Muscle fibres - Biochemical Society Transactions

Organisms, Organs, Cells and Organelles

24 Enriquez,J. A., Femandez-Silva, P., Perez-Martos. A,, LopezPerez. M. J. and Montoya. J. ( I 996) Eur. ]. Biochem. 237, 60 1-6 I0 25 Gaines, G., Rossi, C. and Attarci, G. ( I 987) J. Biol. Chem. 262. I 907- I 9 I 5

26 Soboll, 5. ( 1993) Biochem. SOC. Trans. 2 I , 799-803

Received 25 August I999

Muscle fibres: applications for the study of the metabolic consequences of enzyme deficiencies in skeletal muscle S. Vielhaber*, A. Kudint. R. Schroderf, C.E. Elgert and W. S. Kunzt’ *Department of Neurology, University Magdeburg, Leipziger Str. 44, D 39 I20 Magdeburg, Germany, +Department of Epileptology, University Bonn Medical Center, Sigmund-Freud-Str. 25, D 53 I05 Bonn, Germany, and f Department of Neurology, University Bonn Medical Center, Sigmund-Freud-Str. 25, D 53 I05 Bonn, Germany

pression of the mtDNA mutation in skeletal muscle.

Abstract Mitochondria1 function in saponin-permeabilized muscle fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. We applied these techniques to study metabolic effects of changes in the pattern of mitochondrial enzymes in skeletal muscle of patients with chronic progressive external ophthalmoplegia or KearnsSayre syndrome harbouring large-scale deletions of mitchondrial DNA (mtDNA). In all patients combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits were observed. T h e citrate synthase-normalized activity ratios of these enzymes decreased linearly with increasing mtDNA heteroplasmy. This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For patients with degrees of mtDNA heteroplasmy below about 60 yo we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV. Permeabilized skeletal-muscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. This finding indicates the presence of a ‘metabolic threshold ’ which can be assessed by functional studies of muscle fibres providing the link to the phenotypic ex-

Introduction T h e metabolic consequences of large rearrangements of mitochondrial DNA in skeletal muscle of patients with mitochondrial myopathies remain unclear [1,2]. So, previous attempts to correlate biochemical findings in skeletal muscle of patients with chronic progressive external ophthalmoplegia (CPEO) or Kearns-Sayre syndrome (KSS) to the degree of the rnitochondrial DNA (mtDNA) mutation failed to establish a clear-cut relationship between genotype and the biochemical phenotype [3-81. These problems may be attributed to various reasons. First, the heteroplasmic occurrence of the mtDNA mutation; second, the unpredictable mosaic distribution of mtDNA mutations in the affected tissue ; third, unknown individual effects of various mutations; and fourth, difficulties in the quantitative determination of respiratory chain-enzyme activities and the degree of heteroplasmy of the mtDNA in skeletal muscle. In contrast, in cell cultures containing well-defined amounts of heteroplasmic mtDNA for different mutations, so-called threshold values have been defined beyond which each mutation had an effect on the activity of mtDNA-encoded enzymes. For various tRNA point mutations this threshold has been determined to be above 8 5 % of mutated mtDNA [9]. In contrast, the 4977-bp ‘common deletion ’ had a 5CL5 5 o/o threshold [10,111. In this report we studied the metabolic consequences of deficiencies of enzymes of the mitochondrial respiratory chain in skeletal muscle by applying different techniques: enzyme-activity measurements with improved methods and investigation of saponin-permeabilized muscle fibres

Key words: genotypephenotype relations, mitochondrial (mt) myopathy, mtDNA deletion. Abbreviations used: mt DNA, rnitochondrial DNA: CPEO, chronic progressive external ophthalmoplegia; KSS, Keams-Sayre syndrome: COX, cytochrome c oxidase: CS, citrate synthase. ‘To whom correspondence should be addressed.

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Biochemical Society Transactions (2000) Volume 28, part 2

phosphate buffer, pH 7.4, in the presence of 0.1 yo laurylmaltoside and 200 pM cytochrome c. T o use this high cytochrome c concentration we applied a dual-wavelength photometer (Aminco DW 2000, SLM Instruments, Rochester, NY, U. S.A.) and worked in the P-band of ferrocytochrome c (510-535 nm; credox=5.9 mM-'.cm-'). The activity of rotenone-sensitive NADH :CoQ, oxidoreductases was measured in 100mM phosphate buffer (pH 7.4) containing 1 mM KCN, l00pM CoQ, and 150pM NADH at 340-375 nm according to [14]. The activities of citrate synthase (CS) and succinate :cytochrome c reductase were determined by standard methods as described in ~51.

with respirometric, fluorimetric and imaging techniques.

Materials and methods Muscle biopsy Biopsy specimens were obtained from 18 patients with the clinical picture of CPEO or KSS (age range 28-60 years; median 42.1 years) who underwent a diagnostic biopsy at the Department of Neurology, University Bonn Medical Center, Bonn, Germany (between 1987 and 1999). Skeletal-muscle samples from diagnostic biopsies of 19 patients with questionable myopathic electromyographic abnormalities but no biopsy evidence for a manifest myopathy were used as controls. All patients gave written informed consent prior to biopsy.

Fluorescence microscopy of isolated muscle fibres The isolated fibres were fixed at both ends on a cover slip in a Heraeus flexiperm chamber (Hanau, Germany) and incubated in the medium for measurements. The digital video images were acquired with an inverse fluorescence microscope (model IX-70; Olympus, Tokyo, Japan) equipped with a charge-coupled-device (CCD) camera (model C F 8/1 DXC; Kappa, Gleichen, Germany). The NAD(P)H fluorescence image was obtained using 366 nm excitation and 450 nm long-path emission and the flavoprotein fluorescence image was obtained using 470nm excitation and 525 nm narrow-band emission. The digital ratio images were calculated using the LSM software (Carl Zeiss, Jena, Germany).

Genetic analysis Total DNA was isolated from 10-40 mg of liquidnitrogen-frozen muscle samples by standard methods and Southern blots were performed with 1 pg of DNA digested by either PvuII or the combination of PvuII and BamHI as described previously [121. Solutions The relaxing solution contained 10 mM Ca2+EGTA buffer (0.1 pM free calcium), 20 mM imidazole, 20 mM taurine, 49 mM K+-Mes, 3 mM KH,PO,, 9.5 mM MgCl,, 5 mM ATP and 15 mM phosphocreatine, pH 7.1. The measurements were performed in a medium consisting of 110 mM mannitol, 60 mM KCl, 10 mM KH,PO,, 5 mM MgCl,, 0.5 mM Na,EDTA and 60 mM Tris/ HCl, pH 7.4.

Results With optimized tests (see the Materials and methods section) we measured mitochondrial enzyme activities in muscle-biopsy specimens from 18 patients with large-scale deletions of mtDNA (2-7.5 kb). The results are summarized in Figure 1(A) as plot of NADH :CoQ, reductase/CS ratios versus COX/CS ratios. This linear dependency clearly demonstrates that mitochondrial respiratory chain-enzyme deficiencies can be visualized if the activities are divided by CS activity. Thus COX/CS and NADH :CoQ1 reductase/CS ratios clearly delineated all 18 patients with large-scale deletions from our 19 normal controls (point with error bars). Furthermore, we investigated the putative relationship between the degree of mtDNA heteroplasmy and biochemical parameters of impairment of mitochondrial energy metabolism. We observed inverse correlations

Preparation of muscle fibres About 50 mg of biopsy tissue (M. vastus lateralis) was used for isolation of saponin-permeabilized fibres. Bundles of muscle fibres containing usually 2-4 single fibres were isolated by mechanical dissection. The saponin treatment was performed by incubation of the fibre bundles in relaxing solution (see above for composition) containing 50 ',ug/ml saponin as described in [131. The maximal glutamate + malate and succinate oxidation rates were determined as previously described [12,131. Enzyme activities The activity of cytochrome c oxidase (COX) was measured spectrophotometrically in 100 mM


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mitochondrial function in saponin-permeabilized muscle fibres. T h e treatment of dissected muscle fibres with low amounts of saponin (50 pg) results in an efficient permeabilization of the sarcolemma, leaving the mitochondria and the sarcoplasmic reticulum almost intact. Mitochondria1 function in these fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. A typical experiment visualizing mitochondrial function by fluorescence microscopic detection of the flavoprotein fluorescence/NAD(P)H fluorescence ratio (see [16]) in two individual skeletal-muscle fibres of a CPEO patient is shown in Figure 2. In the endogenous fully oxidized state of both these fibres the ratio of the autofluorescence images is bright (Figure 2A). This is caused by the high flavoprotein a-lipoamide dehydrogenase fluorescence in the oxidized state and the low pyridine nucleotide fluorescence. T h e addition of the mitochondrial substrates octanoylcarnitine and malate caused a partial reduction of the mitochondrial NAD system, leading to a darker ratio image (Figure 2 B). Interestingly, one fibre became nearly black, whereas the intensity of the upper fibre only moderately decreased at its right part. This points already to rate differences of oxidative phosphorylation in both fibres and even within single muscle fibres. Thereafter, we added 1 m M ADP for the maximal stimulation of electron flow through the respiratory chain. Again, the right part of the upper fibre became brighter, while the left part remained dark (Figure 2C). On the addition of cyanide, which blocks selectively COX, both fibres became dark (Figure 2 D). This experiment provided evidence for a partial inhibition of respiratory chain which occurred only in the left part of the upper muscle fibre. Additionally, we applied saponin-permeabilized muscle fibres to study the metabolic effects of changes in the pattern of mitochondria1 enzymes in patients with CPEO or KSS harbouring largescale deletions of mtDNA. As clearly shown in Figure 1(A) in all of our patients we observed combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits. Moreover, the CS-normalized activity ratios of COX and N A D H :CoQ, reductase decreased linearly with increased degree of mtDNA heteroplasmy. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For this we determined the maximal rates of respiration of muscle fibres and applied

between the degrees of mtDNA heteroplasmy and COX/CS ratios as well as NADH :CoQ, oxidoreductase/CS ratios (Figures 1 B and 1C). This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. T o evaluate the metabolic effects of these mitochondrial enzyme deficiencies we studied

Figure I Relationshipbetween the COWCS and NADH:CoQ, oxidoreductaselCS (Q I /CS) ratios (A). and dependency between the degree of heteroplasmy of the mtDNA mutation and the NADH:CoQ, reductase/CS ratio (B) and the COWCS ratio (C) Data points with e m r bars, average of I9 control skeletal-muscle samples.

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inhibitor titration of these rates with the complexI inhibitor rotenone (or amytal) and the complexIV inhibitor azide (see [12]). As shown in Figure 3 for patients with degrees of mtDNA heteroplasmy below about 60% we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV (results not shown). Permeabilized skeletal-muscle fibres from patients with degrees of mtDNA heteroplasmy above 6o % showing enzyme deficiencies exhibited additionally decreased maximal rates of respiration.

Figure 2 Digital ratio video fluorescence images of flavoprotein and NAD(P)H fluorescence of a bundle of saponinpermeabilized muscle fibres of a patient with CPEO A bundle consisting oftwo saponin-permeabilized muscle fibres of a CPEO patient was fixed at both ends in a Heraeus flexiperm chamber and investigated in 300 pi of buffer for measurements (see Materials and methods) on the stage of an Olympus Ix 70 inverted fluorescence microscope. Shown are digital ratio images offlavoprotein and NAD(P)H fluorescence (A) in the endogenous oxidized state, and after the addition of (B) I mM oaanoyl camitine and 5 mM malate, (C) I mM ADP and (D) 4 mM KCN.

Figure 3 Dependency of (A) maximal rates of respiration of saponin-permeabilized muscle fibres and (B) flux control of complex I of respiratory chain on the degree of heteroplasmy of the mtDNA mutation (A) 0, Rates of respiration were determined in the presence of 10 mM succinate. 10 p M rotenone and I mM ADP. 0 . Respiration rates were determined in the presence of 10 mM glutamate, 5 mM malate and I mM ADP. (6)The flux control coefficients were determined from amytal titration experiments of respiration rates of saponin-permeabilized muscle fibres in the presence of 10 mM glutamate, 5 mM malate and I mM ADP essentially as described in [ 191.

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KSS patients consists of fibres with intact mitochondria with mutated mtDNA below the threshold, and fibres with defective mitochondria with mutated mtDNA beyond the mutation threshold of 55% [10,11], it is obvious that due to this mosaicism threshold effects cannot be observed and the phenotypic effects are averaged over the whole heterogeneous fibre population. T o consider the heterogeneity of mtDNA deletions in skeletal muscle from CPEO and KSS patients we applied a functional imaging technique. We observed a heterogeneous distribution of the defect in mitochondrial function not only among individual muscle fibres but even within a single muscle fibre. This result is in agreement with the well-known heterogeneous distribution of COX activity observed by classical muscle histology [3,4]. It indicates, therefore, the presence of a profound metabolic heterogeneity in CPEO or KSS muscle. However, in investigations of large populations of muscle fibres these effects are averaged and tend to disappear if the number of fibres having mitochondrial defects is low. Applying functional investigations of saponin-permeabilized muscle fibres we observed, owing to these averaging effects for patients with degrees of mtDNA heteroplasmy below about 60% at almost normal maximal rates of respiration, an increase in flux control coefficients of complexes I and IV only. Permeabilized skeletalmuscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. These findings, therefore, indicate the presence of a 'metabolic threshold ' which can be assessed by functional studies of muscle fibres. We propose that this 'metabolic threshold ' provides the link to the phenotypic expression of the mtDNA mutation in skeletal muscle of CPEO or KSS patients.

Discussion Previous attempts to establish genotype-phenotype relationships in skeletal-muscle biopsies of CPEO or KSS patients provided controversial results [3-81. In contrast with human skeletal muscle, clear cut interdependencies between the degree of mtDNA heteroplasmy and the mitochondrial respiratory chain defects were reported in studies using cybrids of mtDNA-free cells and mitochondria containing deleted mtDNA [10,11]. Here, a threshold of about 50-55 yo deleted mtDNA was noted to cause a defect in mitochondrial function. In the present study we have applied standardized quantitative methods to evaluate possible correlations of biochemical and mtDNA findings in skeletal muscle of 18 patients with large-scale deletions of mtDNA (deletion size 2-7.5 kb, degree of heteroplasmy 16-78 yo). Applying improved assays we were able to delineate all of our patients with large-scale deletions from controls. Furthermore, we observed that N A D H :CoQ, oxidoreductase/CS ratios and the succinate :cytochrome c reductase/CS ratios (results not shown) correlated with COX/CS ratios. This indicates similar enzyme deficiencies in complexes with different amounts of mitochondrially encoded subunits (complex I, seven subunits; complex 111, one subunit; complex IV, three subunits). In other words, all of our patients have indeed combined complex I 111 IV defects. These findings clearly imply that large-scale deletions (all of which affected at least one tRNA gene) have, irrespective of the individual mutation size or localization, similar effects on all mitochondrially encoded proteins by affecting their biosynthesis. This can be explained by the finding that the steady-state levels of tRNAs in mitochondria are not in large excess, so that a reduction of approximately SO"/, in the amount of one charged tRNA is sufficient to cause a translation defect [17]. We were able to show a linear dependency between the COX/CS ratio, the N A D H :CoQ, reductase/CS ratio and the degree of mtDNA heteroplasmy by means of improved biochemical methods. These findings clearly indicate the absence of any well-defined mutation threshold in human skeletal muscle in its entirety. This result is, however, not in contradiction with the threshold concept [10,11,17,18], which can be applied either at the single fibre level or in a homogeneous cell population. Since skeletal muscle of CPEO and

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The technical assistance of P. Rausch (Department of Epileptology, University Bonn Medical Center, Bonn, Germany) and K. KappesHorn (Department of Neurology,University Bonn Medical Center, Bonn, Germany) is gratefully acknowledged. This work was supported by the BONFOR program of the University of Bonn and a research grant of RhBne-PoulencRorer Germany t o W.S.K.

References I Wallace, D. C. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8739-8746 2 Morgan-Hughes,J. A. and Hanna, M. G. ( 1999) Biochim. Biophys. Ada 1410, 125-145 3 Moreas. C. T.. DiMauro. S., Zeviani, M., Lombes. A,, Shanske, S., Miranda, A. F.. Nakase. H.. Bonilla, E.. Wemeck L. C., Setvidei, S. et al. (1989) N. Engl. J. Med. 320, 1293-1299

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4 Goto. Y.- I., Koga, Y., Horai, S. and Nonaka, I. (I990) I. Neurol. Sci. 100, 63-69 5 Yamamoto, M., Clemens, P. R and Engel, A. G. (I99 I) Neurology 4 I, I 822- I 828 6 Fassati, A., Bordoni, A.. Amboni, P., Fortunato, F., Fagiolari, G.. Bresolin, N., Prelle, A., Comi. G. and Scarlato, G. ( I 994) J.Neurol. Sci. 123, I 4 G I46 7 Lafofet, P., Lombes, A.. Eymard, B., Danan, C., Chevallay, M., Rouche, A., Frachon, P. and Fardeau, M. ( 1995) Neurornusc. Disord. 5, 3 9 9 4 I 3 8 Kiyomoto, B. H., Tengan, C. H., Moraes, C. T., Oliveira. A. S. B. and Gabbai, A. A. ( I 997) J. Neurol. Sci. 152, 16C165 9 Hayashi, J., Ohta, S., Takai, D., Miyabayashi, S., Sakuta, R., Goto, Y. and Nonaka, I. ( I 993) Biochem. Biophys. Res. Commun. 197, 1049- I055 10 Hayashi, J., Ohta, S., Kikuchi, A., Takemitsu. M., Goto, Y.4. and Nonaka, I. ( I 99 I) Proc. Natl. Acad. Sci. U.S.A. 88, 10614-10618 I I Porteous. W. K.. James.A. M., Sheard, P. W., Porteous, C. M., Packer, M. A., Hyslop, S. J.,MeRon, J. V., Pang. C. Y., Wie, Y. H. and Murphy, M: P. (-1 998) Eur.J. Biochem. 257, 192-201

I 2 Kuznetsov, A. V., Winkler, K, Kirches, E., Lins, H., Feistner, H. and Kunz. W. S. ( 1997) Biochim. Biophys. Acta 1360, 142- I50 13 Kunz, W. S., Kuznetsov, A. V., Schulze, W., Eichhom, K., Schild, L., Striggow, F., Bohnensack R, Neuhof, S., Grasshoff, H., Neumann, H. W. and Gellerich, F. N. ( I 993) Biochim. Biophys. Acta I 144,4653 14 Estomell, E., Fato, R, Pallotti, F. and Lenaz, G. ( I 993) FEBS Lett. 332, 127- I 3 I 15 Bergmeyer, H. U. ( I 970) Methoden der enzymatischen Analyse, 2, Auflage, Akademie Verlag, Berlin 16 Kuznetsov, A. V., Maybomda, O., Kunz, D., Winkler, K., Schubert, W. and Kunz, W. S. ( 1998) J. Cell Biol. 140, I09 I - I099 17 Chomyn, A. ( I 998) Am. J. Hum. Genet. 62,745-75 I 18 Moreas, C. T.. Ricci, E.. Bonilla, E., DiMauro, S. and Schon. E. A. ( I 992) Am. J. Hum. Genet. 50,934-949 19 Wiedemann, F. R, Winkler, K., Kuznetsov. A. V.. Bartels, C.. Vielhaber, S., Feistner, H. and Kunz, W. S. ( 1998) J. Neurol. Sci. I56,65-72

Received I 2 August 1999

Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases F. N. Gellerich", S. Trumbeckaite*, J. R. Opalka*, E. Seppett, H. N. Rasmussenf, C. Neuhoffg and S. Zierz* *Muskellabor der Neurologischen Klinik der Martin-Luther-UniversitatHalle-Wittenberg, Julius-Kuhn-Str.7, D06097 Halle/Saale, Germany, +Department of Pathophysiology, University of Tartu, I 8 Ulikooli St., EE-2400 Tartu, Estonia, f Department of Biochemistry, The August Krogh Institute, University of Copenhagen, Univenitetspparken I 3, DK-2 I00 Copenhagen, Denmark, and Slnstitut fur Klinische Pathophysiologie und Experimentelle Medizin im Zentrum fur lnnere Medizin der Univenitat Giessen, Klinikstr. 36, D-35385 Giessen, Germany tosis, and as a bioenergetic consequence the cytosolic phosphorylation potential decreases. Leaky outer membranes can be detected in saponin-skinned fibres with spectrophotometric and oxygraphic methods. This is of special interest in respect to acute impairment of mitochondria during ischaemialreperfusion.

Abstract T h e mitochondrial outer membrane separates the intermembrane space from the cytosol. T h e whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. Experimental evidence is reviewed supporting the hypothesis of dynamic ADP compartmentation within the intermembrane space. T h e outer membrane creates a diffusion barrier for small molecules (adenine nucleotides, creatine phosphate, creatine etc.) causing rate-dependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the outer membrane becomes leaky, cytochrome c and apoptosisinducing factor can be released, leading to apop-

Introduction T h e mitochondrial outer membrane (OM) separates the intermembrane space (IMS) from the cytosol and has to realize the exchange of metabolites, adenine nucleotides, cations and information between mitochondria and the rest of the cell. T h e communication between compartments is possible by the existence of porin pores. An estimated radius of 1.3-2 x lo-' m [ l ] of these pores is sufficient to allow the passage of molecules up to 6 kDa [2]. But larger molecules as peptides and proteins cannot permeate. Based on these findings a recently discovered task of the OM seems to be the entrapment of apoptosis-inducing factor and cytochrome c inside of the mitochondrial outer membrane. For induction of apoptosis

Key words: adenine nucleotides, compartmentation, cytochrome c. dextran, ischaemia.

Abbreviations used: OM, outer membrane; IMS, intermembrane space; Adn-translocator, adenine nucleotide translocator; rnt-HK, mitochondria1 hexokinase: mt-CK, mitochondrial creatine kinase; AK, adenylate kinase; Crp, creatine phosphate; RCI, respiratory control index.


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