Comprehensive morphometric assessment of deltoid muscle development in children: A cross-sectional study.

BACKGROUND: Normative values for different morphometric parameters of muscle fibres during paediatric development, i.e. from 0 to 18 years, are currently unavailable. They would be of major importance to accurately evaluate pathological changes and could be used as reference biomarkers for evaluating treatment response in clinical trials, or physiological adjustments in sports or ageing. METHODS: Data were derived from 482 images with a total of 33 094 fibres from 10 µm cross-sections of snap-frozen muscle from 83 deltoid muscle biopsies from patients, 0-18 years, without neuromuscular pathology stained with ATPase 9.4. Data was acquired and analysed with patented image analysis algorithms from "CARPACCIO.cloud". Several parameters were extracted or calculated, including cross-sectional area (CSA), fibre type, circularity, as well as the Minimum diameter of Feret (MinFeret). FINDINGS: This study illustrates changes in quantitative parameters for muscle morphology over the course of paediatric development and the pivotal changes occurring around puberty. Only fibre size parameters (MinFeret, CSA) are dependent on gender, and only after puberty. All other parameters vary in a similar manner for females and males. The proportion of type 1 fibres is essentially constant from birth to age 10, decreasing to ≈40% by age 18. Circularity decreases with age, to plateau after age 10 for both fibre types. INTERPRETATION: Normative values and reference charts for muscle fibre types in this age range have been generated to allow comparison of data from patients in pathology laboratories working on neuromuscular diseases. FUNDING: BPI FRANCE, PULSALYS, Association de l'Institut de Myologie, French National Research Agency (ANR), LABEX CORTEX of Université de Lyon.

FYCO1 Increase and Effect of Arimoclomol-Treatment in Human VCP-Pathology.

Dominant VCP-mutations cause a variety of neurological manifestations including inclusion body myopathy with early-onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin-dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP-patients. Studying the proteomic signature of VCP-mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP-patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP-patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro-survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP-etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre-clinical testing of this drug in fibroblasts.

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

FDX2 and ISCU Gene Variations Lead to Rhabdomyolysis With Distinct Severity and Iron Regulation.

BACKGROUND AND OBJECTIVES: To determine common clinical and biological traits in 2 individuals with variants in ISCU and FDX2, displaying severe and recurrent rhabdomyolyses and lactic acidosis. METHODS: We performed a clinical characterization of 2 distinct individuals with biallelic ISCU or FDX2 variants from 2 separate families and a biological characterization with muscle and cells from those patients. RESULTS: The individual with FDX2 variants was clinically more affected than the individual with ISCU variants. Affected FDX2 individual fibroblasts and myoblasts showed reduced oxygen consumption rates and mitochondrial complex I and PDHc activities, associated with high levels of blood FGF21. ISCU individual fibroblasts showed no oxidative phosphorylation deficiency and moderate increase of blood FGF21 levels relative to controls. The severity of the FDX2 individual was not due to dysfunctional autophagy. Iron was excessively accumulated in ISCU-deficient skeletal muscle, which was accompanied by a downregulation of IRP1 and mitoferrin2 genes and an upregulation of frataxin (FXN) gene expression. This excessive iron accumulation was absent from FDX2 affected muscle and could not be correlated with variable gene expression in muscle cells. DISCUSSION: We conclude that FDX2 and ISCU variants result in a similar muscle phenotype, that differ in severity and skeletal muscle iron accumulation. ISCU and FDX2 are not involved in mitochondrial iron influx contrary to frataxin.

New insights about myofibrillar myopathies: the role of metalloproteinases 2 and 9 in the pathogenesis

Backgroung: There are few reports suggesting that gene expression and activation of various matrix metalloproteinases (MMPs) are deregulated. MMP-2 and MMP-9 represent the two MMPs, which degrade type IV collagen, the component of basement membrane. Methods: We analysed the involvement of gelatinases, MMP-2 and MMP-9, in the pathogenesis of myofibrillar myopathy (MFM). Muscle specimens from 23 patients well diagnosed with MFM, were immunostained by MMP-2 and MMP-9. We analysed qualitatively the immunoexpression in three compartments: subsarcolemmal (SSC), intracytoplasmic (ICC) and perinuclear (PNC).Results: 95,7% and 100% samples showed MMP-2 and MMP-9 upregulation ICC, respectively. PNC showed MMP-2 (82,6%) and MMP-9 (8,7%) regulation (p<0.001). SSC and ICC did not present statistical significance. There was no correlation between mutated gene and immunohistochemical pattern distribution.Conclusion: Our results suggest that MMP-2 and/or MMP-9 could participate in the pathomechanism of MFM, causing damage of sarcomere and deposition of protein aggregates.

Introdução: Existem poucos relatos sugerindo que a expressão gênica e a ativação de várias metaloproteinases de matriz (MMPs) estão desreguladas. MMP-2 e MMP-9 representam as duas MMPs, que degradam o colágeno tipo IV, o componente da membrana basal.Método: Analisamos o envolvimento das gelatinases, MMP-2 e MMP-9, na patogênese da miopatia miofibrilar (MFM). Amostras de músculos de 23 pacientes bem diagnosticados com MFM foram imunocoradas por MMP-2 e MMP-9. Analisamos qualitativamente a imunoexpressão em três compartimentos: subsarcolemal (SSC), intracitoplasmático (ICC) e perinuclear (PNC).Resultados: 95,7% e 100% das amostras apresentaram ICC de regulação positiva de MMP-2 e MMP-9, respectivamente. PNC mostrou regulação MMP-2 (82,6%) e MMP-9 (8,7%) (p <0,001). SSC e ICC não apresentaram significância estatística. Não houve correlação entre o gene mutado e a distribuição do padrão imunohistoquímico.Conclusão: Nossos resultados sugerem que MMP-2 e / ou MMP-9 podem participar do patomecanismo da MFM, causando dano ao sarcômero e deposição de agregados proteicos.

Clinical phenotype and loss of the slow skeletal muscle troponin T in three new patients with recessive TNNT1 nemaline myopathy.

BACKGROUND: Congenital nemaline myopathies are rare pathologies characterised by muscle weakness and rod-shaped inclusions in the muscle fibres. METHODS: Using next-generation sequencing, we identified three patients with pathogenic variants in the Troponin T type 1 (TNNT1) gene, coding for the troponin T (TNT) skeletal muscle isoform. RESULTS: The clinical phenotype was similar in all patients, associating hypotonia, orthopaedic deformities and progressive chronic respiratory failure, leading to early death. The anatomopathological phenotype was characterised by a disproportion in the muscle fibre size, endomysial fibrosis and nemaline rods. Molecular analyses of TNNT1 revealed a homozygous deletion of exons 8 and 9 in patient 1; a heterozygous nonsense mutation in exon 9 and retention of part of intron 4 in muscle transcripts in patient 2; and a homozygous, very early nonsense mutation in patient 3.Western blot analyses confirmed the absence of the TNT protein resulting from these mutations. DISCUSSION: The clinical and anatomopathological presentations of our patients reinforce the homogeneous character of the phenotype associated with recessive TNNT1 mutations. Previous studies revealed an impact of recessive variants on the tropomyosin-binding affinity of TNT. We report in our patients a complete loss of TNT protein due to open reading frame disruption or to post-translational degradation of TNT.

A Heterozygous Mutation in the Filamin C Gene Causes an Unusual Nemaline Myopathy With Ring Fibers.

Autosomal dominant pathogenic variants in the filamin C gene (FLNC) have been associated with myofibrillar myopathies, distal myopathies, and isolated cardiomyopathies. Mutations in different functional domains of FLNC can cause various clinical phenotypes. A novel heterozygous missense variant c.608G>A, p.(Cys203Tyr) in the actin binding domain of FLCN was found to cause an upper limb distal myopathy (MIM #614065). The muscle MRI findings are similar to those observed in FLNC-myofibrillar myopathy (MIM #609524). However, the muscle biopsy revealed >20% of muscle fibers with nemaline bodies, in addition to numerous ring fibers and a predominance of type 1 fibers. Overall, this case shows some unique and rare aspects of FLNC-myopathy constituting a new morphologic phenotype of FLNC-related myopathies.

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/ß catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.

'Dusty core disease' (DuCD): expanding morphological spectrum of RYR1 recessive myopathies.

Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients' muscle biopsies. We defined "dusty cores" the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&R:15%) and Type1 predominance "plus" (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease.

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

Skeletal muscle biopsy analysis in reducing body myopathy and other FHL1-related disorders.

FHL1 mutations have been associated with various disorders that include reducing body myopathy (RBM), Emery-Dreifuss-like muscular dystrophy, isolated hypertrophic cardiomyopathy, and some overlapping conditions. We report a detailed histochemical, immunohistochemical, electron microscopic, and immunoelectron microscopic analyses of muscle biopsies from 18 patients carrying mutations in FHL1: 14 RBM patients (Group 1), 3 Emery-Dreifuss muscular dystrophy patients (Group 2), and 1 patient with hypertrophic cardiomyopathy and muscular hypertrophy (Group 2). Group 1 muscle biopsies consistently showed RBs associated with cytoplasmic bodies. The RBs showed prominent FHL1 immunoreactivity whereas desmin, αB-crystallin, and myotilin immunoreactivity surrounded RBs. By electron microscopy, RBs were composed of electron-dense tubulofilamentous material that seemed to spread progressively between the myofibrils and around myonuclei. By immunoelectron microscopy, FHL1 protein was found exclusively inside RBs. Group 2 biopsies showed mild dystrophic abnormalities without RBs; only minor nonspecific myofibrillar abnormalities were observed under electron microscopy. Molecular analysis revealed missense mutations in the second FHL1 LIM domain in Group 1 patients and ins/del or missense mutations within the fourth FHL1 LIM domain in Group 2 patients. Our findings expand the morphologic features of RBM, clearly demonstrate the localization of FHL1 in RBs, and further illustrate major morphologic differences among different FHL1-related myopathies.

Expression of myogenic regulatory factors and myo-endothelial remodeling in sporadic inclusion body myositis.

Muscle repair relies on coordinated activation and differentiation of satellite cells, a process that is unable to counterbalance progressive degeneration in sporadic inclusion body myositis (s-IBM). To explore features of myo regeneration, the expression of myogenic regulatory factors Pax7, MyoD and Myogenin and markers of regenerating fibers was analyzed by immunohistochemistry in s-IBM muscle compared with polymyositis, dermatomyositis, muscular dystrophy and age-matched controls. In addition, the capillary density and number of interstitial CD34(+) hematopoietic progenitor cells was determined by double-immunoflourescence staining. Satellite cells and regenerating fibers were significantly increased in s-IBM similar to other inflammatory myopathies and correlated with the intensity of inflammation (R>0.428). Expression of MyoD, visualizing activated satellite cells and proliferating myoblasts, was lower in s-IBM compared to polymyosits. In contrast, Myogenin a marker of myogenic cell differentiation was strongly up-regulated in s-IBM muscle. The microvascular architecture in s-IBM was distorted, although the capillary density was normal. Notably, CD34(+) hematopoietic cells were significantly increased in the interstitial compartment. Our findings indicate profound myo-endothelial remodeling of s-IBM muscle concomitant to inflammation. An altered expression of myogenic regulatory factors involved in satellite cell activation and differentiation, however, might reflect perturbations of muscle repair in s-IBM.

Mutations of the FHL1 gene cause Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare disorder characterized by early joint contractures, muscular dystrophy, and cardiac involvement with conduction defects and arrhythmias. So far, only 35% of EDMD cases are genetically elucidated and associated with EMD or LMNA gene mutations, suggesting the existence of additional major genes. By whole-genome scan, we identified linkage to the Xq26.3 locus containing the FHL1 gene in three informative families belonging to our EMD- and LMNA-negative cohort. Analysis of the FHL1 gene identified seven mutations, in the distal exons of FHL1 in these families, three additional families, and one isolated case, which differently affect the three FHL1 protein isoforms: two missense mutations affecting highly conserved cysteines, one abolishing the termination codon, and four out-of-frame insertions or deletions. The predominant phenotype was characterized by myopathy with scapulo-peroneal and/or axial distribution, as well as joint contractures, and associated with a peculiar cardiac disease characterized by conduction defects, arrhythmias, and hypertrophic cardiomyopathy in all index cases of the seven families. Heterozygous female carriers were either asymptomatic or had cardiac disease and/or mild myopathy. Interestingly, four of the FHL1-mutated male relatives had isolated cardiac disease, and an overt hypertrophic cardiomyopathy was present in two. Expression and functional studies demonstrated that the FHL1 proteins were severely reduced in all tested patients and that this was associated with a severe delay in myotube formation in the two patients for whom myoblasts were available. In conclusion, FHL1 should be considered as a gene associated with the X-linked EDMD phenotype, as well as with hypertrophic cardiomyopathy.

"Necklace" fibers, a new histological marker of late-onset MTM1-related centronuclear myopathy.

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin 1 protein (MTM1) are usually associated with severe neonatal X-linked myotubular myopathy (XLMTM). However, mutations in MTM1 have also been recognized as the underlying cause of "atypical" forms of XLMTM in newborn boys, female infants, female manifesting carriers and adult men. We reviewed systematically the biopsies of a cohort of patients with an unclassified form of centronuclear myopathy (CNM) and identified four patients presenting a peculiar histological alteration in some muscle fibers that resembled a necklace ("necklace fibers"). We analyzed further the clinical and morphological features and performed a screening of the genes involved in CNM. Muscle biopsies in all four patients demonstrated 4-20% of fibers with internalized nuclei aligned in a basophilic ring (necklace) at 3 microm beneath the sarcolemma. Ultrastructurally, such necklaces consisted of myofibrils of smaller diameter, in oblique orientation, surrounded by mitochondria, sarcoplasmic reticulum and glycogen granules. In the four patients (three women and one man), myopathy developed in early childhood but was slowly progressive. All had mutations in the MTM1 gene. Two mutations have previously been reported (p.E404K and p.R241Q), while two are novel; a c.205_206delinsAACT frameshift change in exon 4 and a c.1234A>G mutation in exon 11 leading to an abnormal splicing and the deletion of nine amino acids in the catalytic domain of MTM1. Necklace fibers were seen neither in DNM2- or BIN1-related CNM nor in males with classical XLMTM. The presence of necklace fibers is useful as a marker to direct genetic analysis to MTM1 in CNM.

Apoptosis in mitochondrial myopathies is linked to mitochondrial proliferation.

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.