Free Radical Biology and Medicine 63 (2013) 108–114

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Free Radical Biology and Medicine

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Abbreophthalmyopatwith ragreal-timTNF-α, Ttype 2

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journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

Overexpression of TNF-α in mitochondrial diseases caused bymutations in mtDNA: evidence for signaling through itsreceptors on mitochondria

Gaetano Vattemi a,n, Matteo Marini a, Nicholas R. Ferreri b, Shoujin Hao b,Manuela Malatesta c, Alessandra Meneguzzi d, Valeria Guglielmi a, Cristiano Fava d,Pietro Minuz d, Giuliano Tomelleri a

a Section of Clinical Neurology, Department of Neurological, Neuropsychological, Morphological, and Movement Sciences, University of Verona,37134 Verona, Italyb Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USAc Section of Anatomy and Histology, Department of Neurological, Neuropsychological, Morphological, and Movement Sciences, University of Verona,37134 Verona, Italyd Section of Internal Medicine, Department of Biomedical and Surgical Sciences, University of Verona, 37134 Verona, Italy

a r t i c l e i n f o

Article history:Received 2 January 2013Received in revised form9 April 2013Accepted 16 April 2013Available online 22 April 2013

Keywords:Mitochondrial diseasesTumor necrosis factor-αTNF receptor type 1TNF receptor type 2MitochondriaFree radicals

49/$ - see front matter & 2013 Elsevier Inc. Ax.doi.org/10.1016/j.freeradbiomed.2013.04.025

viations: COX, Cytochrome c oxidase; CPEO,moplegia; MD, Mitochondrial disease; MELAShy with lactic acidosis and stroke-like episodeged red fibers; OXPHOS, Oxidative phosphorye PCR; ROS, Reactive oxygen species; SDH, Suumor necrosis factor-α; TNFR1, TNF receptor

esponding author. Fax: +39 045 8027492.ail address: gaetano.vattemi@univr.it (G. Vatte

a b s t r a c t

Mitochondrial diseases (MDs) are heterogeneous disorders due to impaired respiratory chain functioncausing defective ATP production. Although the disruption of oxidative phosphorylation is central to theMD pathophysiology, other factors may contribute to these disorders. We investigated the expressionand the cellular localization of TNF-α and its receptors, TNFR1 and TNFR2, in muscle biopsies from 15patients with mitochondrial respiratory chain dysfunction. Our data unambiguously demonstrate thatTNF-α is expressed in muscle fibers with abnormal focal accumulations of mitochondria, so-called raggedred fibers, and is delivered to mitochondria where both receptors are localized. Moreover TNF receptorsare differentially regulated in patients' muscle in which the expression levels of TNFR1 mRNA aredecreased and those of TNFR2 mRNA are increased compared with controls. These findings suggest forthe first time that TNF-α could exert a direct effect on mitochondria via its receptors.

& 2013 Elsevier Inc. All rights reserved.

Mitochondrial diseases (MDs)1 constitute a heterogeneousgroup of multisytemic disorders due to impaired respiratory chainfunction causing defective mitochondrial energy production [1,2].The respiratory chain is under dual genetic control, from themitochondrial DNA (mtDNA) and the nuclear DNA; the clinicalphenotype, when associated with mtDNA abnormalities, mainlydepends on the degree of mitochondrial heteroplasmy in high-energy-requiring tissues, primarily skeletal muscle and centralnervous system, and on the biochemical expression of the muta-tions [1,2]. Indeed each cell contains hundreds or thousands of

ll rights reserved.

Chronic progressive external, Mitochondrial encephalo-s; MERRF, Myoclonic epilepsylation; qRT-PCR, Quantitativeccinate dehydrogenase;type 1; TNFR2, TNF receptor

mi).

mtDNA copies, which, at cell division, distribute randomly amongdaughter cells. In normal tissues, all mtDNA molecules are iden-tical (homoplasmy). Mutations of mtDNA usually affect some butnot all mtDNAs within a cell or a tissue (heteroplasmy). Thereforethe clinical expression of a pathogenic mtDNA mutation is largelydetermined by the relative proportion of normal and mutantmtDNA genomes in various tissues. A minimum critical numberof mutant mtDNAs is required to cause mitochondrial dysfunctionin a particular organ or tissue, resulting in a mitochondrial disease(threshold effect).

The pathophysiology of these complex disorders remainslargely unknown and even though the disruption of oxidativephosphorylation (OXPHOS), which causes defective ATP produc-tion, is undoubtedly central to mtDNA diseases, other factors suchas increased oxidative stress, altered calcium handling, and defec-tive turnover of mitochondrial proteins may also contribute [1–4].On muscle tissue ragged red fibers, which are muscle fiberscontaining a high percentage of mutated genome and excessivemitochondrial proliferation, represent the most important hall-mark of OXPHOS pathology. The accumulation of mitochondria in

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the subsarcolemmal region and between myofibrils is thought tobe due to mitochondrial biogenesis in response to a metabolicdeficiency [1,2].

Tumor necrosis factor-α (TNF-α) is a multifunctional proinflam-matory cytokine produced by many cell types, including macro-phages, monocytes, lymphocytes, adipocytes, and cardiomyocytes,in response to inflammation, injury, and other environmental“stresses” [5]. The biological activities of TNF-α range from cellularproliferation and differentiation, to inflammation and mediation ofimmune response, to apoptotic and necrotic cell death [6]. To exertits pleiotropic actions TNF-α must bind to two structurally distincttransmembrane receptors, type 1 (TNFR1) and type 2 (TNFR2),which, in turn, activate several intracellular signaling pathways[7]. Although TNF-α signal transduction is a very complex process,the generation of reactive oxygen species (ROS) can contribute tothe net effects of this cytokine [8,9]. In addition, TNF-α is known tostimulate ROS production in mitochondria by impairing mem-brane permeability and by inhibiting the respiratory chain,thereby causing mitochondrial damage [10,11].

From the above considerations we reasoned that TNF-α mighthave a role in the pathophysiology of mitochondrial encephalo-myopathies. Therefore this investigation was aimed at exploringthe expression and the subcellular distribution of TNF-α and itsreceptors in muscle biopsies from patients affected by mitochon-drial respiratory chain diseases.

Materials and methods

Muscle biopsies

Available muscle biopsy specimens from 15 patients withmitochondrial respiratory chain dysfunction were evaluated. Thecohort included 3 patients with myoclonic epilepsy with raggedred fibers (MERRF) and the A8344G mutation, 5 patients withmitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and the A3243G mutation, and 7 patientswith chronic progressive external ophthalmoplegia (CPEO) and asingle deletion.

Controls were 15 muscle biopsies from subjects who wereultimately deemed to be free of muscle diseases.

Muscle biopsies were performed for diagnostic purpose afterwritten informed consent and the study was approved by the localethical board.

Histology and histochemistry

Muscle samples were snap-frozen in liquid nitrogen-cooledisopentane. Serial 8-μm-thick cryosections were stained withhematoxylin and eosin, modified Gomori trichrome, ATPase (pH4.3, 4.6, and 10.4), succinate dehydrogenase (SDH), cytochrome coxidase (COX), cytochrome c oxidase succinate dehydrogenase(COX–SDH), nicotinamide adenine dinucleotide–tetrazoliumreductase, periodic acid Schiff, Sudan black, and acid phosphatase.

Immunohistochemistry and confocal immunofluorescence microscopy

Immunohistochemical studies were performed on 6.5-μm-thick transverse muscle sections using fluorescence procedures.The following well-characterized antibodies were used: rabbitpolyclonal TNF-α (Santa Cruz Biotechnology, sc-8301, diluted1:100) [12], goat polyclonal TNF-α (R&D Systems, AF-210-NA,diluted 1:100) [13], rabbit polyclonal TNFR1 (Stressgen, CSA-815,diluted 1:500) [14], mouse monoclonal TNFR1 (Bender MedSys-tems, BMS106, diluted 1:50), mouse monoclonal TNFR2 (Santa

Cruz Biotechnology, sc-8041, diluted 1:100) [13], and goat poly-clonal TNFR2 (R&D Systems, AF726, diluted 1:100).

Double immunofluorescence was performed using antibodiesto TNF-α, TNFR1, and TNFR2 in combination with (a) a mousemonoclonal antibody to cytochrome c (BD Pharmingen, 556433,diluted 1:800) or (b) a mouse monoclonal antibody to mitofusin 2(Santa Cruz Biotechnology, sc-100560, diluted 1:250). Confocalimages were acquired with a Zeiss LSM 510 confocal microscope.

To control staining specificity the primary antibody wasomitted or replaced with nonimmune serum at the same concen-tration. Two independent, blinded investigators evaluated therelative staining.

Quantification of ragged red fibers positive for TNF-α, TNFR1, andTNFR2

The total number of ragged red fibers and the number of raggedred fibers positive for TNF-α, TNFR1, and TNFR2 were quantita-tively analyzed in biopsy specimens from 5 of 15 patients, includ-ing 3 patients with CPEO, 1 patient with MELAS, and 1 patient withMERRF. The evaluation of individual muscle fibers with respect totheir status for COX and SDH activity (COX–SDH double stain) aswell as the presence of TNF-α, TNFR1, and TNFR2 immunoreactionwas done on serial muscle sections.

Electron microscopy immunocytochemistry

A small fragment of muscle tissue was fixed in 4% glutaralde-hyde in phosphate buffer, postfixed in 2% osmium tetroxide,dehydrated, and embedded in Spurr resin. This tissue processingallows good morphological preservation, especially of membranes,but the samples need a pretreatment with an oxidizing agentbefore starting the immunocytochemical procedure to improveantibody binding.

Ultrathin sections were placed on nickel grids coated with aFormvar–carbon layer and treated with a 0.2 M aqueous solutionof sodium metaperiodate for 60 min at room temperature, rinsed,and air-dried. The sections were then submitted to a doubleimmunolabeling: they were floated for 3 min on normal goatserum diluted 1:100 in phosphate-buffered saline (PBS) and thenincubated for 17 h at 4 1C with a rabbit polyclonal TNFR1 antibodyand a mouse monoclonal TNFR2 antibody, both diluted 1:10 in PBScontaining 0.1% bovine serum albumin and 0.05% Tween 20; afterbeing rinsed, the sections were floated on normal goat serum andthen reacted for 30 min at room temperature with the respectivesecondary 12- or 6-nm gold-conjugated antibodies diluted 1:10 inPBS. Finally, the sections were rinsed and air-dried. Some sectionswere immunolabeled with goat polyclonal TNF-α, mouse mono-clonal TNFR1, or goat polyclonal TNFR2 according to the proceduredescribed above. All sections were weakly stained with lead citrateand observed in a Philips Morgagni transmission electron micro-scope equipped with a Megaview II camera for digital imageacquisition.

The control for staining specificity was omission of the primaryantibody from test sections.

Quantitative real-time PCR (qRT-PCR) of TNF-α, TNFR1, and TNFR2mRNA accumulation

Trizol reagent (0.5 ml) was added to muscle tissue and totalRNA was separated according to the protocol provided by themanufacturer (Invitrogen, San Diego, CA, USA).

For determination of TNF-α mRNA, a 2-μg aliquot of total RNAwas converted to cDNA using random primers and SuperScript IIreverse transcriptase (Invitrogen). The cDNA (100 ng) from eachRNA sample was placed in a 25-μl RT-PCR mixture using the iTaq

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SYBR Green Supermix with ROX (Bio-Rad). All reactions wereperformed in triplicate using the ABI Prism 7000 thermocycler(Applied Biosystems) under the following conditions: 2 min at95 1C, followed by 50 cycles of 95 1C for 10 s and 60 1C for 30 s.qRT-PCR was used to determine accumulation of mRNA for TNF-αin muscle tissue. The sequences for the human TNF-α-specificprimer pair were forward, 5′-GAGTCAACGGATTTGGTCGT-3′; reverse,

Fig. 1. Confocal fluorescence microscopy images of positive ragged red fibers for TNF-αmuscle fibers with intense cytochrome c (green) staining, and its localization to mitoch(B, red) and of TNFR2 (C and D, red) in two ragged-red fibers and their colocalization toThe same two ragged red fibers are positive for TNF-α and for both TNF receptors (A–D

5′-CCAAAGTAGACCTGCCCAGA-3′. Input cDNAs were normalizedusing the housekeeping gene, GAPDH (forward, 5′-GAGTCAA-CGGATTTGGTCGT-3′; reverse, 5′-TGGAAGATGGTGATGGGATT-3′.Relative TNF-α mRNA accumulation was determined by qRT-PCRusing the 2(−ΔΔCT) method. Statistical analysis was performedwith GraphPad software. The statistical comparison of groups wasdetermined using the t test.

, TNFR1, and TNFR2. (A) Immunoreactivity of TNF-α (red) in two ragged red fibers,ondria, labeled with cytochrome c (green). (B, C, and D) Immunostaining of TNFR1mitochondria, labeled with cytochrome c (B and C, green) or mitofusin 2 (D, green).are serial sections). Bars, 50 μm.

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For determination of TNFR1 and TNFR2 mRNA, a 0.5-μg aliquot oftotal RNA was converted to cDNA using random primers and Power-Script RT (Clontech). The cDNA from each RNA sample was placed in a20-μl RT-PCR mixture using the FastStart DNA Master SYBR Green I kit(Roche) supplemented with 3 mM MgCl2 and Platinum Taq polymer-ase (Invitrogen). qRT-PCR was used to determine accumulation ofmRNA for TNFR1 and TNFR2. The specific primer pairs for human

Fig. 2. Confocal fluorescence microscopy images of positive ragged red fibers for TNF-αwith mitochondrial respiratory chain dysfunction is shown. (A) Localization of TNF-α (of TNFR1 (B, red) and TNFR2 (C, red) to mitochondria, labeled with cytochrome c (gree(D, green). The same two ragged red fibers stain for TNF-α, TNFR1, and TNFR2 (A–D are

TNFR1 and TNRR2 used in this study were designed based onAccession Nos. NM_001065 and NM_001066, respectively (TNFR1,forward, 5′-GGAGCTGTTGGTGGGAATAT-3′; reverse, 5′-GAGGTGGTTT-TCTGAAGCGG-3′; and TNFR2, forward, 5′-GGACGTTCTCCAACACGA-CTT-3′; reverse, 5′-TTCTGGAGTTGGCTGCGTGTG-3′). Input cDNAs werenormalized using the housekeeping gene β-actin, and the efficiencyof primer pair amplification was determined using a standard curve.

, TNFR1, and TNFR2. The immunostaining of muscle sections from a second patientred) to mitochondria, labeled with cytochrome c (green). (B and C) Colocalizationn). (D) Colocalization of TNFR2 (D, red) to mitochondria, labeled with mitofusin 2serial sections). Bars, 50 μm.

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Relative TNFR1 and TNFR2 mRNA accumulation was determined byqRT-PCR using the 2(−ΔΔCT) method.

Controls were muscle biopsies from subjects who were ulti-mately deemed to be free of muscle diseases. For TNF-α mRNA,controls included also muscle biopsies from patients with inflam-matory myopathies.

Statistical analysis

All data are presented as the mean 7 SD. Statistical analyseswere performed using an unpaired t test. Differences with p o 0.05were considered statistically significant.

Results

Immunohistochemistry and confocal immunofluorescence microscopy

TNF-α immunoreactivity was detected in ragged red fibers of all15 patients with mitochondrial respiratory chain dysfunction.A colocalization of TNF-α staining with cytochrome c, an innermitochondrial membrane protein, was observed (Figs. 1A and 2A).

Fig. 3. Ultrastructural immunocytochemistry for TNF-α, TNFR1, and TNFR2. (A andmitochondrial matrix and are occasionally located close to cristae (arrows); some signalanti-TNFR1 (12-nm gold grains) and anti-TNFR2 (6-nm gold grains) antibodies: manymembrane (arrowheads). Bars, 200 nm.

Table 1Quantification of ragged red fibers positive for TNF-α, TNFR1, and TNFR2.

Patient Clinical phenotype Ragged red fibers TNF-α TNFR1 TNFR2

1 CPEO 4.4 39.5 35.7 35.12 CPEO 1.0 57.9 52.6 52.63 CPEO 0.3 50 50 504 MERRF 1.3 42 38 405 MELAS 0.7 66.7 66.7 66.7

The number of muscle fibers with intense SDH stain (ragged red fibers) is indicatedas a percentage of all fibers in the muscle section. The number of ragged red fiberswith immunoreactivity for TNF-α, TNFR1, and TNFR2 is indicated as a percentage ofall ragged red fibers.

Immunostaining for TNFR1 and TNFR2 was also observed in raggedred fibers of all diseased patients and both receptors colocalized withcytochrome c (Figs. 1B, 1C, 2B, and 2C) and with mitofusin 2 (Figs. 1Dand 2D), an outer mitochondrial membrane protein.

On serial sections, nearly all ragged red fibers positive for TNF-αwere also stained for both TNF receptors (Figs. 1A–1D 2A–2D).

The two different antibodies to TNF-α, the two differentantibodies for TNFR1, and the two different antibodies againstTNFR2 produced similar results.

Ragged red fibers were identified by modified Gomori trichromeand SDH stain. The number of ragged red fibers with positiveimmunoreaction for TNF-α, TNFR1, and TNFR2 are reported in Table 1.

Ultrastructural immunocytochemistry

Immunolabeled sections analyzed by transmission electronmicroscopy revealed that TNF-α antibody labeled myofibrils andmitochondria in which the gold grains mostly occurred in themitochondrial matrix and occasionally were found close to cristae(Figs. 3A and 3B). Similarly, both antibodies against TNFR1 andTNFR2 were mainly distributed inside mitochondria and the goldgrains mostly occurred in close proximity to the inner mitochondrialmembrane (Figs. 3C and 3D).

TNF-α, TNFR1, and TNFR2 mRNA

The expression levels of TNF-α were significantly lower inmuscles of control subjects compared with muscles of diseasedpatients, whereas comparable levels of TNF-α were observed inmuscles of patients with MDs and inflammatory myopathies(Fig. 4A). Both TNFR1 and TNFR2 mRNAs were present in controlmuscles (Figs. 4B and 4C). However, the expression levels of TNFR1were decreased in muscle of patients with mitochondrial diseasescompared with control subjects, whereas the expression levels ofTNFR2 were elevated in muscle of diseased patients comparedwith controls (Figs. 4B and 4C). These data suggest that the overall

B) Immunolabeling with anti-TNF-α antibody: gold grains mostly occur in theis also present in myofibrils (open arrows). (C and D) Double immunolabeling withgold grains of both probes occur in close proximity to the inner mitochondrial

Fig. 4. Quantitative real-time PCR of TNF-α, TNFR1, and TNFR2 mRNA. Box plotsshow the expression levels of (A) TNF-α, (B) TNFR1, and (C) TNFR2 in control anddiseased muscles. The expression levels of TNF-α are higher in muscles of patientswith MDs and inflammatory myopathies compared with muscles of controlsubjects. The expression levels of TNFR1 are decreased and those of TNFR2are increased in muscle of MD patients compared with muscles of controls.The differences between the two groups are statistically significant.

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ratio of TNFR1:TNFR2 is substantially altered in patients withdisease.

Discussion

The principal new finding from this study is the proof of TNF-αexpression in muscle biopsies of patients with mitochondrialrespiratory chain dysfunction regardless of the clinical phenotypeand the genetic mutation. We identified TNF-α reactivity in theragged red fibers and, using confocal immunofluorescence micro-scopy and immunoelectron microscopy, observed immunoreactiveTNF-α in the cytoplasm and associated with mitochondria, inparticular in the mitochondrial matrix and next to cristae. Theexpression levels of TNF-α were also determined by quantitativereal-time PCR, which documented a dramatic increase in accumu-lation of TNF-α mRNA in muscle of diseased patients compared

with muscle from control subjects. TNF-α is predominantly pro-duced by macrophages and monocytes, even though nonhemato-poietic noninflammatory cells are also capable of synthesizing thecytokine [5]. TNF-α is constitutively expressed in myoblasts and istransiently upregulated during differentiation stages. TNF-α mRNAexpression in human muscle fibers also increases significantly inresponse to challenge with the calcium ionophore ionomycin andcyclosporin A [15,16]. TNF-α expression in skeletal muscle fibershas been investigated in a few studies; however, it has beenreported only in the cytoplasm of regenerating muscle fibers,suggesting a physiological role for TNF-α in muscle repair andregeneration [17–19].

After showing that TNF-α accumulates within mitochondria,we addressed the issue of whether TNF receptors might be presentwithin this organelle. Using two different and specific antibodiesdirected against type 1 and type 2 receptors, a staining patternthat colocalizes with mitochondrial markers including cytochromec (an inner mitochondrial membrane protein) and mitofusin 2 (anouter mitochondrial membrane protein) was seen by confocalimmunofluorescence microscopy. Immunoelectron microscopyanalysis not only confirmed the subcellular distribution of TNFreceptors but also documented their association with the innermitochondrial membrane of the organelle. In addition, the expres-sion levels of both receptors seem to be differentially regulated; inparticular we found a decrease in mRNA accumulation of TNFR1,whereas TNFR2 expressionwas significantly increased in muscle ofdiseased patients. These findings suggest that the biological effectsof TNF-α in mitochondrial diseases are mainly mediated by its type2 receptor. Two types of TNF receptors have been identified so far,TNFR1 and TNFR2, and both bind TNF-α with almost equal affinity[7]. Although each TNF receptor activates different signalingpathways that vary with cell type, TNF receptors lack intrinsicenzyme activity and require the recruitment of adaptor moleculesto initiate signaling [6]. TNFR1 contains an apoptotic death domainand stimulates oxidant production, whereas TNFR2 does not havea death domain and primarily transduces signals favoring cellsurvival [20]. TNFR1 and TNFR2 subtypes are both expressedconstitutively by cardiac myocytes [21]. It is supposed, at least inthe heart, that the majority of the deleterious effects of TNF-α arerelated to the activation of TNFR1, whereas activation of TNFR2seems to exert a protective role [22].

In summary this study is the first to demonstrate that TNF-α (1) ispresent in the ragged red fibers of patients with mitochondrialrespiratory chain dysfunction, (2) is trafficked to the matrix ofmitochondria, and (3) exerts its biological functions through itsspecific receptors, which are both localized in the inner mitochondrialmembrane and differently regulated. Several intriguing questions stillremain. For instance, what are the cellular effects of TNF-α in themuscle of diseased patients, and how does the protein gain access tothe mitochondrial matrix?

Mitochondrial diseases are heterogeneous disorders due tomutations in mtDNA or nuclear DNA that disrupt respiratory chainfunction [1,2]. In contrast to the growing understanding of themolecular basis of MDs, the pathogenesis of these disorders is stillunclear. Two major metabolic abnormalities derive from mtDNAmutations: (1) defective energy production and (2) increasedoxidative stress [1–4]. Reduced ATP generation, from both uncou-pling of electron transport and decreased synthesis from ADP bymitochondrial ATP synthase, decreases the availability of energysupply generated through the tricarboxylic acid cycle [1–4].Oxidative stress may be increased in muscle fibers harboring amtDNA mutation that causes faulty electron transport and gen-eration of ROS, which, in turn, induce an overexpression ofantioxidant enzymes [3,4]. TNF-α has been implicated in severalaspects of human muscle pathophysiology, such as sarcopenia andapoptosis, metabolism, inflammation, and regeneration [19,23–26].

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However, all these effects are mediated by the binding of TNF-α toits receptors on the cell surface where they trigger the varioussignal-transduction pathways. Interestingly, data from previousstudies support a direct effect of TNF-α on mitochondrial respira-tory function [10,11]. A few studies report that TNF-α can inhibitmitochondrial electron transfer and promote mitochondrialuncoupling independent of its cell surface receptors, and a 60-kDa protein in the inner mitochondrial membrane has beendescribed as a mitochondrial-binding protein for TNF-α[10,11,27]. Recently it has been demonstrated that TNF-α improvesthe rate of State 3 respiration in isolated heart mitochondriasubjected to a stress induced by anoxia and this protective effectis likely to be mediated by ROS and sphingolipids and is indepen-dent of TNF-α binding to its cell surface receptors [28]. Therefore,we propose that a potential function of TNF-α in muscle ofdiseased patients could be to improve respiratory function ofdamaged mitochondria in ragged red fibers via a signalingresponse initiated by TNFR2.

Finally the ability of TNF-α to get to the matrix of mitochondriacould indicate the existence of a specific mechanism of transportfor TNF-α through the outer and the inner mitochondrial mem-brane. However, outer membrane damage with opening of themitochondrial permeability transition pore may occur duringthese muscle disorders and, therefore, could allow access ofTNF-α, at least, to the intermembrane space of the organelle [29].

Further studies will be needed to clarify the mechanism bywhich TNF-α is delivered to the mitochondria and the signalpathway triggered through its receptors at the mitochondrial level

Acknowledgment

The authors are grateful to Marzia Giagnacovo for her skillfultechnical assistance in electron microscopy.

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