Brain, Vol. 122, No. 1, 121-130,
January 1999
© 1999 Oxford University Press
Reduced cytosolic acidification during exercise suggests defective glycolytic activity in skeletal muscle of patients with Becker muscular dystrophy
An in vivo 31P magnetic resonance spectroscopy study
1 MRC Biochemical and Clinical Magnetic Resonance Unit, Oxford University Department of Biochemistry and Oxford Radcliffe Hospital, Oxford, 2 Department of Orthopaedic Surgery, University of Liverpool, Liverpool and 3 Neuromuscular Unit, Department of Paediatrics and Neonatal Medicine, Hammersmith Hospital, London, UK
Correspondence to:
Raffaele Lodi, MD, MRC Biochemical and Clinical Magnetic Resonance Unit, Oxford Radcliffe Hospital, Oxford OX3 9DU, UK E-mail: ral{at}bioch.ox.ac.uk
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Abstract |
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Becker muscular dystrophy is an X-linked disorder due to mutations in the dystrophin gene, resulting in reduced size and/or content of dystrophin. The functional role of this subsarcolemmal protein and the biochemical mechanisms leading to muscle necrosis in Becker muscular dystrophy are still unknown. In particular, the role of a bioenergetic deficit is still controversial. In this study, we used 31P magnetic resonance spectroscopy (31P-MRS) to investigate skeletal muscle mitochondrial and glycolytic ATP production in vivo in 14 Becker muscular dystrophy patients. Skeletal muscle glycogenolytic ATP production, measured during the first minute of exercise, was similar in patients and controls. On the other hand, during later phases of exercise, skeletal muscle in Becker muscular dystrophy patients was less acidic than in controls, the cytosolic pH at the end of exercise being significantly higher in Becker muscular dystrophy patients. The rate of proton efflux from muscle fibres of Becker muscular dystrophy patients was similar to that of controls, pointing to a deficit in glycolytic lactate production as a cause of higher end-exercise cytosolic pH in patients. The maximum rate of mitochondrial ATP production was similar in muscle of Becker muscular dystrophy patients and controls. The results of this in vivo 31P-MRS study are consistent with reduced glucose availability in dystrophin-deficient muscles.
Becker muscular dystrophy; dystrophin; magnetic resonance spectroscopy; energy metabolism; skeletal muscle
PCr = phosphocreatine; Pi = inorganic phosphate; 31P-MRS = phosphorus magnetic resonance spectroscopy
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Introduction |
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TopAbstractIntroductionSubjects and methodsResultsDiscussionAppendix: mathematical details...References |
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Duchenne and Becker muscular dystrophies are X-linked recessive allelic disorders, characterized by progressive muscle wasting and weakness, due to mutations of the gene for dystrophin (Hoffman et al., 1987
). Dystrophin, which is usually absent in Duchenne muscular dystrophy but is present in reduced amounts and/or smaller molecular weight in the milder Becker muscular dystrophy phenotype, is a subsarcolemmal protein linked to F-actin at the N-terminal end and to a group of proteins (dystrophin-associated proteins) at the C-terminus. Dystrophin-associated proteins, by binding merosin, provide a bridge between the intracellular and extracellular environment (Campbell, 1995). Although the structure and localization of dystrophin are well defined, the functional role of this protein and the biochemical mechanisms leading to muscle necrosis in Duchenne muscular dystrophy/Becker muscular dystrophy are still unknown. Evidence that the tricarboxylic acid cycle (Letelier et al., 1993) and some reactions in glycolysis are dependent on the integrity of cytoskeletal organization may suggest a role for defective energy metabolism in muscle fibre degeneration, as shown for neuronal degeneration in neurodegenerative diseases. However, studies on regulation of energy metabolism in dystrophin-deficient muscles have given conflicting results.
In the mdx mouse, the murine model of Duchenne muscular dystrophy, skeletal muscle lactate production during exercise has been reported to be normal (MacLennan et al., 1991; Dunn et al., 1992) while intracellular glycogen content has been reported to be increased (Cullen and Jaros, 1988; MacLennan et al., 1991) and the glucose flux through glycolysis greatly reduced (Even et al., 1994). There are also contradictory studies on the effect of the dystrophin deficit on oxidative phosphorylation in mdx mouse skeletal muscle, mitochondrial respiration having been reported to be normal by some (Hauser et al., 1995; Rezvani et al., 1995) or impaired by others (Glesby et al., 1988; Dunn et al., 1992; Gannoun-Zaki et al., 1995). The few studies in which mitochondrial respiration in vitro was measured in skeletal muscle of Duchenne muscular dystrophy/Becker muscular dystrophy patients have given contrasting results (Olson et al., 1968; Carroll et al., 1985; Chi et al., 1987; Vignos and Lefkowitz, 1979), and no in vivo studies have been reported in which attempts have been made to quantify skeletal muscle glycogenolytic/glycolytic activity and mitochondrial function in patients with dystrophin deficiency.
The aim of the present study was to assess in vivo the rates of oxidative and non-oxidative ATP synthesis in skeletal muscle of patients with Becker muscular dystrophy. To do so, we used phosphorus magnetic resonance spectroscopy (31P-MRS) to study different phases of the response to muscle exercise and post-exercise recovery, thereby allowing quantification of aerobic and anaerobic ATP production (Kemp et al., 1994).
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Subjects and methods |
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Subjects
We recruited 14 patients, all males (age 25 ± 4 years, mean ± SE; range 9–54 years) identified as having Becker muscular dystrophy on the basis of the clinical presentation, muscle biopsy and reduced dystrophin expression as assessed by immunohistochemistry (Table 1
).
All patients were ambulatory [groups I–IV, Archibald and Vignos scale (Archibald and Vignos, 1959)]. All were examined for deletions/duplications of the dystrophin gene, and 10 of them showed an in-frame deletion whereas four did not show any deletion/duplication (Table 1). Controls were 14 age-matched normal male subjects (age 25 ± 4 years, mean ± SE; range 9–58 years); five of these normal subjects were children or adolescents (age 12 ± 1 years; range 9 –15 years) and nine were adults (age 32 ± 4 years; range 21–58 years).
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Informed consent was obtained from each subject or their parent and studies were carried out with the approval of the Central Oxford Research Ethics Committee.
31P-MRS
31P-MRS studies were performed using a 2.0 T superconducting magnet (Oxford Magnet Technology, Eynsham, Oxford, UK) interfaced to a Bruker spectrometer (Bruker, Coventry, UK). Subjects lay supine, with a 6 cm diameter surface coil centred on the maximal circumference of the right calf muscle. Spectra were acquired using a 2 s interpulse delay at rest (64 scans) and during exercise (32 scans) and recovery. As soon as the last 32-scan exercise spectrum was collected, an additional 8-scan spectrum was also recorded, and considered `zero time' of recovery, and the exercise was stopped immediately afterwards. Data were collected for 10 min during recovery (four 8-scan spectra followed by four of 16 scans, three of 32 scans and two of 64 scans). The muscle was exercised by plantar flexion at 0.5 Hz, lifting a weight of 10% of lean body mass (calculated from body weight and skin fold thickness) (Durnin and Womersley, 1974) through a distance of 7 cm. After the first four spectra were collected, the weight was incremented by 2% of lean body mass for each subsequent spectral acquisition. Subjects exercised until they were unable either to move the weight through the required distance or to maintain the required rate. The final, 8-scan, exercise spectrum was acquired at the same workload as the previous 32-scan spectrum.
Data analysis
Relative concentrations of inorganic phosphate (Pi), phosphocreatine (PCr) and ATP were obtained by a time-domain fitting routine (VARPRO, R. de Beer, Delft, The Netherlands) and were corrected for magnetic saturation. Absolute concentrations were obtained assuming that the concentration of cytosolic ATP was 8.2 mM (i.e. mmol/l of intracellular water) (Arnold et al., 1985). Intracellular pH was calculated from the chemical shift of the Pi peak relative to PCr (
Pi, measured in parts per million), as
pH = 6.75 + log10[(Pi – 3.27)/(5.69 – Pi)].
Free cytosolic [ADP] was calculated from pH and [PCr] using a creatine kinase equilibrium constant of 1.66 x 109/M (Veech et al., 1979) and assuming a normal total creatine content of 42.5 mM (Arnold et al., 1985) as
[ADP] = {([total creatine]/[PCr]) –1}x[ATP]/(Keq[H+]).
Measurements of changes in pH and [PCr] from rest to the first exercise spectrum were used to calculate the initial rates of glycolytic ATP synthesis and PCr depletion, whose sum is a reasonable estimate of the initial rate of ATP synthesis (Kemp et al., 1994). We calculate the apparent maximum rate of glycogenolysis, from [Pi] and the glycogenolytic ATP synthesis rate, as an approximate measure of the in vivo activity of glycogen phosphorylase a during the first minute of exercise (in later exercise, glycolysis from circulating glucose may also become important).
During recovery from exercise, the initial rate of PCr re-synthesis is believed to be a good estimate of the end-exercise rate of oxidative ATP synthesis (Kemp et al., 1993b), and this has an approximately hyperbolic (Michaelis–Menten) relationship to its driving force, the cytosolic free [ADP] (Kemp et al., 1993b). Thus the end-exercise [ADP] and initial PCr re-synthesis rate can be used to calculate the maximum rate of oxidative ATP synthesis (Kemp et al., 1993b). Another index of mitochondrial function is the ADP recovery rate [assessed as half-times calculated from the slope of semilogarithmic plots (Arnold et al., 1984)]. The proton efflux rate is calculated from changes in pH and [PCr] (Kemp et al., 1993c).
Statistical analysis
Results are presented as mean ± SE. In normal subjects, phosphorylated compound concentrations and metabolic changes during exercise and recovery from exercise have been shown to be age-dependent. In particular, developmental changes (Younkin et al., 1987) and differences between children and adults have been reported (Taylor et al., 1997). In view of this, statistical comparison between patients and controls was performed using the non-parametric Mann–Whitney U test, where the assumption of a normal data distribution is not made. Statistical significance was taken as P < 0.05.
Assumptions and sources of possible error
Saturation correction
Saturation correction for metabolite concentration calculations was done using correction factors obtained in normal subjects because the acquisition in Becker muscular dystrophy patients of a fully relaxed resting spectrum with a signal-to-noise ratio comparable with that of a 64-scan unsaturated spectrum would have extended the 31P-MRS study time to an unacceptable length. Thus, we cannot exclude relaxation changes in dystrophic muscle metabolites but these are unlikely to have any major effect on our concentration calculations. This is supported by the fact that concentrations of phosphorylated compounds similar to those we find (see Results) have been reported in Becker muscular dystrophy or Duchenne muscular dystrophy patients studied using repetition times longer than our repetition time of 2 s (Younkin et al., 1987; Barbiroli et al., 1992). It should also be noted that concentrations of all metabolites, and in particular of PCr and Pi, are derived from spectral ratios to ATP, and it is extremely unlikely that the dystrophic process would affect differently the relaxation properties of different phosphorylated compounds.
ADP concentration calculation
A widely accepted method for calculating [ADP] as well as [PCr] and [Pi] in skeletal muscle is to assume constant values of [ATP] and [total creatine], obtained from biochemical assay (Arnold et al., 1985), and derive [PCr] and [Pi] from their spectral ratios with ATP and [Cr] by subtraction of [PCr] from [total creatine]. It should be noted that calculation of free [ADP] at rest is particularly prone to error as [PCr] is not very different from [total creatine], and small errors in either value give rise to substantial errors in [Cr] and hence calculated free [ADP].
Exercise load standardization
Normalization of exercise workload is a difficult and unresolved matter. Calculation of lean body mass from body weight and skin fold thickness (Durnin and Womersley, 1974) tends to overestimate it in patients with muscular dystrophy. This method allows for increased skin fold thickness, which is generally increased in dystrophic patients, but cannot allow for fat replacement of degenerated muscle fibres. The effect on our data of a relatively heavier load in Becker muscular dystrophy patients' exercises is addressed in the Discussion.
Initial exercise spectrum
In calculating the initial rate of ATP turnover, we make the assumption that both oxidative ATP synthesis and net proton efflux can, to a first approximation, be ignored. The evidence for this is the similarity of the pH and PCr changes in the first half-minute of exercise at the same rate under ischaemic and aerobic conditions (Kemp et al., 1994). Given the small (and usually positive) changes of pH during this period, the absence of proton efflux is perhaps to be expected. The small contribution of oxidative ATP synthesis is presumably due to a temporary mismatch between oxygen usage and vascular supply. Errors in these assumptions are unlikely to make any difference to the conclusion that initial exercise response is similar in Becker muscular dystrophy patients and controls.
Glycolytic activity during recovery
The interpretation of the recovery data is based on the main assumption that the post-exercise ATP synthesis is entirely oxidative, with glycolysis being switched off at its onset. Though this has not been demonstrated directly, it has been shown in an early report (Taylor et al., 1983), later confirmed by a number of studies, that if ischaemic conditions are maintained there is no PCr re-synthesis or pH change after exercise. This strongly suggests that little if any glycolytic ATP production occurs after the exercise is stopped, although it does not entirely rule it out.
[ADP] driving force of the initial PCr recovery
It has been shown (Argov et al., 1996) that in the late phase of recovery, PCr re-synthesis continues while [ADP] falls below the resting concentration, and that the overall PCr recovery rate has a strong relationship with the cytosolic pH. On the other hand, the initial rate of PCr recovery, which we calculated using the first 32 s of PCr recovery, has been shown to be independent of the cytosolic pH (Lodi et al., 1997a; Walter et al., 1997) and to have an approximately hyperbolic relationship with the [ADP] at the end of exercise (Kemp et al., 1993b). This suggests that [ADP] is the main driving force of PCr re-synthesis in the first part of recovery.
ADP recovery
Post-exercise ADP recovery follows a complex time course which is characterized by an early fast pH-independent phase (Arnold et al., 1984; Argov et al., 1996) and a later slow pH-dependent phase (Argov et al., 1996). To avoid complications due to the biphasic ADP recovery and uncertainties in assessing it precisely in late phases of recovery (late in recovery the cytosolic pH, due to the small intensity of the Pi signal, may be difficult to assess, particularly in dystrophic muscles), we calculated ADP recovery half-times using the first 32 s of ADP recovery. This first fast part of ADP recovery shows, with good approximation, a mono-exponential decline (Arnold et al., 1984; Argov et al., 1996).
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Results |
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Resting muscle
Figure 1
shows resting calf muscle spectra from patient 12 and from an age-matched control. In the patient, the PCr peak was decreased (relative to the ß-ATP peak), and the Pi peak and cytosolic pH were increased. Data from resting muscle from all the patients are reported in
Table
2.
Assuming that ATP concentration is normal in our dystrophic patients (shown in vitro by Samaha et al., 1981), [PCr] was significantly lower than in controls. Although [Pi] was clearly abnormal in some patients (e.g. Fig. 1), in the group as a whole it was not statistically different from normal. Cytosolic pH and calculated free [ADP] were increased in the patient group.
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indicate the three phosphate groups of ATP. The cytosolic pH is calculated from the chemical shift of Pi relative to PCr. The abscissa shows the chemical shift in parts per million (ppm) and ordinate the relative signal intensity.
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Exercise
Start of exercise
There were no significant differences between Becker muscular dystrophy patients and controls in the initial rate of glycolytic ATP production or in the apparent glycogenolytic capacity, which is an approximate measure of the activation of phosphorylase (i.e. the b-to-a conversion) (Table 2
).
End of exercise
Controls exercised longer than Becker muscular dystrophy patients, reaching an even lower end-exercise [PCr] (Table 2). To take account of differences in absolute exercise performances between patient and control groups, end-exercise 31P-MRS variables and exercise duration in Becker muscular dystrophy patients were compared with time points with similar PCr depletion in each age-matched control (Table 2). Patients reached a PCr depletion level (relative to the resting value) similar to controls after a significantly shorter exercise duration. Cytosolic pH and [ADP] were significantly higher than in controls (Table 2, Fig. 1). This was not only due to the higher resting values of pH and [ADP] in patients: the total fall in pH from rest to end-exercise was significantly less in patients (0.10 ± 0.03) than in controls (0.20 ± 0.04; P = 0.03) and the total rise in [ADP] was significantly increased in Becker muscular dystrophy patients (50 ± 6 versus 33 ± 4 µm in controls; P = 0.03) (Fig. 2).
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Recovery from exercise
The rate of PCr re-synthesis after exercise is a sensitive index of the rate of mitochondrial ATP production. Becker muscular dystrophy patients showed an initial rate of PCr recovery not different from controls (Table 2
). Using the ADP control model for mitochondrial respiration, we calculated the maximum rate of mitochondrial ATP synthesis from the initial rate of PCr recovery and the end-exercise [ADP] (Kemp et al., 1993b). Oxidative metabolism was also assessed as post-exercise ADP recovery half-time, a measurement independent of the absolute metabolite concentration. Both of these measures of muscle mitochondrial respiration are independent of the degree of PCr breakdown and cytosolic acidification at the end of exercise. In Becker muscular dystrophy patients, neither the maximum rate of mitochondrial ATP synthesis nor the ADP recovery half-times were significantly different from those in control subjects (Table 2).
The rate of proton efflux at the start of recovery in patients and controls was not significantly different (Table 2), suggesting that there is no up-regulation of proton extrusion mechanisms in dystrophic patients which might be responsible for the lower [H+] during exercise.
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Discussion |
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The mechanism of the progressive muscle fibre necrosis in dystrophin-deficient muscular dystrophies is still unknown, and it is also unresolved whether a deficit in muscle energy production plays a relevant role in the cascade of events starting with dystrophin deficiency and ending with muscle fibre degeneration (Vignos and Lefkowitz, 1979
; Carroll et al., 1985; Chi et al., 1987; Glesby et al., 1988; Dunn et al., 1992; Even et al., 1994; Gannoun-Zaki et al., 1995;Hauser et al., 1995; Rezvani et al., 1995). In this study, we assessed skeletal muscle oxidative and non-oxidative energy production in vivo in patients with Becker muscular dystrophy. In skeletal muscle at rest, the rate of oxidative metabolism is very low and anaerobic ATP production is virtually absent. Therefore, we designed our study to reach a sufficient rate of aerobic and anaerobic metabolism by studying patients with Becker muscular dystrophy who present a relatively limited disability compared with Duchenne muscular dystrophy patients and are more likely than Duchenne muscular dystrophy patients to accomplish the required level of exercise.
Glycogenolytic activity assessed at the start of exercise was similar in patients and controls. The first minute is the only phase of an aerobic exercise where oxidative contribution is negligible in exercise of this type (Kemp et al., 1994) and ATP is produced essentially by either glycogenolytic glycolysis or PCr breakdown. However, glycogenolytic/glycolytic activity can also be estimated indirectly in the later phases of exercise from changes in cytosolic pH. Our patient group showed a higher cytosolic pH than controls for a similar relative exercise intensity, assessed by the degree of PCr breakdown (Table 2). This was not due to the higher resting pH present in Becker muscular dystrophy patients because the fall in pH from the resting value was also significantly reduced in patients when compared with controls with a similar degree of PCr breakdown. Similar findings have been reported in other muscular disorders such as mitochondrial myopathies (Arnold et al., 1985) and myotonic dystrophy (Barnes et al., 1997). However, in mitochondrial myopathies, glycolytic activity is increased and the reduced fall in pH is due to an increased rate of proton extrusion (Arnold et al., 1985). In our Becker muscular dystrophy patients, proton efflux measured during the initial part of post-exercise recovery was not different from controls, indicating that reduced lactate production through glycolysis is the more likely explanation for the reduced acidification. The fact that this is manifest only in the later stages of the exercise suggests that this abnormality lies in the utilization of glucose rather than glycogen, and thus in the glucose transport and/or hexokinase steps.
Two alternative explanations for reduced muscle acidification in Becker muscular dystrophy patients are (i) an increase in the intrinsic intracellular buffering capacity or (ii) a higher proportion of oxidative than glycolytic fibres surviving in dystrophic muscles, resulting in a lower lactate production and lesser acidification during exercise. First, the required increase in buffer capacity in Becker muscular dystrophy muscle would need to be >50%, an implausibly large value. Secondly, the preponderance of surviving Type I (oxidative) fibres in skeletal muscle of Duchenne patients is controversial and still unresolved (Engel, 1986). We do not know the proportion of oxidative and glycolytic fibres in the specific muscle region studied by 31P-MRS in our Becker muscular dystrophy patients because this would require histochemical analysis of muscle from the same volume. However, if in our Becker muscular dystrophy patients the lower acidification during exercise were due to a selective loss of glycolytic fibres, we would expect an increased muscle oxidative capacity, which, as shown in
Table
2 and discussed below, is not the case.
The interpretation of our findings in vivo as due to a glycolytic defect is consistent with a number of observations in vitro of altered glucose metabolism in dystrophin-deficient muscle. The glucose transporter GLUT4, which co-localizes with dystrophin on the inner sarcolemmal membrane (Kahn et al., 1991), is reduced in the diaphragm of mdx mice (Olichon-Berthe et al., 1993). The diaphragm in mdx mice, in contrast to limb muscles, shows progressive degeneration and fibrosis and severe functional deficit (Stedman et al., 1991) similar to that of Duchenne muscular dystrophy/Becker muscular dystrophy skeletal muscles. A preliminary study has found that the reduction in glucose transport in hearts of mdx mice to 50% of the mean normal value is associated with a reduction of nitric oxide synthesis of 63% (Bia et al., 1997). The neuronal type of nitric oxide synthase which interacts with dystrophin and its associated proteins (syntrophins) (Brenman et al., 1996), and increases the rate of glucose transport and metabolism in skeletal muscle (Young et al., 1997), shows a reduced activity also in skeletal muscle of Becker patients (Chao et al., 1996) and mdx mice (Wink et al., 1993). In contrast to these findings of altered glucose transport, hexokinase activity has been found to be normal in dystrophin-deficient muscles, although the possible effect of the higher in vivo pH on hexokinase activity was not explored (Tracey, 1993).
Whether and how the glycolytic deficit in Becker muscular dystrophy relates to the muscle fibre necrosis is difficult to establish. It has been shown that glycolytic ATP production is used mainly for Na+/K+ ATPase activity in cultured myocytes (Hasin and Barry, 1984) and that in the heart, continuation of glycolysis during low flow ischaemia allows maintenance of Na+/K+ ATPase activity and prevents an increase in intracellular Na+ (Cross et al., 1995). These observations may suggest a link between reduced glycolytic ATP production and ionic abnormalities such as increased [Ca2+], reduced [Mg2+], and increased [Na+] and resting cytosolic pH described in dystrophin-deficient muscles (Dunn and Radda, 1991; Dunn et al., 1991). We speculate that during exercise the glycolytic deficit may be responsible for greater ionic imbalance and that this may be related to chronic skeletal muscle injury.
Skeletal muscle oxidative metabolism can be assessed using 31P-MRS during the post-exercise recovery period (Argov and Bank, 1991). When exercise is stopped, mitochondria are the only source of ATP synthesis, and the PCr re-synthesis rate reflects the rate of ATP production through oxidative phosphorylation (Taylor et al., 1983; Arnold et al., 1984). This is the only reliable way of measuring oxidative metabolism in dystrophic patients in vivo that is independent of muscle mass. Muscle mitochondrial ATP production in Becker muscular dystrophy patients was normal, as shown by the initial rate of PCr recovery and the maximum rate of mitochondrial ATP synthesis, which were similar to controls (Table 2), and by the normal post-exercise ADP recovery half-time. The significantly reduced exercise duration in Becker muscular dystrophy patients, required to reach a PCr breakdown level similar to controls, associated with normal muscle oxidative metabolism (Table 2) can be explained by a reduced muscle fibre content in our patients, as shown by fat replacement of calf muscle fibres in some of them on MRI (data not shown). The significantly increased end-exercise [ADP] (Table 2) suggests that the energy demand per single fibre is higher in patients than in controls at the same absolute workload. The normal muscle oxidative metabolism found in our Becker muscular dystrophy patients together with previous observations of normal mitochondrial ATP production in muscles of Becker muscular dystrophy/Duchenne muscular dystrophy carriers (Kemp et al., 1993a) and of patients with limb girdle muscular dystrophy due to a deficit within the sarcoglycan complex (Lodi et al., 1997b) clearly indicate that a defect of different subunits of the dystrophin-associated protein complex does not affect muscle mitochondrial function. It has been suggested that a central event in muscle fibre necrosis in dystrophin-deficient muscles is altered calcium homeostasis, with intramitochondrial Ca2+ accumulation and subsequent impairment of oxidative phosphorylation (Wrogemann and Pena, 1976). However, our present in vivo results rule out a significant role for impaired mitochondrial function in the progressive muscle fibre necrosis in our patients. This is consistent with the finding in mdx mice of normal muscle cytochrome c oxidase activity in the presence of an increased mitochondrial [Ca2+] (Reeve et al., 1997).
31P-MRS abnormalities at rest were largely similar to the abnormalities reported previously in Becker muscular dystrophy, Duchenne muscular dystrophy and symptomatic carriers by ourselves and others (Newman et al., 1982; Barbiroli et al., 1992; Kemp et al., 1993a). In Becker muscular dystrophy patients, we observed a significant reduction in [PCr] and a raised intracellular pH. Cytosolic [Pi] was increased in some patients but did not reach statistical significance. Due to low [PCr] and high pH, calculated free (metabolically active) [ADP] was increased. As ADP is a major regulator of mitochondrial respiration (Chance and Williams, 1955), raised [ADP] at rest frequently is observed in patients with primary mitochondrial myopathies (Arnold et al., 1985). However, high resting [ADP] is not necessarily an index of abnormal mitochondrial control, but could point to increased ATP turnover. Increased [ADP] at rest is observed in the absence of mitochondrial dysfunction in other muscular disorders such as in inclusion body myositis (Lodi et al., 1998). Sarcolemmal abnormalities due to dystrophin deficit are responsible for a number of ionic changes (Dunn and Radda, 1991; Dunn et al., 1991) which may require increased basal ATP consumption.
It can be seen from
Table
2 that the calculated free [ADP] in resting muscle is high, especially for the patient group, when compared with a Km (Michaelis–Menten constant) of 30 µM for ADP. Biopsy data from Duchenne muscular dystrophy patients show that muscle content of ATP is similar in patients and controls (Samaha et al., 1981). On the other hand, we do not have data on the total creatine content, and a reduction in our Becker muscular dystrophy patients cannot be excluded. However, it is important to stress that the possible errors in the calculation of metabolite concentrations do not affect the comparison of oxidative metabolism between Becker muscular dystrophy patients and controls assessed from data collected during recovery from exercise. In particular, a lower [total creatine] in the Becker muscular dystrophy patients would result in an even higher maximum rate of mitochondrial ATP synthesis (as a consequence of a lower calculated [ADP] at the end of exercise; see Subjects and methods), and the post-exercise ADP recovery half-time (Arnold et al., 1984) is a kinetic measurement independent of absolute [ADP].
In conclusion, in the present in vivo study, we have demonstrated abnormalities in patients with Becker muscular dystrophy indicating reduced skeletal muscle glycolytic activity. We have also shown that the glycolytic deficit is consistent with reduced glucose availability rather than a defect of glycogenolysis. Further studies are needed to clarify how the dystrophin deficit leads to altered anaerobic metabolism. In particular, the relationship between nitric oxide production and glucose uptake and utilization should be tested in skeletal muscles of patients with Duchenne and Becker muscular dystrophy.
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Appendix: mathematical details of analysis |
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During exercise, ATP is produced by net hydrolysis of PCr (at rate D mM/min), by glycogenolysis to lactic acid (L mM/min) and by oxidative phosphorylation (Q mM/min). As [ATP] usually remains constant in exercising muscle, total ATP synthesis rate (which is proportional to mechanical power) is given by F = Q + D + L in general, and F
D + L in the first exercise spectrum [where oxidation can, to a first approximation, be ignored under the present conditions (Kemp et al., 1994)]. Protons are produced by glycogenolysis at a rate of 2/3 mole per mole of ATP, and this must equal the rate at which protons are buffered by passive processes and `consumed' as a consequence of net PCr breakdown; in the first exercise spectrum, net proton efflux can be ignored (Kemp et al., 1994), so that L = (3/2)(jD – ßdpH/dt). In this expression, j is a factor describing the proton stoichiometry of the phosphorylation of ADP by PCr with concomitant ATP hydrolysis (Kemp et al., 1994), given approximately by 1/[1 + 10(pH-6.75)] [a recent analysis (Kushmerick, 1997) suggests that this hitherto accepted value of j is an overestimate, but use of the new value would make no significant difference to the comparisons between patients and controls reported in this paper]. This equation also contains ß, the cytosolic buffer capacity: based on 31P-MRS studies of human muscle in vivo (Kemp et al., 1993c), we take this as 20 slykes plus the calculated contribution of [Pi] (a buffer of variable concentration).
The apparent glycogenolytic capacity in early exercise (Lmax mM/min) (an approximate measure of phosphorylase a activity) is calculated as Lmax = L(1 + KPi /[Pi]), where KPi = 26 mM, the [Pi] at which phosphorylase activity is half-maximal. This quantity is designed to estimate the activity of phosphorylase a, based on two plausible assumptions: that phosphorylase activity in vivo is due mainly to phosphorylase a, and that the Pi affinity KPi is constant (at least at the low AMP concentrations calculated in the present experiments) (Ren and Hultman, 1990).
At the start of recovery, the rate of change of pH and the rate of PCr re-synthesis (V mM/min) are used to estimate the end-exercise rate of proton efflux (E mM/min) as E = (j + m)V + ßdpH/dt. In this analysis, E is estimated as the rate at which protons are `generated' by PCr re-synthesis, plus the rate at which they are `liberated' from cellular buffers as the pH rises; the factor m takes account of the small amount of net proton generation resulting from the oxidative generation of CO2. At the start of recovery, the maximum rate of oxidative ATP synthesis (Vmax mM/min) is calculated from the initial V and the end-exercise [ADP] as Vmax = V(1 + Km/[ADP]) (Kemp et al., 1993b), making use of the approximately hyperbolic relationship between oxidation rate and [ADP] and assuming a normal Km of 30 µM (Kemp et al., 1993b) [this analysis is little affected by recent reports that the relationship between oxidative ATP synthesis and [ADP] is more exactly described as sigmoidal (Jeneson et al., 1996)].
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Acknowledgments |
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We would like to thank Dr A. Male for referring some of the patients reported here and the head-teacher and children of St Thomas More Primary School (Kidlington, Oxford) for providing us with control subjects. We also would like to thank Mr D. Manners for his help with the 31P-MRS studies and Mrs E. Gower for help in the organization of the studies. This work was supported by the Medical Research Council. R.L. is a Junior Research Fellow at Wolfson College, Oxford, UK, and is supported by an EEC grant in the framework of the BIOMED programme (contract no. BMH4CT965017). R.L. held a Telethon-Italy fellowship for part of this work.
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Received June 22, 1998. Accepted August 24, 1998.
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