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ATP, phosphocreatine and lactate in exercising muscle in mitochondrial disease and McArdle’s disease
Neuromuscular Disorders 11 (2001) 370±375
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ATP, phosphocreatine and lactate in exercising muscle in mitochondrial disease and McArdle's disease Mervi LoÈfberg a,*, Harri Lindholm b, Hannu NaÈveri c, Anna Majander d, Anu Suomalainen e, Anders Paetau f, Anssi SovijaÈrvi b, Matti HaÈrkoÈnen g, Hannu Somer a a
Institute of Neurosciences, Department of Neurology, Helsinki University Hospital, 00029 HUS, Helsinki, Finland b Department of Medicine, Laboratory of Clinical Physiology, Helsinki University Hospital, Helsinki, Finland c Department of Medicine, Division of Cardiology, Helsinki University Hospital, Helsinki, Finland d Department of Medical Chemistry, Helsinki University Hospital, Helsinki, Finland e National Public Health Institute, Department of Human Molecular Genetics, Helsinki, Finland f Department of Pathology, Helsinki University Hospital, Helsinki, Finland g Department of Clinical Chemistry, Helsinki University Hospital, Helsinki, Finland Received 27 March 2000; received in revised form 19 September 2000; accepted 11 October 2000
Abstract We studied exercise-induced changes in the adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in the skeletal muscle of mitochondrial patients and patients with McArdle's disease. Needle muscle biopsy specimens for biochemical measurement were obtained before and immediately after maximal short-term bicycle exercise test from 12 patients suffering from autosomal dominant and recessive forms of progressive external ophthalmoplegia and multiple deletions of mitochondrial DNA (adPEO, arPEO, respectively), ®ve patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 3243 A ! G point mutation, and four patients with McArdle's disease. Muscle ATP and PCr levels at rest or after exercise did not differ signi®cantly from those of the controls in any patient group. In patients with mitochondrial disease, muscle lactate tended to be lower at rest and increase more during exercise than in controls, the most remarkable rise being measured in patients with adPEO with generalized muscle symptoms and in patients with MELAS point mutation. In McArdle patients, the muscle lactate level decreased during exercise. No correlation was found between the muscle ATP and PCr levels and the respiratory chain enzyme activity. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Muscle ATP concentration; Muscle phosphocreatine concentration; Muscle lactate concentration; Mitochondrial disease; Progressive external ophtalmoplegia; MELAS; McArdle's disease
1. Introduction Skeletal muscle is the most commonly affected tissue in mitochondrial diseases and the manifestations vary from local weakness of extraocular muscles to generalized disabling muscle weakness and fatigability [1]. Exercise intolerance in mitochondrial myopathies has been suggested to be related to impaired oxidative synthesis of adenosine triphosphate (ATP), which is augmented by phosphocreatine (PCr) hydrolysis to adenosine diphosphate (ADP), and by acceleration of anaerobic glycolysis. Further, impaired oxidative metabolism of pyruvate in the mitochondria and accelerated glycolysis result in increased production of lactic acid. * Corresponding author. Tel.: 1358-9-4717-2261; fax: 1358-9-47174089. E-mail address: mervi.lofberg@hus.® (M. LoÈfberg).
Graded exercise testing has an established role in the clinical evaluation of fatigue due to cardiopulmonary diseases. Combined with direct measurement of highenergy phosphate compounds in muscle samples obtained by repeated needle biopsies at various stages of the exercise, it has been widely used in Scandinavian countries to study athletes and patients with exercise intolerance [2±4]. Most of the exercise studies on patients with mitochondrial myopathies have, however, used phosphorous magnetic resonance spectroscopy ( 31P-MRS) to measure the changes in ATP, PCr, inorganic phosphate (Pi), and pH [5±8]. The basic assumptions used in calculation of other muscle metabolite levels in 31P-MRS studies include stable total creatine value and normal ATP level which does not change during exercise. Especially the latter assumption needs to be con®rmed by using a direct measurement for muscle ATP
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M. LoÈfberg et al. / Neuromuscular Disorders 11 (2001) 370±375
concentration in patients with mitochondrial diseases or other metabolic myopathies. The present study was carried out to clarify if some defects of mitochondrial DNA (mtDNA) have an effect on the amount of biochemically measured muscle ATP, PCr, and lactate levels during short-term maximal exercise. Twelve patients with progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions, ®ve patients with an nt 3243 A ! G point mutation in mtDNA, as well as four patients with classical metabolic myopathy caused by muscle phosphorylase enzyme de®ciency were studied with graded bicycle exercise testing with needle muscle biopsy specimens obtained before and immediately after exercise and compared with controls. 2. Patients and methods 2.1. Patients 2.1.1. Patients with autosomal dominant inheritance of PEO (adPEO) AdPEO patients 1±6 (Table 1) belonged to a chromosome 10q24 linked family described previously [9,10]. Two of them had no symptoms, one had ptosis, and three had adPEO1 associated with more generalized muscle symptoms. Patient 7 belonged to another adPEO family, and had PEO as the only symptom. Patient ages varied from 21±58 years (mean 37 years) and they were all male. In histopathological examination of the muscle specimen, all patients had myopathic ®ndings and the number of ragged-red ®bers (RRFs) varied from 3±10% of all muscle ®bers in the sample. All patients had multiple deletions of mtDNA in the Southern blot analysis of the muscle DNA [10]. 2.1.2. Patients with autosomal recessive inheritance of PEO (arPEO) Patients 8 and 9 (Table 1) were siblings and patients 10±12 were members of other arPEO families. One patient had PEO and the rest had PEO and generalized muscle weakness. One patient needed support for walking. Patient ages in this group varied from 43±66 years (mean 51 years) and four out of ®ve were female. Histopathological examination of muscle specimen showed myopathic changes in each patient with RRFs up to 6% of the ®bers and multiple deletions of mtDNA in the Southern blot analysis of muscle DNA. 2.1.3. A classical MELAS patient A 51-year old woman (patient 13 in Table 1) had classical MELAS syndrome with remarkable exercise intolerance, a slightly elevated level of blood lactate, a pronounced increase in cerebrospinal ¯uid (CSF) lactate, and myopathy with 4% RRFs in muscle biopsy specimen. She had an A ! G point mutation at nt 3243 of mtDNA and the percentage of mutated mtDNA in muscle was 90%.
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2.1.4. Pauci-symptomatic MELAS patients We studied also four patients carrying the A ! G point mutation at nt 3243 of mtDNA in 30±60% proportion of total muscle DNA. Patient 14 (Table 1) had right-sided hemiparesis at the age of 35 with no predisposing cause. Patient 15 had severe attacks of migraine and tinnitus, which may be related to MELAS. Patients 16 and 17 had no symptoms. Patient ages varied from 40±51 years (mean 46) and three of them were female. They all had normal blood lactate level, two of them had slightly elevated CSF lactate level, and the histopathological examination of muscle biopsy revealed RRFs in only one out of three patients studied. 2.1.5. McArdle patients Four patients (mean age 35 years, range 27±42 years, two of them female) with muscle phosphorylase de®ciency were studied with the same study protocol. They all had exerciseinduced myalgia and limited exercise capacity and two of them reported frequent muscle cramps at exercise. Serum creatine kinase activity was above normal and the level of blood lactate did not increase in the forearm ischemic exercise test. The examination of muscle specimen revealed myopathy with high glycogen content and the phosphorylase staining was negative. The myophosphorylase activity of these patients was 0.40±1.23 nmol/min/mg protein, the highest value being 1.7% of the mean of the controls (74.0 nmol/min/mg protein) [11]. 2.1.6. Controls As controls, we studied 17 subjects with the same exercise protocol. Thirteen controls were adPEO or arPEO family members who were asymptomatic and had no mtDNA deletions in muscle. The four additional controls were patients, who were referred to the neurological department because of mild exercise intolerance but who had no signs of mitochondrial or neuromuscular disease. The mean age of the control subjects was 46 years (range 20±72 years) and nine of them were female. 2.2. Methods 2.2.1. Ethics The study protocol was approved by the Ethics Committee of the Department of Neurology and informed consent was obtained from all patients and control subjects after the nature of the studies had been fully explained. 2.2.2. Exercise test A graded exercise test using an electrically braked bicycle ergometer (ERG 551, Robert Bosch GmbH, Berlin, Germany) was performed until subjective maximal effort was reached. The initial work load of 10 W increased by 20 W every 2 min until the grade 18±20/20 in the Borg's scale of
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Table 1 Clinical information, biochemical and histochemical results of muscle biopsies of mitochondrial patients a Patient
Age Sex Symptom
S-CK (U/l)
B-Lactate (mmol/l)
CSF-Lactate (mmol/l)
Percentage of Reduced level of RRFs b respiratory chain enzyme activity (% of the controls' mean)
adPEO patients c 1 58 2 35 3 31 4 39 5 33 6 21 7 45
M M M M M M M
PEO1muscle weakness (20 yrs ! ) PEO1muscle cramps PEO1muscle fatigue Slight ptosis No symptoms No symptoms PEO (35 yrs ! )
121 794 276 ± 156 79 112
2.10 2.90 1.10 ± 1.40 1.00 1.00
2.10 ± 1.70 ± ± ± ±
2% 10% 10% 4% 8% 3% 4%
Complex I (43) Complex I (33) Normal Complex I (29) Normal ± Normal
arPEO patients d 8 59 9 54 10 66 11 43 12 34
F F F F M
Ptosis (30yrs ! )1muscle weakness Ptosis (45 yrs ! )1muscle weakness PEO (30 yrs ! )1muscle weakness PEO1muscle weakness (30 yrs ! ) PEO
183 154 430 308 170
0.70 2.30 5.50 0.60 2.00
± ± 3.60 ± ±
5% 4% 6% 3% 4%
Complexes I, II (40, 25) Complexes I, III (39, 3) Complexes I, III (38, 43) Normal Normal
±
2.50
5.30
3%
Complexes I, II, III, IV (18, 31, 25, 36)
Pauci-symptomatic MELAS patients e 14 47 M Right-sided hemiparesis (35 yrs ! ), ataxia 310 15 40 F Migraine, tinnitus 84 16 51 F No symptoms 150 17 44 F No symptoms 134
1.30 0.40 1.20 0.80
2.60 2.20 3.00 1.90
None None 3% ±
Normal ± Normal ±
A classical MELAS patient e 13 51 F Deafness (29 yrs ! ), migraine, stroke-like episodes (50 yrs ! )
a
Reference values: S-CK ,150 U/l for women, ,270 U/l for men; B-Lactate 0.70±1.80 mmol/l; CSF-Lactate 1.10±2.20 mmol/l. RRFs, ragged-red ®bers in muscle specimen. c adPEO, autosomal dominant inheritance of PEO and multiple deletions of mitochondrial DNA. d arPEO, autosomal recessive inheritance of PEO and multiple deletions of mitochondrial DNA. e MELAS, a pauci-symtomatic MELAS patient has A ! G point mutation at nt 3243 of mtDNA, but has not symptoms of a classical MELAS syndrome like a classical MELAS patient. b
perceived exertion [12,13] was achieved. The Wmax (the mean of last 3 min work load) and the percentage of Wmax from predicted normal value [14] were calculated. 2.2.3. Muscle ATP, PCr, and lactate measurements Needle biopsy specimens (Tru-Cut w, Travenol Laboratories, Inc., IL) were taken from the vastus lateralis muscle under local anesthesia of the skin and muscle fascia with lidocaine. One sample was taken before the exercise from one thigh and another, immediately (within 5 s) after the subjective maximum exercise level was reached, from the other thigh. The muscle samples of about 5±10 mg each were frozen immediately in liquid nitrogen and stored in 2758C until analyzed. Perchloric acid extracts of muscle were prepared for determination of muscle metabolites as described previously [2]. Muscle ATP, PCr, and lactate were measured ¯uorometrically in perchloric acid extracts by enzymatic methods according to Lowry [15]. The reagent used for determination of lactate contained 1 mmol/l NAD 1 and 50 mmol/l hydrazine.
2.2.4. Respiratory chain enzyme activities Surgical biopsy sample from the vastus lateralis muscle under local anesthesia with lidocaine was taken usually a few hours after the exercise test. Mitochondria were isolated from muscle biopsy specimens within 2 h. Oxygen consumption was measured polarographically and the respiratory chain enzyme activities (rotenone-sensitive NADH:-cytochrome c oxidoreductase (Complex I1III), antimycin-sensitive succinate:cytochrome c oxidoreductase (Complex II1III), succinate dehydrogenase (Complex II), cytochrome c oxidase (Complex IV)) and citrate synthase activity were analyzed on isolated mitochondria as previously described [10]. To give an estimate of the reduction of activity of the defective complex in Table 1, we calculated the percentage of the patients value from the controls' mean. (In calculations we used (Complex I1III) to describe the activity of Complex I, and (Complex II1III) to describe the activity of Complex III.) 2.3. Statistical analysis The differences of muscle ATP, PCr, and lactate level
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Table 2 Skeletal muscle ATP, PCr, and lactate levels (mmol/kg wet weight) at rest and after exercise in the mitochondrial patients, McArdle patients, and the control subjects (mean ^ SEM) a M-ATP Patient group b
adPEO1 (n 3) adPEO c (n 4) arPEO d (n 5) Classical MELAS patient e (n 1) Pauci-symptomatic MELAS patient e (n 4) McArdle f (n 4) Control (n 17)
M-PCr
M-Lactate
At rest
After exercise
At rest
After exercise
At rest
After exercise
5.28 ^ 0.37 5.44 ^ 1.13 4.26 ^ 0.72 5.02
5.32 ^ 0.71 3.80 ^ 0.28 3.90 ^ 0.34 5.92
19.40 ^ 1.83 17.37 ^ 3.51 15.24 ^ 2.74 17.10
7.41 ^ 2.27 3.92 ^ 0.90 5.00 ^ 0.49 8.60
2.33 ^ 0.25 1.84 ^ 0.35 1.96 ^ 0.47 1.45
20.40 ^ 1.19* 14.61 ^ 2.81 14.31 ^ 2.66 11.20
6.01 ^ 0.81
5.24 ^ 0.75
24.85 ^ 5.79
6.46 ^ 3.01
1.17 ^ 0.37**
20.08 ^ 4.51
4.71 ^ 0.44 4.77 ^ 0.36
4.36 ^ 0.36 4.51 ^ 0.38
14.60 ^ 2.00 19.36 ^ 2.17
3.82 ^ 0.96 5.68 ^ 1.11
2.21 ^ 0.77 2.99 ^ 0.43
1.48 ^ 0.26*** 12.81 ^ 1.36
a
The signi®cance of difference compared to the controls: *P 0:03; **P 0:02; ***P 0:002: adPEO1, patient with autosomal dominant inheritance of PEO and multiple deletions of mitochondrial DNA who also has generalized muscle symptoms. c adPEO , as above but PEO as only symptom. d arPEO, patient with autosomal recessive inheritance of PEO and multiple deletions of mitochondrial DNA. e MELAS, a pauci-symptomatic MELAS patient has A ! G point mutation at nt 3243 of mtDNA, but has not symptoms of classical MELAS syndrome like a classical MELAS patient. f McArdle, patient with muscle phosphorylase enzyme de®ciency (McArdle's disease). b
between the patients and the controls were tested with Mann±Whitney U-test. The correlations between respiratory chain enzyme activities and muscle ATP, PCr, and lactate levels were tested with Spearman Rank Correlation test. 3. Results 3.1. Maximal exercise level achieved The mean Wmax value achieved was 139 W in the adPEO1 and 146 W in the other adPEO patients, 84 W in the arPEO group, and 101 W in the pauci-symptomatic MELAS patients. The percentage of Wmax from predicted normal value in these groups were 74, 73, 55, and 73%, respectively, which were somewhat lower compared to the result of the controls (78%) but the differences were not statistically signi®cant. In contrast, in the McArdle patients the percentage of Wmax from predicted was signi®cantly reduced to 42% of the normal value. The exercise capacity of the patient with classical MELAS syndrome was remarkably reduced with Wmax of 30 W, 23% of the predicted value. 3.2. Skeletal muscle ATP and PCr level There were no signi®cant differences either in the muscle ATP or the PCr level at rest or immediately after the exercise between different patient groups or between patients and the controls. Table 2 presents the detailed results. 3.3. Skeletal muscle lactate level The muscle lactate level at rest was lower in the pauci-
symptomatic MELAS patients than in the controls (P 0:02), but the exercise-induced increase tended to be more remarkable than that of the control subjects (P 0:06) especially when the achieved exercise level is taken into account (Table 2). The muscle lactate level increase was more pronounced in exercise also in the adPEO1 patients compared to the controls (P 0:03), and tended to be higher than in the controls in the arPEO group (P 0:06), as well. The end-exercise muscle lactate level was higher in the adPEO1 patients compared to the controls (P 0:03). In the McArdle patients the muscle lactate after exercise was signi®cantly lower than that of the controls (P 0:002) (the rest value is reported from three patients). 3.4. Respiratory chain enzyme activities Respiratory chain enzyme activities were measured in six adPEO patients, in ®ve arPEO patients, in the classical MELAS patient and in two pauci-symptomatic MELAS patients, and in 15 control subjects. Complex I activity was reduced in two adPEO1 patients, and in one adPEO patient with PEO as his only symptom (Table 1). In two arPEO patients Complex I and III activities were lowered, and surprisingly in one patient the activity of Complex II was reduced together with Complex I (Table 1). No correlation between the respiratory chain enzyme values and muscle ATP, PCr, or lactate levels at rest or after maximal exercise could be found in the patients with familial PEO. A patient with classical MELAS syndrome had reduced activities of all respiratory chain enzymes (Table 1), but normal muscle ATP level could be maintained during exercise with very limited exercise capacity. Reduced activities
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of Complex II in this MELAS patient and in one arPEO patient cannot be directly explained by the defect in their mitochondrial DNA. 4. Discussion We measured the levels of high-energy phosphate compounds and lactate in the resting and maximally exercised muscle of patients with familial PEO and multiple mtDNA deletions, patients with the MELAS nt 3243 A ! G point mutation, and patients with McArdle's disease. We found no signi®cant differences either between the patient groups or between the patients and the control subjects. The achievement of the true maximal effort was supported by the exercise-induced decrease in muscle PCr comparable to controls in all other patient groups, except the classical MELAS, and the adPEO1 patients. These patients achieved the maximal exercise level according to the increase in their muscle lactate. Whether the maintenance of ATP and PCr concentrations requires increased turnover in metabolism, is not answered by this study. A previous study on mitochondrial patients reported that biochemically analyzed muscle ATP and PCr levels at rest were reduced in patients with RRFs and normal in patients without RRFs [16]. In studies using 31P -MRS, the muscle ATP level remained stable during exercise [5], whereas the PCr/ATP ratio at rest, as well as PCr/Pi ratios at rest and at exercise were usually reduced [6,8]. Our data suggest that the energy metabolism of the skeletal muscle is well maintained during short-term exercise in mitochondrial disease patients. The resting skeletal muscle PCr levels in our controls were about 70% of those obtained by 31P -MRS and the end-exercise values about 50% [5,7]. However, they were similar to those measured previously with the same biochemical method in athletes, in controls, and in patients with congestive heart failure [2,4]. Breakdown of PCr inevitably occurred to some extent, although the muscle specimen was frozen in liquid nitrogen in a few seconds after the needle biopsy. In 31P -MRS studies, the PCr values are calculated using a normal concentration for muscle ATP and the assumption of stable total creatine value [5,7]. The muscle ATP levels of our controls and patients correspond to that used in 31P -MRS studies (8 mm in intracellular water corresponding to 5.5 mmol/kg wet weight) to calculate the values of other metabolites and support its validity and relative stability during short-term exercise also in mitochondrial patients. Lactic acidosis in mitochondrial diseases is caused by inef®cient oxidative pyruvate metabolism by mitochondria because of the malfunctioning respiratory chain. However, 31 P-MRS studies have reported a stable or increased pH level in muscle at exercise [5,6,8] with faster recovery than in controls [8]. This suggested that the increased lactate production stimulates buffering systems or extrusion mechanisms that prevent intracellular acidosis [5,6,17]. In
our study, the low resting levels of muscle lactate in familial PEO and MELAS patients support the view. The increased production of lactic acid in our adPEO1 patients and, to a lesser extent, in arPEO and MELAS patients is shown by the large exercise-induced change in the muscle lactate level. In our McArdle patients, the ATP or PCr values at rest and after short-term maximal exercise did not signi®cantly differ from those of the controls. Previous biochemical [18,19] and 31P-MRS [20,21] studies on McArdle patients reported that during maximal ischemic exercise, muscle PCr is rapidly depleted but ATP levels show no change or decline only modestly. The difference between our and previous PCr ®ndings may be explained by the fact that in this exercise protocol, the McArdle patients could use blood borne glucose as signi®cant energy source in the working muscle and maintain higher PCr level than in ischemic exercise. The decrease of the muscle lactate in McArdle patients during exercise is probably related to the effective use of pyruvate in the mitochondria and to the exercise-induced increase in muscle circulation, which exports effectively the minor amounts of lactate that are produced. This result is in accordance with 31P-MRS ®ndings of increasing muscle pH during exercise in McArdle patients [22,23].
References [1] Suomalainen A. Mitochondrial DNA and disease. Ann Med 1997;29:235±246. [2] NaÈveri H, Rehunen S, Kuoppasalmi K, Tulikoura I, HaÈrkoÈnen M. Muscle metabolism during and after strenuous intermittent running. Scand J Clin Lab Invest 1978;38:329±336. [3] Linderholm H, Essen-Gustavsson B, Thornell LE. Low succinate dehydrogenase (SDH) activity in a patient with a hereditary myopathy with paroxysmal myoglobinuria. J Intern Med 1990;228:43±52. [4] NaÈveri HK, Leinonen H, Kiilavuori K, HaÈrkoÈnen M. Skeletal muscle lactate accumulation and creatine phosphate depletion during heavy exercise in congestive heart failure. Cause of limited exercise capacity? Eur Heart J 1997;18:1937±1945. [5] Arnold DL, Taylor DJ, Radda GK. Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann Neurol 1985;18:189±196. [6] Argov Z, Bank WJ, Maris J, Peterson P, Chance B. Bioenergetic heterogeneity of human mitochondrial myopathies: phosphorus magnetic resonance spectroscopy study. Neurology 1987;37:257± 262. [7] Matthews PM, Allaire C, Shoubridge EA, Karpati G, Carpenter S, Arnold DL. In vivo muscle magnetic resonance spectroscopy in the clinical investigation of mitochondrial disease. Neurology 1991;41:114±120. [8] Taylor DJ, Kemp GJ, Radda GK. Bioenergetics of skeletal muscle in mitochondrial myopathy. J Neurol Sci 1994;127:198±206. [9] Suomalainen A, Kaukonen J, Amati P, et al. An autosomal locus predisposing to deletions of mitochondrial DNA. Nat Genet 1995;9:146±151. [10] Suomalainen A, Majander A, Wallin M, et al. Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA: clinical, biochemical, and molecular genetic features of the 10q-linked disease. Neurology 1997;48:1244±1253. [11] LoÈfberg M, JaÈnkaÈlaÈ H, Paetau A, HaÈrkoÈnen M, Somer H. Metabolic
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[18] Rowland LP, Araki S, Carmel P. Contracture in McArdle's disease. Arch Neurol 1965;13:541±544. [19] Ross BD, Radda GK, Gadian DG, Rocker G, Esiri M, Falconer-Smith J. Examination of a case of suspected McArdle's syndrome by 31P nuclear magnetic resonance. N Engl J Med 1981;304:1338±1342. [20] Argov Z, Bank WJ, Maris J, Chance B. Muscle energy metabolism in McArdle's syndrome by in vivo phosphorus magnetic resonance spectroscopy. Neurology 1987;37:1720±1724. [21] De Stefano N, Argov Z, Matthews PM, Karpati G, Arnold DL. Impairment of muscle mitochondrial oxidative metabolism in McArdle's disease. Muscle Nerve 1996;19:764±769. [22] Duboc D, Jehenson P, Tran Dinh S, Marsac C, Syrota A, Fardeau M. Phosphorus NMR spectroscopy study of muscular enzyme de®ciencies involving glycogenolysis and glycolysis. Neurology 1987;37:663±671. [23] Bendahan D, Confort-Gouny S, Kozak-Ribbens G, Cozzone PJ. 31-P NMR characterization of the metabolic anomalies associated with the lack of glycogen phosphorylase activity in human forearm muscle. Biochem Biophys Res Commun 1992;185:16±21.
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ATP, phosphocreatine and lactate in exercising muscle in mitochondrial disease and McArdle's disease Mervi LoÈfberg a,*, Harri Lindholm b, Hannu NaÈveri c, Anna Majander d, Anu Suomalainen e, Anders Paetau f, Anssi SovijaÈrvi b, Matti HaÈrkoÈnen g, Hannu Somer a a
Institute of Neurosciences, Department of Neurology, Helsinki University Hospital, 00029 HUS, Helsinki, Finland b Department of Medicine, Laboratory of Clinical Physiology, Helsinki University Hospital, Helsinki, Finland c Department of Medicine, Division of Cardiology, Helsinki University Hospital, Helsinki, Finland d Department of Medical Chemistry, Helsinki University Hospital, Helsinki, Finland e National Public Health Institute, Department of Human Molecular Genetics, Helsinki, Finland f Department of Pathology, Helsinki University Hospital, Helsinki, Finland g Department of Clinical Chemistry, Helsinki University Hospital, Helsinki, Finland Received 27 March 2000; received in revised form 19 September 2000; accepted 11 October 2000
Abstract We studied exercise-induced changes in the adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in the skeletal muscle of mitochondrial patients and patients with McArdle's disease. Needle muscle biopsy specimens for biochemical measurement were obtained before and immediately after maximal short-term bicycle exercise test from 12 patients suffering from autosomal dominant and recessive forms of progressive external ophthalmoplegia and multiple deletions of mitochondrial DNA (adPEO, arPEO, respectively), ®ve patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 3243 A ! G point mutation, and four patients with McArdle's disease. Muscle ATP and PCr levels at rest or after exercise did not differ signi®cantly from those of the controls in any patient group. In patients with mitochondrial disease, muscle lactate tended to be lower at rest and increase more during exercise than in controls, the most remarkable rise being measured in patients with adPEO with generalized muscle symptoms and in patients with MELAS point mutation. In McArdle patients, the muscle lactate level decreased during exercise. No correlation was found between the muscle ATP and PCr levels and the respiratory chain enzyme activity. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Muscle ATP concentration; Muscle phosphocreatine concentration; Muscle lactate concentration; Mitochondrial disease; Progressive external ophtalmoplegia; MELAS; McArdle's disease
1. Introduction Skeletal muscle is the most commonly affected tissue in mitochondrial diseases and the manifestations vary from local weakness of extraocular muscles to generalized disabling muscle weakness and fatigability [1]. Exercise intolerance in mitochondrial myopathies has been suggested to be related to impaired oxidative synthesis of adenosine triphosphate (ATP), which is augmented by phosphocreatine (PCr) hydrolysis to adenosine diphosphate (ADP), and by acceleration of anaerobic glycolysis. Further, impaired oxidative metabolism of pyruvate in the mitochondria and accelerated glycolysis result in increased production of lactic acid. * Corresponding author. Tel.: 1358-9-4717-2261; fax: 1358-9-47174089. E-mail address: mervi.lofberg@hus.® (M. LoÈfberg).
Graded exercise testing has an established role in the clinical evaluation of fatigue due to cardiopulmonary diseases. Combined with direct measurement of highenergy phosphate compounds in muscle samples obtained by repeated needle biopsies at various stages of the exercise, it has been widely used in Scandinavian countries to study athletes and patients with exercise intolerance [2±4]. Most of the exercise studies on patients with mitochondrial myopathies have, however, used phosphorous magnetic resonance spectroscopy ( 31P-MRS) to measure the changes in ATP, PCr, inorganic phosphate (Pi), and pH [5±8]. The basic assumptions used in calculation of other muscle metabolite levels in 31P-MRS studies include stable total creatine value and normal ATP level which does not change during exercise. Especially the latter assumption needs to be con®rmed by using a direct measurement for muscle ATP
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M. LoÈfberg et al. / Neuromuscular Disorders 11 (2001) 370±375
concentration in patients with mitochondrial diseases or other metabolic myopathies. The present study was carried out to clarify if some defects of mitochondrial DNA (mtDNA) have an effect on the amount of biochemically measured muscle ATP, PCr, and lactate levels during short-term maximal exercise. Twelve patients with progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions, ®ve patients with an nt 3243 A ! G point mutation in mtDNA, as well as four patients with classical metabolic myopathy caused by muscle phosphorylase enzyme de®ciency were studied with graded bicycle exercise testing with needle muscle biopsy specimens obtained before and immediately after exercise and compared with controls. 2. Patients and methods 2.1. Patients 2.1.1. Patients with autosomal dominant inheritance of PEO (adPEO) AdPEO patients 1±6 (Table 1) belonged to a chromosome 10q24 linked family described previously [9,10]. Two of them had no symptoms, one had ptosis, and three had adPEO1 associated with more generalized muscle symptoms. Patient 7 belonged to another adPEO family, and had PEO as the only symptom. Patient ages varied from 21±58 years (mean 37 years) and they were all male. In histopathological examination of the muscle specimen, all patients had myopathic ®ndings and the number of ragged-red ®bers (RRFs) varied from 3±10% of all muscle ®bers in the sample. All patients had multiple deletions of mtDNA in the Southern blot analysis of the muscle DNA [10]. 2.1.2. Patients with autosomal recessive inheritance of PEO (arPEO) Patients 8 and 9 (Table 1) were siblings and patients 10±12 were members of other arPEO families. One patient had PEO and the rest had PEO and generalized muscle weakness. One patient needed support for walking. Patient ages in this group varied from 43±66 years (mean 51 years) and four out of ®ve were female. Histopathological examination of muscle specimen showed myopathic changes in each patient with RRFs up to 6% of the ®bers and multiple deletions of mtDNA in the Southern blot analysis of muscle DNA. 2.1.3. A classical MELAS patient A 51-year old woman (patient 13 in Table 1) had classical MELAS syndrome with remarkable exercise intolerance, a slightly elevated level of blood lactate, a pronounced increase in cerebrospinal ¯uid (CSF) lactate, and myopathy with 4% RRFs in muscle biopsy specimen. She had an A ! G point mutation at nt 3243 of mtDNA and the percentage of mutated mtDNA in muscle was 90%.
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2.1.4. Pauci-symptomatic MELAS patients We studied also four patients carrying the A ! G point mutation at nt 3243 of mtDNA in 30±60% proportion of total muscle DNA. Patient 14 (Table 1) had right-sided hemiparesis at the age of 35 with no predisposing cause. Patient 15 had severe attacks of migraine and tinnitus, which may be related to MELAS. Patients 16 and 17 had no symptoms. Patient ages varied from 40±51 years (mean 46) and three of them were female. They all had normal blood lactate level, two of them had slightly elevated CSF lactate level, and the histopathological examination of muscle biopsy revealed RRFs in only one out of three patients studied. 2.1.5. McArdle patients Four patients (mean age 35 years, range 27±42 years, two of them female) with muscle phosphorylase de®ciency were studied with the same study protocol. They all had exerciseinduced myalgia and limited exercise capacity and two of them reported frequent muscle cramps at exercise. Serum creatine kinase activity was above normal and the level of blood lactate did not increase in the forearm ischemic exercise test. The examination of muscle specimen revealed myopathy with high glycogen content and the phosphorylase staining was negative. The myophosphorylase activity of these patients was 0.40±1.23 nmol/min/mg protein, the highest value being 1.7% of the mean of the controls (74.0 nmol/min/mg protein) [11]. 2.1.6. Controls As controls, we studied 17 subjects with the same exercise protocol. Thirteen controls were adPEO or arPEO family members who were asymptomatic and had no mtDNA deletions in muscle. The four additional controls were patients, who were referred to the neurological department because of mild exercise intolerance but who had no signs of mitochondrial or neuromuscular disease. The mean age of the control subjects was 46 years (range 20±72 years) and nine of them were female. 2.2. Methods 2.2.1. Ethics The study protocol was approved by the Ethics Committee of the Department of Neurology and informed consent was obtained from all patients and control subjects after the nature of the studies had been fully explained. 2.2.2. Exercise test A graded exercise test using an electrically braked bicycle ergometer (ERG 551, Robert Bosch GmbH, Berlin, Germany) was performed until subjective maximal effort was reached. The initial work load of 10 W increased by 20 W every 2 min until the grade 18±20/20 in the Borg's scale of
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Table 1 Clinical information, biochemical and histochemical results of muscle biopsies of mitochondrial patients a Patient
Age Sex Symptom
S-CK (U/l)
B-Lactate (mmol/l)
CSF-Lactate (mmol/l)
Percentage of Reduced level of RRFs b respiratory chain enzyme activity (% of the controls' mean)
adPEO patients c 1 58 2 35 3 31 4 39 5 33 6 21 7 45
M M M M M M M
PEO1muscle weakness (20 yrs ! ) PEO1muscle cramps PEO1muscle fatigue Slight ptosis No symptoms No symptoms PEO (35 yrs ! )
121 794 276 ± 156 79 112
2.10 2.90 1.10 ± 1.40 1.00 1.00
2.10 ± 1.70 ± ± ± ±
2% 10% 10% 4% 8% 3% 4%
Complex I (43) Complex I (33) Normal Complex I (29) Normal ± Normal
arPEO patients d 8 59 9 54 10 66 11 43 12 34
F F F F M
Ptosis (30yrs ! )1muscle weakness Ptosis (45 yrs ! )1muscle weakness PEO (30 yrs ! )1muscle weakness PEO1muscle weakness (30 yrs ! ) PEO
183 154 430 308 170
0.70 2.30 5.50 0.60 2.00
± ± 3.60 ± ±
5% 4% 6% 3% 4%
Complexes I, II (40, 25) Complexes I, III (39, 3) Complexes I, III (38, 43) Normal Normal
±
2.50
5.30
3%
Complexes I, II, III, IV (18, 31, 25, 36)
Pauci-symptomatic MELAS patients e 14 47 M Right-sided hemiparesis (35 yrs ! ), ataxia 310 15 40 F Migraine, tinnitus 84 16 51 F No symptoms 150 17 44 F No symptoms 134
1.30 0.40 1.20 0.80
2.60 2.20 3.00 1.90
None None 3% ±
Normal ± Normal ±
A classical MELAS patient e 13 51 F Deafness (29 yrs ! ), migraine, stroke-like episodes (50 yrs ! )
a
Reference values: S-CK ,150 U/l for women, ,270 U/l for men; B-Lactate 0.70±1.80 mmol/l; CSF-Lactate 1.10±2.20 mmol/l. RRFs, ragged-red ®bers in muscle specimen. c adPEO, autosomal dominant inheritance of PEO and multiple deletions of mitochondrial DNA. d arPEO, autosomal recessive inheritance of PEO and multiple deletions of mitochondrial DNA. e MELAS, a pauci-symtomatic MELAS patient has A ! G point mutation at nt 3243 of mtDNA, but has not symptoms of a classical MELAS syndrome like a classical MELAS patient. b
perceived exertion [12,13] was achieved. The Wmax (the mean of last 3 min work load) and the percentage of Wmax from predicted normal value [14] were calculated. 2.2.3. Muscle ATP, PCr, and lactate measurements Needle biopsy specimens (Tru-Cut w, Travenol Laboratories, Inc., IL) were taken from the vastus lateralis muscle under local anesthesia of the skin and muscle fascia with lidocaine. One sample was taken before the exercise from one thigh and another, immediately (within 5 s) after the subjective maximum exercise level was reached, from the other thigh. The muscle samples of about 5±10 mg each were frozen immediately in liquid nitrogen and stored in 2758C until analyzed. Perchloric acid extracts of muscle were prepared for determination of muscle metabolites as described previously [2]. Muscle ATP, PCr, and lactate were measured ¯uorometrically in perchloric acid extracts by enzymatic methods according to Lowry [15]. The reagent used for determination of lactate contained 1 mmol/l NAD 1 and 50 mmol/l hydrazine.
2.2.4. Respiratory chain enzyme activities Surgical biopsy sample from the vastus lateralis muscle under local anesthesia with lidocaine was taken usually a few hours after the exercise test. Mitochondria were isolated from muscle biopsy specimens within 2 h. Oxygen consumption was measured polarographically and the respiratory chain enzyme activities (rotenone-sensitive NADH:-cytochrome c oxidoreductase (Complex I1III), antimycin-sensitive succinate:cytochrome c oxidoreductase (Complex II1III), succinate dehydrogenase (Complex II), cytochrome c oxidase (Complex IV)) and citrate synthase activity were analyzed on isolated mitochondria as previously described [10]. To give an estimate of the reduction of activity of the defective complex in Table 1, we calculated the percentage of the patients value from the controls' mean. (In calculations we used (Complex I1III) to describe the activity of Complex I, and (Complex II1III) to describe the activity of Complex III.) 2.3. Statistical analysis The differences of muscle ATP, PCr, and lactate level
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Table 2 Skeletal muscle ATP, PCr, and lactate levels (mmol/kg wet weight) at rest and after exercise in the mitochondrial patients, McArdle patients, and the control subjects (mean ^ SEM) a M-ATP Patient group b
adPEO1 (n 3) adPEO c (n 4) arPEO d (n 5) Classical MELAS patient e (n 1) Pauci-symptomatic MELAS patient e (n 4) McArdle f (n 4) Control (n 17)
M-PCr
M-Lactate
At rest
After exercise
At rest
After exercise
At rest
After exercise
5.28 ^ 0.37 5.44 ^ 1.13 4.26 ^ 0.72 5.02
5.32 ^ 0.71 3.80 ^ 0.28 3.90 ^ 0.34 5.92
19.40 ^ 1.83 17.37 ^ 3.51 15.24 ^ 2.74 17.10
7.41 ^ 2.27 3.92 ^ 0.90 5.00 ^ 0.49 8.60
2.33 ^ 0.25 1.84 ^ 0.35 1.96 ^ 0.47 1.45
20.40 ^ 1.19* 14.61 ^ 2.81 14.31 ^ 2.66 11.20
6.01 ^ 0.81
5.24 ^ 0.75
24.85 ^ 5.79
6.46 ^ 3.01
1.17 ^ 0.37**
20.08 ^ 4.51
4.71 ^ 0.44 4.77 ^ 0.36
4.36 ^ 0.36 4.51 ^ 0.38
14.60 ^ 2.00 19.36 ^ 2.17
3.82 ^ 0.96 5.68 ^ 1.11
2.21 ^ 0.77 2.99 ^ 0.43
1.48 ^ 0.26*** 12.81 ^ 1.36
a
The signi®cance of difference compared to the controls: *P 0:03; **P 0:02; ***P 0:002: adPEO1, patient with autosomal dominant inheritance of PEO and multiple deletions of mitochondrial DNA who also has generalized muscle symptoms. c adPEO , as above but PEO as only symptom. d arPEO, patient with autosomal recessive inheritance of PEO and multiple deletions of mitochondrial DNA. e MELAS, a pauci-symptomatic MELAS patient has A ! G point mutation at nt 3243 of mtDNA, but has not symptoms of classical MELAS syndrome like a classical MELAS patient. f McArdle, patient with muscle phosphorylase enzyme de®ciency (McArdle's disease). b
between the patients and the controls were tested with Mann±Whitney U-test. The correlations between respiratory chain enzyme activities and muscle ATP, PCr, and lactate levels were tested with Spearman Rank Correlation test. 3. Results 3.1. Maximal exercise level achieved The mean Wmax value achieved was 139 W in the adPEO1 and 146 W in the other adPEO patients, 84 W in the arPEO group, and 101 W in the pauci-symptomatic MELAS patients. The percentage of Wmax from predicted normal value in these groups were 74, 73, 55, and 73%, respectively, which were somewhat lower compared to the result of the controls (78%) but the differences were not statistically signi®cant. In contrast, in the McArdle patients the percentage of Wmax from predicted was signi®cantly reduced to 42% of the normal value. The exercise capacity of the patient with classical MELAS syndrome was remarkably reduced with Wmax of 30 W, 23% of the predicted value. 3.2. Skeletal muscle ATP and PCr level There were no signi®cant differences either in the muscle ATP or the PCr level at rest or immediately after the exercise between different patient groups or between patients and the controls. Table 2 presents the detailed results. 3.3. Skeletal muscle lactate level The muscle lactate level at rest was lower in the pauci-
symptomatic MELAS patients than in the controls (P 0:02), but the exercise-induced increase tended to be more remarkable than that of the control subjects (P 0:06) especially when the achieved exercise level is taken into account (Table 2). The muscle lactate level increase was more pronounced in exercise also in the adPEO1 patients compared to the controls (P 0:03), and tended to be higher than in the controls in the arPEO group (P 0:06), as well. The end-exercise muscle lactate level was higher in the adPEO1 patients compared to the controls (P 0:03). In the McArdle patients the muscle lactate after exercise was signi®cantly lower than that of the controls (P 0:002) (the rest value is reported from three patients). 3.4. Respiratory chain enzyme activities Respiratory chain enzyme activities were measured in six adPEO patients, in ®ve arPEO patients, in the classical MELAS patient and in two pauci-symptomatic MELAS patients, and in 15 control subjects. Complex I activity was reduced in two adPEO1 patients, and in one adPEO patient with PEO as his only symptom (Table 1). In two arPEO patients Complex I and III activities were lowered, and surprisingly in one patient the activity of Complex II was reduced together with Complex I (Table 1). No correlation between the respiratory chain enzyme values and muscle ATP, PCr, or lactate levels at rest or after maximal exercise could be found in the patients with familial PEO. A patient with classical MELAS syndrome had reduced activities of all respiratory chain enzymes (Table 1), but normal muscle ATP level could be maintained during exercise with very limited exercise capacity. Reduced activities
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of Complex II in this MELAS patient and in one arPEO patient cannot be directly explained by the defect in their mitochondrial DNA. 4. Discussion We measured the levels of high-energy phosphate compounds and lactate in the resting and maximally exercised muscle of patients with familial PEO and multiple mtDNA deletions, patients with the MELAS nt 3243 A ! G point mutation, and patients with McArdle's disease. We found no signi®cant differences either between the patient groups or between the patients and the control subjects. The achievement of the true maximal effort was supported by the exercise-induced decrease in muscle PCr comparable to controls in all other patient groups, except the classical MELAS, and the adPEO1 patients. These patients achieved the maximal exercise level according to the increase in their muscle lactate. Whether the maintenance of ATP and PCr concentrations requires increased turnover in metabolism, is not answered by this study. A previous study on mitochondrial patients reported that biochemically analyzed muscle ATP and PCr levels at rest were reduced in patients with RRFs and normal in patients without RRFs [16]. In studies using 31P -MRS, the muscle ATP level remained stable during exercise [5], whereas the PCr/ATP ratio at rest, as well as PCr/Pi ratios at rest and at exercise were usually reduced [6,8]. Our data suggest that the energy metabolism of the skeletal muscle is well maintained during short-term exercise in mitochondrial disease patients. The resting skeletal muscle PCr levels in our controls were about 70% of those obtained by 31P -MRS and the end-exercise values about 50% [5,7]. However, they were similar to those measured previously with the same biochemical method in athletes, in controls, and in patients with congestive heart failure [2,4]. Breakdown of PCr inevitably occurred to some extent, although the muscle specimen was frozen in liquid nitrogen in a few seconds after the needle biopsy. In 31P -MRS studies, the PCr values are calculated using a normal concentration for muscle ATP and the assumption of stable total creatine value [5,7]. The muscle ATP levels of our controls and patients correspond to that used in 31P -MRS studies (8 mm in intracellular water corresponding to 5.5 mmol/kg wet weight) to calculate the values of other metabolites and support its validity and relative stability during short-term exercise also in mitochondrial patients. Lactic acidosis in mitochondrial diseases is caused by inef®cient oxidative pyruvate metabolism by mitochondria because of the malfunctioning respiratory chain. However, 31 P-MRS studies have reported a stable or increased pH level in muscle at exercise [5,6,8] with faster recovery than in controls [8]. This suggested that the increased lactate production stimulates buffering systems or extrusion mechanisms that prevent intracellular acidosis [5,6,17]. In
our study, the low resting levels of muscle lactate in familial PEO and MELAS patients support the view. The increased production of lactic acid in our adPEO1 patients and, to a lesser extent, in arPEO and MELAS patients is shown by the large exercise-induced change in the muscle lactate level. In our McArdle patients, the ATP or PCr values at rest and after short-term maximal exercise did not signi®cantly differ from those of the controls. Previous biochemical [18,19] and 31P-MRS [20,21] studies on McArdle patients reported that during maximal ischemic exercise, muscle PCr is rapidly depleted but ATP levels show no change or decline only modestly. The difference between our and previous PCr ®ndings may be explained by the fact that in this exercise protocol, the McArdle patients could use blood borne glucose as signi®cant energy source in the working muscle and maintain higher PCr level than in ischemic exercise. The decrease of the muscle lactate in McArdle patients during exercise is probably related to the effective use of pyruvate in the mitochondria and to the exercise-induced increase in muscle circulation, which exports effectively the minor amounts of lactate that are produced. This result is in accordance with 31P-MRS ®ndings of increasing muscle pH during exercise in McArdle patients [22,23].
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