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Bioenergetics of skeletal muscle in mitochondrial myopathy
JOURNAL OF THE
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 127 (1994) 198-206
Bioenergetics of skeletal muscle in mitochondrial myopathy D . J . T a y l o r *, G . J . K e m p , G . K . R a d d a MRC Biochemical and Clinical Magnetic Resonance Unit, John Radcliffe Hospital, Oxford 0X3 9DU, UK Received 17 November 1993; revised 26 July 1994; accepted 4 August 1994
Abstract
31phosphorus nuclear magnetic resonance spectroscopy was used to examine skeletal muscle in 29 patients with mitochondrial myopathy, 9 male and 20 female. Gastrocnemius was investigated in 15 patients and 30 normal subjects and finger flexor muscle (flexor digitorum superficialis, fds) in 24 patients and 35 normal controls. Both muscles were studied in 10 of the patients. Results were abnormal (outside the full range of normal values) in all but 2 patients. In 86% of patients (25/29) abnormalities were detected in resting muscle. In most cases there was a low phosphocreatine/ATP ratio, high calculated free [ADP] and low phosphorylation potential. At rest, abnormality was detected with equal ease in fds and gastrocnemius. Exercise and recovery increased the sensitivity of MRS in detecting abnormal metabolism. Finger flexion was better tolerated by patients than plantar flexion and gave bigger changes in metabolite concentrations and intracellular pH. Thus, results from fds were more easily differentiated from normal. Exercise duration was significantly shorter than in controls while phosphocreatine depletion was more rapid than normal, consistent with a shortfall in mitochondrial ATP synthesis. Nearly all patients (25/27, 93%) showed abnormalities during recovery from exercise. [ADP] was high during exercise and its recovery was delayed, providing increased drive for oxidative phosphorylation. Phosphocreatine resynthesis during recovery (which reflects oxidative ATP synthesis) was slow both in absolute terms and in relation to [ADP]. Recovery of intracellular pH after exercise was significantly more rapid than normal, consistent with an upregulation of proton efflux. Analysis of phosphocreatine and ADP recovery data suggest that mitochondrial control is near-normal in some patients, but confirm that in the more severely affected subjects the maximum capacity of the mitochondria is reduced. Significant correlations of MRS abnormalities with the degree of muscle weakness were found at rest, during exercise and, especially, in recovery. There was no obvious relationship between the MRS findings and clinical features such as eye signs, seizures, intellectual impairment or muscle pain, or with the genetic lesion.
Keywords: Skeletal muscle; Metabolism; Mitochondria; Oxidative phosphorylation; Regulation; ATP turnover; Myopathy; Magnetic resonance spectroscopy
I. Introduction
There has been considerable progress in understanding the biochemistry (Scholte 1988) and genetics (Morgan-Hughes et al. 1990; DiMauro and Moraes 1993) of mitochondrial cytopathies, but many of the pathophysiological mechanisms remain obscure. Skele-
Abbreviations: rid, free induction decay; PCr, phosphocreatine; PHi, intracellular pH; fds, flexor digitorum superficialis; MRS, phosphorus magnetic resonance spectroscopy; MM, mitochondrial myopathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; CPEO, chronic progressive external ophthalmoplegia; KSS, Kearns-Sayre-Shy syndrome. * Corresponding author. Tel.: ( + 44-(865) 221870; Fax: (+44-865) 221112. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 2 2 - 5 1 0 X ( 9 4 ) 0 0 1 9 1 - X
tal muscle, which can increase its ATP turnover by two orders of magnitude during exercise, is the most commonly affected tissue. In exercising muscle, ATP is produced by glycolysis, mitochondrial oxidation and transfer of high energy phosphate from phosphocreatine (PCr) to ADP, while recovery after exercise is fuelled by oxidative metabolism only. 31p MRS measurements of changes in intracellular pH (pH i) and the concentrations of PCr and Pi during exercise and recovery contain information about all of these processes. We and others have assessed the utility of MRS measurements in the diagnosis of mitochondrial disease (Arnold et al, 1985; Argov et al. 1987; Matthews et al. 1991; Barbiroli et al. 1993). In this paper we compare the bioenergetic and pH i abnormalities in two different muscles, finger flexors and gastrocnemius.
7 41 38 70 7
34 19 16 12 24
36 37 12 26
27
27 39 42 19 24
46 50 14 17 22
23 65 26 28
1 2 3 4 5
6 7 8 9 10
11 12 13 14
15
16 17 18 19 20
21 22 23 24 25
26 27 28 29
F F F F
F M F M F
F F F M M
M
F F F M
F F M F F
F M M F F
Sex
N Y N N
Y Y N Y N
Y Y Y N Y
N
Y Y Y N
N Y Y Y Y
Y Y Y Y N
N N N N
N N Y N N
N N Y N N
N
N N Y N
N N N Y N
N N N N N
+ + + +
+ + + + +
+ + + + +
+
+ + + +
+ + + +
-
+ + + + + +
+ + + + + U CPEO MM MM
CPEO KSS MELAS CPEO MM
CPEO CPEO U MM CPEO
U
CPEO CPEO KSS U
NORMAL KSS CPEO MELAS CPEO
KSS CPEO CPEO CPEO MM
Y U Y Y
Y Y Y Y Y
Y Y U Y U
Y
Y N Y Y
U U Y Y Y
Y Y U U Y
High c Syndrome
blood lactate
Weakness
Eye signs
Seizures
Clinical features b
Biopsy data
Y U Y Y
Y Y Y Y Y
Y Y Y Y Y
Y
N Y Y Y
U Y N Y Y
Y Y Y Y Y
Abnormal changes in biopsy d
+ + + +
/ / / + / + /
/ + - / +/ +/ / +
I I I&IV
I&II
complex I U complex I complex I
complex U complex complex complex
complex IV cyt b complex I&II U U
/ / / + / / +
+ + + +
/ / / /
+/ + / / + +/ +/ +
+ + + +
/ / + / / / -
/ / + / / /
+/ +/ + /
+/ + /
+/ + / + / + +/ +/
+ + + +
/ +
- / +/ + / + +/ +
+ / +/ + +/
/ + + / +/ +/ / +
Reco f/g
- /
- / + / / +/ +/
+ + + -
/ +
- / +/ + / +/ +
- / +/ +/
/ +/ +/ +/ / +
Ex f/g
MRS abnormality f Rest f/g
U - / complex IV? +/ + complex I / + malate-aspartate +/ + shuttle pyruvate oxidation / +
U U complex IV comples I&IV U
complex I&IV complex l&ll U U complex I&IV
Biochemical abnormalities ~
nt 3260 mutation 5.0 kb deletion no mutation detected no mutation detected
4.977 kb deletion no studies nt 3243 mutation no mutation detected nt 3302 mutation
nt 3243 mutation nt 3243 mutation no studies no studies no mutation detected
no studies
no studies no studies 4.977 kb deletion no studies
nt 11778 point mutation 8.962 kb deletion + duplic'n 4.977 kb deletion nt 3243 mutation 4.977 kb deletion
no studies nt 3243 mutation no studies no studies no mutation detected
Mitochondrial D N A
1,2 9 2,18 2,18
2,6,14 2,6,15,16 17
1,2
13 8 2 2 6
12
10 2,11
2
4 5,9 7,8 6 9
1,2 2 2 3
Ref. g
(1982).
a Key: Y, yes; N, no; U, unknown. b Eye signs: progressive external opthalmoplegia a n d / o r ptosis. Weakness: graded from - (apparently normal strength) to + + + (severe weakness). Syndrome: see text. U = does not fit any category, or not enough information to classify. Other features: Myalgia present in cases 4, 11, 14, 15, 22, 25 and 26; impaired intellect in cases 7, 9, 13, 18 and 23. c Lactate: blood level high at rest a n d / o r in response to exercise. d Abnormal changes in biopsy: ragged red fibres or other abnormalities suggestive of mitochondrial myopathy on light or electron microscopy. e Biochemical abnormality: histochemically, muscle from patient 12 had fibres with no cytochrome oxidase, but isolated mitochondria showed normal respiration and cytochrome content. Patient 29 was assumed to have same defect present in her sister (patient 28). f MRS abnormality: f = fds, g = gastrocnemius. + or - refers to presence or absence of abnormality. Reference 1, Dr. J. Morgan-Hughes, personal communication; 2, Arnold et al. (1985); 3, Dr. J. Leonard, personal communication; 4, Dr. P.M. Matthews, personal communication; 5, Poulton et al. (1993); 9, Dr. J. Poulton, personal communication; 7, Turnbull et al. (1985); 8, Prof. D.M. Turnbull, personal communication; 6, Poulton et al. (1994); 10, Poulton et al. (1991); 11, Hayes et al. (1987); 12, Edwards et al. (1985); 13, Dr. R.M. Lane, personal communication; 14, Hayes et al. (1985); 15, Gadian et al. (1982); 16, Land et al. (1981); 17, Bindoff et al. (1993); 18, Radda et al.
Age (yrs)
Case No.
Table 1 Patients ~
7~
I
"-4
"x.
200
D.J. Taylor et al. / Journal of the Neurological Sciences 127 (1994) 198-206
2. Subjects and methods Subjects
The patients studied are described in Table 1, which gives a summary of the clinical features and laboratory results, as well as of the MRS results to be described in this paper. Patients were consecutive referrals from neurological specialists and had a clinical assessment consistent with mitochondrial myopathy. Biochemical analysis of muscle biopsies from 4 of the patients had been performed in our own laboratory using standard methods. The remainder had histological, biochemical or genetic analysis carried out by the centre from which they had been referred. References in which these patients have previously been described are given in Table 1. Patients were assigned, when possible, to categories based on the following criteria (Table 1): (1) MM, mitochondrial myopathy. Biopsy findings consistent with abnormal mitochondria; myopathic features such as easy fatiguability and exercise intolerance, but with no eye signs or other organ system apparently involved. (2) CPEO, chronic progressive external ophthalmoplegia. The features listed for MM may also be present. (3) KSS, Kearns-Sayre-Shy syndrome. As for CPEO but with involvement of other systems, typically pigmentary retinopathy, heart block, cerebellar ataxia and CSF protein above 100 m g / d l . There may be dementia, sensorineural hearing loss, endocrine pancreatic dysfunction and short stature. (4) MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. Seizures are triggered by the stroke like episodes, and sometimes dementia as well as motor and sensory deficits develop. A proximal myopathy of variable severity, retinitis pigmentosa, Fanconi anaemia, short stature and deafness may be present. Control subjects were healthy male and female volunteers (ages 20-80 years). Investigations were approved by the local ethics committee, and all subjects gave informed consent. 3;p M R S
MR spectra were collected in 1.9 Tesla superconducting magnets (Oxford Instruments, UK; 30 cm bore for finger flexors, 60 cm bore for gastrocnemius) interfaced to Biospec spectrometers (Oxford Research Systems, UK). Spectra of flexor digitorum superricialis (fds) of the dominant arm were obtained as described in Arnold et al. (1985), using a 2.5-cm surface coil placed over the muscle. Exercise was by bulb squeeze at a rate of 22 min-~ against 100 mm Hg for 5.0 rain, then against 300 mm Hg for 2.5 rain. A series of 6 spectra, each composed of 32 free induction decays (rids) with an interpulse delay of 2 sec, were ceilected during exercise. The midpoint of the first exercise spectrum was 0.5 min. Recovery data were collected
for a minimum of 10 min: 4 spectra of 16 fids followed by 4 of 32 and 2 of 64. The midpoints of the first 3 recovery spectra were 0.27, 0.83 and 1.4 rain. Spectra from the gastrocnemius were collected as previously described (Hands et al. 1986), using a 6-cm diameter surface coil beneath the muscle. Exercise was performed by plantar flexion at a rate of 30 min-J using a foot pedal to raise of weight of 10% lean body mass for 8.8 rain followed by 16% lean body mass for 3.8 min. Spectra collected during exercise were composed of 32 rids. The midpoint of the first exercise spectrum was 0.50 min. Recovery data were collected as 4 spectra of 8 rids followed by 4 of 16 rids, 3 of 32 and 2 of 64. The midpoints of the first 3 recovery spectra were 0.13, 0.51 and 0.87 min. Spectral peak areas were measured by manual triangulation. Cytosolic [P~] and [PCr] were calculated as mmol/1 cell water (mM) from the peak area ratios of Pi//3-ATP and PCr//3-ATP, pH i from the chemical shift of Pi relative to PCr, and free [ADP] as g m o l / l cell water ( g M ) from pH~ and [PCr] (Arnold et al. 1985). For statistical purposes, the phosphorylation potential is expressed as its reciprocal, ([Pi] [ADP])/ [ATP]. Recovery half times were determined by graphical interpolation. Initial rates of PCr resynthesis ( m M / m i n ) after exercise were calculated from [PCr] in the last exercise and first recovery spectra. To exhibit abnormalities in [ADP] and PCr resynthesis at the start of recovery, apparent maximal mitochondrial capacity, Vm~,x (mM/min), was calculated using the hyperbolic relationship between the initial rate of PCr synthesis and [ADP] assuming a normal Knl for ADP (30 # M ) as Vm~,x=(d[PCr]/dt){1 + (Km/[ADP])} (Kemp et al. 1993a). When signal-to-noise was adequate, the rate of pH~ recovery in the near-linear portion of the timecourse was calculated (Taylor et al. 1983). Statistics
Results in the Tables are presented as means with absolute ranges. Student's t-test (unpaired 2-tailed) was used to assess statistical difference between groups. To assess abnormalities in individual patients, we used the stringent criterion that an individual result was taken as abnormal only if it fell outside the entire range of the control values. Abnormalities were all in the direction of the change of the mean, except where indicated. Correlations between the NMR findings and muscle weakness were made using Spearman's correlation coefficient.
3. Results Resting muscle
Results were abnormal in 25 of the 29 patients (86%). In 4 out of the 24 studies on fds all values fell
D.J. Taylor et al. /Journal of the Neurological Sciences 127 (1994) 198-206
201
Table 2 Resting muscle a No. of
fds
p (MM v cont)
% abnormal
p
studies c
MM 24
control 35
MM 15
control 30
fds
gast
fds
gast
(r) b
pHi
7.04 (6.90-7.12) 0.39 (0.19-0.71) 2.51 (1.80-3.37) 6.9 (2.5-17.3) 22 (3-50) 13 (1.5-53)
7.02 (6.98-7.10) 0.34 (0.25-0.47) 3.08 (2.79-3.36) 9.5 (6.3-13.6) 6 (1-12) 2.7 (0.3-4.8)
7.05 (6.98-7.16) 0.39 (0.23-0.58) 2.56 (2.09-3.17) 6.8 (4.0-10.7) 22 (4-45) 11.1 (1.5-20.7)
7.01 (6.89-7.10) 0.31 (0.17-0.45) 2.91 (2.53-3.47) 9.8 (6.6-15.9) 14 (0-21) 5.7 (0-9.9)
0.01
0.004
8
13
0.0l
0.002
17
27
0.0001
0.001
79
60
0.0002
0.0001
50
47
0.0001
0.003
75
70
0.0001
0.0001
83
53
0.60 (0.11) 0.17 (0.29) 0.003 ( - 0.59) 0.04 (-0.42) 0.0005 (0.65) 0.005 (0.55)
Pi/ATP PCr/ATP PCr/P~ ADP (tzM) 1/(phosph potential) ×106 (M 1)
gastrocnemius
Numbers in parentheses refer to ranges. b Significance (p) and correlation coefficient (r) of fds data with respect to degree of weakness as shown in Table 1. c Total number of patients studied, 29: fds only in 14; gastrocnemius only in 5; both muscles in 10. a
within normal limits, while for gastrocnemius 3 out of 15 could not be distinguished from normal. Normal results were obtained for both muscles in only 1 of the 10 patients in whom both fds and gastrocnemius were investigated The data were grouped by muscle and results are shown in Table 2. In fds the phosphorylation potential was the best discriminator of abnormality with 20 out of 24 results being lower than the normal range. PCr/ATP and [ADP] were also abnormal in most studies. The mean pH i was significantly higher than normal in gastrocnemius but was not useful for identifying mitochondrial disease in individual patients. In gastrocnemius the abnormality most frequently found was elevated [ADP] (12 out of 15 studies), while more than half also had low PCr/ATP and phosphorylation potential. In spite of a significantly increased mean Pi/ATP, this ratio was not a good discriminator in most patients. Only 7 patients out of the total of 29 had a Pi/ATP ratio above the normal range. Table 3 Exercising muscle
The similarity in p values for the comparison between the patient and control groups for fds and for gastrocnemius suggests that detection of abnormality was independent of the muscle examined. When data from fds and gastrocnemius were compared for the 10 patients in whom both muscles were studied, 22 individual values for the variables listed in Table 2 (excluding pH i) were abnormal, while in fds this number was 25. Significant correlations were observed between the degree of weakness (Table 1) and PCr/ATP, [ADP] and l/(phosphorylation potential) in fds. Those having greater weakness showed greater abnormality (Table 2). Exercise Exercise data were from 27 of the 29 patients. Using the criteria listed in Table 3 (duration of exercise, pHi, PCr/(PCr + Pi) and [ADP]), 21 of the 27 (78%) showed abnormalities. Duration is very non-specific, however,
a
No. of studies c
fds MM 21
control 20
MM 12
control 17
fds
Duration (min)
6.1 (3.8-7.5) 6.77 (6.22-7.05) 0.37 (0.19-0.80) 83 (12-202)
7.5 (7.5) 6.74 (6.34-7.00) 0.42 (0.24-0.65) 45 (9-76)
8.5 (3.8-12.5) 6.93 (6.62-7.07) 0.53 (0.16-0.78) 70 (25-141)
12.5 (12.5) 6.90 (6.69-7.06) 0.55 (0.24-0.76) 47 (25-89)
End exercise pH PCr/(PCr + P~) ADP (/zM)
gastrocnemius
p
% abnormal
p
gast
fds
gast
(r) b
0.0001
0.0001
57
83
0.72
0.04
5
9
0.33
0.78
23
17
0.001
0.05
52
25
0.34 (0.22) 0.78 (0.06) 0.79 (-0.006) 0.08 (0.39)
a Numbers in parentheses refer to ranges. b Significance (p) and correlation coefficient (r) of fds data with respect to degree of weakness as shown in Table 1. c Total number of patients studied, 27: fds only in 15; gastrocnemius only in 6; both muscles in 6.
202
D.Z Taylor et aL /Journal of the Neurological Sciences 127 (1994) 198-200
a n d if this c r i t e r i o n is excluded, the n u m b e r a b n o r m a l d r o p s to 14 of 27 (52%). D a t a f r o m the two muscles a r e shown in T a b l e 3. E n d - o f - e x e r c i s e [PCr] a n d p H i w e r e similar in p a t i e n t s a n d controls but, n o t surprisingly, p a t i e n t s t e n d e d to r e a c h this m e t a b o l i c state faster. [ADP] was significantly g r e a t e r t h a n n o r m a l in fds but, overall, fewer a b n o r m a l i t i e s w e r e d e t e c t e d d u r i n g exercise t h a n at rest o r in recovery. W e f o u n d that m o r e u s a b l e d a t a w e r e c o l l e c t e d from fds b e c a u s e finger flexion was b e t t e r t o l e r a t e d t h a n p l a n t a r flexion, a n d l a r g e r c h a n g e s in m e t a b o l i t e c o n c e n t r a t i o n s a n d p H i w e r e a t t a i n e d . F o r e x a m p l e , o u t o f the 10 p a t i e n t s in w h o m N M R e x a m i n a t i o n s w e r e c a r r i e d out on b o t h muscles, all w e r e able to p e r f o r m several m i n u t e s of the fds exercise b u t 3 w e r e u n a b l e to carry out any of the g a s t r o c n e m i u s exercise protocol. D a t a from exercise a n d recovery w e r e actually a c q u i r e d in all 24 fds studies, but 3 of t h e m a r e not i n c l u d e d in the results b e c a u s e a d i f f e r e n t exercise p r o t o c o l was used.
PCr
ATP
Pi
~ CASE No25
Recovery
T h e r e w e r e recovery d a t a for a total of 27 p a t i e n t s , a n d of these, 25 h a d a b n o r m a l M R S findings (93%). O n e of the r e m a i n i n g 2 p a t i e n t s h a d c l e a r a b n o r m a l i ties at rest, b u t t h e o t h e r was e n t i r e l y n o r m a l with r e s p e c t to all of t h e M R S results. D a t a f r o m t h e two muscles a r e shown in T a b l e 4. R e c o v e r y half times for PCr, Pi a n d A D P w e r e significantly slower t h a n normal, reflecting inhibition o f P C r r e p l e t i o n and, thus, A T P synthesis. T h e r e l a t i o n s h i p b e t w e e n t h e end-ofexercise [ A D P ] a n d t h e initial r a t e of P C r resynthesis is p l o t t e d for fds in Fig. 1, showing t h a t in t h e m i t o c h o n drial m y o p a t h y g r o u p t h e r a t e was lower t h a n n o r m a l for a given [ADP]. U n d e r c o n d i t i o n s of c o n s t a n t p H i, P C r / P i can be t a k e n as a m e a s u r e o f [ADP], so Fig. 1 Table 4 Recovery from exercise a Number of studies d fds
Half time (s) PCr Pi
ADP Initial d[PCr]/dt (mM/min) p H i recovery rate (pH unit/min) Vmax,assuming K m is normal (mM/min)
SUBJECT
Fig. 1. Magnetic resonance spectra of resting skeletal muscle in case 25 and in a control subject. c a r r i e s i n f o r m a t i o n similar to a plot o f w o r k rate against P i / P C r in a e r o b i c exercise, which is a b n o r m a l in mitoc h o n d r i a l m y o p a t h y ( A r g o v et al. 1987). T h e p H i d e c r e a s e in g a s t r o c n e m i u s was very m o d e s t ( m e a n , 0.11 p H units at the e n d of exercise), so it was not possible to m e a s u r e p H i recovery accurately. In fds, in t h e 10 p a t i e n t s in w h o m it could be m e a s u r e d , the r a t e o f recovery of p H i was clearly g r e a t e r t h a n n o r m a l in 7 of t h e m ( T a b l e 4), suggesting that p r o t o n efflux is i n d e e d u p r e g u l a t e d in m i t o c h o n d r i a l m y o p a -
gastrocnemius
p
% abnormal b
p
MM 21
control 20
MM 10
control 17
fds
gast
fds
gast
(r)"
92 (30-240) 65 (15-150) 53 (15-225) 12 (5-37) 0.20 (0.09-0.40) 19 (5-48)
44 (15-69) 28 (10-42) 12 (14-17) 21 (10-36) 0.10 (0.08-0.12) 38 (21-61)
64 (30-190) 48 (15-190) 21 (8-40) 10 (5-16) n.a.
31 (12-51) 30 (13-42) 12 (9-20) 15 (8-21) n.a.
0.002
0.01
40
0.001
0.17
0.03
0.005
0.001
0.009
(I.003
n.a.
15 (6-24)
24 (16-33)
0.0001
0.0003
55 {11/20} 50 {9/18} 67 {10/15} 24 {8/21} 70 {7/10} 67 {14/21}
0.001 (0.67) 0.01 (0.60) 0.94 (0.02) 0.006 (-0.60) 0.05 ( - 0.63) 0.004 ( - 0.60)
30 60 50 n.a. 70
Numbers in parentheses refer to ranges. b Numbers in accolades refer to the number of abnormal findings/total number of results. Inadequate signal-to-noise prevented some variables from being measured, so these values are not available (n.a.). c Significance (p) and correlation coefficient (r) of fds data with respect to degree of weakness as shown in Table 1. d Total number of patients studied, 27: fds only in 17; gastrocnemius only in 6; both muscles in 4. a
D.Z Taylor et al. /Journal o f the Neurological Sciences 127 (1994) 198-206
203
Correlation of MRS and clinical findings ~
4o
E
3O
O
•
Ooo o
2O
i
|
0.
•
•
OO
O
o
i
i
i
i
i
50
100
150
200
250
[ADP] (p,M)
Fig. 2. Mitochondrial function during recovery. Initial recovery of PCr ( m M / min) as a function of end-exercise [ADP] in patients (e) and controls (©). The solid line shows the theoretical relationship with K m = 30 ~ M and Vmax = 40 m M / m i n .
thy. Significant correlations were observed between the degree of weakness (Table 1) and halftimes for recovery of PCr and P~, initial rate of PCr resynthesis and Vmax.
Correlation of NMR findings with biochemical and genetic abnormalities Muscle mitochondria from the majority of the patients showed a deficiency in complex I activity, often in association with deficiencies of other complexes. Results from fds of patients with complex I deficiency only (n = 4) were compared with complex I + IV deficiencies (n = 3) and with a combined group of complex I + IV and complex I + II deficiencies. There were no statistically significant differences between these groups. Of the 29 patients, mitochondrial DNA (mtDNA) analysis had been performed on 19 (Table 1). Of the 14 patients in whom a mtDNA abnormality was detected, all but 1 had MRS abnormalities. The exception was a patient with the 11778 bp point mutation associated with Leber's hereditary optic neuropathy. Her exercise and recovery results could not be included because a different exercise protocol had been used. It is of interest, however, that the patient's [ADP] was high during exercise. Furthermore, both of this patient's sons (not reported here), who had the same genetic lesion, had abnormalities in resting muscle and 1 also in exercise. A comparison of the MRS results for fds from the 6 patients with mtDNA defects in the gene for tRNA l~u (cases 2, 9, 16, 17, 25 and 26) and those from the 5 patients with the large deletions (cases 7,8,10, 21 and 27 revealed no significant differences between the groups (results were similar if only subjects with the 3243 point mutation and the 4.977 kb deletion were compared). MRS studies were abnormal in the 5 remaining patients in whom mtDNA results were negative.
The probability of detecting abnormal energetics was greater the more pronounced the muscle weakness (Tables 2-4), but there were no significant differences found associated with the presence or absence of muscle pain, external ophthalmoplegia or ptosis, seizures or intellectual impairment. There was little relationship between MRS results and the clinical presentation. The 5 MM patients showed very variable results, and included 3 of the most abnormal studies. The 12 CPEO patients varied from normal to very abnormal; most mildly abnormal studies fell into this group (for example 6 out of 7 normal Vm~x values). Results from the 4 KSS patients were all moderately abnormal, and those from the 2 MELAS patients were very abnormal.
Overall The results show that although abnormalities are detectable by MRS in the resting muscle of most patients with mitochondrial disease, the monitoring of exercise and recovery can also contribute to the detection of abnormal mitochondrial metabolism. Results from 4 of the fds studies were normal for resting muscle, but in 2 of them both exercise and recovery values were outside the normal range. For gastrocnemius, results from recovery were abnormal in 2 studies for which both rest and exercise values were within normal limits. Of the 27 patients with data for rest, exercise and recovery, 25 were abnormal in at least 2 of these 3 states, while 13 patients were abnormal for all 3. In one patient, data from rest, exercise and recovery in both muscles were all within normal limits.
4. Discussion
Detection of abnormality in mitochondrial myopathy There is considerable scatter and overlap of data from the patient and control groups. In spite of this, in only 1 of 29 patients was no NMR abnormality detected. Matthews et al. (1991) concluded from their study of gastrocnemius muscle that when used in conjunction with standard non-invasive t e s t s 31p NMR is at least as sensitive for the diagnosis of mitochondrial disease as histopathologic examination of muscle biopsy specimens. Our data are consistent with this. Matthews et al. looked only at resting gastrocnemius and found high Pi/ATP to be a common feature in mitochondrial disease, while in our study Pi/ATP was high in only a quarter of patients. The reason for this difference is not known, but the results from the present study make it clear that Pi/ATP is not a reliable indicator of mitochondrial disease, as they suggested, nor is it due to a difference between fds and gastrocnemius. It is not likely to be due to the difference in interpulse delay in
204
D.J. Tayloret aL /Journal of the Neurological Sciences 127 (1994) 198-206
the two studies since P~ and PCr are in the same cellular compartment and have very similar T~ relaxation times. Low P C r / A T P at rest was found in both studies (and by us in both fds and gastrocnemius). This finding is in itself relatively non-specific, and because the P C r / A T P ratio is ultimately incorporated into the calculations of [ADP] and phosphorylation potential it accounts for many of the abnormal results in resting muscle. In contrast, recovery of PCr, Pi and ADP after exercise are dynamic processes which rely on oxidative metabolism and are under State 3 respiratory control. Abnormalities in recovery, therefore, give more specific indications of mitochondrial dysfunction than abnormalities at rest. Half times for PCr or PCr/P~ recovery have often been used to assess mitochondrial function, and we find that sensitivity is increased by taking into account the relationship of PCr recovery rate to [ADP]. This relationship is discussed further below. We have addressed the issue raised previously of whether the sensitivity of MRS for detection of abnormal mitochondrial metabolism might be increased by investigating more than one muscle group (Matthews et ai. 1991). In resting muscle (in which respiratory control is near State 4 (Chance et al. 1985), abnormality in our patients was detected equally easily in fds and gastrocnemius. In exercise there were few patients who could be clearly differentiated from normal except for high [ADP]. This was elevated in half of all fds studies but only a quarter of gastrocnemius studies. Duration of exercise for both fds and gastrocnemius was generally shorter in patients than in control subjects, but this is not a specific finding for mitochondrial myopathy. It would be reasonable to expect the physiological stress of increased ATP demand to reveal marginal abnormalities, and our inability to differentiate clearly the patients' response from normal probably arises for several reasons: firstly, blood flow, muscle mass and other factors which affect metabolism during exercise are not easily defined precisely (and for fds the nominal work load was the same for all subjects and not tailored to individual differences); secondly, "mitochondrial reserve" permits some degree of mitochondrial impairment to be tolerated without corresponding impairment of exercise response (see below). In recovery it was easier to obtain reliable data from fds because patients tolerated finger flexion better than plantar flexion, and the substantial changes in PCr, Pi and pH i necessary to measure recovery rates were achieved more easily in fds. The degree of mitochondrial impairment necessary in order to be detectable by 3~p MRS is difficult to assess. This is partly because the patients were a heterogeneous group with respect to their mitochondrial defects. Also, they were referred from a variety of sources, so different studies had been performed on
them and the biopsies. In addition, the degree of abnormality detected in vivo by histological, histochemical or biochemical tests may not be an accurate reflection of biochemical activity in vivo. The percentage of ragged red fibres in the biopsies of patients who showed abnormalities was as low as < 5%, while in one case in whom MRS abnormalities were found, 5% of fibres were reported to have no histochemically detectable cytochrome oxidase but the isolated mitochondria showed normal respiration. We found no relationship between MRS abnormalities and clinical features with the exception of weakness, and no relationship between the MRS findings and the genetic abnormality. This is not surprising given the current poor understanding of the relationships between genetic defect, biochemical abnormality and clinical manifestations. It may be asked how specific these findings are for primary mitochondrial disease. It is clear, for example, that Vm~× and other recovery indices are impaired in other conditions as a result of decreased mitochondrial content or impaired substrate and oxygen supply (Kemp et al. 1993a), suggesting that these are markers of mitochondrial function. However, other features such as raised [ADP] in exercise (Kemp et al. 1993a), rapid pH recovery and abnormalities of resting muscle are not necessarily seen. Also, low P C r / A T P and high P i / A T P in resting muscle are not specific for mitochondrial myopathy, but dystrophic and inflammatory processes are associated with high concentrations of phosphodiesters a n d / o r phosphomonoesters (visible in the 31p NMR spectra) and often with high intracellular pH (Matthews et al. 1991; Kemp et al. 1993b). Metabolite concentrations in resting muscle
In 3 of the patients, [ATP] and [total creatine] had been measured and were within normal limits. These patients all showed abnormalities in resting and recovering muscle. Therefore, we have assumed that P i / A T P and P C r / A T P can be used to calculate [Pi] and [PCr] (Arnold et al. 1985). The regulation of phosphorus metabolite concentrations in muscle, which is not well understood, must depend on many processes including membrane transport, mitochondrial function and the constraints imposed by the presence of creatine kinase, an enzyme which operates at or near equilibrium: PCr + ADP + H + ~ ATP + creatine At steady state the concentrations of all mitochondrial regulators must adopt values at which the rate of ATP synthesis is adequate to basal needs. The most important mitochondrial regulators in skeletal muscle are [ADP] and, at the concentrations that prevail in resting muscle, also [P~] (Balaban 1990). The concentrations of [Pi] and [ADP] must be defined by the properties of state 4 mitochondrial control. In addition, [Pi] is defined by sarcolemmal Pi transport, and in-
D.J. Taylor et al. /Journal of the Neurological Sciences 127 (1994) 198-206
205
creased [Pi] appears to require increased Na, Pi cotransport (Kemp et al. 1992). PCr, pH i and ADP are in equilibrium via creatine kinase (Veech et al. 1979), and pH i is probably controlled mainly by sarcolemmal H ÷ efflux, an important component of which is the N a ÷ / H + antiport (Madshus 1988). The relationships between these processes are far from fully understood. However, abnormalities in [ADP] and [PCr] can be viewed as direct reflections of altered mitochondrial control, with [ADP] functioning as the error signal in the feedback loop that matches ATP synthesis to demand (Chance et al. 1985). Increased Pi/ATP, which has been a more prominent feature of mitochondrial myopathic muscle in other studies (Matthews et al. 1991), is at present unexplained except as an adaptation to lower phosphorylation potential and further increase mitochondrial drive.
Upregulation of proton efflux can be considered an appropriate response to a mitochondrial defect which permits high [ADP] to be maintained in the face of increased lactic acid production without the need for excessive PCr depletion. Thus, we have demonstrated that 31p MRS can, in conjunction with clinical assessment and standard laboratory methods, be helpful in the diagnosis of mitochondrial myopathy. It can also be used to provide quantitative information about ATP turnover in normal and diseased muscle. An assessment of the rate of PCr resynthesis with respect to [ADP] increases the rate of detection of mitochondrial disease, and a comparison of the rate of PCr resynthesis with end-exercise [ADP] can be used to quantify the effective size of the mitochondrial defect.
Mitochondrial A TP synthesis during recovery Data from skeletal muscle during exercise and recovery are consistent with control by [ADP] acting at the adenine nucleotide translocase of the mitochondrial membrane according to a hyperbolic relationship with apparent K m of ~ 30 /zM (see discussion and references in Kemp et al. (1993a)). The recovery data from normal muscle can be seen to be consistent with this model when the rate of PCr resynthesis (which is a quantitative measure of oxidative ATP synthesis rate) is plotted against [ADP] (Fig. 1). In the patients, PCr recovery is slow despite a normal or increased stimulus to oxidative phosphorylation, in particular a raised [ADP]. Fig. 1 shows that patients with slow PCr recovery exhibit a strikingly abnormal relationship between oxidative ATP production (as reflected in PCr repletion) and [ADP]. It can be seen from Table 4 that this altered relationship is consistent with a reduced Vma~. However, an increased g m cannot be excluded. In some of our patients, mitochondrial control could not be distinguished from normal, particularly in the gastrocnemius muscle. Animal studies show that there is sufficient mitochondrial reserve in skeletal muscle to provide adequate oxidative ATP synthesis at mitochondrial activities 40-50% of normal (Cooper et al. 1988). Clearly, an enhanced rise in [ADP] during exercise is one way in which this can be achieved. For example, with a K m of 30 /zM, the difference between end-exercise [ADP] in patients and controls reported in Table 2 could completely compensate for an approximately 30% decrease in mitochondrial capacity.
Acknowledgements
Control of pH i during exercise In spite of increased lactic acid production in mitochondrial myopathy, pH i is prevented from falling excessively during exercise. The data in Table 4 support the concept, based on rapid pH i recovery (Arnold et al. 1985), that this is due to increased proton efflux.
G.J.K. acknowledges personal support from the Muscular Dystrophy Group of Great Britain and Northern Ireland. We would like to thank Prof. D.M. Turnbull, Dr. J. Poulton, Dr. R. Lane, Dr. P. Matthews, Dr. J. Morgan-Hughes and Dr. J. Leonard for making available the results of mtDNA studies.
References Argov, Z., W.J. Bank, J. Maris, P. Peterson and B. Chance (1987) Bioenergetic heterogeneity of human mitochondrial myopathies: phosphorus magnetic resonance spectroscopy study. Neurology, 37: 257-262. Arnold, D.L., D.J. Taylor and G.K. Radda (1985) Investigations of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann. Neurol., 18: 189-196. Balaban, R. (1990) Regulation of oxidative phosphorylation in the mammalian cell. Am. J. Physiol., 258: C377-C389. Barbiroli, B., P. Montagna, P. Martinelli, R. Lodi, S. Iotti, P. Cortelli, R. Funicello and P. Zaniol (1993) Defective brain energy metabolism shown by in vivo 31p MR spectroscopy in 28 patients with mitochondrial cytopathies. J. Cereb. Blood Flow Metab., 13: 469-474. Bindoff, L.A., N. Howell, J. Poulton, D.A. McCullough, K.J. Morton, R.N. Lightowlers, C.M. Turnbull and K. Weber (1993) Abnormal RNA processing associated with a novel tRNA mutation in mitochondrial DNA. potential disease mechanism. J. Biol. Chem., 268: 19559-19564. Chance, B., J.S. Leigh, B.J. Clark, J. Maris, J. Kent, S. Nioka and D. Smith (1985) Control of oxidative metabolism and oxygen delivery in human skeletal muscle: A steady state analysis of the work/energy cost transfer function. Proc. Natl. Acad. Sci. USA, 82: 8384-8388. Cooper, J.M., R.K.H. Petty, R.A.J. Challis, M.J. Brosnan, E.A. Shoubridge, G.K. Radda, J.A. Morgan-Hughes and J.B. Clark (1988) An animal model of mitochondrial myopathy: a biochemical and physiological investigation of rats treated in vivo with the NADH-CoQ reductase inhibitor, diphenyleneiodonium. J. Neurol. Sci., 83: 335-347. DiMauro, S. and C.T. Moraes (1993) Mitochondrial encephalomyopathies. Arch. Neurol., 50: 1197-1208.
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D.J. Taylor et al. /Journal of the Neurological Sciences 127 (1994) 198-206
Hands, L.J., P.J. Bore, G. Galloway, P.J. Morris and G.K. Radda (1986) Muscle metabolism in patients with peripheral vascular disease investigated by 31P nuclear magnetic resonance spectroscopy. Clin. Sci., 71: 283-290. Hayes, D.J., D.J. Taylor, P.J. Bore, D. Hilton-Jones, D.L. Arnold, M.V. Squier, A.E. Gent and G.K. Radda (1987) An unusual metabolic myopathy: a malate-aspartate shuttle defect. J. Neurol. Sci., 82: 27-39. Hayes, D.J., D. Hilton-Jones, D.L. Arnold, G. Galloway, P. Styles, J. Duncan and G.K. Radda (1985) A mitochondrial encephalopathy. A combined 31p magnetic resonance and biochemical investigation. J. Neurol. Sci., 71: 105-118. Kemp, G.J., K.E. Polgreen and G.K. Radda (1992) Skeletal muscle Pi transport and [Pi] studied in L6 myoblasts and rabbit muscle membrane vesicles. Biochim. Biophys. Acta, 1137: 10-18. Kemp, G.J., C.H. Thompson, D.J. Taylor, L.J. Hands, B. Rajagopalan and G.K. Radda (1993a) Quantitative analysis by 31p NMR of abnormal mitochondrial oxidation in skeletal muscle during recovery from exercise. NMR Biomed., 6: 302-310. Kemp, G.J., D.J. Taylor, J.F. Dunn, S.P. Frostick and G.K. Radda (1993b) Cellular energetics of dystrophic muscle. J. Neurol. Sci., 116: 201-206. Land, J.M, J.M. Hockaday, J.T. Hughes and B.D. Ross (1981) Childhood mitochondrial myopathy with ophthalmoptegia. J. Neurol. Sci., 51: 371-382. Madshus, I.H. (1988) Regulation of intracellular pH in eukaryotic cells. Biochem. J., 250: 1-8. Matthews, P.M., C. Allaire, E.A. Shoubridge, G. Karpati, S. Carpenter and D.L. Arnold (1991) In vivo muscle magnetic resonance spectroscopy in the clinical investigation of mitochondrial disease. Neurology, 41:114-120.
Morgan-Hughes, J.A., A.H.V. Schapira, J.M. Cooper, I.J. Holt, A.E. Harding and J.G. Clark (1990) The molecular pathology of respiratory-chain dysfunction in human mitochondrial myopathies. Biochim. Biophys. Acta, 1018: 217-222. Poulton, J., M.E. Deadman, D.M. Turnbull, B. Lake and R.M. Gardiner (1991) Detection of mitochondrial DNA deletions in blood using the polymerase chain reaction: non-invasive diagnosis of mitochondrial myopathy. Clin. Genet., 39: 33-38. Poulton, J., M.E. Deadman, L. Bindoff, K. Morton, J. Land and G. Brown (1993) Families of mt DNA re-arrangements can be detected in patients with mtDNA deletions: duplications may be a transient intermediate form. Hum. Mol. Genet., 2: 23-30. Poulton, J., K.J. Morton, K. Weber, G.K. Brown and L. Bindoff (1994) Are duplications of mitochondrial DNA characteristic of Kearns-Sayre syndrome? Hum. Mol. Genet., in press. Radda, G.K., P.J. Bore, D.G. Gadian, B.D. Ross, P. Styles, D.J. Taylor and J. Morgan-Hughes (1982) 31-P NMR examination of two patients with NADH-CoQ reductase deficiency. Nature, 295: 608-609. Scholte, H.R. (1988) The biochemical basis of mitochondrial diseases. J. Bioenerg. Biomembr., 20: 161-191. Taylor, D.J., P.J. Bore, D.G. Gadian and G.K. Radda (1983) Bioenergetics of intact human muscle. A 31p nuclear magnetic resonance study. Mol. Biol. Med., 1: 77-94. Turnbull, D.M., M.A. Johnson, D.J. Dick, N.E.F. Cartlidge and H.S.A. Sherratt (1985) Partial cytochrome oxidase deficiency without subsarcolemmal accumulation of mitochondria in chronic progressive external ophthalmoplegia. J. Neurol. Sci., 70: 93-100. Veech, R.L., J.W.R. Lawson, N.W. Cornell and H.A. Krebs (1979) Cytosolic phosphorylation potential. J. Biol. Chem., 254: 65386547.
NEUROLOGICAL SCIENCES ELSEVIER
Journal of the Neurological Sciences 127 (1994) 198-206
Bioenergetics of skeletal muscle in mitochondrial myopathy D . J . T a y l o r *, G . J . K e m p , G . K . R a d d a MRC Biochemical and Clinical Magnetic Resonance Unit, John Radcliffe Hospital, Oxford 0X3 9DU, UK Received 17 November 1993; revised 26 July 1994; accepted 4 August 1994
Abstract
31phosphorus nuclear magnetic resonance spectroscopy was used to examine skeletal muscle in 29 patients with mitochondrial myopathy, 9 male and 20 female. Gastrocnemius was investigated in 15 patients and 30 normal subjects and finger flexor muscle (flexor digitorum superficialis, fds) in 24 patients and 35 normal controls. Both muscles were studied in 10 of the patients. Results were abnormal (outside the full range of normal values) in all but 2 patients. In 86% of patients (25/29) abnormalities were detected in resting muscle. In most cases there was a low phosphocreatine/ATP ratio, high calculated free [ADP] and low phosphorylation potential. At rest, abnormality was detected with equal ease in fds and gastrocnemius. Exercise and recovery increased the sensitivity of MRS in detecting abnormal metabolism. Finger flexion was better tolerated by patients than plantar flexion and gave bigger changes in metabolite concentrations and intracellular pH. Thus, results from fds were more easily differentiated from normal. Exercise duration was significantly shorter than in controls while phosphocreatine depletion was more rapid than normal, consistent with a shortfall in mitochondrial ATP synthesis. Nearly all patients (25/27, 93%) showed abnormalities during recovery from exercise. [ADP] was high during exercise and its recovery was delayed, providing increased drive for oxidative phosphorylation. Phosphocreatine resynthesis during recovery (which reflects oxidative ATP synthesis) was slow both in absolute terms and in relation to [ADP]. Recovery of intracellular pH after exercise was significantly more rapid than normal, consistent with an upregulation of proton efflux. Analysis of phosphocreatine and ADP recovery data suggest that mitochondrial control is near-normal in some patients, but confirm that in the more severely affected subjects the maximum capacity of the mitochondria is reduced. Significant correlations of MRS abnormalities with the degree of muscle weakness were found at rest, during exercise and, especially, in recovery. There was no obvious relationship between the MRS findings and clinical features such as eye signs, seizures, intellectual impairment or muscle pain, or with the genetic lesion.
Keywords: Skeletal muscle; Metabolism; Mitochondria; Oxidative phosphorylation; Regulation; ATP turnover; Myopathy; Magnetic resonance spectroscopy
I. Introduction
There has been considerable progress in understanding the biochemistry (Scholte 1988) and genetics (Morgan-Hughes et al. 1990; DiMauro and Moraes 1993) of mitochondrial cytopathies, but many of the pathophysiological mechanisms remain obscure. Skele-
Abbreviations: rid, free induction decay; PCr, phosphocreatine; PHi, intracellular pH; fds, flexor digitorum superficialis; MRS, phosphorus magnetic resonance spectroscopy; MM, mitochondrial myopathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; CPEO, chronic progressive external ophthalmoplegia; KSS, Kearns-Sayre-Shy syndrome. * Corresponding author. Tel.: ( + 44-(865) 221870; Fax: (+44-865) 221112. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 2 2 - 5 1 0 X ( 9 4 ) 0 0 1 9 1 - X
tal muscle, which can increase its ATP turnover by two orders of magnitude during exercise, is the most commonly affected tissue. In exercising muscle, ATP is produced by glycolysis, mitochondrial oxidation and transfer of high energy phosphate from phosphocreatine (PCr) to ADP, while recovery after exercise is fuelled by oxidative metabolism only. 31p MRS measurements of changes in intracellular pH (pH i) and the concentrations of PCr and Pi during exercise and recovery contain information about all of these processes. We and others have assessed the utility of MRS measurements in the diagnosis of mitochondrial disease (Arnold et al, 1985; Argov et al. 1987; Matthews et al. 1991; Barbiroli et al. 1993). In this paper we compare the bioenergetic and pH i abnormalities in two different muscles, finger flexors and gastrocnemius.
7 41 38 70 7
34 19 16 12 24
36 37 12 26
27
27 39 42 19 24
46 50 14 17 22
23 65 26 28
1 2 3 4 5
6 7 8 9 10
11 12 13 14
15
16 17 18 19 20
21 22 23 24 25
26 27 28 29
F F F F
F M F M F
F F F M M
M
F F F M
F F M F F
F M M F F
Sex
N Y N N
Y Y N Y N
Y Y Y N Y
N
Y Y Y N
N Y Y Y Y
Y Y Y Y N
N N N N
N N Y N N
N N Y N N
N
N N Y N
N N N Y N
N N N N N
+ + + +
+ + + + +
+ + + + +
+
+ + + +
+ + + +
-
+ + + + + +
+ + + + + U CPEO MM MM
CPEO KSS MELAS CPEO MM
CPEO CPEO U MM CPEO
U
CPEO CPEO KSS U
NORMAL KSS CPEO MELAS CPEO
KSS CPEO CPEO CPEO MM
Y U Y Y
Y Y Y Y Y
Y Y U Y U
Y
Y N Y Y
U U Y Y Y
Y Y U U Y
High c Syndrome
blood lactate
Weakness
Eye signs
Seizures
Clinical features b
Biopsy data
Y U Y Y
Y Y Y Y Y
Y Y Y Y Y
Y
N Y Y Y
U Y N Y Y
Y Y Y Y Y
Abnormal changes in biopsy d
+ + + +
/ / / + / + /
/ + - / +/ +/ / +
I I I&IV
I&II
complex I U complex I complex I
complex U complex complex complex
complex IV cyt b complex I&II U U
/ / / + / / +
+ + + +
/ / / /
+/ + / / + +/ +/ +
+ + + +
/ / + / / / -
/ / + / / /
+/ +/ + /
+/ + /
+/ + / + / + +/ +/
+ + + +
/ +
- / +/ + / + +/ +
+ / +/ + +/
/ + + / +/ +/ / +
Reco f/g
- /
- / + / / +/ +/
+ + + -
/ +
- / +/ + / +/ +
- / +/ +/
/ +/ +/ +/ / +
Ex f/g
MRS abnormality f Rest f/g
U - / complex IV? +/ + complex I / + malate-aspartate +/ + shuttle pyruvate oxidation / +
U U complex IV comples I&IV U
complex I&IV complex l&ll U U complex I&IV
Biochemical abnormalities ~
nt 3260 mutation 5.0 kb deletion no mutation detected no mutation detected
4.977 kb deletion no studies nt 3243 mutation no mutation detected nt 3302 mutation
nt 3243 mutation nt 3243 mutation no studies no studies no mutation detected
no studies
no studies no studies 4.977 kb deletion no studies
nt 11778 point mutation 8.962 kb deletion + duplic'n 4.977 kb deletion nt 3243 mutation 4.977 kb deletion
no studies nt 3243 mutation no studies no studies no mutation detected
Mitochondrial D N A
1,2 9 2,18 2,18
2,6,14 2,6,15,16 17
1,2
13 8 2 2 6
12
10 2,11
2
4 5,9 7,8 6 9
1,2 2 2 3
Ref. g
(1982).
a Key: Y, yes; N, no; U, unknown. b Eye signs: progressive external opthalmoplegia a n d / o r ptosis. Weakness: graded from - (apparently normal strength) to + + + (severe weakness). Syndrome: see text. U = does not fit any category, or not enough information to classify. Other features: Myalgia present in cases 4, 11, 14, 15, 22, 25 and 26; impaired intellect in cases 7, 9, 13, 18 and 23. c Lactate: blood level high at rest a n d / o r in response to exercise. d Abnormal changes in biopsy: ragged red fibres or other abnormalities suggestive of mitochondrial myopathy on light or electron microscopy. e Biochemical abnormality: histochemically, muscle from patient 12 had fibres with no cytochrome oxidase, but isolated mitochondria showed normal respiration and cytochrome content. Patient 29 was assumed to have same defect present in her sister (patient 28). f MRS abnormality: f = fds, g = gastrocnemius. + or - refers to presence or absence of abnormality. Reference 1, Dr. J. Morgan-Hughes, personal communication; 2, Arnold et al. (1985); 3, Dr. J. Leonard, personal communication; 4, Dr. P.M. Matthews, personal communication; 5, Poulton et al. (1993); 9, Dr. J. Poulton, personal communication; 7, Turnbull et al. (1985); 8, Prof. D.M. Turnbull, personal communication; 6, Poulton et al. (1994); 10, Poulton et al. (1991); 11, Hayes et al. (1987); 12, Edwards et al. (1985); 13, Dr. R.M. Lane, personal communication; 14, Hayes et al. (1985); 15, Gadian et al. (1982); 16, Land et al. (1981); 17, Bindoff et al. (1993); 18, Radda et al.
Age (yrs)
Case No.
Table 1 Patients ~
7~
I
"-4
"x.
200
D.J. Taylor et al. / Journal of the Neurological Sciences 127 (1994) 198-206
2. Subjects and methods Subjects
The patients studied are described in Table 1, which gives a summary of the clinical features and laboratory results, as well as of the MRS results to be described in this paper. Patients were consecutive referrals from neurological specialists and had a clinical assessment consistent with mitochondrial myopathy. Biochemical analysis of muscle biopsies from 4 of the patients had been performed in our own laboratory using standard methods. The remainder had histological, biochemical or genetic analysis carried out by the centre from which they had been referred. References in which these patients have previously been described are given in Table 1. Patients were assigned, when possible, to categories based on the following criteria (Table 1): (1) MM, mitochondrial myopathy. Biopsy findings consistent with abnormal mitochondria; myopathic features such as easy fatiguability and exercise intolerance, but with no eye signs or other organ system apparently involved. (2) CPEO, chronic progressive external ophthalmoplegia. The features listed for MM may also be present. (3) KSS, Kearns-Sayre-Shy syndrome. As for CPEO but with involvement of other systems, typically pigmentary retinopathy, heart block, cerebellar ataxia and CSF protein above 100 m g / d l . There may be dementia, sensorineural hearing loss, endocrine pancreatic dysfunction and short stature. (4) MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. Seizures are triggered by the stroke like episodes, and sometimes dementia as well as motor and sensory deficits develop. A proximal myopathy of variable severity, retinitis pigmentosa, Fanconi anaemia, short stature and deafness may be present. Control subjects were healthy male and female volunteers (ages 20-80 years). Investigations were approved by the local ethics committee, and all subjects gave informed consent. 3;p M R S
MR spectra were collected in 1.9 Tesla superconducting magnets (Oxford Instruments, UK; 30 cm bore for finger flexors, 60 cm bore for gastrocnemius) interfaced to Biospec spectrometers (Oxford Research Systems, UK). Spectra of flexor digitorum superricialis (fds) of the dominant arm were obtained as described in Arnold et al. (1985), using a 2.5-cm surface coil placed over the muscle. Exercise was by bulb squeeze at a rate of 22 min-~ against 100 mm Hg for 5.0 rain, then against 300 mm Hg for 2.5 rain. A series of 6 spectra, each composed of 32 free induction decays (rids) with an interpulse delay of 2 sec, were ceilected during exercise. The midpoint of the first exercise spectrum was 0.5 min. Recovery data were collected
for a minimum of 10 min: 4 spectra of 16 fids followed by 4 of 32 and 2 of 64. The midpoints of the first 3 recovery spectra were 0.27, 0.83 and 1.4 rain. Spectra from the gastrocnemius were collected as previously described (Hands et al. 1986), using a 6-cm diameter surface coil beneath the muscle. Exercise was performed by plantar flexion at a rate of 30 min-J using a foot pedal to raise of weight of 10% lean body mass for 8.8 rain followed by 16% lean body mass for 3.8 min. Spectra collected during exercise were composed of 32 rids. The midpoint of the first exercise spectrum was 0.50 min. Recovery data were collected as 4 spectra of 8 rids followed by 4 of 16 rids, 3 of 32 and 2 of 64. The midpoints of the first 3 recovery spectra were 0.13, 0.51 and 0.87 min. Spectral peak areas were measured by manual triangulation. Cytosolic [P~] and [PCr] were calculated as mmol/1 cell water (mM) from the peak area ratios of Pi//3-ATP and PCr//3-ATP, pH i from the chemical shift of Pi relative to PCr, and free [ADP] as g m o l / l cell water ( g M ) from pH~ and [PCr] (Arnold et al. 1985). For statistical purposes, the phosphorylation potential is expressed as its reciprocal, ([Pi] [ADP])/ [ATP]. Recovery half times were determined by graphical interpolation. Initial rates of PCr resynthesis ( m M / m i n ) after exercise were calculated from [PCr] in the last exercise and first recovery spectra. To exhibit abnormalities in [ADP] and PCr resynthesis at the start of recovery, apparent maximal mitochondrial capacity, Vm~,x (mM/min), was calculated using the hyperbolic relationship between the initial rate of PCr synthesis and [ADP] assuming a normal Knl for ADP (30 # M ) as Vm~,x=(d[PCr]/dt){1 + (Km/[ADP])} (Kemp et al. 1993a). When signal-to-noise was adequate, the rate of pH~ recovery in the near-linear portion of the timecourse was calculated (Taylor et al. 1983). Statistics
Results in the Tables are presented as means with absolute ranges. Student's t-test (unpaired 2-tailed) was used to assess statistical difference between groups. To assess abnormalities in individual patients, we used the stringent criterion that an individual result was taken as abnormal only if it fell outside the entire range of the control values. Abnormalities were all in the direction of the change of the mean, except where indicated. Correlations between the NMR findings and muscle weakness were made using Spearman's correlation coefficient.
3. Results Resting muscle
Results were abnormal in 25 of the 29 patients (86%). In 4 out of the 24 studies on fds all values fell
D.J. Taylor et al. /Journal of the Neurological Sciences 127 (1994) 198-206
201
Table 2 Resting muscle a No. of
fds
p (MM v cont)
% abnormal
p
studies c
MM 24
control 35
MM 15
control 30
fds
gast
fds
gast
(r) b
pHi
7.04 (6.90-7.12) 0.39 (0.19-0.71) 2.51 (1.80-3.37) 6.9 (2.5-17.3) 22 (3-50) 13 (1.5-53)
7.02 (6.98-7.10) 0.34 (0.25-0.47) 3.08 (2.79-3.36) 9.5 (6.3-13.6) 6 (1-12) 2.7 (0.3-4.8)
7.05 (6.98-7.16) 0.39 (0.23-0.58) 2.56 (2.09-3.17) 6.8 (4.0-10.7) 22 (4-45) 11.1 (1.5-20.7)
7.01 (6.89-7.10) 0.31 (0.17-0.45) 2.91 (2.53-3.47) 9.8 (6.6-15.9) 14 (0-21) 5.7 (0-9.9)
0.01
0.004
8
13
0.0l
0.002
17
27
0.0001
0.001
79
60
0.0002
0.0001
50
47
0.0001
0.003
75
70
0.0001
0.0001
83
53
0.60 (0.11) 0.17 (0.29) 0.003 ( - 0.59) 0.04 (-0.42) 0.0005 (0.65) 0.005 (0.55)
Pi/ATP PCr/ATP PCr/P~ ADP (tzM) 1/(phosph potential) ×106 (M 1)
gastrocnemius
Numbers in parentheses refer to ranges. b Significance (p) and correlation coefficient (r) of fds data with respect to degree of weakness as shown in Table 1. c Total number of patients studied, 29: fds only in 14; gastrocnemius only in 5; both muscles in 10. a
within normal limits, while for gastrocnemius 3 out of 15 could not be distinguished from normal. Normal results were obtained for both muscles in only 1 of the 10 patients in whom both fds and gastrocnemius were investigated The data were grouped by muscle and results are shown in Table 2. In fds the phosphorylation potential was the best discriminator of abnormality with 20 out of 24 results being lower than the normal range. PCr/ATP and [ADP] were also abnormal in most studies. The mean pH i was significantly higher than normal in gastrocnemius but was not useful for identifying mitochondrial disease in individual patients. In gastrocnemius the abnormality most frequently found was elevated [ADP] (12 out of 15 studies), while more than half also had low PCr/ATP and phosphorylation potential. In spite of a significantly increased mean Pi/ATP, this ratio was not a good discriminator in most patients. Only 7 patients out of the total of 29 had a Pi/ATP ratio above the normal range. Table 3 Exercising muscle
The similarity in p values for the comparison between the patient and control groups for fds and for gastrocnemius suggests that detection of abnormality was independent of the muscle examined. When data from fds and gastrocnemius were compared for the 10 patients in whom both muscles were studied, 22 individual values for the variables listed in Table 2 (excluding pH i) were abnormal, while in fds this number was 25. Significant correlations were observed between the degree of weakness (Table 1) and PCr/ATP, [ADP] and l/(phosphorylation potential) in fds. Those having greater weakness showed greater abnormality (Table 2). Exercise Exercise data were from 27 of the 29 patients. Using the criteria listed in Table 3 (duration of exercise, pHi, PCr/(PCr + Pi) and [ADP]), 21 of the 27 (78%) showed abnormalities. Duration is very non-specific, however,
a
No. of studies c
fds MM 21
control 20
MM 12
control 17
fds
Duration (min)
6.1 (3.8-7.5) 6.77 (6.22-7.05) 0.37 (0.19-0.80) 83 (12-202)
7.5 (7.5) 6.74 (6.34-7.00) 0.42 (0.24-0.65) 45 (9-76)
8.5 (3.8-12.5) 6.93 (6.62-7.07) 0.53 (0.16-0.78) 70 (25-141)
12.5 (12.5) 6.90 (6.69-7.06) 0.55 (0.24-0.76) 47 (25-89)
End exercise pH PCr/(PCr + P~) ADP (/zM)
gastrocnemius
p
% abnormal
p
gast
fds
gast
(r) b
0.0001
0.0001
57
83
0.72
0.04
5
9
0.33
0.78
23
17
0.001
0.05
52
25
0.34 (0.22) 0.78 (0.06) 0.79 (-0.006) 0.08 (0.39)
a Numbers in parentheses refer to ranges. b Significance (p) and correlation coefficient (r) of fds data with respect to degree of weakness as shown in Table 1. c Total number of patients studied, 27: fds only in 15; gastrocnemius only in 6; both muscles in 6.
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a n d if this c r i t e r i o n is excluded, the n u m b e r a b n o r m a l d r o p s to 14 of 27 (52%). D a t a f r o m the two muscles a r e shown in T a b l e 3. E n d - o f - e x e r c i s e [PCr] a n d p H i w e r e similar in p a t i e n t s a n d controls but, n o t surprisingly, p a t i e n t s t e n d e d to r e a c h this m e t a b o l i c state faster. [ADP] was significantly g r e a t e r t h a n n o r m a l in fds but, overall, fewer a b n o r m a l i t i e s w e r e d e t e c t e d d u r i n g exercise t h a n at rest o r in recovery. W e f o u n d that m o r e u s a b l e d a t a w e r e c o l l e c t e d from fds b e c a u s e finger flexion was b e t t e r t o l e r a t e d t h a n p l a n t a r flexion, a n d l a r g e r c h a n g e s in m e t a b o l i t e c o n c e n t r a t i o n s a n d p H i w e r e a t t a i n e d . F o r e x a m p l e , o u t o f the 10 p a t i e n t s in w h o m N M R e x a m i n a t i o n s w e r e c a r r i e d out on b o t h muscles, all w e r e able to p e r f o r m several m i n u t e s of the fds exercise b u t 3 w e r e u n a b l e to carry out any of the g a s t r o c n e m i u s exercise protocol. D a t a from exercise a n d recovery w e r e actually a c q u i r e d in all 24 fds studies, but 3 of t h e m a r e not i n c l u d e d in the results b e c a u s e a d i f f e r e n t exercise p r o t o c o l was used.
PCr
ATP
Pi
~ CASE No25
Recovery
T h e r e w e r e recovery d a t a for a total of 27 p a t i e n t s , a n d of these, 25 h a d a b n o r m a l M R S findings (93%). O n e of the r e m a i n i n g 2 p a t i e n t s h a d c l e a r a b n o r m a l i ties at rest, b u t t h e o t h e r was e n t i r e l y n o r m a l with r e s p e c t to all of t h e M R S results. D a t a f r o m t h e two muscles a r e shown in T a b l e 4. R e c o v e r y half times for PCr, Pi a n d A D P w e r e significantly slower t h a n normal, reflecting inhibition o f P C r r e p l e t i o n and, thus, A T P synthesis. T h e r e l a t i o n s h i p b e t w e e n t h e end-ofexercise [ A D P ] a n d t h e initial r a t e of P C r resynthesis is p l o t t e d for fds in Fig. 1, showing t h a t in t h e m i t o c h o n drial m y o p a t h y g r o u p t h e r a t e was lower t h a n n o r m a l for a given [ADP]. U n d e r c o n d i t i o n s of c o n s t a n t p H i, P C r / P i can be t a k e n as a m e a s u r e o f [ADP], so Fig. 1 Table 4 Recovery from exercise a Number of studies d fds
Half time (s) PCr Pi
ADP Initial d[PCr]/dt (mM/min) p H i recovery rate (pH unit/min) Vmax,assuming K m is normal (mM/min)
SUBJECT
Fig. 1. Magnetic resonance spectra of resting skeletal muscle in case 25 and in a control subject. c a r r i e s i n f o r m a t i o n similar to a plot o f w o r k rate against P i / P C r in a e r o b i c exercise, which is a b n o r m a l in mitoc h o n d r i a l m y o p a t h y ( A r g o v et al. 1987). T h e p H i d e c r e a s e in g a s t r o c n e m i u s was very m o d e s t ( m e a n , 0.11 p H units at the e n d of exercise), so it was not possible to m e a s u r e p H i recovery accurately. In fds, in t h e 10 p a t i e n t s in w h o m it could be m e a s u r e d , the r a t e o f recovery of p H i was clearly g r e a t e r t h a n n o r m a l in 7 of t h e m ( T a b l e 4), suggesting that p r o t o n efflux is i n d e e d u p r e g u l a t e d in m i t o c h o n d r i a l m y o p a -
gastrocnemius
p
% abnormal b
p
MM 21
control 20
MM 10
control 17
fds
gast
fds
gast
(r)"
92 (30-240) 65 (15-150) 53 (15-225) 12 (5-37) 0.20 (0.09-0.40) 19 (5-48)
44 (15-69) 28 (10-42) 12 (14-17) 21 (10-36) 0.10 (0.08-0.12) 38 (21-61)
64 (30-190) 48 (15-190) 21 (8-40) 10 (5-16) n.a.
31 (12-51) 30 (13-42) 12 (9-20) 15 (8-21) n.a.
0.002
0.01
40
0.001
0.17
0.03
0.005
0.001
0.009
(I.003
n.a.
15 (6-24)
24 (16-33)
0.0001
0.0003
55 {11/20} 50 {9/18} 67 {10/15} 24 {8/21} 70 {7/10} 67 {14/21}
0.001 (0.67) 0.01 (0.60) 0.94 (0.02) 0.006 (-0.60) 0.05 ( - 0.63) 0.004 ( - 0.60)
30 60 50 n.a. 70
Numbers in parentheses refer to ranges. b Numbers in accolades refer to the number of abnormal findings/total number of results. Inadequate signal-to-noise prevented some variables from being measured, so these values are not available (n.a.). c Significance (p) and correlation coefficient (r) of fds data with respect to degree of weakness as shown in Table 1. d Total number of patients studied, 27: fds only in 17; gastrocnemius only in 6; both muscles in 4. a
D.Z Taylor et al. /Journal o f the Neurological Sciences 127 (1994) 198-206
203
Correlation of MRS and clinical findings ~
4o
E
3O
O
•
Ooo o
2O
i
|
0.
•
•
OO
O
o
i
i
i
i
i
50
100
150
200
250
[ADP] (p,M)
Fig. 2. Mitochondrial function during recovery. Initial recovery of PCr ( m M / min) as a function of end-exercise [ADP] in patients (e) and controls (©). The solid line shows the theoretical relationship with K m = 30 ~ M and Vmax = 40 m M / m i n .
thy. Significant correlations were observed between the degree of weakness (Table 1) and halftimes for recovery of PCr and P~, initial rate of PCr resynthesis and Vmax.
Correlation of NMR findings with biochemical and genetic abnormalities Muscle mitochondria from the majority of the patients showed a deficiency in complex I activity, often in association with deficiencies of other complexes. Results from fds of patients with complex I deficiency only (n = 4) were compared with complex I + IV deficiencies (n = 3) and with a combined group of complex I + IV and complex I + II deficiencies. There were no statistically significant differences between these groups. Of the 29 patients, mitochondrial DNA (mtDNA) analysis had been performed on 19 (Table 1). Of the 14 patients in whom a mtDNA abnormality was detected, all but 1 had MRS abnormalities. The exception was a patient with the 11778 bp point mutation associated with Leber's hereditary optic neuropathy. Her exercise and recovery results could not be included because a different exercise protocol had been used. It is of interest, however, that the patient's [ADP] was high during exercise. Furthermore, both of this patient's sons (not reported here), who had the same genetic lesion, had abnormalities in resting muscle and 1 also in exercise. A comparison of the MRS results for fds from the 6 patients with mtDNA defects in the gene for tRNA l~u (cases 2, 9, 16, 17, 25 and 26) and those from the 5 patients with the large deletions (cases 7,8,10, 21 and 27 revealed no significant differences between the groups (results were similar if only subjects with the 3243 point mutation and the 4.977 kb deletion were compared). MRS studies were abnormal in the 5 remaining patients in whom mtDNA results were negative.
The probability of detecting abnormal energetics was greater the more pronounced the muscle weakness (Tables 2-4), but there were no significant differences found associated with the presence or absence of muscle pain, external ophthalmoplegia or ptosis, seizures or intellectual impairment. There was little relationship between MRS results and the clinical presentation. The 5 MM patients showed very variable results, and included 3 of the most abnormal studies. The 12 CPEO patients varied from normal to very abnormal; most mildly abnormal studies fell into this group (for example 6 out of 7 normal Vm~x values). Results from the 4 KSS patients were all moderately abnormal, and those from the 2 MELAS patients were very abnormal.
Overall The results show that although abnormalities are detectable by MRS in the resting muscle of most patients with mitochondrial disease, the monitoring of exercise and recovery can also contribute to the detection of abnormal mitochondrial metabolism. Results from 4 of the fds studies were normal for resting muscle, but in 2 of them both exercise and recovery values were outside the normal range. For gastrocnemius, results from recovery were abnormal in 2 studies for which both rest and exercise values were within normal limits. Of the 27 patients with data for rest, exercise and recovery, 25 were abnormal in at least 2 of these 3 states, while 13 patients were abnormal for all 3. In one patient, data from rest, exercise and recovery in both muscles were all within normal limits.
4. Discussion
Detection of abnormality in mitochondrial myopathy There is considerable scatter and overlap of data from the patient and control groups. In spite of this, in only 1 of 29 patients was no NMR abnormality detected. Matthews et al. (1991) concluded from their study of gastrocnemius muscle that when used in conjunction with standard non-invasive t e s t s 31p NMR is at least as sensitive for the diagnosis of mitochondrial disease as histopathologic examination of muscle biopsy specimens. Our data are consistent with this. Matthews et al. looked only at resting gastrocnemius and found high Pi/ATP to be a common feature in mitochondrial disease, while in our study Pi/ATP was high in only a quarter of patients. The reason for this difference is not known, but the results from the present study make it clear that Pi/ATP is not a reliable indicator of mitochondrial disease, as they suggested, nor is it due to a difference between fds and gastrocnemius. It is not likely to be due to the difference in interpulse delay in
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D.J. Tayloret aL /Journal of the Neurological Sciences 127 (1994) 198-206
the two studies since P~ and PCr are in the same cellular compartment and have very similar T~ relaxation times. Low P C r / A T P at rest was found in both studies (and by us in both fds and gastrocnemius). This finding is in itself relatively non-specific, and because the P C r / A T P ratio is ultimately incorporated into the calculations of [ADP] and phosphorylation potential it accounts for many of the abnormal results in resting muscle. In contrast, recovery of PCr, Pi and ADP after exercise are dynamic processes which rely on oxidative metabolism and are under State 3 respiratory control. Abnormalities in recovery, therefore, give more specific indications of mitochondrial dysfunction than abnormalities at rest. Half times for PCr or PCr/P~ recovery have often been used to assess mitochondrial function, and we find that sensitivity is increased by taking into account the relationship of PCr recovery rate to [ADP]. This relationship is discussed further below. We have addressed the issue raised previously of whether the sensitivity of MRS for detection of abnormal mitochondrial metabolism might be increased by investigating more than one muscle group (Matthews et ai. 1991). In resting muscle (in which respiratory control is near State 4 (Chance et al. 1985), abnormality in our patients was detected equally easily in fds and gastrocnemius. In exercise there were few patients who could be clearly differentiated from normal except for high [ADP]. This was elevated in half of all fds studies but only a quarter of gastrocnemius studies. Duration of exercise for both fds and gastrocnemius was generally shorter in patients than in control subjects, but this is not a specific finding for mitochondrial myopathy. It would be reasonable to expect the physiological stress of increased ATP demand to reveal marginal abnormalities, and our inability to differentiate clearly the patients' response from normal probably arises for several reasons: firstly, blood flow, muscle mass and other factors which affect metabolism during exercise are not easily defined precisely (and for fds the nominal work load was the same for all subjects and not tailored to individual differences); secondly, "mitochondrial reserve" permits some degree of mitochondrial impairment to be tolerated without corresponding impairment of exercise response (see below). In recovery it was easier to obtain reliable data from fds because patients tolerated finger flexion better than plantar flexion, and the substantial changes in PCr, Pi and pH i necessary to measure recovery rates were achieved more easily in fds. The degree of mitochondrial impairment necessary in order to be detectable by 3~p MRS is difficult to assess. This is partly because the patients were a heterogeneous group with respect to their mitochondrial defects. Also, they were referred from a variety of sources, so different studies had been performed on
them and the biopsies. In addition, the degree of abnormality detected in vivo by histological, histochemical or biochemical tests may not be an accurate reflection of biochemical activity in vivo. The percentage of ragged red fibres in the biopsies of patients who showed abnormalities was as low as < 5%, while in one case in whom MRS abnormalities were found, 5% of fibres were reported to have no histochemically detectable cytochrome oxidase but the isolated mitochondria showed normal respiration. We found no relationship between MRS abnormalities and clinical features with the exception of weakness, and no relationship between the MRS findings and the genetic abnormality. This is not surprising given the current poor understanding of the relationships between genetic defect, biochemical abnormality and clinical manifestations. It may be asked how specific these findings are for primary mitochondrial disease. It is clear, for example, that Vm~× and other recovery indices are impaired in other conditions as a result of decreased mitochondrial content or impaired substrate and oxygen supply (Kemp et al. 1993a), suggesting that these are markers of mitochondrial function. However, other features such as raised [ADP] in exercise (Kemp et al. 1993a), rapid pH recovery and abnormalities of resting muscle are not necessarily seen. Also, low P C r / A T P and high P i / A T P in resting muscle are not specific for mitochondrial myopathy, but dystrophic and inflammatory processes are associated with high concentrations of phosphodiesters a n d / o r phosphomonoesters (visible in the 31p NMR spectra) and often with high intracellular pH (Matthews et al. 1991; Kemp et al. 1993b). Metabolite concentrations in resting muscle
In 3 of the patients, [ATP] and [total creatine] had been measured and were within normal limits. These patients all showed abnormalities in resting and recovering muscle. Therefore, we have assumed that P i / A T P and P C r / A T P can be used to calculate [Pi] and [PCr] (Arnold et al. 1985). The regulation of phosphorus metabolite concentrations in muscle, which is not well understood, must depend on many processes including membrane transport, mitochondrial function and the constraints imposed by the presence of creatine kinase, an enzyme which operates at or near equilibrium: PCr + ADP + H + ~ ATP + creatine At steady state the concentrations of all mitochondrial regulators must adopt values at which the rate of ATP synthesis is adequate to basal needs. The most important mitochondrial regulators in skeletal muscle are [ADP] and, at the concentrations that prevail in resting muscle, also [P~] (Balaban 1990). The concentrations of [Pi] and [ADP] must be defined by the properties of state 4 mitochondrial control. In addition, [Pi] is defined by sarcolemmal Pi transport, and in-
D.J. Taylor et al. /Journal of the Neurological Sciences 127 (1994) 198-206
205
creased [Pi] appears to require increased Na, Pi cotransport (Kemp et al. 1992). PCr, pH i and ADP are in equilibrium via creatine kinase (Veech et al. 1979), and pH i is probably controlled mainly by sarcolemmal H ÷ efflux, an important component of which is the N a ÷ / H + antiport (Madshus 1988). The relationships between these processes are far from fully understood. However, abnormalities in [ADP] and [PCr] can be viewed as direct reflections of altered mitochondrial control, with [ADP] functioning as the error signal in the feedback loop that matches ATP synthesis to demand (Chance et al. 1985). Increased Pi/ATP, which has been a more prominent feature of mitochondrial myopathic muscle in other studies (Matthews et al. 1991), is at present unexplained except as an adaptation to lower phosphorylation potential and further increase mitochondrial drive.
Upregulation of proton efflux can be considered an appropriate response to a mitochondrial defect which permits high [ADP] to be maintained in the face of increased lactic acid production without the need for excessive PCr depletion. Thus, we have demonstrated that 31p MRS can, in conjunction with clinical assessment and standard laboratory methods, be helpful in the diagnosis of mitochondrial myopathy. It can also be used to provide quantitative information about ATP turnover in normal and diseased muscle. An assessment of the rate of PCr resynthesis with respect to [ADP] increases the rate of detection of mitochondrial disease, and a comparison of the rate of PCr resynthesis with end-exercise [ADP] can be used to quantify the effective size of the mitochondrial defect.
Mitochondrial A TP synthesis during recovery Data from skeletal muscle during exercise and recovery are consistent with control by [ADP] acting at the adenine nucleotide translocase of the mitochondrial membrane according to a hyperbolic relationship with apparent K m of ~ 30 /zM (see discussion and references in Kemp et al. (1993a)). The recovery data from normal muscle can be seen to be consistent with this model when the rate of PCr resynthesis (which is a quantitative measure of oxidative ATP synthesis rate) is plotted against [ADP] (Fig. 1). In the patients, PCr recovery is slow despite a normal or increased stimulus to oxidative phosphorylation, in particular a raised [ADP]. Fig. 1 shows that patients with slow PCr recovery exhibit a strikingly abnormal relationship between oxidative ATP production (as reflected in PCr repletion) and [ADP]. It can be seen from Table 4 that this altered relationship is consistent with a reduced Vma~. However, an increased g m cannot be excluded. In some of our patients, mitochondrial control could not be distinguished from normal, particularly in the gastrocnemius muscle. Animal studies show that there is sufficient mitochondrial reserve in skeletal muscle to provide adequate oxidative ATP synthesis at mitochondrial activities 40-50% of normal (Cooper et al. 1988). Clearly, an enhanced rise in [ADP] during exercise is one way in which this can be achieved. For example, with a K m of 30 /zM, the difference between end-exercise [ADP] in patients and controls reported in Table 2 could completely compensate for an approximately 30% decrease in mitochondrial capacity.
Acknowledgements
Control of pH i during exercise In spite of increased lactic acid production in mitochondrial myopathy, pH i is prevented from falling excessively during exercise. The data in Table 4 support the concept, based on rapid pH i recovery (Arnold et al. 1985), that this is due to increased proton efflux.
G.J.K. acknowledges personal support from the Muscular Dystrophy Group of Great Britain and Northern Ireland. We would like to thank Prof. D.M. Turnbull, Dr. J. Poulton, Dr. R. Lane, Dr. P. Matthews, Dr. J. Morgan-Hughes and Dr. J. Leonard for making available the results of mtDNA studies.
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