Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.

The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 +/- 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 +/- 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.     

Mitochondrial DNA mutations as a fundamental mechanism in physiological declines associated with aging

The hypothesis that mitochondrial DNA damage accumulates and contributes to aging was proposed decades ago. Only recently have technological advancements, which facilitate microanalysis of single cells or portions of cells, revealed that mtDNA deletion mutations and, perhaps, single nucleotide mutations accumulate to physiologically relevant levels in the tissues of various species with age. Although a link between single nucleotide mutations and physiological consequences in aging tissue has not been established, the accumulation of deletion mutations in skeletal muscle fibres has been associated with sarcopenia. Different, and apparently random, deletion mutations are specific to individual fibres. However, the mtDNA deletion mutation within a phenotypically abnormal region of a fibre is the same, suggesting a selection, amplification and clonal expansion of the initial deletion mutation. mtDNA deletion mutations within a muscle fibre are associated with specific electron transport system abnormalities, muscle fibre atrophy and fibre breakage. These data point to a causal relationship between mitochondrial DNA mutations and the age-related loss of muscle mass.     

Mitochondrial abnormalities are more frequent in muscles undergoing sarcopenia.

The hypothesis that the accumulation of electron transport system (ETS) abnormalities and sarcopenia are linked was investigated. Vastus lateralis, soleus, and adductor longus muscles were studied in 5-, 18-, and 36-mo-old male Fischer 344 x Brown Norway F(1) hybrid rats. A significant decrease in soleus and vastus lateralis muscle mass was observed with age. Adductor longus was resistant to muscle mass loss. Multiple serial sections were analyzed for the activities of cytochrome-c oxidase (COX) and succinate dehydrogenase (SDH). The number of fibers exhibiting a COX(-)/SDH(++) phenotype increased with age in both vastus lateralis and soleus muscles. No ETS-abnormal fibers were identified in adductor longus at any age. Cross-sectional area of ETS-abnormal fibers decreased in the abnormal region (region displaying COX(-)/SDH(++) phenotype), whereas control fibers did not. The vastus lateralis muscle, which undergoes a high degree of sarcopenia, exhibited more ETS abnormalities and associated fiber loss than the soleus and adductor longus muscles, which are more resistant to sarcopenia, suggesting a direct association between ETS abnormalities and fiber loss.     

Mitochondrial death effectors: Relevance to sarcopenia and disuse muscle atrophy

Accelerated apoptosis in skeletal muscle is increasingly recognized as a potential mechanism contributing to the development of sarcopenia of aging and disuse muscle atrophy. Given their central role in the regulation of apoptosis, mitochondria are regarded as key players in the pathogenesis of myocyte loss during aging and other atrophying conditions. Oxidative damage to mitochondrial constituents, impaired respiration and altered mitochondrial turnover have been proposed as potential triggering events for mitochondrial apoptotic signaling. In addition, iron accumulation within mitochondria may enhance the susceptibility to apoptosis during the development of sarcopenia and possibly acute muscle atrophy, likely through exacerbation of oxidative stress. Mitochondria can induce myocyte apoptosis via both caspase-dependent and independent pathways, although the apoptogenic mediators involved may be different depending on age, muscle type and specific atrophying conditions. Despite the considerable advances made, additional research is necessary to establish a definite causal link between apoptotic signaling and the development of sarcopenia and acute atrophy. Furthermore, a translational effort is required to determine the role played by apoptosis in the pathogenesis of sarcopenia and disuse-induced muscle loss in human subjects.     

Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).     

Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys

Rhesus monkey vastus lateralis muscle was examined histologically for age-associated electron transport system (ETS) abnormalities: fibers lacking cytochrome c oxidase activity (COX(-)) and/or exhibiting succinate dehydrogenase hyperreactivity (SDH(++)). Two hundred serial cross-sections (spanning 1600 microm) were obtained and analyzed for ETS abnormalities at regular intervals. The abundance and length of ETS abnormal regions increased with age. Extrapolating the data to the entire length of the fiber, up to 60% of the fibers were estimated to display ETS abnormalities in the oldest animal studied (34 years) compared to 4% in a young adult animal (11 years). ETS abnormal phenotypes varied with age and fiber type. Middle-aged animals primarily exhibited the COX(-) phenotype, while COX(-)/SDH(++) abnormalities were more common in old animals. Transition region phenotype was affected by fiber type with type 2 fibers first displaying COX(-) and then COX(-)/SDH(++) while type 1 fibers progressed from normal to SDH(++) and then to COX(-)/SDH(++). In situ hybridizations studies revealed an association of ETS abnormalities with deletions of the mitochondrial genome. By measuring cross-sectional area along the length of ETS abnormal fibers, we demonstrated that some of these fibers exhibit atrophy. Our data suggest mitochondrial (mtDNA) deletions and associated ETS abnormalities are contributors to age-associated fiber atrophy.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.     

Cystein cathepsin and Hsp90 activities determine the balance between apoptotic and necrotic cell death pathways in caspase-compromised U937 cells

Caspase-inhibited cells induced to die may exhibit the traits of either apoptosis or necrosis or both, simultaneously. However, mechanisms regulating the commitment to these distinct forms of cell death are barely identified. We found that staurosporine induced both apoptotic and necrotic traits in U937 cells exposed to the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone. Morphology and flow cytometry revealed that individual cells exhibited either apoptotic or necrotic traits, but not the mixed phenotype. Inhibition of cathepsin activity by benzyloxycarbonyl-Phe-Ala-fluoromethylketone rendered caspase-compromised cells resistant to staurosporine-induced apoptosis, but switched the cell death form to necrosis. Inhibition of heat shock protein 90 kDa (Hsp90) chaperon activity by geldanamycin conferred resistance to necrosis in caspase-compromised cells but switched the cell death form to apoptosis. Combination of benzyloxycarbonyl-Phe-Ala-fluoromethylketone and geldanamycin halted the onset of both forms of cell death by saving mitochondrial trans-membrane potential and preventing acidic volume (lysosomes) loss. These effects of benzyloxycarbonyl-Phe-Ala-fluoromethylketone and/or geldanamycin on cell death were restricted to caspase-inhibited cells exposed to staurosporine but influenced neither only the staurosporine-provoked apoptosis nor hydrogen peroxide (H2O2)-generated necrosis. Our results demonstrate that the staurosporine-induced death pathway bifurcates in caspase-compromised cells and commitment to apoptotic or necrotic phenotypes depends on cathepsin protease or Hsp90 chaperon activities.     

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Palmitate-induced apoptosis in cardiomyocytes is mediated through alterations in mitochondria: prevention by cyclosporin A

Palmitate, a C16 fatty acid found in high concentrations in the blood in acute myocardial infarction, induces apoptotic cell death. To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic chick heart and were treated with palmitate. Concentration-dependent loss of cell viability was established by loss of the ability of palmitate-treated cells to exclude propidium iodide (PI), metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and retain fluorescein diacetate (FDA). Dual staining with PI and FDA and subsequent analysis by FACS established that palmitate-induced cell death was predominantly necrosis whereas apoptosis occurred in 13% of all dead cells. The low proportion of palmitate-induced apoptosis was confirmed by evaluation of the DNA content or PI fluorescent staining of the DNA of permeabilized cardiomyocytes. A critical role for mitochondria in the pathogenesis of palmitate-induced cell death was demonstrated, for the first time, based on palmitate-induced reduction of mitochondrial activity as assessed by the mitochondrial-selective dye chloromethyl-X-Rosamine and the presence of a greater amount of the mitochondrial marker cytochrome C in the cytosol of palmitate-treated cardiomyocytes than in control cells. Further, cyclosporin that inhibits the development of mitochondrial transition pores blocked palmitate-induced alteration in mitochondrial function and palmitate-induced cell death. We further demonstrated the selectivity of cyclosporin A for the prevention of apoptotic cell death in the heart as there was no alteration in necrotic cell death produced by palmitate with cyclosporin pretreatment. Our data demonstrate the nature of palmitate-induced cell death in cardiomyocytes (both apoptotic and necrotic), propose a mitochondrial basis for its pathogenesis and show that cyclosporin A prevents palmitate-induced apoptotic cardiomyocyte cell death.     

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.     

Mitochondrial DNA deletion mutations: a causal role in sarcopenia.

Mitochondrial DNA (mtDNA) deletion mutations accumulate with age in tissues of a variety of species. Although the relatively low calculated abundance of these deletion mutations in whole tissue homogenates led some investigators to suggest that these mutations do not have any physiological impact, their focal and segmental accumulation suggests that they can, and do, accumulate to levels sufficient to affect the metabolism of a tissue. This phenomenon is most clearly demonstrated in skeletal muscle, where the accumulation of mtDNA deletion mutations remove critical subunits that encode for the electron transport system (ETS). In this review, we detail and provide evidence for a molecular basis of muscle fiber loss with age. Our data suggest that the mtDNA deletion mutations, which are generated in tissues with age, cause muscle fiber loss. Within a fiber, the process begins with a mtDNA replication error, an error that results in a loss of 25-80% of the mitochondrial genome. This smaller genome is replicated and, through a process not well understood, eventually comprises the majority of mtDNA within the small affected region of the muscle fiber. The preponderance of the smaller genomes results in a dysfunctional ETS in the affected area. As a consequence of both the decline in energy production and the increase in oxidative damage in the region, the fiber is no longer capable of self-maintenance, resulting in the observed intrafiber atrophy and fiber breakage. We are therefore proposing that a process contained within a very small region of a muscle fiber can result in breakage and loss of muscle fiber from the tissue.     

Tumor necrosis factor alpha signaling in skeletal muscle: effects of age and caloric restriction

Past the age of 50 years, aging individuals lose muscle mass at an approximate rate of 1-2% per year. This age-related muscle atrophy, termed sarcopenia, can have significant effects on individual health and quality of life and can also impact the socioeconomic status. Sarcopenia is due to both a decrease in the number of fibers and the atrophy of the remaining fibers. The mechanisms causing loss of fibers have not been clearly defined, but may likely involve apoptosis. Elevated levels of circulating tumor necrosis factor alpha (TNF-alpha) and adaptations in TNF-alpha signaling in aged skeletal muscle may be contributing factors for the activation of apoptosis. These adaptations may be fiber-type specific, which could explain the selective loss of type II fibers, vs. type I fibers, in the aging process. Caloric restriction, a proven antiaging intervention, is known to attenuate the loss of muscle mass and function with age. Furthermore, caloric restriction has been shown to attenuate the age-associated adaptations in TNF-alpha signaling in skeletal muscle, which may be a possible mechanism by which CR prevents apoptosis and the loss of muscle fibers with age. The potential role of TNF-alpha in the progression of sarcopenia will be discussed, as well as the effects of life-long caloric restriction on TNF-alpha signaling.     

Evidence for necrosis,but not apoptosis,in human hepatoma cells with knockdown of mitochondrial aquaporin-8

We previously found that mitochondrial aquaporin-8 (mtAQP8) channels facilitate mitochondrial H₂O₂ release in human hepatoma HepG2 cells and that their knockdown causes oxidant-induced mitochondrial dysfunction and loss of viability. Here, we studied whether apoptosis or necrosis is involved as the mode of cell death. We confirmed that siRNA-induced mtAQP8 knockdown significantly decreased HepG2 viability by MTT assay, LDH leakage, and trypan blue exclusion test. Analysis of mitochondrial proapoptotic Bax-to-antiapoptotic Bcl_XL ratio, mitochondrial cytochrome c release and caspase-3 activation showed no alterations in mtAQP8-knockdown cells. This indicates a primary mechanism of cell death other than the intrinsic mitochondrial apoptotic pathway. Thus, nuclear staining with DAPI did not reveal any increase of apoptotic features, i.e. chromatin condensation or nuclear fragmentation. Flow cytometry studies after double cell staining with annexin V and propidium iodide confirmed lack of apoptosis and suggested necrosis as the primary mechanism of death in mtAQP8-knockdown HepG2 cells. Necrosis was further supported by the increased nuclear delocalization and extracellular release of the High Mobility Group Box 1 protein. The knockdown of mtAQP8 in another human hepatoma-derived cell line, i.e. HuH-7 cells, also induced necrotic but not apoptotic death. Our data suggest that mtAQP8 knockdown induces necrotic cell death in human neoplastic hepatic cells, a finding that might be relevant to therapeutic strategies against hepatoma cells.     

The histone deacetylase inhibitor butyrate improves metabolism and reduces muscle atrophy during aging

Sarcopenia, the loss of skeletal muscle mass and function during aging, is a major contributor to disability and frailty in the elderly. Previous studies found a protective effect of reduced histone deacetylase activity in models of neurogenic muscle atrophy. Because loss of muscle mass during aging is associated with loss of motor neuron innervation, we investigated the potential for the histone deacetylase (HDAC) inhibitor butyrate to modulate age‐related muscle loss. Consistent with previous studies, we found significant loss of hindlimb muscle mass in 26‐month‐old C57Bl/6 female mice fed a control diet. Butyrate treatment starting at 16 months of age wholly or partially protected against muscle atrophy in hindlimb muscles. Butyrate increased muscle fiber cross‐sectional area and prevented intramuscular fat accumulation in the old mice. In addition to the protective effect on muscle mass, butyrate reduced fat mass and improved glucose metabolism in 26‐month‐old mice as determined by a glucose tolerance test. Furthermore, butyrate increased markers of mitochondrial biogenesis in skeletal muscle and whole‐body oxygen consumption without affecting activity. The increase in mass in butyrate‐treated mice was not due to reduced ubiquitin‐mediated proteasomal degradation. However, butyrate reduced markers of oxidative stress and apoptosis and altered antioxidant enzyme activity. Our data is the first to show a beneficial effect of butyrate on muscle mass during aging and suggests HDACs contribute to age‐related muscle atrophy and may be effective targets for intervention in sarcopenia and age‐related metabolic disease.     

Abnormalities of mitochondrial functioning can partly explain the metabolic disorders encountered in sarcopenic gastrocnemius

Aging triggers several abnormalities in muscle glycolytic fibers including increased proteolysis, reactive oxygen species (ROS) production and apoptosis. Since the mitochondria are the main site of substrate oxidation, ROS production and programmed cell death, we tried to know whether the cellular disorders encountered in sarcopenia are due to abnormal mitochondrial functioning. Gastrocnemius mitochondria were extracted from adult (6 months) and aged (21 months) male Wistar rats. Respiration parameters, opening of the permeability transition pore and ROS production, with either glutamate (amino acid metabolism) or pyruvate (glucose metabolism) as a respiration substrate, were evaluated at different matrix calcium concentrations. Pyruvate dehydrogenase and respiratory complex activities as well as their contents measured by Western blotting analysis were determined. Furthermore, the fatty acid profile of mitochondrial phospholipids was also measured. At physiological calcium concentration, state III respiration rate was lowered by aging in pyruvate conditions (-22%), but not with glutamate. The reduction of pyruvate oxidation resulted from a calcium-dependent inactivation of the pyruvate dehydrogenase system and could provide for the well-known proteolysis encountered during sarcopenia. Matrix calcium loading and aging increased ROS production. They also reduced the oxidative phosphorylation. This was associated with lower calcium retention capacities, suggesting that sarcopenic fibers are more prone to programmed cell death. Aging was also associated with a reduced mitochondrial superoxide dismutase activity, which does not intervene in toxic ROS overproduction but could explain the lower calcium retention capacities. Despite a lower content, cytochrome c oxidase displayed an increased activity associated with an increased n-6/n-3 polyunsaturated fatty acid ratio of mitochondrial phospholipids. In conclusion, we propose that mitochondria obtained from aged muscle fibers display several functional abnormalities explaining the increased proteolysis, ROS overproduction and vulnerability to apoptosis exhibited by sarcopenic muscle. These changes appear to be related to modifications of the fatty acid profile of mitochondrial lipids.