Neuromuscular Disorders 12 (2002) 247257 www.elsevier.

com/locate/nmd

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 reected 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 conned 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 specic muscles undergo cycles of necrosis and imperfect regeneration, and necrotic cells tend to be replaced by fatty or brous tissue. Dystrophin acts to form a link between the actin cytoskeleton and specic transmembrane proteins [3] and is therefore thought to have a structural role within muscle bres. 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 conned to the neuromuscular junction [6]. In the dystrophin-decient mdx mouse, however, it continues to be associated with the sarcolemma for some weeks after birth, and its disappearance coincides with the rst signs of bre 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 ndings 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 signicantly reduced force output associated with the muscle wasting and the muscle exhibits a slower phenotype than normal as evident from muscle function data

0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(01)00278-4

248

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

(slower twitch, less fatigue, slower maximal speed of shortening), myosin composition (greater proportion of slower myosin heavy chain isoforms) and histology (all bres stain positively for NADH reductase activity). Similar changes are observed in DMD muscle [1315]. 31 P magnetic resonance spectroscopy (MRS) has identied abnormalities of metabolism in DMD in vivo. Resting skeletal muscle is characterized by low phosphocreatine (PCr)/adenosine triphosphate (ATP) and high inorganic phosphate (Pi)/ATP, and calculated free [adenosine diphosphate] ([ADP]) [1618]. These results suggest that energy metabolism is compromised in the absence of dystrophin, while the high intracellular pH in DMD is a reection of altered ionic homeostasis. The mdx mouse, which shares genetic homology with DMD [2], exhibits qualitatively similar but quantitatively milder abnormalities by MRS, consistent with its much milder phenotype [10,1921]. The MRS ndings in mdx are similar to those in carriers of DMD and in Beckers muscular dystrophy (BMD) [18,22], 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. 31P MRS studies from resting muscle of BMD, DMD and DMD carriers have abnormalities consistent with impaired oxidative metabolism, but data from exercise and recovery in BMD and carriers suggest that oxidative function may be normal [23]. However, data consistent with low glycolytic activity has been found in BMD [22]. The aim of the experiments presented here was to characterise and compare mdx, 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.

2. Materials and methods 2.1. Animals Mice used in this study were utr-dys (n 25, 62 ^ 8 days old, 17.9 ^ 3.1 g), their mdx littermates (n 41, 65 ^ 6 days old, 24.2 ^ 3.9 g), and 41 normal C57Bl/10 controls (n 41, 62 ^ 4 days old, 24.3 ^ 4.3 g). The numbers of animals used in each experiment are shown in Tables 14. Animals were anaesthetized with a mixture of fentanyl citrate (0.53 mg kg 21), uanisone (0.0167 mg kg 21) and midazolam (0.0167 mg kg 21) in distilled water. Each mouse was placed in an acrylic cradle and the left knee and ankle joints immobilized. The calcaneal tendon was attached to an isometric force transducer at the base of the cradle. Isometric force production of the hindlimb muscles was measured with a MacLab computer system connected to the force transducer. 2.2. Electrical stimulation The ischiatic nerve of the left hindlimb was isolated surgically and two platinum electrodes were attached distal to the tibial nerve branch. These were xed in position using surgical thread and attached to a Digitimer DS7 stimulus isolator which in turn was connected to a MacLab computer system. Delivery of 100 ms stimulus pulses to the ischiatic nerve evoked simultaneous activation of the hindlimb muscles. Because tension generation was measured solely from the calcaneal tendon, force measurement represented activation of the gastrocnemius and soleus muscles. The gastrocnemius is several times larger in cross-sectional area (CSA) than soleus (Fig. 1) and therefore contributes

Fig. 1. Quantication of gastrocnemius cross-sectional area (CSAg). Key anatomical features of a transverse 1H NMR image of C57 mouse hindlimb are shown in the left-hand panel. To estimate gastrocnemius area, line A (right-hand panel) was drawn connecting the medial face of the tibia (T) through the bula (F), with line B drawn perpendicular to line A and through the bula. Quadrants 1 and 2, excluding subcutaneous fat, were calculated as CSAg. ANT, anterior muscle compartment; G, gastrocnemius muscle. The soleus muscle is approximately dened by the white outline in the left-hand panel.

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

249

the majority of tension generated. Resting tension was set at 50 g, and stimulation intensity was determined by delivering a series of single stimuli of increasing current. The current was increased from 1 mA to a point where no further increase in force was seen (supramaximal stimulus), typically 30 mA. These conditions were used in the ensuing stimulation experiments. A single supramaximal twitch was used for assessment of contractile function. During collection of 31P MRS data, the hindlimb was activated using a series of intermittent stimulation trains, each consisting of eight individual pulses at 30 Hz. A rest period of 1.75 s followed each train. This cycle was repeated for a total of 9.9 min, to coincide with the acquisition of eight 31P MRS spectra. Data from twitch contractions and the initial 30 Hz tetanic contraction were analysed using MacLab software. Time to peak contraction, peak tension and time taken to relax to one half peak tension were measured. The intermittent stimulation protocol elicited a series of semi-fused tetanic contractions. These were analysed by measuring the peak tension of every tenth contraction. In order to assess fatigue, data were normalized to the maximum potentiated force. In all groups maximum potentiation occurred after 60 s stimulation. This was conrmed by separate analysis of successive twitches in the rst 2 min. 2.3. 31P MRS 2.3.1. Data acquisition Data were acquired using a 9.4 Tesla vertical bore Oxford Instruments magnet and a Varian Inova spectrometer. Following surgery as described above, an 8-mm wide and 11-mm long curved rectangular section surface coil was positioned around the hindlimb over the gastrocnemius muscle. The probe was then inserted into the magnet bore which was maintained at 318C. The coil was tuned to 31P resonant frequency and eld homogeneity adjusted using the 1H free induction decay. Half height line widths of the water peak were typically 60 Hz (range 4080 Hz). The coil positioning was chosen to obtain signal predominantly from the gastrocnemius muscle, but there will inevitably have been some contribution from muscles in the anterior compartment. 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. 31 P MRS pulse parameters were: pulse width, 10 ms; acquisition time, 0.315 s; repetition time, 2.325 s; spectral width, 6500 Hz; 2048 data points. A 128-scan spectrum from resting muscle was acquired (total time 4.9 min) followed by a continuous series of 32-scan spectra of 1.23 min each. The rst two of the 32-scan spectra were collected at rest, the next eight during stimulation (9.9 min), and the nal 12 during recovery from stimulation (14.2 min). In order to assess the effects of magnetic saturation on signal intensity, a spectrum of 64 scans, repetition time 15 s, was

acquired in 15 of the mice immediately following the collection of the 128-scan spectrum. 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 quantication of phosphorus metabolite concentrations. Gastrocnemius muscles for use in subsequent 1H spectroscopy studies were frozen in isopentane cooled to near freezing point. 2.3.2. Data analysis Relative concentrations of Pi, PCr and ATP were obtained using a time-domain tting routine [24] with the AMARES algorithm and MRUI software [25,26]. For ATP, the signal from the b-phosphate group was used (see Fig. 3). 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. 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, which was taken as 0.64 ml g 21 [27]. Intracellular pH was determined by the chemical shift between Pi and PCr peaks [22]. Free cytosolic [ADP] was calculated from the creatine kinase equilibrium expression as described previously [28] using an equilibrium constant of 1:66 109 [29]: ADP {free creatineATP}={PCrH1 Keq } 1

The phosphorylation potential ([ATP]/[ADP][Pi]), a measure of the energy available to the cell, is expressed as its reciprocal in order to convert the parameter to a normal distribution. Recovery rates of PCr and ADP were calculated from the data points at the end of stimulation and in the rst 2 min of recovery and were modelled on mono-exponential recovery kinetics [30,31]. The initial rate of proton efux during recovery was calculated from the changes in PCr and pH [32] from the end of stimulation to the rst data point in recovery (midpoint, 37 s). 2.4. 1H MRS Fat and water content of isolated gastrocnemius from C57 and utr-dys were assessed using 1H MRS. Each muscle sample was placed in a 5-mm glass tube lled with D2O in a conventional nuclear magnetic resonance (NMR) probe tuned to the 1H resonance at 400.26 MHz. The D2O substantially reduced the susceptibility artefact resulting from the tissueair interface, which otherwise impaired shimming. Data were acquired using four 34-ms pulses, an acquisition time of 0.5 s, and a repetition time of 15.534 s. Spectral width was 5000 Hz, and each spectrum was acquired as 4992 data points.

250

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

2.5. 1H MRI 2.5.1. Data acquisition The size of the gastrocnemius was determined using 1H magnetic resonance imaging (MRI). Mice were anaesthetized using a 3% concentration of halothane mixed with two-thirds N2O and one-third O2 delivered at 1.5 l min 21. Anaesthesia was maintained using 1% halothane. The mouse was supported on a bed heated to 328C and its left hindlimb inserted into a 10-mm AldermanGrant coil. The leg was held in position with surgical thread tied around the calcaneus. The animal was placed in a 7 Tesla vertical bore Oxford Instruments magnet, and data were collected using a Varian INOVA system. The probe was tuned to 1H resonance and magnetic eld homogeneity adjusted using the 1H free induction decay. The lower hindlimb was then imaged in longitudinal section using a T1-weighted spin-echo sequence. Typical acquisition parameters were a repetition time of 500 ms, sweep width of 50 kHz, echo time of 20 ms and eight averages. From this image, the position of a series of transverse images between knee joint and ankle was determined. Images of 1 mm slice thickness with a separation of 0.3 mm were then acquired. The eld of view was 11 11 mm with a matrix size of 128 128 pixels giving a nominal in-plane resolution of 86 mm. Total imaging time was 8.7 min. 2.5.2. 1H Image analysis Images were converted into Image Browser program format (Varian) for quantication and normalized to maximum signal intensity. 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. It was not possible to distinguish the boundary between the anterior muscle compartment and the hindlimb muscle compartment in all but two mice, so in all mice CSAg was calculated using the bula as a reference for the most anterior extent of the gastrocnemius. A region of interest was then selected encompassing all leg tissue posterior to this point (Fig. 1). The method slightly underestimated the true CSAg by about 10%, but as soleus
Table 1 Biochemical analysis of hindlimb muscle Variable Units Group C57 n ATP TCr TCr/ATP Wet/dry wt. a ATP TCr
a

was included in the calculated area, we did not consider this error to be signicant, and correction was not made for it. 2.6. Biochemical analysis of muscle Frozen tissue was crushed using a percussion mortar cooled with liquid nitrogen, and extracted using perchloric acid. [ATP] was analysed using the method of Passoneau and Lowry [33] and total creatine ([TCr], phosphocreatine 1 free creatine) according to Conn et al. [34]. The ratio of dry to wet tissue was determined by freeze-drying weighed muscle samples. 2.7. Statistical analysis Data are expressed as the mean ^ SD, except where stated. Statistical signicance (taken as P , 0:05) was assessed using the non-parametric MannWhitney U-test.

3. Results 3.1. Biochemical analysis Concentrations of ATP and TCr based on wet and dry weights of tissue are given in Table 1. On the basis of wet weight, the concentrations changed in the order C57 . mdx . utr-dys; mean [TCr]/[ATP] increased in this same order, suggesting that the changes in [ATP] were greater than those in [TCr]. This was conrmed when the metabolite concentrations were expressed in terms of tissue dry weight. The wet/dry weight ratios, also given in Table 1, show that the water content of utr-dys muscle was more than 10% greater than in the other two groups. Our methods are unable to distinguish the tissue location of this additional water, and in the calculations of intracellular metabolite concentrations we have assumed that it is extracellular (Section 2). If in fact it is intracellular, then the sarcoplasmic concentrations of ATP, 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.

Statistical signicance (P) mdx 14 6.6 ^ 1.3 22.3 ^ 2.1 3.6 ^ 0.9 4.33 ^ 0.36 28.5 ^ 5.7 97 ^ 10 utr-dys 8 5.2 ^ 1.4 20.3 ^ 2.2 4.3 ^ 1.5 4.83 ^ 0.30 25.3 ^ 6.9 98 ^ 11 mdx vs. C57 utr-dys vs. mdx utr-dys vs. C57

mmol (kg wet wt.) 21 mmol (kg wet wt.) 21

mmol (kg dry wt.) 21 mmol (kg dry wt.) 21

14 8.1 ^ 1.0 25.3 ^ 2.4 3.2 ^ 0.4 4.22 ^ 0.16 34.1 ^ 4.2 107 ^ 11

0.01 0.001 0.4 0.60 0.03 0.01

0.04 0.09 0.30 0.047 0.26 0.74

, 0.001 0.002 0.09 0.01 0.005 0.14

n 5 for each group.

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

251

3.3. Bioenergetics of resting muscle Representative 31P rest spectra are shown in Fig. 3 and the data are given in Table 2. Spectra in Fig. 3 are scaled to identical b-ATP peak heights in order to compare the relative concentrations of PCr in the three groups. The metabolite ratios PCr/ATP and PCr/Pi decreased in the order C57 . mdx . utr-dys as Pi/ATP increased. When the intracellular metabolite concentrations were calculated using these ratios and the biochemically determined [ATP], it was found that although [PCr] did decrease signicantly in mdx and was even lower in utr-dys, [Pi] was increased only in utr-dys. Data from utr-dys were more variable than in the other two groups reecting both a lower signal-tonoise ratio due to the smaller muscle bulk and a wider variation in phenotype. In resting muscle the Pi signal consisted of a single peak in C57, mdx and approximately half of the utr-dys mice, but the peak was broader in the two experimental groups compared to controls. Spectra from the remaining utr-dys mice exhibited two Pi peaks with the second, smaller peak at a lower (more acidic) chemical shift. For the data shown in Table 2, the pH for utr-dys was calculated from the midpoint of the major, more alkaline peak if two distinct Pi peaks were present. The pH for utr-dys was not different from C57

Fig. 2. 1H spectra of ex-vivo C57 and utr-dys hindlimb muscle. Spectra are normalized to water peak signal intensity shown inset. The same spectra are magnied in the main gure. The proportion of fat to water in both muscles was less than 1%. The origin of the peak at 3.7 ppm is uncertain.

3.2. Muscle fat content Fig. 2 illustrates 1H NMR spectra from isolated gastrocnemius muscles. In both C57 and utr-dys the fat signal intensity relative to that of water was , 1%, and utr-dys muscle contained less total fat than C57 as shown by the lower fat/water signal intensity ratio. This indicates that our calculations of absolute metabolite concentrations were not affected by large or differing muscle fat content.

Fig. 3. 31P NMR spectra of mouse hindlimb. Spectra from resting muscle are 128 scans; those from stimulation and recovery are 32 scans (see Section 2). Spectra from each mouse are normalized to the height of the b-ATP signal intensity in resting muscle. The vertical lines are positioned at 4.94 ppm, which is the chemical shift corresponding to a pH of 7.10, the pH of resting muscle in C57. The position of the Pi peak is pH dependent, with decreased pH indicated by a reduced chemical shift relative to the PCr peak.

252

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

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. pot. c 41 7.10 ^ 0.06 2.90 ^ 0.48 0.32 ^ 0.17 13.2 ^ 9.1 12.7 ^ 1.6 39.5 ^ 3.7 36.7 ^ 6.0 2.7 ^ 6.0 4.1 ^ 2.2 10 ^ 17 3^7 mdx 41 7.18 ^ 0.06 2.52 ^ 0.23 0.40 ^ 0.16 7.5 ^ 3.0 10.3 ^ 2.0 34.8 ^ 3.3 26.0 ^ 2.4 8.8 ^ 2.4 4.2 ^ 1.6 33 ^ 13 13 ^ 6 utr-dys 25 7.06 ^ 0.14 2.07 ^ 0.61 1.23 ^ 1.27 5.3 ^ 9.0 8.1 ^ 2.2 31.7 ^ 3.4 16.3 ^ 3.9 15.5 ^ 3.9 5.7 ^ 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 signicantly different from each other (most at P , 0:002) except for pH in utr-dys vs. C57 (P 0:4) and Pi in mdx vs. C57 (P 0:6). b Number of animals as in Table 1. c Reciprocal of phosphorylation potential.

signicantly different between groups (Fig. 4 and Table 3), but after about 2 min, C57 muscle continued to acidify to a pH of 6.6 while the pH in mdx and utr-dys muscle decreased only to about 6.8. The mean maximum difference in pH between rest and stimulation, reached at 3 min of stimulation, was 0.46 for C57, 0.34 for mdx and 0.28 for utr-dys. For the remaining period of stimulation, pH in mdx drifted upward but it increased markedly in C57 so that at the point when contraction ceased, there was little difference between mdx and C57 (6.94 in mdx, 6.87 in C57, Table 3). In utr-dys, pH remained stable so that at the end of the stimulation period it was 6.79. Initial rates of proton efux calculated from the end of stimulation to the rst data point in recovery were lower in mdx mice than C57 and lower still in utr-dys. However, as shown in Fig. 4, pH recovery over the 10-min period following stimulation was slow only for utr-dys (0.03 ^ 0.01 units min 21 in C57 and mdx but only 0.01 ^ 0.01 units min 21 in utr-dys). At the start of contraction, the rate of [PCr] decrease was 3 times greater in C57 than in mdx and 4 times greater than

but, as previously reported [19], mdx muscle was more alkaline. Calculated [ADP] was signicantly elevated in both mdx and utr-dys muscle. It was more than 3 times greater in mdx muscle and 5 times greater in utr-dys muscle than in C57. As a consequence of increased [ADP] and increased [Pi], phosphorylation potential in utr-dys was signicantly decreased (presented in Table 2 as 1/phosphorylation potential). The 31P MRS peak from phosphomonoesters (PME) was prominent in utr-dys (Fig. 3), just as it is in human dystrophinopathies, but the signicance of this is not known. PME signals can originate from a variety of compounds: phosphocholine and phosphoethanolamine (involved in membrane synthesis), sugar phosphates such as glucose-6phosphate (an intermediate in glycolysis) and inosine monophosphate (a product of ATP breakdown). 3.4. Bioenergetics in stimulation and recovery P NMR spectra are shown in Fig. 3, while Fig. 4 illustrates the time course of changes in intracellular pH, [PCr] and [ADP] in response to stimulation. Additional data are given in Table 3. The small muscle mass limits signal to noise in these experiments, particularly in the utr-dys animals, and this is reected in the errors. Furthermore, the basic time resolution is 72 s, and so the recovery times shown in Table 3 were obtained by tting the recovery data to an exponential. These factors limit the precision of the measurements and should be considered when interpreting the data. In all groups, PCr declined rapidly as Pi increased. There was no signicant change in [ATP] throughout the experiment as determined by b-ATP signal intensity. Following cessation of stimulation, PCr and Pi recovered rapidly. Initially the rate of muscle acidication in stimulation was not
31

Fig. 4. Biochemical response to stimulation. The black bar in the top graph denotes the stimulation period and the vertical line the end of stimulation. Data points are mean ^ SEM.

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257 Table 3 Muscle bioenergetics during stimulation and recovery Variable Units Group C57 n Stimulation, 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 signicance (P) mdx vs. C57 utr-dys vs. mdx

253

utr-dys vs. C57

2 0.15 ^ 0.14 2 27.7 ^ 5.2 113 ^ 18

2 0.16 ^ 0.12 2 15.6 ^ 3.4 85 ^ 22

2 0.23 ^ 0.21 2 6.8 ^ 7.2 80 ^ 60

0.6 0.001 0.01

0.9 0.05 0.7

0.9 0.002 0.19

mmol l 21 mmol l 21

6.87 ^ 0.10 11.6 ^ 1.9 136 ^ 32

6.94 ^ 0.10 10.5 ^ 2.2 129 ^ 26

6.79 ^ 0.13 9.6 ^ 3.0 89 ^ 42

0.1 0.3 0.7

0.045 0.7 0.05

0.5 0.1 0.02

Recovery from stimulation PCr, t1/2 s ADP, t1/2 s H 1, initial efux rate mmol l 21 min 21
a

64 ^ 14 31 ^ 10 5.4 ^ 3.4

60 ^ 11 30 ^ 7 2.4 ^ 2.9

58 ^ 17 34 ^ 9 1.1 ^ 2.8

0.9 1.0 0.50

0.2 0.6 0.02

Initial rate of change during stimulation was calculated using data from resting muscle and the rst data point in stimulation (midpoint, 37 s).

in utr-dys (Fig. 4 and Table 3). By minute 4, [PCr] in all groups reached a near steady state at approx. 10 mmol l 21, so that by the end of stimulation there was no difference between them. As shown in Table 3, it was impossible to distinguish differences in the half time of [PCr] recovery between groups. However, it can be seen that both C57 and mdx recovered to their pre-stimulation [PCr] by the end of the experiment, but that utr-dys only achieved about 80% repletion. Calculated [ADP] in C57 and mdx rose steadily during stimulation and reached similar values, but in utr-dys the [ADP] remained much lower than in the other two groups. Similar to the recovery of [PCr], it was impossible to distin-

guish differences in recovery half time for [ADP], a measure of oxidative activity [30] in the three groups of mice. 3.5. Muscle morphology and contraction characteristics CSAh and CSAg were reduced in utr-dys (Table 4), and peak tension scaled to both was signicantly lower than in mdx or C57 (data shown for CSAg). Force production from a single 30 Hz stimulation train showed mean group differences similar to those for twitch tension, although they did not reach signicance. The ratio of twitch tension to 30 Hz tetanic contraction was virtually identical in the three groups. As shown previously in vitro for soleus, 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 signicance (P) mdx utr-dys mdx vs. C57 utr-dys vs. mdx utr-dys vs. C57

cm 2 cm 2

5 0.39 ^ 0.05 0.15 ^ 0.02

5 0.45 ^ 0.05 0.16 ^ 0.02

6 0.32 ^ 0.05 0.12 ^ 0.02

0.1 0.6

0.01 0.006

0.045 0.03

g cm 22 g cm 22 ms ms

11 102 ^ 18 576 ^ 153 0.18 ^ 0.02 47 ^ 6 87 ^ 6

14 92 ^ 25 565 ^ 150 0.17 ^ 0.05 44 ^ 6 80 ^ 8

6 67 ^ 12 429 ^ 118 0.17 ^ 0.06 52 ^ 7 118 ^ 4 b 0.2 0.5 0.7 0.2 0.02 0.04 0.9 0.76 0.02 0.01 0.004 0.1 0.96 0.3 0.01

Muscle function data normalized for CSAg. n 3.

254

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

of dysfunction related to the absence of rst dystrophin and then utrophin, and show that the maintenance of cellular homeostasis is the prime feature of this disorder rather than energetic functionality per se. 4.1. Intracellular metabolite concentrations and pH Previous MRS studies have shown that resting muscle from mdx and human dystrophinopathies exhibit many of the same abnormalities [18,19,22]. PCr/ATP and PCr/Pi are decreased and Pi/ATP is elevated. We found that utrdys also showed these abnormalities but that the changes were more severe than in mdx. The results from utr-dys are similar to those in human DMD, while the degree of abnormality in mdx more closely resembles the milder form of human dystrophin deciency, BMD. Due to problems associated with fatty inltration and brosis, it has been difcult to determine absolute concentrations of metabolites in human dystrophy. The similarity of the MRS-detectable changes between DMD, BMD and the animal models suggests that our ndings of low ATP and TCr in mdx and utr-dys are also present in the human diseases. There is a shift in bre-type distribution in DMD toward type I, oxidative, bres [14], and in utr-dys this shift is complete so that only type I bres are present [35,36]. A similar, but less marked transition occurs in mdx mice [37]. Slow-twitch (type 1) muscles tend to have lower [ATP] and [PCr] than fast muscles [3840]. MRS of rat muscle has shown that regions with a higher proportion of type I bres have a lower PCr/Pi and an increased Pi/ATP ratio [41]. However the differences we nd in metabolite ratios between control, mdx and utr-dys are too great to be due solely to bre type changes. The increased width of the Pi peak in mdx and utr-dys at rest is indicative of a wider variation in bre pH than in C57. The mdx mice showed the high intracellular pH also found in human dystrophinopathy [18] but, surprisingly, in utr-dys muscle the pH was no different from normal. In some of the utr-dys mice there was an additional, more acid Pi peak from a second, distinct bre population. Our methods are unable to determine whether these differences are due to different muscle groups, to abnormal sarcolemmal proton transport as discussed below or to degeneration per se. Another limitation is that it is not possible to differentiate between a Pi peak consisting of a small number of bres with high [Pi] or a large number of bres with relatively low [Pi]. Neither this lower pH nor the presence of the second Pi peak was due to ischaemia, which could have been caused, for example, by pressure of the 31P NMR coil on the leg surface. Preliminary experiments showed that if the muscle were made ischaemic, Pi increased over time as a single peak as the pH decreased. These parameters, however, were stable in the experiments presented here. 4.2. Oxidative metabolism The present results conrm our and others interpretation of data from human dystrophinopathy [18,22,23], that

Fig. 5. Force production during stimulation normalized to CSAg (see Fig. 1). Values are mean ^ SEM.

and extensor digitorum longus muscles [35], we also found that the times taken to reach peak twitch tension and to relax to half peak tension were signicantly slower in utr-dys than in mdx or C57. In response to the series of 30 Hz tetanic trains applied during collection of 31P MRS data, mdx and C57 muscle showed little difference in force development scaled to CSAg (Fig. 5). Over the rst 60 s there was an increase of 124 ^ 5% for mdx and 132 ^ 9% for C57 relative to the initial force of contraction, followed thereafter by a steady decline in force production to about half of the maximum. In contrast, utr-dys muscle displayed a sharp drop in force production followed after about 1 min by slowly developing fatigue. By the end of stimulation there was little difference in force production per unit CSA in the groups.

4. Discussion MRS has proved to be a powerful modality for investigating dystrophic muscle in the human, but a full characterization of the biochemistry is hampered by the difculties in obtaining biopsy material. In order to better understand the biochemical, physiological and functional parameters associated with dystrophin-related proteins, we have here combined MRS measurements with wet biochemistry and muscle contraction measurements in two dystrophic mouse models and controls. Our main ndings include substantial variations in the energetic status of this muscle at rest, but relatively normal energy production during exercise and recovery. One difculty with most in vivo MRS methods that have been employed for both human and model studies is that precise localization of the signal to specic muscle groups has not been possible, and bre-type heterogeneity will always be present. In spite of these technical limitations, our ndings clearly document a progressive severity

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257

255

oxidative activity is relatively normal during recovery when ATP turnover is high. They also conrm that [ADP] is raised at rest. This is consistent with abnormally high ATP turnover, 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 [4244]. However, the actual ADP concentration at rest appears to be startlingly high, being near the Km for oxidative ATP synthesis. It therefore seems likely that other factors such as the efciency of ATP utilization contribute to the level of mitochondrial activity. During stimulation, utr-dys muscle produced less force than C57 or mdx for a given cross-sectional area, consistent with lower net PCr depletion and a lower [ADP]. Although [ATP] was reduced by about 30% in utr-dys compared to control, the intracellular concentration was about 8 mmol l 21 and this was unaffected by exercise. 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]. After stimulation, [ADP] in all three groups of mice decreased rapidly to the pre-stimulation level. 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. (1))[31]. 4.3. Glycolytic metabolism Force production per unit CSA was almost identical in C57 and mdx during stimulation, making it possible to compare the relative contributions of glycolysis and oxidative phosphorylation to energy production in these two groups of mice. During stimulation, the fall in pH is due almost entirely to the lactic acid produced by glycolysis, modied by proton consumption from the net decrease in [PCr] (see Eq. (1)) and by proton efux. During stimulation the net decrease in [PCr] and the change in pH were both less in mdx than in C57 and proton efux was not greater than normal (discussed below). 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 ndings in the skeletal muscles of BMD patients showing reduced acidication during incremental aerobic exercise [22]. 4.4. Contraction characteristics and performance A marked feature of the response to stimulation was a lack of force potentiation in utr-dys mice in the rst minute. Force potentiation is thought to be due the increase in activity of the enzyme myosin light chain kinase, which regulates myosin phosphorylation and hence the number of active myosin-actin cross-bridges [46]. Fast muscles show greater potentiation than slow [47], and the much smaller degree of potentiation in utr-dys muscle is probably at least partly reection of a shift in bre type composition [36]. 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 bres. The overall pattern of force development in utr-dys muscle is very similar to that of individual slow motor units when stimulated intermittently [48]. A similar pattern of force production was found when an intermittent stimulation protocol was used in a DMD patient study [13]. During the rst few contractions in our experiments there was a pronounced force drop in utr-dys muscle. This phenomenon has been termed sag [49], but its cause is unknown. It is frequency dependent, and its appearance in utr-dys muscle may reect a change in force-frequency characteristics that also are associated with bre type [35]. 4.5. Regulation of intracellular pH In resting muscle of the mdx mouse the abnormal concentrations of Na 1, Ca 21 and H 1 clearly show that ionic homeostasis is abnormal [42,43]. The low rate of proton efux and slow pH recovery we found in utr-dys are additional indicators of this. 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. In previous studies it was found that pH recovery in mdx was slow after muscle stimulation [10,20,21], and that upregulation of utrophin reversed these changes [10]. Our preliminary experiments conrmed the ndings in mdx (data not shown). In the experiments presented here, a milder stimulation protocol was used, 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. The ndings strongly suggest that abnormalities in proton efux are related to the associations between the transporters and other constituents of the sarcolemmal membrane, which are more easily disrupted by muscle contraction when dystrophin is absent and are even more fragile in the absence of utrophin. It has been shown that in response to eccentric contractions, utr-dys muscle sustains a greater degree of membrane damage than does mdx [35]. Our failure to elicit slow pH recovery in BMD patients and DMD carriers [18,22] is likely to be due to the less damaging nature of muscle contraction in voluntary exercise. The results of any stimulation protocol are likely to be inuenced by muscle blood ow. Muscle contraction disturbs blood ow due a rise in intramuscular pressure [51,52]. The stimulation pattern that we employed is intermittent and was chosen in order to allow brief restoration of blood ow between contractions. The skeletal muscle of mdx mice is decient in neuronal-type nitric oxide synthase (nNOS) expression and activity [53,54], which results in abnormal modulation of a-adrenergic vasoconstriction during muscle activity [55]. If this were to lead to decient

256

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257 utrophin leads to major functional improvements in dystrophin-decient muscles of mice. Nat Med 1997;3(11):12161221. Goudemant JF, Deconinck N, Tinsley JM, et al. Expression of truncated utrophin improves pH recovery in exercising muscles of dystrophic mdx mice: a 31P NMR study. Neuromusc Disord 1998;8(6):371379. Deconinck AE, Rafael JA, Skinner JA, et al. Utrophin-dystrophindecient mice as a model for Duchenne muscular dystrophy. Cell 1997;90(4):717727. Grady RM, Teng H, Nichol MC, Cunningham JC, Wilkinson RS, Sanes JR. Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 1997;90(4):729738. Scott OM, Vrbova G, Hyde SA, Dubowitz V. Responses of muscles of patients with Duchenne muscular dystrophy to chronic electrical stimulation. J Neurol Neurosurg Psychiatry 1986;49(12):14271434. Webster C, Silberstein L, Hays AP, Blau HM. Fast muscle bers are preferentially affected in Duchenne muscular dystrophy. Cell 1988;52(4):503513. Morris CJ, Raybould JA. Histochemically demonstrable bre abnormalities in normal skeletal muscle and in muscle from carriers of Duchenne muscular dystrophy. J Neurol Neurosurg Psychiatry 1971;34(3):348352. Newman RJ, Bore PJ, Chan L, et al. Nuclear magnetic resonance studies of forearm muscle in Duchenne dystrophy. Br Med J Clin Res Ed 1982;284(6322):10721074. Younkin DP, Berman P, Sladky J, Chee C, Bank W, Chance B. 31P NMR studies in Duchenne muscular dystrophy: age-related metabolic changes. Neurology 1987;37(1):165169. Kemp GJ, Taylor DJ, Dunn JF, Frostick SP, Radda GK. Cellular energetics of dystrophic muscle. J Neurol Sci 1993;116(2):201206. Dunn JF, Frostick S, Brown G, Radda GK. Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle. Biochim Biophys Acta 1991;1096(2):115120. Dunn JF, Tracey I, Radda GK. A 31P-NMR study of muscle exercise metabolism in mdx mice: evidence for abnormal pH regulation. J Neurol Sci 1992;113(1):108113. Dunn JF, Tracey I, Radda GK. Exercise metabolism in Duchenne muscular dystrophy: a biochemical and 31P-nuclear magnetic resonance study of mdx mice. Proc R Soc Lond B Biol Sci 1993;251(1332):201206. Lodi R, Kemp GJ, Muntoni F, et al. Reduced cytosolic acidication during exercise suggests defective glycolytic activity in skeletal muscle of patients with Becker muscular dystrophy. An in vivo 31P magnetic resonance spectroscopy study. Brain 1999;122(Pt 1):121 130. Barbiroli B, Funicello R, Ferlini A, Montagna P, Zaniol P. Muscle energy metabolism in female DMD/BMD carriers: a 31P-MR spectroscopy study. Muscle Nerve 1992;15(3):344348. van der Veen JW, de Beer R, Luyten PR, van Ormondt D. Accurate quantication of in vivo 31P NMR signals using the variable projection method and prior knowledge. Magn Reson Med 1988;6(1):92 98. van den Boogart A. Quantitative data analysis of in vivo MRS data sets. Magn Reson Chem 1997;35:S146S152. van den Boogart A. MRUI Manual v96.3; a users guide to the magnetic resonance user interface software package. Delft: University of Delft, 1997. Sjogaard G, Saltin B. Extra- and intracellular water spaces in muscles of man at rest and with dynamic exercise. Am J Physiol 1982;243(3):R271R280. Kemp GJ, Radda GK. Quantitative interpretation of bioenergetic data from 31P and 1H magnetic resonance spectroscopic studies of skeletal muscle: an analytical review. Magn Reson Q 1994;10(1):4363. Veech RL, Lawson JW, Cornell NW, Krebs HA. Cytosolic phosphorylation potential. J Biol Chem 1979;254(14):65386547. Arnold DL, Matthews PM, Radda GK. Metabolic recovery after exer-

muscle perfusion during the protocol we would have expected to see a greater muscle acidosis in mdx mice during stimulation. The absence of this indicates that nNOS abnormalities are unlikely to affect the interpretation of the mdx results. A deciency of nNOS in utr-dys skeletal muscle has not, to the authors knowledge, been reported. However, cardiac data indicate that nNOS activity is depressed to a similar degree in both mdx and utr-dys [56]. If a similar situation exists in skeletal muscle, nNOS deciency would not account for the differences in exercise metabolism seen in mdx and utr-dys mice in the present study. 4.6. Conclusions We have found that the degree of abnormality in muscle metabolism and function is greater in the absence of both utrophin and dystrophin than in the absence of dystrophin alone. Considering the severity of the disorders in the two models it is clear that, in terms of skeletal muscle metabolism, utr-dys shows more of the phenotypic characteristics of DMD than mdx does. In spite of the striking changes in metabolite concentrations, ionic homeostasis and electrophysiology, we found no evidence that basic energetic processes in muscle are impaired in the absence of dystrophin or utrophin. In bringing together in vivo MRS, electrophysiology and direct biochemical analysis we have been able to investigate metabolic phenotype in a detail that is not possible in human subjects. Acknowledgements We wish to thank Mr Paul Cassidy, Dr Andrew Blamire and Dr Carsten Liess for technical assistance. References
[1] Emery AEH. Duchenne muscular dystrophy. Oxford: Oxford University Press, 1993. [2] Hoffman EP, Brown Jr RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51(6):919928. [3] Ervasti JM, Campbell KP. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 1993;122(4):809823. [4] Carlson CG. The dystrophinopathies: an alternative to the structural hypothesis. Neurobiol Dis 1998;5(1):315. [5] Tinsley JM, Blake DJ, Roche A, et al. Primary structure of dystrophin-related protein. Nature 1992;360(6404):591593. [6] Blake DJ, Tinsley JM, Davies KE. Utrophin: a structural and functional comparison to dystrophin. Brain Pathol 1996;6(1):3747. [7] Khurana TS, Watkins SC, Chafey P, et al. Immunolocalization and developmental expression of dystrophin related protein in skeletal muscle. Neuromusc Disord 1991;1(3):185194. [8] Takemitsu M, Ishiura S, Koga R, et al. Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice. Biochem Biophys Res Commun 1991;180(3):11791186. [9] Deconinck N, Tinsley J, De Backer F, et al. Expression of truncated

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18] [19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29] [30]

M.A. Cole et al. / Neuromuscular Disorders 12 (2002) 247257 cise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31P NMR. Magn Reson Med 1984;1(3):307315. Argov Z, De Stefano N, Arnold DL. ADP recovery after a brief ischemic exercise in normal and diseased human muscle a 31P MRS study. NMR Biomed 1996;9(4):165172. Kemp GJ, Taylor DJ, Styles P, Radda GK. The production, buffering and efux of protons in human skeletal muscle during exercise and recovery. NMR Biomed 1993;6(1):7383. Passoneau JV, Lowry OH. Enzymatic analysis: a practical guide. Totowa, NJ: Humana Press, 1993. Conn RB. Flourometric determination of creatine. Clin Chem 1960;6:537548. Deconinck N, Rafael JA, Beckers-Bleukx G, et al. Consequences of the combined deciency in dystrophin and utrophin on the mechanical properties and myosin composition of some limb and respiratory muscles of the mouse. Neuromusc Disord 1998;8(6):362370. Rafael JA, Townsend ER, Squire SE, Potter AC, Chamberlain JS, Davies KE. Dystrophin and utrophin inuence ber type composition and post-synaptic membrane structure. Hum Mol Genet 2000;9(9):13571367. Carnwath JW, Shotton DM. Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J Neurol Sci 1987;80(1):3954. Edstrom L, Hultman E, Sahlin K, Sjoholm H. The contents of highenergy phosphates in different bre types in skeletal muscles from rat, guinea-pig and man. J Physiol 1982;332:4758. Hintz CS, Chi MM, Fell RD, et al. Metabolite changes in individual rat muscle bers during stimulation. Am J Physiol 1982;242(3):C218C228. Meyer RA, Brown TR, Kushmerick MJ. Phosphorus nuclear magnetic resonance of fast- and slow-twitch muscle. Am J Physiol 1985;248(3 Pt 1):C279C287. Challiss RA, Blackledge MJ, Radda GK. Spatial heterogeneity of metabolism in skeletal muscle in vivo studied by 31P-NMR spectroscopy. Am J Physiol 1988;254(3 Pt 1):C417C422. Dunn JF, Radda GK. Total ion content of skeletal and cardiac muscle in the mdx mouse dystrophy: Ca 21 is elevated at all ages. J Neurol Sci 1991;103(2):226231. Dunn JF, Bannister N, Kemp GJ, Publicover SJ. Sodium is elevated in

257

[44]

[31]

[45] [46]

[32]

[33] [34] [35]

[47]

[48]

[49]

[36]

[50] [51] [52]

[37]

[38]

[53]

[39]

[40]

[54]

[41]

[55]

[42]

[56]

[43]

mdx muscles: ionic interactions in dystrophic cells. J Neurol Sci 1993;114(1):7680. Dunn JF, Burton KA, Dauncey MJ. Ouabain sensitive Na 1 /K(1)ATPase content is elevated in mdx mice: implications for the regulation of ions in dystrophic muscle. J Neurol Sci 1995;133(12):1115. Fitts RH. Cellular mechanisms of muscle fatigue. Physiol Rev 1994;74(1):4994. Grange RW, Vandenboom R, Houston ME. Physiological signicance of myosin phosphorylation in skeletal muscle. Can J Appl Physiol 1993;18(3):229242. Moore RL, Stull JT. Myosin light chain phosphorylation in fast and slow skeletal muscles in situ. Am J Physiol 1984;247(5 Pt 1):C462 C471. Burke RE, Levine DN, Tsairis P, Zajac FEd. Physiological types and histochemical proles in motor units of the cat gastrocnemius. J Physiol 1973;234(3):723748. Burke RE, Rudomin P, Zajac FEd. The effect of activation history on tension production by individual muscle units. Brain Res 1976;109(3):515529. Juel C. Lactateproton cotransport in skeletal muscle. Physiol Rev 1997;77(2):321358. Barcroft H, Millen JLE. The blood ow through muscle during sustained contraction. J Physiol 1939;97:1731. Walloe L, Wesche J. Time course and magnitude of blood ow changes in the human quadriceps muscles during and following rhythmic exercise. J Physiol 1988;405:257273. Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 1995;82(5):743 752. Chang WJ, Iannaccone ST, Lau KS, et al. Neuronal nitric oxide synthase and dystrophin-decient muscular dystrophy. Proc Natl Acad Sci USA 1996;93(17):91429147. Thomas GD, Sander M, Lau KS, Huang PL, Stull JT, Victor RG. Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-decient skeletal muscle. Proc Natl Acad Sci USA 1998;95(25):1509015095. Bia BL, Cassidy PJ, Young ME, et al. Decreased myocardial nNOS, increased iNOS and abnormal ECGs in mouse models of Duchenne muscular dystrophy. J Mol Cell Cardiol 1999;31(10):18571862.