Brain Advance Access originally published online on March 14, 2006
Brain 2006 129(5):1249-1259; doi:10.1093/brain/awl061
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Apoptosis in mitochondrial myopathies is linked to mitochondrial proliferation
1 Institut National de la Santé et de la Recherche Médicale, U582, 2 Université Pierre et Marie Curie, 3 AP HP, CHU Pitié-Salpêtrière, Institut de Myologie, Paris, 4 AP HP, Hôpital Ambroise Paré, Physiologie, Boulogne-Billancourt, 5 UFR Médicale Paris-Ile-de-France-Ouest and 6 Hôpital Morvan, Service d'Anatomo-pathologie, Brest, France
Correspondence to: Anne Lombès, Inserm U582, Institut de Myologie, Groupe hospitalier Pitié-Salpêtrière, 47-83 Boulevard de l'Hôpital, 75013 Paris Cedex, France E-mail: a.lombes{at}myologie.chups.jussieu.fr
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Summary |
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 Top Summary IntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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Increased susceptibility to apoptosis has been shown in many models of mitochondrial defects but its relevance to human diseases is still discussed. We addressed the presence of apoptosis in muscle from patients with mitochondrial DNA (mtDNA) disorders. Taking advantage of the mosaic pattern of muscle morphological anomalies associated with heteroplasmic mtDNA alterations, we have used an in situ approach to address the relationship between apoptosis and respiratory defect, mitochondrial proliferation and mutation load. Different patterns of mitochondrial morphological alterations were provided by the analysis of muscles with large mtDNA deletion (16 cases) or with the MELAS mutation (4 cases). The patient's age at biopsy ranged from 0.4 to 66 years and the muscle mutant mtDNA proportion from 32 to 82%. Apoptotic muscle fibres were observed in a small proportion of muscle fibres of 16 out of the 20 biopsies by three different detection methods for different steps of apoptosis: caspase 3 activation, fragmentation of nuclear DNA [terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay] or overexpression of the pro-apoptotic factor Bax. Analysis of apoptotic features in parallel to cytochrome c oxidase (COX) and succinate dehydrogenase activity of more than 34 000 individual muscle fibres showed that apoptosis occurred only in muscle fibres with mitochondrial proliferation (ragged red fibres, RRF) irrespective of their COX activity. Molecular analyses of single muscle fibres evidenced that, as expected, the presence of COX defect was associated with higher proportion of mutant mtDNA and lower amount of normal mtDNA. Within COX-defective fibres, the presence of mitochondrial proliferation was associated with increase of the mtDNA content but without change in the ratio between normal and mutant mtDNA molecules, thus showing that mitochondrial proliferation was accompanied by similar amplification of normal and mutant mtDNA molecules. Within RRF, apoptosis was associated with higher mutation proportion, suggesting that it was provoked by severe respiratory defect in the same time as increased mitochondrial mass. In conclusion, apoptosis most probably contributes to mitochondrial pathology. It is tightly linked to mitochondrial proliferation and high mutation load. When considering training therapeutics, one will have to take into account the possibility to induce apoptosis in parallel to mitochondrial proliferation.
Key Words: apoptosis; mitochondrial myopathies; mitochondrial DNA; deletion
Abbreviations: COX = cytochrome c oxidase; MELAS = mitochondrial encephalopathy with lactic acidosis and stroke-like episodes; mtDNA = mitochondrial DNA; PBS = phosphate-buffered saline; RRF = ragged red fibres; SDH = succinate dehydrogenase; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling
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Received September 19, 2005. Revised February 10, 2006. Accepted February 19, 2006.
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Introduction |
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TopSummaryIntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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Apoptosis is an evolutionarily-conserved form of cell death occurring in various physiological conditions such as morphogenesis and tissue homeostasis but also in diverse diseases including many neurodegenerative disorders (Leonard and Schapira, 2000
). Mitochondria play a key role in triggering or amplifying apoptotic events (Desagher and Martinou, 2000). Mitochondrial factors that may trigger apoptosis are Ca2+ overload, increased exposure to reactive oxygen species or severe decline in energetic capacity, the latter two being associated with mitochondrial myopathies (Bernardi et al., 2001). Several pro-apoptotic proteins are located in the mitochondrial intermembrane space. Among them, the cytosolic release of cytochrome c and Smac/Diablo leads to cascade activation of cytosolic caspases and subsequent apoptotic cell death with its morphological characteristics: cell shrinkage, plasma membrane blebbing, apoptotic bodies, and condensation and fragmentation of nuclear DNA (Saraste and Pulkki, 2000). Commitment to apoptosis is an end-point phenomenon, which depends on the permanent balance between pro- and anti-apoptotic factors, among which the Bcl-2 family of proteins (Bax, Bcl-2, ...) plays a major role.
Increased susceptibility to apoptosis has been shown in cellular and animal models of mitochondrial diseases (Geromel et al., 2001; Wang et al., 2001; Zhang et al., 2003; Carrozzo et al., 2004; Kujoth et al., 2005). Its relevance to human diseases is more controversial and difficult to address because most affected organs are not readily accessible to relevant investigation. Muscle biopsy, however, is most often available from affected patients and muscle symptoms are frequent in human mitochondrial diseases. At the beginning of the disease, they mostly consist in intolerance to exercise, which is probably directly due to the defective energy production. However, progressive muscle weakness and amyotrophy are often observed later during the patients' evolution and may be responsible for a severe handicap. This muscle evolution is not accompanied by specific morphological alterations that may explain the loss of muscle such as necrosis, inflammatory infiltrates or muscle fibre atrophy. The hypothesis of a progressive loss of muscle fibres by apoptosis may therefore be proposed. Previous reported studies have, however, produced contradictory results and therefore did not solve the issue on the prevalence of apoptosis and its relation to the mitochondrial defect (Mirabella et al., 2000; Sciacco et al., 2001; Fagiolari et al., 2002; Ikezoe et al., 2002; Umaki et al., 2002; Formichi et al., 2003). While some studies reported prominent apoptosis in mitochondrial myopathies (Mirabella et al., 2000; Ikezoe et al., 2002; Umaki et al., 2002; Formichi et al., 2003), others did not find any (Sciacco et al., 2001; Fagiolari et al., 2002). When present, apoptotic muscle fibres greatly varied in their proportion [from 0 to 1% to more than 75% of the muscle fibres (Mirabella et al., 2000; Ikezoe et al., 2002)]. That proportion correlated with the severity of the muscle disease in some series (Mirabella et al., 2000) but not in others (Formichi et al., 2003). In one report (Mirabella et al., 2000), apoptosis was localized in cytochrome c oxidase (COX) defective fibres, independent of their mitochondrial proliferation status, whereas it was mostly seen in COX-negative fibres with important mitochondrial proliferation in two other reports (Ikezoe et al., 2002; Umaki et al., 2002). All these studies on apoptosis in mitochondrial myopathies have used in situ methods, the technical difficulties of which, especially the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, are important and probably underlie some of the contradiction in the reported results. Immunoreaction of Bcl-2 or Bax analyses the balance of pro- and anti-apoptotic factors, respectively. Immunoreaction specific for the activated form of caspase 3 (after its cleavage), or the TUNEL assay, which shows DNA strand breaks, reveals mechanisms occurring only when the cell is committed to apoptosis.
Mitochondrial diseases are caused by very diverse genetic alterations located either on nuclear DNA or on mitochondrial DNA (mtDNA) (DiMauro, 2004). The presence of apoptosis has been studied mostly in diseases associated with mtDNA alterations (Mirabella et al., 2000; Sciacco et al., 2001; Fagiolari et al., 2002; Ikezoe et al., 2002; Umaki et al., 2002; Formichi et al., 2003). MtDNA deleterious alterations are most often heteroplasmic, that is, mutant mtDNA molecules coexist with a residual population of wild-type mtDNA molecules within each cell. The mutation proportion may greatly vary between cells in the same tissue and between organs of the same individual. This induces a great deal of complexity in the genotype/phenotype relationship (Chinnery et al., 1997, 2001; Jeppesen et al., 2003) and may underlie some of the contradiction of earlier reports on apoptosis. Heteroplasmy, however, also provides a unique possibility to directly assess in situ the deleterious consequences of mtDNA alterations. On the one hand, extraction of total DNA from a single muscle fibre allows one to analyse the proportion of mutant mtDNA molecules. On the other hand, the respiratory status of the same single muscle fibre can be evaluated using the two histochemical reactions of COX and succinate dehydrogenase (SDH) activities (Petruzzella et al., 1994; Prelle et al., 1994). The three catalytic subunits of COX, fourth complex of the respiratory chain, are mtDNA-encoded. COX activity, therefore, is directly dependent on the mtDNA status. SDH is the proximal part of complex II of the respiratory chain, all subunits of which are nuclear DNA-encoded. SDH activity, therefore, does not depend on the mtDNA and has often been used to demonstrate mitochondrial proliferation in the presence of an mtDNA defect (Tanji and Bonilla, 2000; Taylor et al., 2004). The presence of a significant link between a high proportion of mutated mtDNA and a respiratory defect in individual muscle fibres is considered to be the demonstration of the deleterious potential of the mutation (Sternberg et al., 2001; Coulbault et al., 2005). Although the residual amount of normal mtDNA, which depends on both the proportion of the mutation and the total mtDNA copy number, may in fact represent the critical parameter for the cell respiratory capacity, it has much less frequently been analysed than the proportion of the mutation (Hammans et al., 1992; Oldfors et al., 1992; Sciacco et al., 1994).
We have looked for the presence of apoptosis in mitochondrial myopathies using morphological techniques showing end-point phenomena such as caspase 3 activation or fragmentation of nuclear DNA (TUNEL assay) but also overexpression of the pro-apoptotic factor Bax. Taking advantage of the mosaic pattern of morphological mitochondrial alterations in muscle biopsies from patients with a deleterious heteroplasmic mtDNA alteration, we have used an in situ approach to address the relationship between apoptosis and respiratory defect (as revealed by COX histochemical reaction), mitochondrial proliferation (as revealed by SDH histochemical reaction) and mutation load (assessed by quantitative PCR analysis of the amount and proportion of both normal and mutant mtDNA).
The combination of these techniques allowed us to demonstrate the presence of apoptosis and to show its relationship to mitochondrial proliferation, amount of residual normal mtDNA and proportion of mutant mtDNA.
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Patients, material and methods |
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TopSummaryIntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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Patients
Muscle biopsies were obtained from 16 patients with a large mtDNA deletion and 4 patients with the A3243G point mutation of the mitochondrial tRNAleu(UUR) [mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) mutation]. MtDNA deletions were revealed by Southern blot analysis of the patients' total muscle DNA, while the A3243G mutation was shown by a previously described denaturing gradient gel electrophoresis-based method (Sternberg et al., 1998
) and then identified by direct sequencing with a 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The clinical and genetic data for these patients are summarized in Table 1. All patients were in a stable clinical situation at the time of biopsy and during the following months. Muscle biopsies from 10 subjects, from 0.2 to 49 years of age, who were deemed to be free of any muscle disorder by clinical, biological and morphological investigations, were used as controls for the morphological analyses.
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Methods
Muscle morphological analyses
Muscle specimens from the deltoid or quadriceps muscles were obtained by open biopsy, immediately frozen in isopentane cooled by liquid nitrogen and stored at –80°C. Serial transversal muscle sections were cut with a cryostat microtome, dried at room temperature and stored at –80°C.
Histochemical detection of COX and of SDH activities were performed on 10 µm thick sections according to standard procedures.
Three different immunohistochemical procedures were applied to assess the apoptotic status of muscle on 8 µm thick sections. For immunoreaction of active caspase 3 and Bax, muscle sections were fixed in ice-cold acetone for 15 min, incubated at room temperature with 0.3% H2O2 in phosphate-buffered saline (PBS) for 15 min, then in PBS + 3% bovine serum albumin (BSA) for 30 min and then overnight at 4°C with rabbit anti-active caspase 3 (BD Pharmingen, Tokyo, Japan) or mouse anti-Bax (B-9, sc-7480, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies diluted 1/400 or 1/100, respectively, in PBS + 3% BSA. After washing in PBS, the sections were incubated for 30 min at room temperature with the secondary antibody [multi-link biotinylated goat anti-rabbit or anti-mouse IgG, respectively (Vector Labs, Burlingame, CA, USA)] diluted 1/200 in PBS + 3% BSA. After washing in PBS, incubation with the streptavidin-peroxidase and revelation were performed according to the manufacturer's instructions (Vector Labs). The TUNEL assay was performed with NBT/BCIP substrate according to the protocol associated with the In Situ Alkaline Phosphatase Cell Death Detection Kit (Boehringer, Mannheim, Germany) but for a 1/10 dilution of the terminal deoxynucleotidyl transferase + fluorescein-labelled nucleotide mixture in TUNEL diluting buffer (Boehringer) (Duan et al., 2003).
All histological analyses were performed on serial muscle sections, allowing evaluation of individual muscle fibres with respect to their status for COX and SDH activity as well as the presence of Bax, caspase 3 and TUNEL immunoreaction. When mtDNA analyses were performed on individual muscle segments, serial sections were used for active caspase 3 immunoreaction (8 µm thick section) and combined COX + SDH histochemical reactions (16 µm thick section) (Oldfors et al., 1995) in order to establish the apoptosis status of individual muscle fibre segments dissected out of the section stained for COX + SDH.
Ultrastructural analysis was performed on muscle samples from Patients 13 and 15 with mtDNA deletion and Patients 17 and 20 with MELAS mutation. Fixation was in 2.5% glutaraldehyde in phosphate buffer, post-fixation in 2% osmium tetroxide solution in the same buffer and embedding in epoxy resin. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined using a Jeol 1010 electron microscope.
Genetic analyses
DNA extraction
DNA extraction from whole muscle was performed using standard methods based upon treatment by proteinase K and SDS, phenol/chloroform extraction and isopropanol precipitation. DNA from single muscle fibre segments dissected out of 16 µm muscle sections was extracted by incubation at 55°C for 2 h and then at 95°C for 10 min in 10 µl of cell lysis buffer (50 mM Tris–HCl pH 8.5, 1 mM EDTA, 0.5% Tween-20, 200 µg/ml proteinase K) (He et al., 2002).
Quantification of the diverse mtDNA species
Identification of deletion breakpoints was performed by PCR amplification of the mtDNA region encompassing the deletion breakpoints followed by its direct sequencing in both directions on an automated sequencer (Applied Biosystems) with the Dynamic Terminator Cycle Sequencing Kit (Amersham Biosciences). This technique allowed the identification of a small mtDNA region included in the mtDNA deleted region of Patients 1–16 and therefore ‘constantly deleted’ in that series of patients (see Supplementary Fig. 1).
Quantification by real-time PCR was performed using LightCycler FastStart Reaction SYBR Green I kit (Roche), following the manufacturer's instructions with assay conditions set up such that the amplification had an efficiency around 95% and was specific, giving one single peak on the fusion curve of the amplified fragments (see Supplementary Fig. 2 and Table 1). For quantification of each mtDNA species, serial dilutions of the linearized plasmid vector pGEM-T Easy (Promega) containing the target gene as an insert were used as the standard (Supplementary Table 1). Each quantification was performed in duplicate. Results were expressed as mean and standard error of the mean (SEM) of copies of mtDNA species. This number was used as such for the DNA extracted from individual muscle fibres. It was related to the amount of nuclear DNA in DNA samples extracted from whole muscle fragment and expressed as copies per picogram nuclear DNA. Quantification of nuclear DNA was also performed by real-time PCR with serial dilutions of total DNA from normal fibroblasts as the standards (Supplementary Table 1).
In samples with the MELAS point mutation, real-time PCR amplification (see Supplementary Fig. 1) was followed by digestion with the Apa I restriction enzyme and migration on a 3% agarose gel. Homoduplex molecules from wild-type mtDNA and heteroduplex molecules were not digested and migrated as a 299 base pairs (bp) band, while homoduplex molecules from mutant mtDNA were digested by the enzyme giving 2 bands (178 and 121 bp) (see Supplementary Fig. 3). Percentage of the mutation in each sample was calculated by densitometry of the bands with ChemiImager Tm5500 (Alpha Innotech, San Leandro, CA, USA) and comparison with densitometry of standard samples.
Statistical analyses
Only non-parametric tests were used. Comparison of quantitative parameters between groups was performed using the Mann–Whitney test. Correlation analyses were made with the Spearman rank order correlation test. A P-value below 0.05 was considered significant.
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Results |
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TopSummaryIntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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Significant signs of apoptosis were observed in most patients' biopsies
To search for the presence of apoptosis in the muscle of patients with mitochondrial myopathies, the diverse assays of apoptosis were first set up in order to give negative results in 10 control muscles. Immunoreaction for activated caspase 3 as well as for Bax was totally negative in control muscles, but the TUNEL assay showed very rare (<0.1%) positive myonuclei in some of the control muscles. With that restriction in mind, we then proceeded to look for the presence of apoptosis in the muscle of the 20 patients with mitochondrial myopathy.
The positive immunoreaction for activated caspase 3 and Bax had a diffuse, reticular or granular pattern with higher reaction in the subsarcolemmal region in some muscle fibres (Fig. 1, activated caspase 3 and Bax). TUNEL-positive myonuclei could be prominent in the muscle of some patients (Fig. 2). Their proportion greatly varied between patients as did the proportion of fibres with positive immunoreaction for activated caspase 3 or Bax. The proportions of apoptotic fibres obtained with the three different assays are shown in Table 2. These proportions were tightly correlated (P < 0.001 with correlation coefficient 0.858 and 0.895 for the percentage of activated caspase 3-positive muscle fibres versus Bax-positive muscle fibres or TUNEL-positive myonuclei, respectively), confirming that the three assays were analysing the same phenomenon and with similar sensibility. That result allowed us to use only activated caspase 3 immunoreaction as representative of apoptosis in the following in situ muscle analyses.
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The presence of apoptosis in muscle was confirmed by ultrastructural analysis of the muscle from two patients with a large mtDNA deletion and two with the MELAS mutation. Myonuclei with typical apoptotic alteration associating nuclear fragmentation and condensed chromatin were observed (Fig. 3). Other anomalies were alterations of the size and structure of mitochondria. There was no sign of necrosis.
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In conclusion, apoptotic features were present in 16 out of the 20 patients. The four patients without apoptotic muscle fibres were Patients 1 and 2, youngest patients of the series presenting with a Pearson's syndrome, and Patients 7 and 8, presenting with chronic progressive external ophthalmoplegia (CPEO) and the lowest muscle mutant mtDNA proportion among adult patients (Table 1). Apoptosis involved a variable but always a small proportion of muscle fibres (Table 2).
Morphological signs of apoptosis and of mitochondrial proliferation were tightly linked
The mosaic pattern of morphological anomalies associated with heteroplasmic mtDNA alteration brings a unique opportunity to investigate the relationship between apoptosis and mitochondrial anomalies on serial cross-sections. Individual muscle fibre segments were separately evaluated with respect to their respiratory function (evaluated by COX histochemical activity), mitochondrial proliferation (evaluated by SDH histochemical activity) and apoptosis (evaluated by the positive activated caspase 3 and/or Bax immunoreaction). A characteristic of mitochondrial myopathy associated with the MELAS mutation is the occurrence of mitochondrial proliferation in fibres with positive COX activity (see Fig. 1, MELAS, COX, arrows) (Hammans et al., 1992). It differs from the mitochondrial myopathy associated with large mtDNA deletion where mitochondrial proliferation is only seen in COX-negative fibres. Analysis of the two different genetic mitochondrial myopathies therefore allowed us to differentiate the influence of mitochondrial proliferation from that of respiratory defect.
The results of the analysis of more than 34 000 individual muscle fibres are summarized in Tables 3 and 4. In the muscles with an mtDNA deletion (Table 3), apoptosis was observed only in a subgroup of the muscle fibres with mitochondrial proliferation [ragged red fibres (RRF)]. Mitochondrial proliferation was observed only in COX-negative fibres and RRF, which were a subgroup (from 11 to 80%) of COX-negative fibres. COX-positive muscle fibres never showed any mitochondrial proliferation or any sign of apoptosis. In the muscle with the MELAS mutation (Table 4), apoptosis also occurred only in RRF. Although these RRF were most often COX-negative fibres, some had kept significant residual COX activity (Table 4). In conclusion, in both types of mitochondrial myopathies, apoptosis occurred only in the presence of marked mitochondrial proliferation and despite relative preservation of COX activity in some RRF.
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At the level of whole muscle fragment, the link between apoptosis and mitochondrial proliferation was further suggested by the tight correlation found between the proportion of activated caspase 3-, Bax- or TUNEL-positive fibres and the proportion of RRF (P < 0.001 for the three parameters). The correlation between apoptosis and respiratory defect appeared less tight (P = 0.043, 0.022 and 0.055 for the relationship between the proportion of Bax, TUNEL or activated caspase 3, respectively, and the proportion of COX-negative fibres). Furthermore, that correlation might only be indirect through the link between the proportion of COX-negative fibres and that of RRF (P = 0.037).
In conclusion, both at the level of whole muscle fragment and at that of individual muscle fibres, apoptosis was tightly linked to mitochondrial proliferation but less so to respiratory defect.
Respiratory defect was associated with lower content of normal mtDNA, and mitochondrial proliferation was not associated with preferential proliferation of mutant mtDNA at the level of single muscle fibres
The pattern of morphological alterations could have been solely due to quantitative aspects of the genetic defect. To address this possibility, the amount of residual normal mtDNA and of total mtDNA as well as the proportion of mtDNA alteration were measured in 300 single muscle fibres of known morphological status with respect to apoptosis, mitochondrial proliferation and respiratory defect. Three muscles with a large mtDNA deletion (Patients 3, 5 and 14) and one with the MELAS mutation (Patient 20) were analysed in order to compare different age at muscle biopsy and different mtDNA alteration.
Whatever their mitochondrial proliferation status, COX-negative fibres had significantly lower amount of normal mtDNA copy number and higher proportion of mutant mtDNA than COX-positive fibres (P < 0.001 for the two mtDNA parameters when results from the four muscles were analysed as a group or separately for each muscle) (Fig. 4).
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Mitochondrial proliferation was associated with higher amounts of total but also normal mtDNA. This was significant when the results from the four muscles were analysed as a group (P < 0.001 for both parameters) and when results from the muscle of Patient 5 and Patient 14, respectively, were separately analysed (P = 0.008 and 0.031 for the amount of total mtDNA and P = 0.026 and 0.013 for the amount of normal mtDNA). It did not reach statistical significance for Patient 3 and 20. In the four patients' muscle, the increase of the amount of normal mtDNA associated with mitochondrial proliferation was insufficient to reach the amount of normal mtDNA in COX-positive muscle fibres (Fig. 4).
Mitochondrial proliferation did not change the mutation proportion as shown by the lack of significant difference of the proportion of mutant mtDNA in COX-negative RRF versus COX-negative fibres without mitochondrial proliferation (P = 0.63, 0.64, 0.84, 0.76 and 0.55 for the muscle of Patients 3, 5, 14 and 20 analysed separately and as a group, respectively).
Within RRF, the presence of apoptosis correlated significantly with higher proportion of mutant mtDNA when the results from the four muscles were analysed as a group (P = 0.049) or when those from Patient 5 muscle were analysed separately (P = 0.003). In two other muscles (Patients 14 and 20) apoptosis was associated with higher proportion of mutant mtDNA but without reaching statistical significance (Fig. 4). In contrast, the presence of apoptosis did not correlate with the amount of residual normal or of total mtDNA in any of the muscles taken separately or analysed as a group.
At the level of whole muscle fragment, the link between apoptosis and high mutation load was strengthened by the significant correlation found between the proportion of apoptotic muscle fibres and proportion of mutant mtDNA (P = 0.003, 0.007, and 0.04, respectively, for the proportion of activated caspase 3-positive or Bax-positive or TUNEL-positive fibres, respectively). The proportion of mutant mtDNA also correlated with the proportion of RRF (P = 0.004) but not with that of COX-defective fibres (P = 0.594). On the other hand, the residual amount of normal mtDNA did not correlate with the proportion of any of these morphological alterations (P = 0.700, 0.846, 0.906 for the three apoptotic features and P = 0.831 and 0.719 for RRF and COX defect, respectively).
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Discussion |
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TopSummaryIntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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Pathophysiology of mitochondrial disorders is likely to differ depending on their underlying cause. We chose to study apoptosis in muscle harbouring a large-size mtDNA deletion or the MELAS mutation for the following reasons. Both deleterious alterations of the mtDNA induce a global defect in mitochondrial translation, which has been reported to induce apoptosis (Wang et al., 2001
; Liu et al., 2004). These two mtDNA alterations are commonly found in mitochondrial myopathies and their study has therefore potential interest for many patients. Patients with these mtDNA alterations have been analysed in previous reports on apoptosis in mitochondrial myopathies. We could therefore compare our data with reported ones. The recurrence of the mtDNA alterations permitted to gather 20 unrelated patients, thus decreasing the bias from inter-individual variation. Lastly, the occurrence of mitochondrial proliferation in COX-positive fibres, which is encountered in myopathy due to the MELAS mutation, provided an opportunity to separately analyse the influence of respiratory defect and mitochondrial proliferation. Taking advantage of the heteroplasmic nature of these alterations of the mtDNA, we used an in situ approach to search for the presence of apoptosis and to analyse its relationship with respiratory defect, mitochondrial proliferation and mutation load in single fibres. Although the relationship of apoptosis and morphological alterations have been previously analysed, this report is the first to address its link to the mutation load of single fibres. The main findings of this study are the existence of significant apoptosis in the muscle of most patients, its tight correlation with mitochondrial proliferation and the fact that mitochondrial proliferation does not seem to modify the mutant mtDNA proportion.
Previous contradictory results with respect to the presence of apoptosis, its severity and relationship to respiratory defect, mitochondrial proliferation and mutation load in whole muscle fragment (Mirabella et al., 2000; Sciacco et al., 2001; Fagiolari et al., 2002; Ikezoe et al., 2002; Formichi et al., 2003) could have been partly due to technical difficulties involved in the in situ evaluation of apoptosis. Indeed, immunohistochemical techniques give essentially qualitative results (absence or presence of the abnormal signal in single muscle fibres), which are subsequently used for the evaluation of the proportion of abnormal fibres in muscle. The initial setting up of the signal : noise ratio represents, obviously, an essential part of the technique, which may considerably shift the threshold of the abnormal signal and modify its observed frequency accordingly. The three assays used in this paper gave negative results in a range of control muscles. In diseased muscles, they gave negative results in most COX-negative RRF, thus showing that positive immunoreaction was not solely due to the increase of mitochondrial proteins. Furthermore, in individual muscle fibres, the results of the three assays were most often in agreement, showing the identity of their threshold and also suggesting that apoptosis execution was complete in muscle contrarily to earlier reports (Ikezoe et al., 2002; Umaki et al., 2002).
Apoptosis affected a small proportion of the patients' muscle fibres, raising the question of its provoking factors. It was only observed in fibres with mitochondrial proliferation. The link between apoptosis and mitochondrial proliferation remains to be directly addressed. It could be that mitochondrial proliferation occurred in the most severely affected fibres. However, the presence of apoptosis in some COX-positive RRF in muscles with the MELAS mutation suggested that apoptosis was essentially linked to mitochondrial proliferation. Furthermore, the hierarchy of significance found in the correlation studies suggested an indirect link between apoptosis and respiratory chain defect through mitochondrial proliferation. Mitochondrial proliferation may also just make induction of apoptosis easier by the sole increase of the cell content of pro-apoptotic intra-mitochondrial molecules (Bax, Smac/Diablo...), which is not totally compensated for by the probably concomitant increase of intra-mitochondrial anti-apoptotic molecules (Bcl-2).
Genetic analyses of individual muscle fibres confirmed that, as previously reported (Hammans et al., 1992; Oldfors et al., 1992; Sciacco et al., 1994), higher proportion of mutant mtDNA but also lower amount of residual normal mtDNA were associated with respiratory defect in individual muscle fibres. They also showed that mitochondrial proliferation equivalently increased normal and mutant mtDNA molecules without changing their ratio. This lack of significant influence of mitochondrial proliferation on the mutation proportion could have been due to the difficulty to show an increase of the already very high proportion of mtDNA mutation found in COX-defective fibres, whatever their mitochondrial proliferation (see Fig. 4). However, contrarily to most previous studies, we did not analyse the mutation proportion itself but we separately quantified the amount of normal and mutant mtDNA molecules. This allowed us to clearly show that mitochondrial proliferation increased similarly both mtDNA species.
Genetic analyses conducted at the level of the whole muscle fragment DNA confirmed that apoptosis was tightly linked to the proportion of mtDNA mutation and to mitochondrial proliferation. In parallel, however, they did not show a correlation between the muscle proportion of mutation or normal mtDNA content and its COX defect. That observation could suggest that evaluation of the muscle respiratory competency by COX histochemistry was inadequate. Indeed, quantification of COX defect by histochemistry took into account only totally defective muscle fibres, thus leaving out the numerous partially COX-defective fibres. Furthermore, COX activity is a very partial parameter of respiratory competency as exemplified by the COX-positive RRF encountered in muscle with the MELAS mutation. Finally, individual patients might considerably differ with respect to the threshold of mutation above which a respiratory defect appears as shown by the very diverse mutation proportion found in COX-positive muscle fibres (see Fig. 4). The diverse size of muscle fibres, which was not taken into account, could also represent an important parameter for the evaluation of the threshold of normal mtDNA content below which a respiratory defect appears. This is shown by the increasing amount of normal mtDNA in the COX-positive fibres with increasing age at the muscle biopsy (Fig. 4).
As occurrence of apoptosis was still a matter of dispute at the beginning of our work, the design of our project was built to address the presence of apoptosis and its relevance to morphological anomalies encountered in mitochondrial myopathies as well as to mutation load. Once proven to be significantly present, the relevance of apoptosis to muscle symptoms could not be directly addressed owing to the lack of proper evaluation of the patients' muscle symptoms. In particular, none of them had had a quantitative evaluation of their muscle strength and bulk. Indeed, the symptoms of the MELAS patients were dominated by their cerebral involvement and little attention was given to their muscle. The 16 patients with an mtDNA deletion had, however, been assessed with respect to the presence or absence of permanent limb or axial muscle weakness. The six patients with clinical myopathy had a higher proportion of apoptotic muscle fibres than those without (median value 2.00 versus 0.25, P = 0.073). This difference did not reach significance but was, however, striking considering the small number of patients and the loss of power due to the qualitative rather than quantitative analysis of muscular symptoms.
The rapid time course of apoptosis, at least as evaluated in cultured cells (Wyllie et al., 1980), renders highly significant the presence of apoptosis at a given time point. Even a very low proportion of apoptotic muscle fibres should provoke rapid loss of muscle bulk in the absence of compensation. Amyotrophy and muscle weakness are, however, late and slowly progressive symptoms in mitochondrial myopathy, implying either that apoptosis is not always completed in muscle as proposed (Ikezoe et al., 2002; Umaki et al., 2002) or, more probably, that dead muscle fragments are continuously replaced by satellite cells. We are currently trying to solve this issue by the analysis of the satellite cells status in mitochondrial myopathies.
Apoptosis will have to be taken into account when considering training therapeutic trials. Endurance training has been shown to induce significant improvement of the patients' intolerance to exercise (Taivassalo et al., 1998, 2001; Taivassalo and Haller, 2004). This clinical and physiological improvement was accompanied by significant mitochondrial proliferation (50% increase of mitochondrial mass) and improved respiratory chain activities (Taivassalo et al., 2001). Two-thirds of the patients showed also an increase of their muscle mutant mtDNA proportion, which was considered an important drawback of that otherwise quite effective treatment (Taivassalo et al., 2001). This induced increase of mutant mtDNA proportion might have solely been due to random bias in the small group of nine patients as, in our studies, mitochondrial proliferation did not seem to favour mutant mtDNA in individual muscle fibres. Also, according to our data, the induced increase in muscle mitochondrial mass was likely to increase apoptosis in muscle. Evaluation of apoptosis should therefore be included in the evaluation of therapeutic trials, which trigger mitochondrial proliferation.
In conclusion, apoptosis may clearly contribute to mitochondrial pathology. It is tightly linked to mitochondrial proliferation and has therefore to be investigated when considering training therapeutics that are likely to induce muscle mitochondrial proliferation. However, although high proportion of a heteroplasmic mtDNA mutation is associated with mitochondrial proliferation and apoptosis, the former does not seem to modulate the proportion of the mutation within individual muscle fibres. The mechanisms inducing mitochondrial proliferation in only some of the COX-deficient fibres remain to be elucidated and represent an essential step towards therapeutic attempts of mitochondrial diseases.
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Supplementary material |
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TopSummaryIntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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Supplementary data are available at Brain Online.
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Notes |
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* These authors contributed equally to this work.
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Acknowledgements |
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This work has been supported by grants from the AFM (Association Française contre les Myopathies). K.A. is the recipient of a post-doctoral fellowship from INSERM. We wish to thank Professor Michel Fardeau, Dr Gillian Butler-Brown and Dr Marc Fiszman for their critical reading of the manuscript.
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TopSummaryIntroductionPatients, material and methodsResultsDiscussionSupplementary materialReferences |
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