Skeletal muscle bioenergetics in myotonic dystrophy

Journal of the Neurological Sciences. 116 ~1993) 193-200

[ q3

1993 Elsevier Science Publishers B.V All rights reserved 0022-510X/93/$06.00 JNS 03985

Skeletal muscle bioenergetics in myotonic dystrophy D.J. Taylor a, G.J.

Kemp

a, C.G. Woods

b J.H. Edwards

b and G.K. Radda a

a MRC Biochemical and Clinical Magnetic Resonance Unit. John Radcliffe Hospital, Oxford. UK and b Department of Medical Genetics. Churchill Hospital and Genetics Laboratory, Department of Biochemistry, Oxford. UK

(Received 16 July 1992) (Revised. received 11 November. 1992) (Accepted 17 November. 1992) Key words: 31Phosphorus nuclear magnetic resonance: Spectroscopy; 31P-MRS; Myotonic dystrophy: In vivo: Human: Skeletal

muscle Summary

Skeletal muscle function of 15 patients with myotonic dystrophy (dystrophia myotonica, DM) was investigated using 3ap magnetic resonance spectroscopy to evaluate bioenergetics and intracellular pH at rest and during exercise and recovery. Results from DM patients, normal controls and mitochondrial myopathy patients were compared in order to assess the possible contribution of abnormal mitochondrial metabolism to muscle dysfunction in DM. In resting DM muscle, intracellular pH (pH ~) was normal, but there were significant elevations in the concentration ratios of PJATP, phosphomonoesters/ATP and phosphodiesters/ATP. In patients with the most severe exercise intolerance the phosphocreatine/ATP ratio was also reduced. Resting muscle of 11 mitochondrial myopathy patients showed similar changes to those of the most exercise-intolerant DM patients. In exercising DM muscle, energy stores were rapidly depleted as in mitochondrial myopathy. Muscle acidified in all subjects, but in DM the decrease in pH~ was less than in normal muscle. Recovery half-times for phosphocreatine. Pj and ADP were normal in DM but slow in mitochondrial myopathy. The initial rate of phosphocreatine repletion after exercise was rapid in DM, consistent with high [ADP], but slow in mitochondrial myopathy in spite of elevated [ADP]~Because recovery is an oxidative process, we conclude that there was no decrease in the oxidative capacity of the muscles in this group of DM patients. In the subjects in whom it could be measured, the rate of recovery of intracellular pH was greater in the 3 DM patients (0.t4.0.15 and 0.16 U/min) than in the 7 normal controls (0.08-0.12 U/min, mean 0.10). The results do not rule out a minor abnormality m glycogenolysis, but they suggest that the failure to acidify normally during exercise is probably due to rapid proton efflux.

Introduction

Myotonic dystrophy (dystrophia myotonica, DM) is a multisystem disorder which has recently been shown to result from expansion of an unstable CTG triplet repeat in a gene located on chromosome 19 (Brook et al. 1992; Fu et al. 1992; Mahadevan et al. 1992) whose product is likely to be a cAMP-dependent protein kinase (Brook et al. 1992). Although the pathophysiological mechanisms have not yet been fully defined, this has important consequences for the cell (reviewed

Correspondence to." Dr. D.J. Taylor, MRC Biochemical and Clinical Magnetic Resonance Unit, John Radcliffe Hospital. Oxford. OX3 9DU. U.K. Tel.: (0865) 221870: Fax: (0865) 221112.

in Rudel and Lehmann-Horn 1985). In muscle the concentrations of Na*, C1- and K - are altered (Edstrtm and Wroblewski, 1989), and increased K effiux during exercise has been described (Wevers et al. 1990). There is greater Na* channel activity in the sarcolemma of reseated muscle fibre segments and in cultured muscle ceils voltage-sensitive Ca 2+ influx and intracellular free Ca 2÷ concentrations are increased (Jacobs et al. 1991). The maintenance of ionic concentration gradients across the cell membrane requires energy, and abnormal muscle bioenergetics might provide part of the explanation for the myotonia and weakness in DM. In support of this, defective oxidative metabolism was implied by the presence of ragged red fibres in muscle biopsies from about half the patients in two studies (Ono et al. 1986; Nakagawa et al. 1989).

194 and the decreased m i t o c h o n d r i a l enzyme activities in muscle h o m o g e n a t e s from almost all p a t i e n t s in the latter study. Muscle wasting is obviously a major factor in the weakness and exercise intolerance eventually f o u n d in DM, but it is not known if changes in bioenergetics are also important. In order to investigate the possible c o n t r i b u t i o n of defective energetics and ionic abnormalities to muscle dysfunction in myotonic dystrophy, wc used 31phosphorus m a g n e t i c r e s o n a n c e spectroscopy ( M R S ) to assess the response of the muscle to exercise. Results are c o m p a r e d with findings from normal controls a n d from patients with previously-diagnosed m i t o c h o n d r i a l disorders. A preliminary report of some of these findings has b e e n p r e s e n t e d (Taylor et al. 1991)).

Subjects

and

Methods

~;P-MRS Spectra were o b t a i n e d from the flexor digitorum superficialis muscle in the d o m i n a n t forearm. Investigations were carried out as previously described ( A r n o l d et al. 1985) using a F o u r i e r transform spect r o m e t e r (Oxford Research Systems, Coventry, UK) interfaced with a 1.9 Tesla, 25 cm bore s u p e r c o n d u c t ing m a g n e t (Oxford M a g n e t Technology, Oxford, UK). The subject lay supine with the arm a b d u c t e d to 90 ° and positioned in the bore of the m a g n e t . T h e pulsing conditions of 2-s interpulse delay and 20-#s pulse length were chosen to optimise the 3tp signal. T h e 90 ° pulse at the centre of the 2.5 cm d i a m e t e r surface coil was 16 #s. A s p e c t r u m composed of a m i n i m u m of 128 a c c u m u l a t e d free i n d u c t i o n decays (f.i.d.s; collection time >_ 256 s) from resting muscle provided the baseline values for each individual exercise study. Each exercise s p e c t r u m was collected over 32 or 64 s. Data were acquired for 12 rain after the subject stopped exercising (4 spectra of 16 f.i.d.s followed by 4 of 32 and 2 of 64). Metabolite ratios were calculated from the peak areas of p h o s p h o c r e a t i n e (PCr), inorganic phosphate (P,). /3-ATP, p h o s p h o m o n o e s t e r s (PME), and p h o s p h o d i e s t e r s (PDE), and p H i was d e t e r m i n e d from the chemical shift difference of the P~ and PCr peaks as previously detailed ( A r n o l d et al. 1985). PCr is usually expressed as the ratio of P C r / ( P C r + Pi ) because P C r + P, r e m a i n s c o n s t a n t d u r i n g exercise and the ratio corrects for changes in signal intensity due to any m o v e m e n t of the muscle away from the coil. Free cytoplasmic A D P was calculated from the creatine kinase e q u i l i b r i u m reaction (Veech et al. 1979) assuming a creatine c o n c e n t r a t i o n of 42.5 m m o l / 1 of intracellular water and an A T P c o n c e n t r a t i o n of 8.2 m m o l / l of intraccllular water (see A r n o l d et al. 1985).

TABLE 1 PATIENT DETAILS Patient

Age

Sex

Muscle features

EMG

Eye signs

1

53

M

+ + +

+ + +

ND

2 3 4 5 6 7 8 9 If) 11 * 12 13 14 15

56 47 38 49 26 24 53 34 54 41 27 46 52 57

M F F M M F F F M M M M M M

+++ +++ + + +++ ++ ++ + + ++ ++ + + + +

+ ND + + +++ ++ ~ NI) ++ ND ++~ ND + +

+ + + ¢ND ND + + + ND ~~ + ~ ++ +

Min of exercise 0

0 1.9 2.3 2.3 3.8 5.0 6.3 7.5 10.0 11.3 11.3 12.5 12.5 ** 15.1)

Key to symbols used: (1) For muscle features, + = mild myotonia only. + + = moderate myotonia and weakness of grip, + + + myotonia and severe weakness of grip: (2) For EMG the degree of abnormality is indicated by the number of plusses; (3) For eye signs, + = lens opacity on slit lamp examination, + + = mikt cataract, + + + = severe cataract; (4) ND, not done; (5)*, Also suffering from Tourene's syndrome: (6) **. exercise data unusable for technical reasons.

Recovery half times (t~/2) werc calculated from the semilog t r a n s f o r m a t i o n of the first 4 data points in recovery, pH~ recovery was expressed as the rate of increase in pH i d u r i n g the linear p o r t i o n of the recovery curve (approx. 2 - 8 min after the end of exercise). pH~ recovery could be d e t e r m i n e d only for patients and controls in which the pH~ immediately following exercise was 6.6 or less. Initial rates of PCr resynthesis were d e t e r m i n e d from the calculated PCr c o n c e n t r a tion at the e n d of exercise and the m i d p o i n t of the first post-exercise data a c c u m u l a t i o n period (11.27 rain).

Subjects DM.

F i f t e e n patients (5 w o m e n a n d 10 m e n ) aged 2 4 - 5 7 years (mean, 44 years) were studied by 3~P-MRS. Cases were selected from those in whom the history a n d clinical findings, s u p p o r t e d by E M G a n d slit lamp examination, were entirely consistent with the diagnosis, including a family history consistent with d o m i n a n t inheritance. In most cases more than one relative had b e e n e x a m i n e d by us, or had hospital records consistent with the diagnosis. Clinical details are shown in T a b l e 1. Mitochondrial myopathy. All 11 patients in this group ( 1 2 - 4 6 years, m e a n 29 years; 6 w o m e n a n d 5 men) had good clinical a n d histological or biochemical evidence of defective m i t o c h o n d r i a l metabolism. T e n patients showed ragged red fibres in the muscle biopsy; the r e m a i n i n g p a t i e n t was f o u n d to have a m i t o c h o n -

t95 drial D N A deletion (Dr. P.M. Matthews, personal communication). Muscle from 5 of the patients showed low N A D H - C o Q reductase activity, 1 had low m a l a t e aspartate shuttle activity, 2 had a low concentration of coenzyme Q and another had high 0 2 consumption. One patient had no further biochemical studies on his muscle, but resting plasma lactate was 6.8 mM. Normal controls. Subjects were healthy men and women age 20-60 years (mean 35 years). The investigations were approved by the Oxford Ethics Committee. Informed consent was obtained from all subjects.

Exercise protocols (1) This was performed by 13 normal controls, all of the DM patients who could exercise (n = 13) and 4 patients with mitochondrial myopathy. The fingers of the dominant arm pulled a weight of 750 g through a distance of 5 cm at a rate of 40 per minute (0.25 W) for 5 min. The weight was then increased by 250 g (0.08 W) at 1.25 min intervals for a maximum duration of exercise of 15 min. If the subject became unable to move the weight the full distance at the required rate. the exercise was terminated (maximum duration, 15 min). (2) A second exercise regime was identical to Protocol 1 except that the load at the beginning of exercise was increased by 1 - 2 kg so that the changes in PCr and Pi were as great as in the patient group. This protocol was used for a group of 6 normal subjects. The heavier work load led to changes in PCr and Pi as extensive as those in the patients, making it possible to compare patients and controls under conditions of similar PCr depletion. (3) D a t a from 7 of the 11 mitochondriai myopathy patients were from a study carried out previously (Arnold et al. 1985). The exercise was similar to Protocol 1 except that a s p h y g m o m a n o m e t e r bulb was squeezed at a rate of 22 per min to a preset pressure of 100 m m H g for 5.0 min followed by 2.5 min at 300 m m H g . Most patients were not able to complete the exercise (mean duration, 4.8 __ 1.2 min).

Mitochondrial myopathy. Exercise data from the 7 patients who used Protocol 3 and from the 4 who carried out Protocol t were not significantly different from each other (see Results); they were therefore treated as a single group for comparison with DM patients and normal subjects.

Results

Resting muscle DM. The total phosphorus signal was lower than normal in most of the patients. This is illustrated in Fig. 1: Spectrum B was collected over 512 s but has a lower signal-to-noise than the Spectrum A from normal muscle which was collected over only 256 s. consistent with replacement of muscle fibres by fat a n d / o r fibrous tissue. The forearm finger flexors in both Group 1 and 2 patients had normal p H i . I n G r o u p 1 there was also normal concentration of PCr relative to A T P (Table 2). This suggests that the absolute concentrations of A T P and total creatlne in the muscle fibres were normal, since in conditions where the absolute concentrations in muscle are known to b e abnormal (such as hypothyroidism in humans (see Taylor et al. 1992) and dystrophin deficiency in the mouse (Dunn et al. 1991)), the metabolite concentration ratios determined by MRS are also abnormal. N o r m a l PCr con-

4 B

/k

2

Statistics and presentation of data Data are presented as the m e a n + SD. Significance was calculated using the non-parametric Mann-Whitney U-test. DM. Data were divided into two groups based on the patients' ability to exercise. The 9 whose duration of exercise (Protocol 1) was at least 4 min composed G r o u p 1 (for technical reasons, exercise and recovery data from one patient could not be used). Patients whose exercise duration on Protocol 1 was limited to less than 2.5 min duration (n = 5) formed G r o u p 2: two of these subjects were too weak to perform any exercise

A

A

Fig. 1. 3~p-MR spectra from flexor digitorum superficialis of a normal subject (A) and a DM patient (B). The x-axis represents the frequency of the signals from the phosphorus nuclei. Peak assignments: (l) PME: (2) Pi, (3) phosphodiesters P D E ; (4) PCr; (5-7) y, a and fl phosphate groups, respectively, of ATP. The a peak also contains signal from NAD(P) + and NAD(P)H. The y-aras represents signal intensity in arbitral~/units.

196 TABLE 2 RESTING

SKELETAL

MUSCLE Patients

Controls

Variable

Mitochondrial myopathv

DM

(7olumn: Group: II:

PHi PCr/ATP Pi/ATP PCr/P i PME/ATP PDE/ATP ADP (/xM) ((Phosphorylation Potential) ~). 1(1(' ~'

C 2 5

D

17

B 1 10

7.04 + (1.03 2.93 + 0.19 0.33 + (I.04 9.0 + 1.4 0.04_+0.05 0.01 -+0.02 9 +4

7.05 + 0.01 2.93 _+ 0.19 (I.42 _+ 0.11 * 7.5 + 1.9 * 0.11 + 0 . 1 3 (1.16+0.12 * 9 +4

7.113 -+ 0.04 2.56 -+ 0 . ,~9 ~ ** 0.55 + 0.04 ** 5.3 ± 1.3 ** //.29+0.24 ( I . 4 4 - + 0 . 1 8 ** 18 +8 *

7.04 + 2.46 + 0.41 + 7.1 ~ 0.12~ 0.14+ 24 -~

4.5

5./1 + 2 . 1

14.8

15.3 + 1 4 . 9 *

A

_+2.3

11

+ 6 . 6 **

0.06 (I.49 ** 0.13 * 4.2 * 11.05 ** (/.09 ** 16 *

** P < 0.(I3 c o m p a r e d to C o l u m n A. ** P < 0.008 c o m p a r e d to C o l u m n A. ~' P h o s p h o r y l a t i o n p o t e n t i a l = [ A T P ] / ( [ A D P ] [ P i ] ) .

centration and pH~ signifies that the free (metabolically active) ADP concentration and the phosphorylation potential were also normal. Pi and PDE signals were elevated compared to ATP, so the absolute intra-

cellular concentration of these compounds was probably high. Group 2 patients had Pi/ATP and PDE/ATP ratios that were even higher than in the Group 1 patients. In

TABLE 3 EXERCISE

AND RECOVERY

Variable

Controls

Patients DM

Column: Exercise Protocol ;1 Duration (mini P C r / I P C r + Pi) 1st min lasl min P H i , 1st rain last rain S l o p e of p H i vs I o g [ P C r / ( P C r + Pi] ADP. lastmin(v,M)

A

Mitochondrial myopathy

B

i 13 12.8 (I.78 (/.40 7.(17 6.44 1.67 26

+ 2.1 * + 0.06 _+ 0.17 + t).(13 + 0.17 + I).55

* * * **

+ 12 **

C

2 6 4.0 1/.54 0.27 6.99 6.18 2.63

D

+ 1.7 *

I 9 9.3

+ 3.8

+ + + + +

0.60 (I,25 7.07 6.77 0.57

+ + + + +

i1.//6 0.10 0.1 l * 0.18 ** 0.81 * *

0.20 (). 12 0.06 0.25 (I.27

I o r 3 ;' 11 5.3 + 1.8 O.58 {).32 7.(13 ~.77

+ 0.13 + 0.14 -+ 0.10 + I).23

19

+ II**

81

+37

82

71 33 < 16

+20 * + 1(1

38 23 _< 16

+25 + 8

28

+ 9

96 + 44 ** 62 +41 * 77 + 8 4 ** (10-225) 12 + 5 7:.

+ 20

Recovery

tl/2(sl,

PCr 1", ADP

51 32 < 18

_+22 _+ 15

Initial rate o f P C r r e s y n t h e s i s (raM/mini

19

+ 7 *

Statistical c o m p a r i s o n b

A vs. C

16

+ 4 *

B. vs. C

" 7 o n P r o t o c o l 1, 4 on P r o t o c o l 3 (see M e t h o d s ) . M e a n values for e a c h o f the two protocols are given in the text. h Significance: * P < 0.04; ** P <_ (/.006.

Cvs. D

t97 addition the P C r / A T P ratio was low. This suggests that the concentration of free ADP was high and the phosphorylation potential low. Mitochondrial myopathy. Abnormalities in resting muscle were similar to those found in the DM patients: high Pi/ATP, P D E / A T P and [ADP], low P C r / A T P . PCr/P~ and phosphorylation potential; pH~ was normal (Table 2).

Response to exerctse DM. Somewhat surprisingly, patients were not limited in our exercise regime by their myotonia even though myotonia was elicited by finger clenching in each patient on clinical examination. This may have been due in part to the type of exercise (rhythmic opening and closing of the fingers without clenching) and to the warm-up effect, which can allow normal muscle activity after the first few contractions (Cooper et al. 1988). The duration of exercise was less than normal, but the degree of PCr depletion was greater (Table 3). In order to make more meaningful comparisons between the patients and controls, 6 studies were performed on control subjects in which a heavier load was used (Protocol 2) so that the decrease in PCr and increase in P~ were not significantly different from those in the patients (Table 3, Columns B and C). The pH i decreased significantly in all patients during exercise (Table 3), indicating that glycogenolysis and glycolysis were functional. Mean p H at the end of exercise was 6.74 in the patients, significantly higher than the values of 6.45 for normal subjects carrying out the same exercise (Protocol 1) and 6.18 for normal subjects exercising at the heavier workload (Protocol 2). Mean PCr depletion in the patients was almost identical to controls working against the heavier load. Thus, in the patients the pH i decrease was less than normal for the change in PCr, so the cytoplasmic free [ADP] was higher (Table 3, Column C vs. Columns A and B; Fig. 2). When the changes in pH i and PCr are expressed as the slope of the straight line relationship between pH i and log[PCr/(PCr + Pi)] during exercise, this slope is significantly lower in patients (Table 3. Column C vs. Columns A and B). Mitochondrial myopathy. In the 4 mitochondrial myopathy patients investigated using Protocol 1 the mean values at the end of exercise of P C r / ( P C r -~ Pi), pH i and ADP (/xM) were 0.30. 6.70 and 89, respectively, not significantly different from the corresponding values of 0.25, 6.77 and 81 in DM. Corresponding results from 7 additional mitochondrial myopathy patients who had been investigated previously using a slightly different exercise regime (Protocol 3) were 0.33, 6.81 and 79, not significantly different from either the other 4 mitochondrial myopathy patients or the DM patients. The data from all 11 mitochondrial my-

50 40

•

•

e

30" Initial Rate (mM/min) 20

°0

•

•

oOO0)

&),~

O* ,,

10"

D

~

/1

a

0

e

B []

2O

ADP/ Initial Rate

15

O A

10-

D []

5 00 &D&~

..,,, ~ • c o t 0

.

20

•

[]

• •

&

II .

40

.

.

,

60

80

100

,

120

End of Exercise [ADPI (~M) Fig. 2. PCr recovery after exercise for each subject. (A) Initial rate of PCr repletion plotted against [ADP] at the end of exercise (B) H a n e s plot of the same data. showing [ADP]/tinitial rate of PCr repletion) as a function of [ADP]. In (B). the negative x-intercept is an estimate of K m (see text) and l / s l o p e is an estimate of maximum rate, Symbols: e. controls (Protocol l); A DM (Protocol 1): L~ mitochondrial myopathy (Protocol 1); L] mitochondrial myopathy (Protocol 2).

opathy patients are therefore presented as a single group in Table 3 and as individual data points in Fig. 2 (see below).

Recot~ery from exercise DM. Recovery was not slow in the DM muscle when judged by the half times (t]/2) for the r e t u r n of PCr, Pi and ADP to their resting values (Table 3, Column C). Indeed. control muscle which depleted PCr as extensively in exercise as DM muscle had a significantly slower PCr recovery half time (Table 3, Column B vs. C). The absolute PCr recovery rate. which is a measure of the net oxidative ATP synthesis rate. was also not reduced in DM (Table 3, Column C). Fig. 2A shows the approximately hyperbolic relationship between initial PCr repletion rate and [ADP] expected on the basis of metabolic control of mitochondrial ATP synthesis by [ADP] (Chance et al. 1985). Fig. 2B shows a Hanes plot of these data. Although there was little overlap in normal controls and DM patients with respect to their end-of-exercise [ADP], in the Hanes plot

198 the data points in the two groups can be seen to fall approximately on the same straight line. From this plot the K m for (ADP] activation of ATP synthesis as measured from the rate of PCr resynthesis gives a value of 20/~mol/1. Because of low signal-to-noise ratio and the modest fall in pH~ during exercise, it was possible to measure the overall rate of recovery of pH~ in only 3 of the patients. In these 3 it was significantly higher (0.14, 0.15 and 0.16 units/rain; P - - 0 . 0 1 ) than the 8 normal controls in whom it could be measured (0.10_+0.02; range: 0.08-0.12). Mitochondrial myopathy. Mean recovery half-times (t~/2) for PCr, Pi and ADP were at least twice as long for mitochondrial myopathy patients as for DM patients or for normal controls on Protocol 1 (Table 3, Column D). Even though end-of-exercise ADP concentrations were higher, initial PCr resynthesis rates were significantly slower than in both DM (Fig 2; Table 3, Column D vs. C. P = 0.001) and normal controls (Fig 2; Table 3, Column D vs. A, P = 0.009). The rate of recovery of pH~ could be measured in 6 of the 11 mitochondrial myopathy patients. Some of the individual rates were dearly fast, but the range was wide (0.08-0.23 units/mink the mean rate of 0.15 _+ 0.06 units/rain was not significantly different from normal (0.10 _+ 0.02, P = 0.11).

Discussion

There are several features of DM which might contribute to abnormal muscle cell energetics, Firstly, the myotonia prolongs contraction, utilising additional energy. Secondly, the decreased Na + gradient across the cell membrane reduces the free energy available for Na+-dependent transport processes. Thirdly, if mitochondrial metabolism, glycogenolysis or glycolysis is defective, ATP production will be affected. Degenerative processes in the muscle might also be expected to lead to changes. In DM patients with no more than moderate exercise intolerance the resting muscle was bioenergetically normal except for a mildly elevated P J A T P . In patients with severe exercise intolerance the P C r / A T P ratio and the phosphorylation potential were reduced, indicating that the energy available for contraction and other cellular work was decreased. In all cases the p H i in resting muscle remained within the normal range. This is of particular interest because in Duchenne and Becker dystrophies, one of the characteristic MRS-observable abnormalities is an increased pH~ (Kemp et al. 1991). High pH~ is also one of the major features in degenerating muscle secondary to denervation (Frostick et al. 1987). Thus the changes we find in DM cannot be attributed solely to degenerative changes in

the muscle fibres, and must be due, at least in part, to specific disease-related processes. The normal pH i in resting muscle from DM implies that the 'set-point' of the processes mediating active proton effiux, for example the N a + / H ~ antiporter, is not altered. However, the relationship between the control of resting pH~ and of pH~ during exercise is not clearly understood. Mechanisms involved in the regulation of pH in muscle disease are only beginning to be investigated, largely because of the difficulty of measuring pH in vivo and of separating the contributions of passive buffering and active proton effiux. By far the largest source of protons in exercising muscle is lactic acid produced by glycolysis with glycogen as the source of glucosyl units. This pathway was obviously intact in DM because muscle acidified significantly with exercise in all patients. The decrease in pill in DM muscle was, however, less than in normal muscle. Coupled with such a marked depletion of PCr, this must mean either that proton production was low or proton disposal more efficient than normal. Using MRS, we have shown abnormal rates of pH~ recovery in studies of hypertension (Dudley et al. 1990), hypothyroidism (Taylor et al. 1992) and mitochondrial disease (Arnold et al. 1985), probably reflecting altered activity of the Na ~/H + antiporter (Juel 1988). Although a decrease in proton production in DM cannot be ruled out entirely, the faster recovery of pH~ suggests that the latter mechanism is responsible for all or part of the diminished pH~ response to exercise. A major defect in glycogenolyis is unlikely because glycogen accumulation is not a feature of DM muscle, while it is a major finding in the known defects of glycogen utilisation such as phosphorylase and debrancher deficiencies. The high P~/ATP strongly suggests that the absolute concentration of cytoplasmic P~ was high in DM muscle at rest. Pi is an important biochemical regulator, but little is known about the control of its concentration in tissues. It is increased in resting muscle in several disorders such as mitochondrial myopathy (Arnold et al. 1985), hypothyroidism (Taylor et al. 1992) and Duchenne dystrophy (Kemp et al. 1991). Because the transport of Pi into the muscle against its electrochemical gradient is dependent on the Na ~ gradient across the cell membrane, the decreased Na ~ gradient in DM must work against an increase in intracellular P,. Thus other as yet undefined mechanisms must be responsible for the increase in cytosolic P~ concentration. Structural defects in mitochondrial DNA, abnormal histological appearance of these organelles or decreases in mitochondrial enzyme activities arc being reported in an increasing number of muscle disorders. These include hypothyroid myopathy (Khaleeli et al. 1983), chronic heart failure (Mancini et al. 1989), polymyalgia rheumatica (Hfirld et al. 1990) and venous insufficiency (Taheri et al. 1990) as well as DM (Ono et

199

al. 1986: Nakagawa et al. 1989). In each of these conditions, it may be asked whether mitochondrial dysfunction has a significant effect on muscle energetics. In D M the abnormal metabolite ratios at rest and the rapid loss of PCr and high (ADP] in exercise are consistent with (Arnold et al. 1985), but not specific for (Radda et al. 1988; Taylor et al. 19901, mitochondrial disease. Because recovery is an oxidative process, recovery times for phosphorus metabolites are sensitive indicators of mitochondrial dysfunction (Arnold et al. 1985). In the present study PCr, Pi and A D P recovered slowly in the mitochondrial myopathy patients, but in D M recovery was either normal or rapid. End-of-exercise [ADP], which was elevated in both D M and mitochondrial myopathy, is a driving force for A T P synthesis at the start of recovery, and the presence of creatine kinase in skeletal muscle means that PCr repletion represents net A T P synthesis (Taylor et al. 1983). Therefore, we compared the relationship between end-of-exercise [ADP] and the initial rate o f PCr repletion in patients and controls. This initial rate was slow in the mitochondrial myopathy group, indicating that even the elevated A D P was unable to stimulate a normal rate of A T P synthesis, while in DM muscle the rate was normal with respect to [ADP]. Normal and DM muscle had a K m for [ADP] of about 20 /xM, a value not dissimilar to other values found in this laboratory for human (30 /xM; Taylor et al. 19891 and rat muscle (at least 30 ,t~M: Brindle et al. 1989). In conclusion, our data reveal abnormalities in intracellular Pi concentration and in muscle proton accumulation during exercise, but no evidence for impaired mitochondrial or glycogenolytic function in DM. The more rapid recovery of pH in the patients suggests that the decrease in intracellular acidification of muscle during exercise is a result of enhanced proton effiux. It would be of great interesl to know how the genetic defect in DM. thought to involve a cAMP-dependent protein kinase (Brook et al. 1992 k results in the abnormalities in metabolism and proton handling which we observe. Acknowledgements

This work was supported by the Medical Research Council of Great Britain. the British Heart Foundation, the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Department of Health. We would like to thank Professor W. Pryse-Phillips, Dr. P.M. Matthews and Dr. K. Mills for EMG studies and Professor A. Bron and Mr. M Harris for eye examinations.

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