarcolemma. q 2002 Elsevier Science B.V. All rights reserved.
Keywords: Dystrophin; Utrophin; Skeletal muscle; Magnetic resonance spectroscopy; Magnetic resonance imaging; Energy metabolism

1. Introduction Duchenne muscular dystrophy (DMD) occurs in 1 in every 3500 male births [1]. This X-linked recessive disorder is due to mutation of the gene coding for the protein dystrophin, resulting in its absence in DMD [2]. There is abnormal muscle development in which specific muscles undergo cycles of necrosis and imperfect regeneration, and necrotic cells tend to be replaced by fatty or fibrous tissue. Dystrophin acts to form a link between the actin cytoskeleton and specific transmembrane proteins [3] and is therefore thought to have a structural role within muscle fibres. The integral nature of this protein complex and the cell membrane suggests that dystrophin could also be important in other functions, for example in aggregation of ion channels and neurotransmitter receptors in the sarcolemma [4]. The protein utrophin shares 80% functional homology
* Corresponding author. Tel.: 144-1865-221868; fax: 144-1865221112. E-mail address: pstyles@bioch.ox.ac.uk (P. Styles). 1 Present address: Department of Molecular and Cellular Biochemistry, The Ohio State University Medical School, 1645 Neil Avenue, Columbus, OH 43210, USA. 2 Present address: Dipartimento di Medicina Clinica e Biotecnologia Applicata, University of Bologna, Bologna, Italy.

with dystrophin [5]. It is found at the sarcolemma before birth. Normally, utrophin is replaced by dystrophin during development and becomes confined to the neuromuscular junction [6]. In the dystrophin-deficient mdx mouse, however, it continues to be associated with the sarcolemma for some weeks after birth, and its disappearance coincides with the first signs of fibre necrosis [7,8]. Utrophin may be able to ameliorate much of the muscle pathology associated with the absence of dystrophin; expression of a truncated form of utrophin at very high levels in mdx results in nearnormal muscle [9,10]. The hypothesis that utrophin compensates for the lack of dystrophin in mdx is supported by findings from a mouse lacking in both dystrophin and utrophin (utr-dys). This model exhibits many of the pathophysiological features that are present in DMD but absent in mdx [11,12]. In contrast to the hindlimb hypertrophy of mdx, utr-dys shows profound muscle wasting and displays kyphosis as a result of weakness in the paraspinal muscles. The life span of utr-dys is severely shortened to between 6 and 20 weeks, compared with about 2 years for their mdx littermates. Ten-week-old utr-dys muscle has two main physiological characteristics which show marked similarities to DMD. There is significantly reduced force output associated with the muscle wasting and the muscle exhibits a slower phenotype than normal as evident from muscle function data

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DMD and DMD carriers have abnormalities consistent with impaired oxidative metabolism. 24. Key anatomical features of a transverse 1H NMR image of C57 mouse hindlimb are shown in the left-hand panel. Similar changes are observed in DMD muscle [13–15]. fluanisone (0. ANT. slower maximal speed of shortening).2 ^ 3. G. less fatigue. Each mouse was placed in an acrylic cradle and the left knee and ankle joints immobilized.1. The numbers of animals used in each experiment are shown in Tables 1–4. and 41 normal C57Bl/10 controls (n ¼ 41.9 g). These were fixed in position using surgical thread and attached to a Digitimer DS7 stimulus isolator which in turn was connected to a MacLab computer system. Resting skeletal muscle is characterized by low phosphocreatine (PCr)/adenosine triphosphate (ATP) and high inorganic phosphate (Pi)/ATP.19–21]. their mdx littermates (n ¼ 41. The soleus muscle is approximately defined by the white outline in the left-hand panel. line A (right-hand panel) was drawn connecting the medial face of the tibia (T) through the fibula (F).3 ^ 4. The calcaneal tendon was attached to an isometric force transducer at the base of the cradle.0167 mg kg 21) and midazolam (0. 31 P magnetic resonance spectroscopy (MRS) has identified abnormalities of metabolism in DMD in vivo. Materials and methods 2. The mdx mouse. 17. utr-dys and normal mice with respect to skeletal muscle bioenergetics and intracellular pH in order to clarify our understanding of the role of energy metabolism in the pathophysiology of dystrophinopathy. and calculated free [adenosine diphosphate] ([ADP]) [16–18]. excluding subcutaneous fat. Cole et al. 62 ^ 8 days old. . which shares genetic homology with DMD [2].9 ^ 3. exhibits qualitatively similar but quantitatively milder abnormalities by MRS. with line B drawn perpendicular to line A and through the fibula. To estimate gastrocnemius area. 24. Quantification of gastrocnemius cross-sectional area (CSAg).3 g). 31P MRS studies from resting muscle of BMD.0167 mg kg 21) in distilled water. anterior muscle compartment. The gastrocnemius is several times larger in cross-sectional area (CSA) than soleus (Fig. but data from exercise and recovery in BMD and carriers suggest that oxidative function may be normal [23]. were calculated as CSAg. Because tension generation was measured solely from the calcaneal tendon. 65 ^ 6 days old. Quadrants 1 and 2.A. force measurement represented activation of the gastrocnemius and soleus muscles. Isometric force production of the hindlimb muscles was measured with a MacLab computer system connected to the force transducer.53 mg kg 21). Delivery of 100 ms stimulus pulses to the ischiatic nerve evoked simultaneous activation of the hindlimb muscles. in which the function and/or quantity of dystrophin is reduced (but not absent) because of any of a number of known mutations in the dystrophin gene. The MRS findings in mdx are similar to those in carriers of DMD and in Becker’s muscular dystrophy (BMD) [18.248 M. 2. However.1 g). Animals were anaesthetized with a mixture of fentanyl citrate (0. These results suggest that energy metabolism is compromised in the absence of dystrophin. data consistent with low glycolytic activity has been found in BMD [22]. gastrocnemius muscle.22]. Electrical stimulation The ischiatic nerve of the left hindlimb was isolated surgically and two platinum electrodes were attached distal to the tibial nerve branch. Animals Mice used in this study were utr-dys (n ¼ 25. 62 ^ 4 days old. 1) and therefore contributes Fig. 2. myosin composition (greater proportion of slower myosin heavy chain isoforms) and histology (all fibres stain positively for NADH reductase activity). while the high intracellular pH in DMD is a reflection of altered ionic homeostasis. / Neuromuscular Disorders 12 (2002) 247–257 (slower twitch. consistent with its much milder phenotype [10. 1.2. The aim of the experiments presented here was to characterise and compare mdx.

to coincide with the acquisition of eight 31P MRS spectra.9 min. the next eight during stimulation (9. Recovery rates of PCr and ADP were calculated from the data points at the end of stimulation and in the first 2 min of recovery and were modelled on mono-exponential recovery kinetics [30. 2.3.2 min). data were normalized to the maximum potentiated force. This was confirmed by separate analysis of successive twitches in the first 2 min. 2.A. The initial rate of proton efflux during recovery was calculated from the changes in PCr and pH [32] from the end of stimulation to the first data point in recovery (midpoint. In all groups maximum potentiation occurred after 60 s stimulation. 1H MRS Fat and water content of isolated gastrocnemius from C57 and utr-dys were assessed using 1H MRS. each consisting of eight individual pulses at 30 Hz. 2. The probe was then inserted into the magnet bore which was maintained at 318C. 3). a spectrum of 64 scans.2. . Data were processed using 30 Hz line broadening and corrected for the effects of magnetic saturation as determined by the difference in relative signal intensities at the two different interpulse delays. and each spectrum was acquired as 4992 data points. During collection of 31P MRS data.4. Each muscle sample was placed in a 5-mm glass tube filled with D2O in a conventional nuclear magnetic resonance (NMR) probe tuned to the 1H resonance at 400.26].4 Tesla vertical bore Oxford Instruments magnet and a Varian Inova spectrometer. Free cytosolic [ADP] was calculated from the creatine kinase equilibrium expression as described previously [28] using an equilibrium constant of 1:66 £ 109 [29]: ½ADPŠ ¼ {½free creatineŠ½ATPŠ}={½PCrŠ½H1 Š½Keq Š} ð1Þ The phosphorylation potential ([ATP]/[ADP][Pi]). and a repetition time of 15. spectral width. Absolute concentrations of PCr and Pi expressed as mmol l 21 of intracellular water were calculated from the PCr/ ATP and Pi/ATP ratios and the biochemically-determined ATP concentration (described below) corrected for intracellular water content. The intermittent stimulation protocol elicited a series of semi-fused tetanic contractions. / Neuromuscular Disorders 12 (2002) 247–257 249 the majority of tension generated. A single supramaximal twitch was used for assessment of contractile function.534 s.3. A 128-scan spectrum from resting muscle was acquired (total time 4. a measure of the energy available to the cell. Time to peak contraction. In order to assess the effects of magnetic saturation on signal intensity. 31P MRS 2. 6500 Hz.325 s. For ATP. Data acquisition Data were acquired using a 9. peak tension and time taken to relax to one half peak tension were measured. Half height line widths of the water peak were typically 60 Hz (range 40–80 Hz). Cole et al. repetition time 15 s. This cycle was repeated for a total of 9. The D2O substantially reduced the susceptibility artefact resulting from the tissue–air interface.9 min). 37 s). A rest period of 1.1. Data from twitch contractions and the initial 30 Hz tetanic contraction were analysed using MacLab software. was acquired in 15 of the mice immediately following the collection of the 128-scan spectrum. The current was increased from 1 mA to a point where no further increase in force was seen (supramaximal stimulus).64 ml g 21 [27]. The first two of the 32-scan spectra were collected at rest. These conditions were used in the ensuing stimulation experiments. Gastrocnemius muscles for use in subsequent 1H spectroscopy studies were frozen in isopentane cooled to near freezing point. In order to assess fatigue. which was taken as 0. is expressed as its reciprocal in order to convert the parameter to a normal distribution. Resting tension was set at 50 g. 31 P MRS pulse parameters were: pulse width. typically 30 mA.26 MHz.M. The coil positioning was chosen to obtain signal predominantly from the gastrocnemius muscle. Spectral width was 5000 Hz. but there will inevitably have been some contribution from muscles in the anterior compartment. and stimulation intensity was determined by delivering a series of single stimuli of increasing current. At the end of the experiment the animal was killed by cervical dislocation and the right (unstimulated) gastrocnemius was excised and snap frozen for subsequent biochemical quantification of phosphorus metabolite concentrations. acquisition time. an 8-mm wide and 11-mm long curved rectangular section surface coil was positioned around the hindlimb over the gastrocnemius muscle. 2048 data points. Intracellular pH was determined by the chemical shift between Pi and PCr peaks [22]. which otherwise impaired shimming.9 min) followed by a continuous series of 32-scan spectra of 1. Data analysis Relative concentrations of Pi.5 s. an acquisition time of 0. repetition time. PCr and ATP were obtained using a time-domain fitting routine [24] with the AMARES algorithm and MRUI software [25. Data were acquired using four 34-ms pulses. 10 ms. and the final 12 during recovery from stimulation (14. Considerations of signal-to-noise and required time resolution meant that in these in vivo studies it was not feasible to implement more precise methods for signal localization. the hindlimb was activated using a series of intermittent stimulation trains.31]. These were analysed by measuring the peak tension of every tenth contraction.315 s. The coil was tuned to 31P resonant frequency and field homogeneity adjusted using the 1H free induction decay. the signal from the b-phosphate group was used (see Fig.3.75 s followed each train.23 min each. 0. Following surgery as described above. 2.

6.1 ^ 4. If in fact it is intracellular. total creatine and some of the metabolites determined from the MRS data would be even more abnormal in utr-dys than shown in the tables. so in all mice CSAg was calculated using the fibula as a reference for the most anterior extent of the gastrocnemius.4 0.5 ^ 5. and data were collected using a Varian INOVA system. The lower hindlimb was then imaged in longitudinal section using a T1-weighted spin-echo sequence.4 3.2 107 ^ 11 0.2 ^ 1.1 3.0 25.30 25. The probe was tuned to 1H resonance and magnetic field homogeneity adjusted using the 1H free induction decay.047 0.3 ^ 2. This was confirmed when the metabolite concentrations were expressed in terms of tissue dry weight.04 0.) 21 mmol (kg dry wt.33 ^ 0. the position of a series of transverse images between knee joint and ankle was determined. 1H Image analysis Images were converted into Image Browser program format (Varian) for quantification and normalized to maximum signal intensity. The method slightly underestimated the true CSAg by about 10%. Our methods are unable to distinguish the tissue location of this additional water. the concentrations changed in the order C57 . Statistical significance (taken as P . a ATP TCr a was included in the calculated area. Mice were anaesthetized using a 3% concentration of halothane mixed with two-thirds N2O and one-third O2 delivered at 1.3 ^ 2. Statistical significance (P) mdx 14 6. 0. and extracted using perchloric acid.) 21 mmol (kg wet wt.03 0.26 0. mean [TCr]/[ATP] increased in this same order.3 22. suggesting that the changes in [ATP] were greater than those in [TCr].16 34.22 ^ 0. The field of view was 11 £ 11 mm with a matrix size of 128 £ 128 pixels giving a nominal in-plane resolution of 86 mm. 0:05) was assessed using the non-parametric Mann–Whitney U-test.3 mm were then acquired. The leg was held in position with surgical thread tied around the calcaneus. but as soleus Table 1 Biochemical analysis of hindlimb muscle Variable Units Group C57 n ATP TCr TCr/ATP Wet/dry wt.2.7 97 ^ 10 utr-dys 8 5. C57 utr-dys vs.5 l min 21. / Neuromuscular Disorders 12 (2002) 247–257 2. phosphocreatine 1 free creatine) according to Conn et al. Results 3.6 ^ 1. On the basis of wet weight. 1H MRI 2.) 21 14 8. sweep width of 50 kHz.5 4.9 4. except where stated.6 ^ 0. Biochemical analysis of muscle Frozen tissue was crushed using a percussion mortar cooled with liquid nitrogen. echo time of 20 ms and eight averages.3 ^ 6.2 4. show that the water content of utr-dys muscle was more than 10% greater than in the other two groups. 2.09 0.250 M. . [ATP] was analysed using the method of Passoneau and Lowry [33] and total creatine ([TCr]. C57 mmol (kg wet wt.7 min.74 . 1). also given in Table 1.1. Typical acquisition parameters were a repetition time of 500 ms.7.14 n ¼ 5 for each group. mdx .005 0. Images of 1 mm slice thickness with a separation of 0.36 28.09 0. and correction was not made for it. It was not possible to distinguish the boundary between the anterior muscle compartment and the hindlimb muscle compartment in all but two mice.01 0. 3.001 0. From this image. Total imaging time was 8. Anaesthesia was maintained using 1% halothane.2 ^ 0.3 ^ 2. Biochemical analysis Concentrations of ATP and TCr based on wet and dry weights of tissue are given in Table 1.4 20. Data acquisition The size of the gastrocnemius was determined using 1H magnetic resonance imaging (MRI).A. A region of interest was then selected encompassing all leg tissue posterior to this point (Fig. The animal was placed in a 7 Tesla vertical bore Oxford Instruments magnet.9 98 ^ 11 mdx vs. 2.30 0. The ratio of dry to wet tissue was determined by freeze-drying weighed muscle samples.1.001 0.5. utr-dys.5. The mouse was supported on a bed heated to 328C and its left hindlimb inserted into a 10-mm Alderman–Grant coil.4 4. and in the calculations of intracellular metabolite concentrations we have assumed that it is extracellular (Section 2).83 ^ 0. mdx utr-dys vs. Statistical analysis Data are expressed as the mean ^ SD.01 0.01 0.) 21 mmol (kg dry wt.3 ^ 1. then the sarcoplasmic concentrations of ATP. we did not consider this error to be significant. [34]. The wet/dry weight ratios. Cross-sectional area of hindlimb muscle (CSAh) and of the gastrocnemius alone (CSAg) were calculated from the slice with the largest CSAh that was distal to both the knee and the area of fat associated with the popliteal fossa.1 ^ 1. Cole et al.002 0.5.60 0. 2.

2. Data from utr-dys were more variable than in the other two groups reflecting both a lower signal-tonoise ratio due to the smaller muscle bulk and a wider variation in phenotype. those from stimulation and recovery are 32 scans (see Section 2). Cole et al. Spectra are normalized to water peak signal intensity shown inset. The origin of the peak at 3. Spectra from resting muscle are 128 scans. Bioenergetics of resting muscle Representative 31P rest spectra are shown in Fig. and utr-dys muscle contained less total fat than C57 as shown by the lower fat/water signal intensity ratio. which is the chemical shift corresponding to a pH of 7. The proportion of fat to water in both muscles was less than 1%. 2 illustrates 1H NMR spectra from isolated gastrocnemius muscles. but the peak was broader in the two experimental groups compared to controls. mdx . utr-dys as Pi/ATP increased. The metabolite ratios PCr/ATP and PCr/Pi decreased in the order C57 . The same spectra are magnified in the main figure. 1H spectra of ex-vivo C57 and utr-dys hindlimb muscle. 3.10. 3 are scaled to identical b-ATP peak heights in order to compare the relative concentrations of PCr in the three groups.A. more alkaline peak if two distinct Pi peaks were present.3.94 ppm. 3 and the data are given in Table 2. Muscle fat content Fig. / Neuromuscular Disorders 12 (2002) 247–257 251 3. 1%. Fig. Spectra from the remaining utr-dys mice exhibited two Pi peaks with the second. Spectra from each mouse are normalized to the height of the b-ATP signal intensity in resting muscle. Spectra in Fig. it was found that although [PCr] did decrease significantly in mdx and was even lower in utr-dys. the pH for utr-dys was calculated from the midpoint of the major. The position of the Pi peak is pH dependent.7 ppm is uncertain. the pH of resting muscle in C57. In both C57 and utr-dys the fat signal intensity relative to that of water was . This indicates that our calculations of absolute metabolite concentrations were not affected by large or differing muscle fat content. When the intracellular metabolite concentrations were calculated using these ratios and the biochemically determined [ATP]. The pH for utr-dys was not different from C57 Fig. 3. with decreased pH indicated by a reduced chemical shift relative to the PCr peak. In resting muscle the Pi signal consisted of a single peak in C57. [Pi] was increased only in utr-dys. The vertical lines are positioned at 4. smaller peak at a lower (more acidic) chemical shift.2. . For the data shown in Table 2. mdx and approximately half of the utr-dys mice.M. 31P NMR spectra of mouse hindlimb.

Initially the rate of muscle acidification in stimulation was not 31 Fig. particularly in the utr-dys animals. Additional data are given in Table 3. At the start of contraction. PME signals can originate from a variety of compounds: phosphocholine and phosphoethanolamine (involved in membrane synthesis). phosphorylation potential in utr-dys was significantly decreased (presented in Table 2 as 1/phosphorylation potential). For the remaining period of stimulation.3 26. Biochemical response to stimulation.5 ^ 3.6 39.87 in C57. but the significance of this is not known. and this is reflected in the errors.9 15.94 in mdx.34 for mdx and 0.6 while the pH in mdx and utr-dys muscle decreased only to about 6.18 ^ 0. There was no significant change in [ATP] throughout the experiment as determined by b-ATP signal intensity. As a consequence of increased [ADP] and increased [Pi].40 ^ 0.2 ^ 9.23 0. 6.79.8 ^ 2. The small muscle mass limits signal to noise in these experiments.0 34. c 41 7. sugar phosphates such as glucose-6phosphate (an intermediate in glycolysis) and inosine monophosphate (a product of ATP breakdown). there was little difference between mdx and C57 (6.1 ^ 2.7 ^ 1.32 ^ 0.01 ^ 0.A. In utr-dys. pH remained stable so that at the end of the stimulation period it was 6. 3).7 ^ 6.3 ^ 2. 4. Bioenergetics in stimulation and recovery P NMR spectra are shown in Fig.7 ^ 3. Initial rates of proton efflux calculated from the end of stimulation to the first data point in recovery were lower in mdx mice than C57 and lower still in utr-dys.0 ^ 2.27 5.06 2.5 ^ 3. was 0. The 31P MRS peak from phosphomonoesters (PME) was prominent in utr-dys (Fig. pH in mdx drifted upward but it increased markedly in C57 so that at the point when contraction ceased.1 ^ 2. just as it is in human dystrophinopathies. Table 3). 4.28 for utr-dys. as previously reported [19]. 4 illustrates the time course of changes in intracellular pH. Following cessation of stimulation.4 16.0 10. However.06 ^ 0.252 M.7 ^ 3.16 7.03 ^ 0. PCr declined rapidly as Pi increased.06 2. pot. Furthermore. Cole et al. The mean maximum difference in pH between rest and stimulation.7 36.90 ^ 0. mdx muscle was more alkaline. the basic time resolution is 72 s.6 33 ^ 13 13 ^ 6 utr-dys 25 7. In all groups.0 4. Calculated [ADP] was significantly elevated in both mdx and utr-dys muscle. but after about 2 min. C57 (P ¼ 0:6). 0.2 ^ 1. reached at 3 min of stimulation.01 units min 21 in C57 and mdx but only 0. These factors limit the precision of the measurements and should be considered when interpreting the data. PCr and Pi recovered rapidly.1 12. significantly different between groups (Fig.07 ^ 0.4 4.10 ^ 0.8 ^ 3. The black bar in the top graph denotes the stimulation period and the vertical line the end of stimulation. It was more than 3 times greater in mdx muscle and 5 times greater in utr-dys muscle than in C57.0 2. C57 muscle continued to acidify to a pH of 6.8.48 0.46 for C57.14 2.17 13.9 5. while Fig. b Number of animals as in Table 1. [PCr] and [ADP] in response to stimulation.0 8. the rate of [PCr] decrease was 3 times greater in C57 than in mdx and 4 times greater than but. and so the recovery times shown in Table 3 were obtained by fitting the recovery data to an exponential. 4 and Table 3).5 ^ 3. / Neuromuscular Disorders 12 (2002) 247–257 Table 2 Intracellular metabolite ratios and concentrations in resting muscle a Variable Units Group C57 n pH PCr/ATP Pi/ATP PCr/Pi ATP b TCr b PCr free Cr Pi ADP 1/phos. 3.2 52 ^ 24 45 ^ 39 mmol l 21 mmol l 21 mmol l 21 mmol l 21 mmol l 21 mmol l 21 £ 10 26 M a Values for all groups of animals were significantly different from each other (most at P . . as shown in Fig. Data points are mean ^ SEM.4 8.4.3 ^ 3. 3.3 ^ 9.52 ^ 0.61 1. C57 (P ¼ 0:4) and Pi in mdx vs. 0:002) except for pH in utr-dys vs.01 units min 21 in utr-dys).23 ^ 1. c Reciprocal of phosphorylation potential.2 10 ^ 17 3^7 mdx 41 7.2 31. pH recovery over the 10-min period following stimulation was slow only for utr-dys (0.7 ^ 6.

50 0. .4 60 ^ 11 30 ^ 7 2.9 1.05 0.10 10. Similar to the recovery of [PCr]. However.02 Recovery from stimulation PCr.4 ^ 3.9 136 ^ 32 6.15 ^ 0. but that utr-dys only achieved about 80% repletion.7 ^ 5.002 0.45 ^ 0. C57 cm 2 cm 2 5 0. C57 utr-dys vs. C57 2 0.79 ^ 0.9 0. diaphragm Table 4 Hindlimb morphology and muscle function Variable Units Group C57 Morphology n CSAh CSAg Response to stimulation n Peak twitch tension a Peak 30 Hz tetanic tension a Twitch/tetanus ratio Time to peak tension Relaxation time to 1/2 peak tension a b Statistical significance (P) mdx utr-dys mdx vs.01 Muscle function data normalized for CSAg.045 0.7 0. The ratio of twitch tension to 30 Hz tetanic contraction was virtually identical in the three groups.1 0.1 0.7 0. although they did not reach significance.01 0. 3.2 0.05 0.21 2 6.006 0.5.2 80 ^ 60 0. a measure of oxidative activity [30] in the three groups of mice.01 0.7 0. Cole et al.16 ^ 0.02 6 0.0 89 ^ 42 0.7 0.76 0.5 ^ 2. initial efflux rate mmol l 21 min 21 a 64 ^ 14 31 ^ 10 5.5 0.6 0.19 mmol l 21 mmol l 21 6.05 0.02 0.2 129 ^ 26 6.2 113 ^ 18 2 0. it was impossible to distin- guish differences in recovery half time for [ADP].9 58 ^ 17 34 ^ 9 1.05 44 ^ 6 80 ^ 8 6 67 ^ 12 429 ^ 118 0.04 0.14 2 27.M.02 5 0.A.32 ^ 0.94 ^ 0.004 0.001 0.96 0.02 0.12 ^ 0. in utr-dys (Fig. it was impossible to distinguish differences in the half time of [PCr] recovery between groups.6 ^ 3.2 0.02 47 ^ 6 87 ^ 6 14 92 ^ 25 565 ^ 150 0.1 0. it can be seen that both C57 and mdx recovered to their pre-stimulation [PCr] by the end of the experiment.17 ^ 0.17 ^ 0. mdx utr-dys vs.9 0.2 0.02 Initial rate of change during stimulation was calculated using data from resting muscle and the first data point in stimulation (midpoint. t1/2 s H 1. C57 utr-dys vs.8 ^ 7. 37 s). Calculated [ADP] in C57 and mdx rose steadily during stimulation and reached similar values.4 ^ 2.02 0. t1/2 s ADP.12 2 15. so that by the end of stimulation there was no difference between them. Muscle morphology and contraction characteristics CSAh and CSAg were reduced in utr-dys (Table 4).1 0.05 0.6 ^ 1. n ¼ 3. mdx 253 utr-dys vs. but in utr-dys the [ADP] remained much lower than in the other two groups.6 0.15 ^ 0.05 0.23 ^ 0. As shown previously in vitro for soleus.13 9.045 0. / Neuromuscular Disorders 12 (2002) 247–257 Table 3 Muscle bioenergetics during stimulation and recovery Variable Units Group C57 n Stimulation.1 ^ 2. As shown in Table 3. Force production from a single 30 Hz stimulation train showed mean group differences similar to those for twitch tension.06 52 ^ 7 118 ^ 4 b 0.9 0.10 11.3 0.87 ^ 0. 4 and Table 3).0 0.01 0.18 ^ 0.3 0.16 ^ 0. initial rate of change a pH U min 21 PCr mmol l 21 min 21 ADP mmol l 21 min 21 End of stimulation pH PCr ADP 10 mdx 8 utr-dys 9 Statistical significance (P) mdx vs. and peak tension scaled to both was significantly lower than in mdx or C57 (data shown for CSAg).4 85 ^ 22 2 0.8 0. 10 mmol l 21. [PCr] in all groups reached a near steady state at approx. By minute 4.03 g cm 22 g cm 22 ms ms 11 102 ^ 18 576 ^ 153 0.5 0.6 ^ 3.6 0.39 ^ 0.

that Fig.2. In spite of these technical limitations. 4. physiological and functional parameters associated with dystrophin-related proteins. Cole et al. However the differences we find in metabolite ratios between control.1. Preliminary experiments showed that if the muscle were made ischaemic. The mdx mice showed the high intracellular pH also found in human dystrophinopathy [18] but. in utr-dys muscle the pH was no different from normal. Our methods are unable to determine whether these differences are due to different muscle groups. 5). utr-dys muscle displayed a sharp drop in force production followed after about 1 min by slowly developing fatigue. In some of the utr-dys mice there was an additional. BMD and the animal models suggests that our findings of low ATP and TCr in mdx and utr-dys are also present in the human diseases. Our main findings include substantial variations in the energetic status of this muscle at rest. Due to problems associated with fatty infiltration and fibrosis. A similar. Intracellular metabolite concentrations and pH Previous MRS studies have shown that resting muscle from mdx and human dystrophinopathies exhibit many of the same abnormalities [18. Another limitation is that it is not possible to differentiate between a Pi peak consisting of a small number of fibres with high [Pi] or a large number of fibres with relatively low [Pi]. oxidative. while the degree of abnormality in mdx more closely resembles the milder form of human dystrophin deficiency. and in utr-dys this shift is complete so that only type I fibres are present [35.22]. Values are mean ^ SEM. 4. 5. One difficulty with most in vivo MRS methods that have been employed for both human and model studies is that precise localization of the signal to specific muscle groups has not been possible. / Neuromuscular Disorders 12 (2002) 247–257 of dysfunction related to the absence of first dystrophin and then utrophin. followed thereafter by a steady decline in force production to about half of the maximum. we also found that the times taken to reach peak twitch tension and to relax to half peak tension were significantly slower in utr-dys than in mdx or C57. The increased width of the Pi peak in mdx and utr-dys at rest is indicative of a wider variation in fibre pH than in C57. by pressure of the 31P NMR coil on the leg surface.36].254 M. for example. PCr/ATP and PCr/Pi are decreased and Pi/ATP is elevated. and extensor digitorum longus muscles [35]. 4. which could have been caused. The results from utr-dys are similar to those in human DMD. however. The similarity of the MRS-detectable changes between DMD. In order to better understand the biochemical. Oxidative metabolism The present results confirm our and others’ interpretation of data from human dystrophinopathy [18. we have here combined MRS measurements with wet biochemistry and muscle contraction measurements in two dystrophic mouse models and controls. By the end of stimulation there was little difference in force production per unit CSA in the groups. it has been difficult to determine absolute concentrations of metabolites in human dystrophy. 1). Neither this lower pH nor the presence of the second Pi peak was due to ischaemia. mdx and utr-dys are too great to be due solely to fibre type changes. and show that the maintenance of cellular homeostasis is the prime feature of this disorder rather than energetic functionality per se. These parameters. MRS of rat muscle has shown that regions with a higher proportion of type I fibres have a lower PCr/Pi and an increased Pi/ATP ratio [41]. There is a shift in fibre-type distribution in DMD toward type I. In contrast. but relatively normal energy production during exercise and recovery. more acid Pi peak from a second.A. Force production during stimulation normalized to CSAg (see Fig. Pi increased over time as a single peak as the pH decreased. Over the first 60 s there was an increase of 124 ^ 5% for mdx and 132 ^ 9% for C57 relative to the initial force of contraction. to abnormal sarcolemmal proton transport as discussed below or to degeneration per se.23]. distinct fibre population. We found that utrdys also showed these abnormalities but that the changes were more severe than in mdx.22. our findings clearly document a progressive severity . but a full characterization of the biochemistry is hampered by the difficulties in obtaining biopsy material. BMD.19. Discussion MRS has proved to be a powerful modality for investigating dystrophic muscle in the human. were stable in the experiments presented here. surprisingly. but less marked transition occurs in mdx mice [37]. and fibre-type heterogeneity will always be present. In response to the series of 30 Hz tetanic trains applied during collection of 31P MRS data. Slow-twitch (type 1) muscles tend to have lower [ATP] and [PCr] than fast muscles [38–40]. mdx and C57 muscle showed little difference in force development scaled to CSAg (Fig. fibres [14].

The stimulation pattern that we employed is intermittent and was chosen in order to allow brief restoration of blood flow between contractions. In previous studies it was found that pH recovery in mdx was slow after muscle stimulation [10. They also confirm that [ADP] is raised at rest. making it possible to compare the relative contributions of glycolysis and oxidative phosphorylation to energy production in these two groups of mice. The overall pattern of force development in utr-dys muscle is very similar to that of individual slow motor units when stimulated intermittently [48]. (1)) and by proton efflux. Glycolytic metabolism Force production per unit CSA was almost identical in C57 and mdx during stimulation. and the much smaller degree of potentiation in utr-dys muscle is probably at least partly reflection of a shift in fibre type composition [36]. / Neuromuscular Disorders 12 (2002) 247–257 255 oxidative activity is relatively normal during recovery when ATP turnover is high. being near the Km for oxidative ATP synthesis. If this were to lead to deficient . the intracellular concentration was about 8 mmol l 21 and this was unaffected by exercise. A similar pattern of force production was found when an intermittent stimulation protocol was used in a DMD patient study [13]. 4. a milder stimulation protocol was used. the actual ADP concentration at rest appears to be startlingly high.54]. [ADP] in all three groups of mice decreased rapidly to the pre-stimulation level. 4. Control of [H 1] is by lactate-H 1 transport and Na 1/H 1 exchange across the sarcolemma [50]. The strong association between dystrophin and its related proteins in the sarcolemma suggests that the function and/ or abundance of these transporters are affected in dystrophinopathy. During stimulation. This strongly suggests that the relative contribution of glycolysis to total ATP synthesis was less in mdx than in C57 and is consistent with out findings in the skeletal muscles of BMD patients showing reduced acidification during incremental aerobic exercise [22]. Muscle contraction disturbs blood flow due a rise in intramuscular pressure [51.5. The findings strongly suggest that abnormalities in proton efflux are related to the associations between the transporters and other constituents of the sarcolemmal membrane.4. It has been shown that in response to eccentric contractions.20. Contraction characteristics and performance A marked feature of the response to stimulation was a lack of force potentiation in utr-dys mice in the first minute. During stimulation. which are more easily disrupted by muscle contraction when dystrophin is absent and are even more fragile in the absence of utrophin. It therefore seems likely that other factors such as the efficiency of ATP utilization contribute to the level of mitochondrial activity. Force potentiation is thought to be due the increase in activity of the enzyme myosin light chain kinase. Our failure to elicit slow pH recovery in BMD patients and DMD carriers [18. and under these conditions pH recovery in mdx was normal (although the calculated proton extrusion rate at the beginning of recovery was low) but in utr-dys there was a very low initial proton extrusion rate and slow overall pH recovery. modified by proton consumption from the net decrease in [PCr] (see Eq. The increased fatigue resistance of utr-dys muscle found when a more intense stimulation protocol is used [35] is also consistent with an increase in the proportion of type 1 fibres.M. Regulation of intracellular pH In resting muscle of the mdx mouse the abnormal concentrations of Na 1. which results in abnormal modulation of a-adrenergic vasoconstriction during muscle activity [55]. During the first few contractions in our experiments there was a pronounced force drop in utr-dys muscle. utr-dys muscle sustains a greater degree of membrane damage than does mdx [35].21]. In utr-dys the incomplete restitution of PCr in the later part of the observed recovery period is entirely consistent with the effects of low muscle pH on the creatine kinase equilibrium (Eq. Cole et al. After stimulation. Low ATP is therefore unlikely to be the cause of contractile abnormalities because only when [ATP] falls below 1 mmol l 21 are contraction speed and force affected [45]. The low rate of proton efflux and slow pH recovery we found in utr-dys are additional indicators of this.A. However. utr-dys muscle produced less force than C57 or mdx for a given cross-sectional area.52]. It is frequency dependent. This is consistent with abnormally high ATP turnover. the fall in pH is due almost entirely to the lactic acid produced by glycolysis.3. consistent with lower net PCr depletion and a lower [ADP]. The results of any stimulation protocol are likely to be influenced by muscle blood flow. and its appearance in utr-dys muscle may reflect a change in force-frequency characteristics that also are associated with fibre type [35].22] is likely to be due to the less damaging nature of muscle contraction in voluntary exercise. In the experiments presented here. Fast muscles show greater potentiation than slow [47]. and that upregulation of utrophin reversed these changes [10]. which in turn is linked to increased activity of ion pumps necessary to maintain ionic balance in the presence of the high intracellular concentrations of cations such as Na 1 and Ca 21 [42–44]. which regulates myosin phosphorylation and hence the number of active myosin-actin cross-bridges [46]. Ca 21 and H 1 clearly show that ionic homeostasis is abnormal [42. The skeletal muscle of mdx mice is deficient in neuronal-type nitric oxide synthase (nNOS) expression and activity [53.43]. Our preliminary experiments confirmed the findings in mdx (data not shown). 4. (1))[31]. but its cause is unknown. Although [ATP] was reduced by about 30% in utr-dys compared to control. During stimulation the net decrease in [PCr] and the change in pH were both less in mdx than in C57 and proton efflux was not greater than normal (discussed below). This phenomenon has been termed ‘sag’ [49].

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