Neurology, Institute of Neurology, London, UK. Summary ... production and post-exercise ADP recovery rates were ... Table 1 Clinical data of patients with s-IBM.
Brain (1998), 121, 2119–2126
Normal in vivo skeletal muscle oxidative metabolism in sporadic inclusion body myositis assessed by 31P-magnetic resonance spectroscopy Raffaele Lodi,1 Doris J. Taylor,1 Sarah J. Tabrizi,5 David Hilton-Jones,2 Marian V. Squier,3 Aneke Seller,4 Peter Styles1 and Anthony H. V. Schapira5,6 1MRC
Biochemical and Clinical Magnetic Resonance Unit, Oxford Radcliffe Hospital, 2Department of Clinical Neurology, University of Oxford, 3Department of Neuropathology, Radcliffe Infirmary, 4DNA Laboratory, Churchill Hospital, Oxford, 5University Department of Clinical Neurosciences, Royal Free Hospital School of Medicine and 6University Department of Clinical Neurology, Institute of Neurology, London, UK
Correspondence to: Raffaele Lodi, MD, MRC Biochemical and Clinical Magnetic Resonance Unit, Oxford Radcliffe Hospital, Oxford OX3 9DU, UK
Summary Sporadic inclusion body myositis (s-IBM) is a chronic inflammatory myopathy of unknown pathogenesis. The common findings of ragged red fibres, cytochrome c oxidase-negative fibres and multiple mitochondrial DNA deletions in the muscle of patients with s-IBM have suggested that a deficit of energy metabolism may be of pathogenic relevance. To test this hypothesis we used 31P magnetic resonance spectroscopy to assess in vivo skeletal muscle mitochondrial function in the calf muscles of 12 patients with definite s-IBM. Eleven patients showed multiple mitochondrial DNA deletions in skeletal muscle and 67% showed ragged red fibres and/or cytochrome c oxidase-negative fibres. T1-weighted MR images showed
increased signal intensity in the calf muscle of all patients except one. The involvement of calf muscle was confirmed by 31P magnetic resonance spectroscopy of resting muscle, which disclosed abnormalities in metabolite ratios in all patients. However, muscle oxidative metabolism assessed during recovery from exercise was normal in patients with s-IBM, as maximum rates of mitochondrial ATP production and post-exercise ADP recovery rates were within the normal range in all cases. We conclude that muscle mitochondrial abnormalities are a secondary process and unlikely to play a significant role in the pathogenesis of s-IBM.
Keywords: inclusion body myositis; skeletal muscle; oxidative metabolism; magnetic resonance spectroscopy; MRI Abbreviations: Km 5 Michaelis–Menten constant; mtDNA 5 mitochondrial DNA; PCR 5 polymerase chain reaction; PCr 5 phosphocreatine; Pi 5 inorganic phosphate; 31P-MRS 5 31P-magnetic resonance spectroscopy; s-IBM 5 sporadic inclusion body myositis; TCr 5 total creatine; TE 5 echo time; TR 5 repetition time; Vmax 5 maximum rate of mitochondrial ATP production
Introduction Sporadic inclusion body myositis (s-IBM) is a slowly progressive inflammatory myopathy which occurs most often in men over the age of 50 years. Diagnosis of s-IBM is based on typical clinical and histological findings. Weakness and atrophy affect both proximal and distal muscle groups with a characteristic early involvement of the quadriceps and deep finger flexors (Dalakas, 1991; Griggs et al., 1995; Sekul et al., 1997). Inflammatory infiltrates, partial invasion of nonnecrotic fibres, rimmed vacuoles, amyloid deposits and 15– 18-nm tubulofilaments are found in muscle biopsy specimens © Oxford University Press 1998
(Griggs et al., 1995). Despite the well-defined clinical and pathological features, the aetiology and pathogenesis of the disease are still unknown. Besides the typical pathological changes, ultrastructural mitochondrial abnormalities (Carpenter et al., 1978) and ragged red fibres are frequently reported in muscle of patients with s-IBM (Carpenter and Karpati, 1984). Two recent studies showed that cytochrome c oxidase deficient fibres and ragged red fibres are associated with multiple deletions of mitochondrial DNA (mtDNA) in the skeletal muscle of
2120
R. Lodi et al. Table 1 Clinical data of patients with s-IBM Case
Sex
Age (years)
Age at onset Weakness and wasting (years) Proximal Distal
1 2 3 4 5 6 7 8 9 10 11 12
M M M M M M F M F M M F
62 60 58 72 33 47 73 65 60 56 63 50
57 47 55 68 29 25 60 62 50 48 59 44
1 1 1 1 1 1 1 1 1 1 1 1
1 1 – 1 – 1 1 1 1 1 – –
COX-negative Ragged red fibres ribres
mtDNA deletions
1 1 n.a. 1 – – – 1 1 1 1 –
1 1 1 1 – 1 1 1 1 1 1 1
1 – 1 1 – – – 1 1 1 1 –
COX 5 cytochrome c oxidase; 1 5 present; – 5 absent; n.a. 5 not available.
s-IBM patients (Oldfors et al., 1993; Santorelli et al., 1996). Mitochondrial changes are also reported in polymyositis and dermatomyositis (Rifai et al., 1995) but the degree of abnormality is lower than in s-IBM and such changes are regarded as an unusual finding in both conditions (Watkins and Cullen, 1987; Griggs et al., 1995; Rifai et al., 1995; Blume et al., 1997). Although cytochrome c oxidase-negative fibres and ragged red fibres are typical findings in patients with mitochondrial myopathies, due to defects of mtDNA (DiMauro and Moraes, 1993), it is not clear whether a defect of oxidative metabolism plays a role in the pathogenesis of s-IBM and is involved in the progressive skeletal muscle degeneration. Mitochondrial abnormalities and mtDNA deletions are not present in all s-IBM patients (Santorelli et al., 1996) and clinical features in s-IBM differ from those described in patients with primary mitochondrial disorders due to multiple mtDNA deletions (Zeviani et al., 1989; Hirano et al., 1994). Moreover, ragged red fibres (Rifai et al., 1995), cytochrome c oxidase-deficient fibres (Muller-Hocker, 1990) and mtDNA deletions (Simonetti et al., 1992) accumulate with age even in normal subjects. Skeletal muscle energy metabolism can be conveniently assessed in vivo using phosphorus magnetic resonance spectroscopy (31P-MRS). By means of 31P-MRS, defects of mitochondrial respiration have been demonstrated in skeletal muscle of patients with primary mitochondrial disorders in the absence of any symptoms or signs (Taylor et al., 1994; Lodi et al., 1997c) and even of morphological or in vitro biochemical abnormalities (Cortelli et al., 1991). In the present study we used 31P-MRS to establish whether an in vivo deficit of skeletal muscle mitochondrial function is an underlying feature of s-IBM and, thus, whether it might play a relevant role in the pathogenesis of this disorder.
Patients and methods Patients We studied 12 s-IBM patients [nine men and three women, aged 58 6 11 years ( mean 6 SD)] with a mean age at
disease onset of 50 6 13 years (Table 1). Diagnosis was made of definite s-IBM in all cases, on the basis of clinical presentation and histological examination of muscle biopsy specimens from quadriceps or deltoid muscles, according to recently published diagnostic criteria (Griggs et al., 1995). Sensory and motor nerve conduction studies were normal in all patients. Electromyography showed myopathic changes in all cases. An increased number of polyphasic long-duration and high-amplitude motor unit potentials, which are likely to be due to local primary muscle changes rather than to reinnervation (Luciano and Dalakas, 1997), were detected in Patients 2 and 4. Serial frozen sections of muscle were analysed inter alia using modified Gomori-trichrome stain to look for ragged red fibres and using succinate dehydrogenase and cytochrome c oxidase stains to assess mitochondrial enzyme activity.
mtDNA analysis Muscle biopsies, stored at –70°C, were quickly transferred to Dounce homogenizers pre-cooled with liquid N2. Further liquid N2 was added and the muscle ground to a fine powder. Between 0.5 and 2.0 ml of lysis buffer (10 mM Tris–HCl, pH 7.4; 25 mM Na EDTA, pH 8.0; and 10 mM NaCl), depending on the size of the sample, plus proteinase K and SDS (sodium dodecyl sulphate) to final concentrations of 2 mg/ml and 1% w/v, respectively, were then added. The muscle suspensions were incubated overnight at 55°C, followed by phenol/chloroform extraction. DNA was precipitated from the aqueous layer by addition of twice the volume of ice cold ethanol plus one-tenth volume of 3 M sodium acetate at pH 5.5. The DNA was resuspended in 10 mM Tris–HCl and 1 mM EDTA at pH 8.0. The extracted DNA was analysed for the presence of major mitochondrial rearrangements, essentially by the method of Li et al. (1995). However, the Perkin Elmer XL polymerase chain reactions (PCR) kit used by the authors was replaced with reagents purchased from Bioline UK and the cycling profile was
In vivo oxidative metabolism in s-IBM simplified. PCRs were performed in a final volume of 50 µl with 0.25 µM of each primer (L1, nt 2695–2720; H3, nt 16459–16436) (Li et al., 1995), 2.5 mM MgCl2, 0.25 mM each dNTP, 1 3 Bio-optiperform III buffer and 1.5 units BIO-X-ACT Taq polymerase. PCRs were carried out in a Perkin Elmer Geneamp 2400 thermal cycler and were hotstarted at 80°C by the addition of 20–50 ng DNA and denatured at 95°C for 1.5 min before cycling at 94°C for 10 s, and 68°C for 10 min plus 30 s for each subsequent cycle (25 cycles in total). PCR products (5–10 µl) were separated on 0.7% Seakem gels (FMC Bioproducts, Maine, USA).
Magnetic resonance A 2.0-T superconducting magnet (Oxford Magnet Technology, Eynsham, Oxford, UK) interfaced to a Bruker spectrometer (Bruker, Coventry, UK) was used for 31P-MRS and MRI. 31P-MRS
Subjects lay supine with a 6-cm diameter surface coil centred on the maximal circumference of the right calf muscle. Spectra were acquired using a 2-s inter-pulse delay at rest (64 scans), and during exercise (32 scans) and recovery. As soon as the last 32-scan exercise spectrum was collected, an additional eight scan spectrum was also recorded, to be considered ‘zero time’ for the recovery phase, and the exercise was stopped immediately afterwards. Data were collected for 10 min during recovery (four eight-scan spectra followed by four of 16 scans, three of 32 scans and two of 64 scans). The muscle was exercised by plantar flexion at 0.5 Hz, lifting a weight of 10% of lean body mass [calculated from body weight and skin-fold thickness (Durnin and Womersley, 1974)] through a distance of 7 cm. After four spectra, the weight was incremented by 2% of lean body mass for each subsequent spectral acquisition. Relative concentrations of inorganic phosphate (Pi), phosphocreatine (PCr) and ATP were obtained by a timedomain fitting routine (VARPRO, R. de Beer, Delft, Netherlands) and were corrected for magnetic saturation. Absolute concentrations were obtained by assuming that the concentration of ATP in normal muscle is 8.2 mM (i.e. mmol/l of intracellular water) (Arnold et al., 1985). Intracellular pH was calculated from the chemical shift of the Pi peak relative to PCr, δPi , measured in p.p.m. (parts per million), as pH 5 6.75 1 log10[(δPi – 3.27)/(5.69 – δPi)]. Free cytosolic [ADP] was calculated from pH and [PCr] using a creatine kinase equilibrium constant (Keq) of 1.66 3 109 M–1 (Veech et al., 1979) and assuming a normal total creatine content (TCr) of 42.5 mM (i.e. mmol/l of intracellular water) (Arnold et al., 1985) as [ADP] 5 ([ATP][Cr])/ ([PCr][H1]Keq), where [Cr] is the concentration of creatine calculated as [TCr] – [PCr]. For statistical purposes, the reciprocal of the
2121
phosphorylation potential, [Pi] 3 [ADP])/[ATP], was used. PCr recovery half times were calculated from the slope of semi-logarithmic plots. Initial rates of PCr resynthesis after exercise (V) were calculated (in mM/min) from the exponential rate constant of PCr recovery (k 5 0.693/t1/2) and the total fall in [PCr] during exercise (∆[PCr]) as V 5 k 3 ∆[PCr]. The maximum rate of mitochondrial ATP production, Vmax, was calculated (in mM/min) using a hyperbolic relationship between the PCr recovery rate and [ADP] and a normal Michaelis–Menten constant (Km) for ADP of 30 µM (Kemp et al., 1993b), from the initial rate of post-exercise PCr resynthesis and end-exercise [ADP] ([ADPend]): Vmax 5 V{1 1 (Km/[ADPend])} (Kemp et al., 1993b). Another sensitive index of mitochondrial function, the half-time of post-exercise ADP recovery (ADP t1/2), was calculated from the slope of semi-logarithmic plots (Arnold et al., 1984).
MRI MRI of the right leg was carried out using an 18-cm-diameter quadrature birdcage coil. Three sagittal gradient-echo images [repetition time (TR) 5 130 ms; echo time (TE) 5 10 ms; matrix size 5 128 3 128; field of view 5 30 cm] were obtained to identify the maximal calf thickness. T1-weighted images were obtained in the transaxial orientation. A spinecho pulse sequence (TR 5 600 ms; TE 5 20 ms) was used, with a 256 3 256 matrix, field of view 5 25 cm and eight 10-mm sections separated by 10 mm gaps. Based on the signal intensity patterns of the images lateral and medial gastrocnemius and soleus muscles were examined for the detection of abnormalities using a semi-quantitative grading system as described (Lodi et al., 1997b). Briefly, the scores were: 0 5 normal signal intensity; 1 5 mild hyperintensity (involving less than one-third of the total muscle cross sectional area); 2 5 moderate hyperintensity (up to two-thirds of the total muscle area); and 3 5 severe hyperintensity (more than two-thirds of muscle area). The score grading was performed on the axial image obtained at the maximal calf circumference, without reference to clinical data. We defined a ‘Calf Muscle Score’ as the sum of scores from the medial and lateral gastrocnemius, and soleus muscles. Control subjects were 12 healthy sex- and age-matched volunteers (nine men and three women aged 58 6 11 years). Individual results were taken as abnormal when they fell outside the entire range of the control values. Linear regression analysis was used to calculate correlation coefficients. Statistically significant results, determined by Student’s unpaired t test, were taken as those with P , 0.05. Informed consent was obtained from each patient and normal volunteer, and studies were carried out with approval of the Central Oxford Research Ethics Committee.
2122
R. Lodi et al.
Fig. 1 T1-weighted axial images of the leg (TR 5 600 ms; TE 5 20 ms), at the level of the centre of the phosphorus coil, from Patient 8 (B) and a sex- and age-matched control subject (A).
Results Ragged red fibres and/or cytochrome c oxidase-deficient fibres were demonstrated in muscle biopsy specimens of 67% (n 5 8) of s-IBM patients, and multiple mtDNA deletions were detected by a PCR-based method in muscle tissue of 11 of the 12 patients (Table 1). Figure 1B shows a T1-weighted leg image from Patient 8 and Fig. 1A that from a sex- and age-matched control subject. In the patient’s image, the mild signal intensity increase of soleus and lateral gastrocnemius muscle is associated with a severe signal intensity increase in the medial gastrocnemius. Signal hyperintensity on T1-weighted images in muscles of s-IBM patients has been shown to be due mainly to replacement of degenerated muscle fibres with fat (Reimers et al., 1994; Sekul et al., 1997), but oedema-like abnormalities may also contribute to it (Reimers et al., 1994; Sekul et al., 1997). In all cases but Patient 3, MRI disclosed variable degrees of signal hyperintensity in calf muscle (Calf Muscle Score range 5 1–8 for the patients). The Calf Muscle Score was zero in all control subjects. Figure 2 shows typical resting calf muscle spectra from a patient (Patient 8 in Fig. 2B) and from a sex- and agematched control subject (Fig. 2A). In the patient, the PCr peak is decreased and Pi peak is increased (relative to the β-ATP peak). Lower signal-to-noise in the patient’s spectrum is due to the lower muscle fibre content in the volume of tissue investigated by the coil. Data from resting muscle are reported in Table 2. In s-IBM patients, the PCr to ATP and Pi to ATP ratios were significantly reduced and increased, respectively, while cytosolic pH was significantly increased. The low [PCr] (data not shown) and the high pH resulted in a calculated free [ADP] significantly increased in patients. The phosphorylation potential was below the normal range in all patients (represented by the reciprocal of the phosphorylation potential in Table 2). Patients and control subjects exercised inside the magnet, reaching a similar end-exercise PCr concentration (expressed as a percentage of the resting level in Table 3). The mean end-exercise free [ADP], although increased in s-IBM patients, was not statistically different from that in control
Fig. 2 Resting calf muscle phosphorus MR spectra (TR 5 2 s; number of scans 5 64) from Patient 8 (B) and a sex- and agematched control subject (A). Pi 5 inorganic phosphate; PCr 5 phosphocreatine. The cytosolic pH is calculated from the chemical shift of Pi relative to PCr. Abscissa shows the chemical shift in parts per million (p.p.m.) and ordinate the relative signal intensity.
subjects (Table 3). In all s-IBM patients an end-exercise [ADP] well above the Km value of 30 µM (Kemp et al., 1993b) is expected to have fully stimulated the mitochondrial ATP production. Under these conditions the rate of PCr resynthesis after exercise is a very sensitive index of the rate of mitochondrial ATP production. All the s-IBM patients showed an initial rate of PCr recovery, V, within the normal range (Table 3). Using the ADP control model for mitochondrial respiration (Chance and Williams, 1955) we calculated the maximum rate of mitochondrial ATP synthesis, Vmax, from V and the end-exercise [ADP] (Kemp et al., 1993b). Oxidative metabolism was also assessed as postexercise ADP recovery half time, a measurement independent of the absolute metabolite concentration. Both Vmax and the ADP recovery rate are convenient assessments of muscle mitochondrial respiration, as they are independent of the degree of PCr breakdown and cytosolic acidification at the end of exercise (Lodi et al., 1997a). Mitochondrial Vmax did not differ between patients and control subjects, i.e. all s-IBM
In vivo oxidative metabolism in s-IBM Table 2
31P-MRS
Case
2123
data from resting muscle 31P-MRS
data
Calculated values
pH
PCr/ATP
Pi/ATP
[ADP] (µM)
1/PP 3 106 (mM)
1 2 3 4 5 6 7 8 9 10 11 12
7.06 7.10 7.01 7.01 7.05 7.08 7.04 7.07 7.05 7.07 7.06 7.02
3.88 3.50 3.90 3.63 3.46 3.58 3.75 3.81 3.92 3.21 3.73 4.00
0.80 1.20 0.45 0.66 0.61 0.74 0.78 0.86 1.06 1.05 0.96 0.51
19 30 17 21 28 27 21 21 18 36 22 15
15.3 36.3 7.6 14.2 17.2 20.3 16.3 18.0 19.0 37.3 21.3 7.8
s-IBM patients (n 5 12) Control subjects (n 5 12)
7.05 6 0.03
3.70 6 0.23
0.81 6 0.23
23 6 6
19.2 6 9.3
7.02 6 0.02 (6.97–7.05)
4.21 6 0.29 (3.70–4.55)
0.48 6 0.10 (0.29–0.66)
12 6 5 (7–21)
5.5 6 1.2 (3.4–7.2)
P-value
0.008
0.0001
0.0002
0.0001
0.0001
[ADP] 5 free cytosolic ADP; PP 5 phosphorylation potential. Patient and control group data are expressed as means 6 SD (range in parentheses).
patients showed a Vmax within the normal range (Table 3). There was no correlation between Vmax and the degree of MRI abnormalities of calf muscle expressed as Calf Muscle Score (r 5 0.1, P 5 0.7) (Fig. 3). Normal mitochondrial respiration was confirmed by the ADP recovery half time, which was within the normal range in all patients (Table 3). ADP recovery half time did not correlate with the Calf Muscle Score (data not shown).
Discussion Pathogenesis of s-IBM is still unknown. A number of observations have shown that mitochondrial alterations and abnormalities of mtDNA are common findings in the skeletal muscle of patients with s-IBM (Oldfors et al., 1993, 1995; Rifai et al., 1995; Santorelli et al., 1996). In primary mitochondrial disorders these changes are associated with a deficit of in vivo muscle mitochondrial ATP production as assessed by 31P-MRS (Arnold et al., 1985; Taylor et al., 1994; Lodi et al., 1997c). Our results confirmed the frequent occurrence of mitochondrial changes such as ragged red fibres, cytochrome c oxidase-negative fibres and multiple mtDNA deletions in muscle biopsy specimens of patients with s-IBM. On the other hand, in vivo muscle energy metabolism was unaltered in all 12 of the s-IBM patients we studied using 31P-MRS. Abnormalities of mitochondrial respiration can be detected using 31P-MRS, by stressing skeletal muscle with an aerobic incremental exercise and following the recovery phase (Barbiroli, 1992). When exercise is stopped, mitochondria are the only source of ATP synthesis and the PCr resynthesis rate reflects very precisely the rate of ATP production through
oxidative phosphorylation (Taylor et al., 1983; Arnold et al., 1984). We did not detect any deficit of the muscle oxidative metabolism in s-IBM patients as shown by the initial rate of PCr recovery, V, and mitochondrial Vmax which were within the normal range in all cases (Table 3). This result is not due to the variable degree of involvement of calf muscle by the pathological process in different patients as no correlation was found between mitochondrial Vmax and the degree of calf muscle MRI abnormalities (Fig. 3). Normal muscle mitochondrial respiration in s-IBM was confirmed by the post-exercise ADP recovery half time, which was within the normal range in all patients. In contrast to the finding of a normal rate of oxidative ATP production during recovery from exercise, all patients showed abnormalities at rest. In the patient group we observed a significant reduction in the PCr to ATP ratio, an increase in the Pi to ATP ratio and a raised intracellular pH. The accepted method for calculating [ADP], [PCr] and [Pi] in skeletal muscle is to assume constant values for [ATP] and [TCr], obtained from biochemical assay (Arnold et al., 1985), and to derive [PCr] and [Pi] from their spectral ratios with ATP and [Cr] by subtraction of [PCr] from [TCr] (see also Patients and methods section). Using this traditional method, we observed a significant reduction in [PCr] associated with an apparently stoichiometric increase in Pi concentration in the patient group (data not shown). As result of the low [PCr] and high cytosolic pH, the free (metabolically active) [ADP], calculated from the creatine kinase reaction (Veech et al., 1979), was significantly increased in s-IBM patients. [ADP] and [Pi] are incorporated into the calculation of the phosphorylation potential, for which all s-IBM patients were below the normal range (Table 2).
2124
R. Lodi et al. Table 3
31P-MRS
Case
end-exercise and recovery data End-exercise
Recovery
PCr (%)
[ADP] (µM)
V (mM/min)
Vmax (mM/min)
ADP t1/2 (s)
1 2 3 4 5 6 7 8 9 10 11 12
24 35 62 24 52 31 51 64 40 73 51 39
108 104 56 135 74 82 54 66 79 66 81 84
30 18 26 26 38 18 20 31 25 30 25 26
60 48 51 33 72 25 35 43 41 37 39 31
8.4 6.4 8.4 8.0 7.4 14.6 11.4 11.5 7.5 5.2 11.2 11.1
s-IBM patients (n 5 12) Control subjects (n 5 12)
46 6 16
82 6 23
31 6 10
43 6 13
9.3 6 2.8
42 6 16 (23–66)
68 6 40 (35–150)
33 6 14 (14–69)
45 6 11 (18–58)
10.6 6 3.4 (4.0–14.9)
P-value
0.5
0.3
0.7
0.7
0.3
[ADP] 5 free cytosolic ADP; V 5 initial rate of PCr post-exercise resynthesis; Vmax 5 maximum rate of mitochondrial ATP production. Patient and control group data are expressed as mean 6 SD (range in parentheses).
As ADP is a major regulator of respiration (Chance and Williams, 1955), raised [ADP] at rest is frequently observed in patients with primary mitochondrial myopathies (Matthews et al., 1991; Taylor et al., 1994). However, high resting [ADP] is not necessarily an index of abnormal mitochondrial control; it could point to increased ATP turnover. Increased [ADP] at rest is observed in the absence of mitochondrial dysfunction in patients with Duchenne and Becker muscular dystrophies and in manifesting carriers of Duchenne muscular dystrophy (Kemp et al., 1993a, 1997). One possible explanation for the high resting ADP might be an increased demand at rest for ATP in muscles of both s-IBM and Duchenne/Becker muscular dystrophy patients. Sarcolemmal abnormalities due to dystrophin deficit in Duchenne/Becker muscular dystrophy are responsible for a number of ionic changes which may require increased basal ATP consumption. We can speculate that this might also be the case in s-IBM, where sarcolemmal damage due to the cell-mediated inflammatory process may result in increased basal ATP consumption. The high resting cytosolic pH, present in Duchenne/Becker muscular dystrophy and s-IBM, but only occasionally reported in mitochondrial myopathies (Arnold et al., 1985; Matthews et al., 1991; Taylor et al., 1994; Lodi et al., 1997c), is a clear index of abnormal ionic regulation, and in Duchenne/Becker muscular dystrophy it has been interpreted as a consequence of an increase in the Na1/H1 antiport set point due to high sarcoplasmic [Ca21] (Frelin et al., 1988). It can be seen from the data in Table 2 that the calculated free [ADP] in resting muscle is high, especially for the
Fig. 3 Maximum rate of mitochondrial ATP production (Vmax) as a function of the Calf Muscle Score (obtained adding the scores from the lateral and medial gastrocnemius, and the soleus muscles, see Patients and methods section) in s-IBM patients.
patient group, when compared with a Km of 30 µM for ADP. It should be noted that calculation of free [ADP] at rest is particularly prone to error as [PCr] is not very different from [TCr], and small errors in either value give rise to substantial errors in [Cr], and hence to calculated free [ADP]. However, the differences in PCr to ATP ratios and pH between the two groups strongly suggest that [ADP] at rest probably is raised in s-IBM relative to control subjects. Because calculating [ADP] accurately depends on a precise knowledge of [TCr] and [ATP], and this would require biochemical assay of muscle from each subject from the same volume as that studied by 31P-MRS, these uncertainties cannot be resolved from the data available in the present study. As discussed above, the high pH is suggestive of sarcolemmal changes
In vivo oxidative metabolism in s-IBM which would lead to altered ion handling. It might reasonably be expected that these alterations would be accompanied by concentration changes in the metabolites involved in cellular energetics, such as creatine and these phosphorylated compounds. Since PCr to ATP, Pi to ATP and PCr to Pi ratios are altered in s-IBM, the concentration of one or more of these metabolites must be abnormal. It is important to stress that the possible errors in the calculation of metabolite concentrations do not affect the comparison of oxidative metabolism in s-IBM patients with that in control subjects, assessed from data collected during recovery from exercise. In particular, a lower [TCr] in the s-IBM patients would result in an even higher Vmax, as a consequence of a lower calculated [ADP] at the end of exercise, and the post-exercise ADP recovery half time (Arnold et al., 1984) is a kinetic measurement independent of absolute [ADP]. Despite a previous report identifying deficiencies in individual respiratory chain enzyme activities (Santorelli et al., 1996), we could find no evidence of a defect in overall muscle oxidative phosphorylation in our s-IBM patient group. The normal mitochondrial respiration found in the present study in all s-IBM patients is not due to lack of involvement of the muscles we investigated. In addition to myopathic electromyographic changes present in the calf muscles of all our s-IBM patients, all cases but one showed MRI abnormalities in their calf muscles, and 31P-MRS abnormalities of the same muscles at rest were also detected in all patients. This is robust evidence that the muscles showing a normal mitochondrial respiration were not spared by the pathological process. As we investigated a relatively large volume of muscle, we cannot exclude an oxidative phosphorylation defect in a small proportion of muscle fibres. However, resting 31P-MRS abnormalities indicate that muscle biochemical changes other than deficits of mitochondrial respiration are a feature of the skeletal muscle in s-IBM, and that mitochondrial alterations seen on muscle biopsy are likely to be a secondary manifestation. This is consistent with a preliminary report showing that mtDNA abnormalities are present in muscle, but not in lymphocytes, of patients with s-IBM, and that there is no correlation between mtDNA abnormalities and muscle biopsy changes (Sanders et al., 1997). There is no known treatment for s-IBM. Patients with s-IBM do not usually respond to immunosuppressants (Danon et al., 1982), and a recent controlled trial of high doses of intravenous immunoglobulin did not show clear benefit (Dalakas et al., 1997). Our in vivo results indicate that in s-IBM there is no rationale for therapies enhancing mitochondrial function (Bendahan et al., 1992; De Stefano et al., 1995), despite the high frequency of skeletal muscle morphological mitochondrial abnormalities and mtDNA deletions.
Acknowledgements We wish to thank the physicians of the National Hospital for Neurology and Neurosurgery and the Royal Free Hospital
2125
for allowing us to study their patients, also Dr J. Poulton for the mtDNA studies, Dr S. Kumar and D. Manners for their help with the MRI studies, Dr G. J. Kemp for helpful discussion and Mrs E. Gower for helping in the organization of the studies. This work was supported by the Medical Research Council. R.L. is a Junior Research Fellow at Wolfson College, Oxford, UK, and is supported by an EEC grant in the framework of the BIOMED programme (Contract n. BMH4CT965017). S.J.T. is a Medical Research Council Training Fellow.
References Arnold DL, Matthews PM, Radda GK. Metabolic recovery after exercise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31P NMR. Magn Reson Med 1984; 1: 307–15. Arnold DL, Taylor DJ, Radda GK. Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann Neurol 1985; 18: 189–96. Barbiroli B. 31P MRS of human skeletal muscle. In: De Certains JD, Bove´e WMMJ, Podo F, editors. Magnetic resonance spectroscopy in biology and medicine. Functional and pathological tissue characterization. Oxford: Pergamon Press; 1992. p. 369–86. Bendahan D, Desnuelle C, Vanuxem D, Confort-Gouny S, FigarellaBranger D, Pellissier JF, et al. 31P NMR spectroscopy and ergometer exercise test as evidence for muscle oxidative performance improvement with coenzyme Q in mitochondrial myopathies [see comments]. Neurology 1992; 42: 1203–8. Comment in: Neurology 1993; 43: 628–9. Blume G, Pestronk A, Frank B, Johns DR. Polymyositis with cytochrome oxidase negative muscle fibres. Early quadriceps weakness and poor response to immunosuppressive therapy. Brain 1997; 120: 39–45. Carpenter S, Karpati G, editors. Pathology of skeletal muscle. New York: Churchill Livingstone; 1984. Carpenter S, Karpati G, Heller I, Eisen A. Inclusion body myositis: a distinct variety of idiopathic inflammatory myopathy. Neurology 1978; 28: 8–17. Chance B, Williams GR. Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 1955; 217: 409–27. Cortelli P, Montagna P, Avoni P, Sangiorgi S, Bresolin N, Moggio M, et al. Leber’s hereditary optic neuropathy: genetic, biochemical, and phosphorus magnetic resonance spectroscopy study in an Italian family. Neurology 1991; 41: 1211–5. Dalakas MC. Polymyositis, dermatomyositis and inclusion-body myositis [see comments]. [Review]. N Engl J Med 1991; 325: 1487–98. Comment in: N Engl J Med 1992; 326: 1293. Dalakas MC, Sonies B, Dambrosia J, Sekul E, Cupler E, Sivakumar K. Treatment of inclusion-body myositis with IVIg: a double-blind, placebo-controlled study [see comments]. Neurology 1997; 48: 712–6. Comment in: Neurology 1997; 48: 567–8.
2126
R. Lodi et al.
Danon MJ, Reyes MG, Perurena OH, Masdeu JC, Manaligod JR. Inclusion body myositis. A corticosteroid-resistant idiopathic inflammatory myopathy. Arch Neurol 1982; 39: 760–4. De Stefano N, Matthews PM, Ford B, Genge A, Karpati G, Arnold DL. Short-term dichloroacetate treatment improves indices of cerebral metabolism in patients with mitochondrial disorders. Neurology 1995; 45: 1193–8.
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: 114–20. Muller-Hocker J. Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm of man without muscular disease: an agerelated alteration. J Neurol Sci 1990; 100: 14–21.
DiMauro S, Moraes CT. Mitochondrial encephalomyopathies. [Review]. Arch Neurol 1993; 50: 1197–208.
Oldfors A, Larsson NG, Lindberg C, Holme E. Mitochondrial DNA deletions in inclusion body myositis. Brain 1993; 116: 325–36.
Durnin JV, Womersley J. Body fat assessed from total body density and its estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br J Nutr 1974; 32: 77–97.
Oldfors A, Moslemi AR, Fyhr IM, Holme E, Larsson NG, Lindberg C. Mitochondrial DNA deletions in muscle fibers in inclusion body myositis. J Neuropathol Exp Neurol 1995; 54: 581–7.
Frelin C, Vigne P, Ladoux A, Lazdunski M. The regulation of the intracellular pH in cells from vertebrates. [Review]. Eur J Biochem 1988; 174: 3–14.
Reimers CD, Schedel H, Fleckenstein JL, Nagele M, Witt TN, Pongratz DE, et al. Magnetic resonance imaging of skeletal muscles in idiopathic inflammatory myopathies of adults. J Neurol 1994; 241: 306–14.
Griggs RC, Askanas V, DiMauro S, Engel A, Karpati G, Mendell JR, et al. Inclusion body myositis and myopathies. [Review]. Ann Neurol 1995; 38: 705–13.
Rifai Z, Welle S, Kamp C and Thornton CA. Ragged red fibers in normal aging and inflammatory myopathy [see comments]. Ann Neurol 1995; 37: 24–9. Comment in: Ann Neurol 1995; 38: 273–4.
Hirano M, Silvestri G, Blake DM, Lombes A, Minetti C, Bonilla E, et al. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): clinical, biochemical and genetic features of an autosomal recessive mitochondrial disorder. Neurology 1994; 44: 721–7.
Sanders MK, Hassanzedah S, Lin H, Dalakas MC and Epstein ND. Detection of mutations in the mitochondrial genome from muscle and lymphocytes of patients with inclusion body myositis (IBM) [abstract]. Neurology 1997; 48 (3 Suppl): A124.
Kemp GJ, Taylor DJ, Dunn JF, Frostick SP, Radda GK. Cellular energetics of dystrophic muscle. J Neurol Sci 1993a; 116: 201–6.
Santorelli FM, Sciacco M, Tanji K, Shanske S, Vu TH, Golzi V, et al. Multiple mitochondrial DNA deletions in sporadic inclusion body myositis: a study of 56 patients. Ann Neurol 1996; 39: 789–95.
Kemp GJ, Taylor DJ, Radda GK. Control of phosphocreatine resynthesis during recovery from exercise in human skeletal muscle. NMR Biomed 1993b; 6: 66–72. Kemp GJ, Lodi R, Taylor DJ, Thompson CH, Male A, Muntoni F, et al. Muscle bioenergetics in Becker muscular dystrophy studied by 31P magnetic resonance spectroscopy. 14th Annual Meeting European Society for Magnetic Resonance in Medicine and Biology. Brussels, 1997. p. 44. Li YY, Hengstenberg C, Maisch B. Whole mitochondrial genome amplification reveals basal level multiple deletions in mtDNA of patients with dilated cardiomyopathy. Biochem Biophys Res Commun 1995; 210: 211–8. Lodi R, Kemp GJ, Iotti S, Radda GK, Barbiroli B. Influence of cytosolic pH on in vivo assessment of human muscle mitochondrial respiration by phosphorus magnetic resonance spectroscopy. Magma 1997a; 5: 165–71. Lodi R, Muntoni F, Taylor J, Kumar S, Sewry C, Blamire A, et al. Correlative MR and 31P-MR spectroscopy study in sarcoglycan deficient limb muscular dystrophy. Neuromuscul Disord 1997b; 8: 505–11. Lodi R, Taylor DJ, Tabrizi SJ, Kumar S, Sweeney M, Wood NW, et al. In vivo skeletal muscle mitochondrial function in Leber’s hereditary optic neuropathy assessed by 31P-magnetic resonance spectroscopy. Ann Neurol 1997c; 42: 573–9. Luciano CA, Dalakas MC. Inclusion body myositis: no evidence for a neurogenic component. Neurology 1997; 48: 29–33.
Sekul EA, Chow C, Dalakas MC. Magnetic resonance imaging of the forearm as a diagnostic aid in patients with sporadic inclusion body myositis. Neurology 1997; 48: 863–6. Simonetti S, Chen X, DiMauro S, Schon EA. Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR. Biochim Biophys Acta 1992; 1180: 113–22. Taylor DJ, Bore PJ, Styles P, Gadian DG, Radda GK. Bioenergetics of intact human muscle. A 31P nuclear magnetic resonance study. Mol Biol Med 1983; 1: 77–94. Taylor DJ, Kemp GJ, Radda GK. Bioenergetics of skeletal muscle in mitochondrial myopathy. J Neurol Sci 1994; 127: 198–206. Veech RL, Lawson JW, Cornell NW, Krebs HA. Cytosolic phosphorylation potential. J Biol Chem 1979; 254: 6538–47. Watkins SC, Cullen MJ. A qualitative and quantitative study of the ultrastructure of regenerating muscle fibres in Duchenne muscular dystrophy and polymyositis. J Neurol Sci 1987; 82: 181–92. Zeviani M, Servidei S, Gellera C, Bertini E, DiMauro S, DiDonato S. An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 1989; 339: 309–11.
Received January 15, 1998. Revised May 22, 1998. Accepted June 25, 1998
