Peter J. Adhihetty

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Age-related loss of muscle mass and strength (sarcopenia) leads to a decline in physical function and frailty in the elderly. Among the many proposed underlying causes of sarcopenia, mitochondrial dysfunction is inherent in a variety of aged tissues. The intent of this study was to examine the effect of aging on key groups of regulatory proteins involved in mitochondrial biogenesis and how this relates to physical performance in two groups of sedentary elderly participants, classified as high- and low-functioning based on the Short Physical Performance Battery test. Muscle mass was decreased by 38% and 30% in low-functioning elderly (LFE) participants when compared to young and high-functioning elderly participants, respectively, and positively correlated to physical performance. Mitochondrial respiration in permeabilized muscle fibers was reduced (41%) in the LFE group when compared to the young, and this was associated with a 30% decline in cytochrome c oxidase activity. Levels of key metabolic regulators, SIRT3 and PGC-1α, were significantly reduced (50%) in both groups of elderly participants when compared to young. Similarly, the fusion protein OPA1 was lower in muscle from elderly subjects; however, no changes were detected in Mfn2, Drp1 or Fis1 among the groups. In contrast, protein import machinery components Tom22 and cHsp70 were increased in the LFE group when compared to the young. This study suggests that aging in skeletal muscle is associated with impaired mitochondrial function and altered biogenesis pathways and that this may contribute to muscle atrophy and the decline in muscle performance observed in the elderly population.

It is well established that contracting skeletal muscles produce free radicals. Given that radicals are known to play a prominent role in the pathogenesis of several diseases, the 1980s-90s dogma was that contraction-induced radical production was detrimental to muscle because of oxidative damage to macromolecules within the fibre. In contrast to this early outlook, it is now clear that both reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in cell signalling pathways involved in muscle adaptation to exercise and the remodelling that occurs in skeletal muscle during periods of prolonged inactivity. This review will highlight two important redox sensitive signalling pathways that contribute to ROS and RNS-induced skeletal muscle adaptation to endurance exercise. We begin with a historical overview of radical production in skeletal muscles followed by a discussion of the intracellular sites for ROS and RNS production in muscle fibres. We will then provide a synopsis of the redox-sensitive NF-B and PGC-1α signalling pathways that contribute to skeletal muscle adaptation in response to exercise training. We will conclude with a discussion of unanswered questions in redox signalling in skeletal muscle in the hope of promoting additional research interest in this field.

Muscle loss during aging and disuse is a highly prevalent and disabling condition, but knowledge about cellular pathways mediating muscle atrophy is still limited. Given the postmitotic nature of skeletal myocytes, the maintenance of cellular homeostasis relies on the efficiency of cellular quality control mechanisms. In this scenario, alterations in mitochondrial function are considered a major factor underlying sarcopenia and muscle atrophy. Damaged mitochondria are not only less bioenergetically efficient, but also generate increased amounts of reactive oxygen species, interfere with cellular quality control mechanisms, and display a greater propensity to trigger apoptosis. Thus, mitochondria stand at the crossroad of signaling pathways that regulate skeletal myocyte function and viability. Studies on these pathways have sometimes provided unexpected and counterintuitive results, which suggests that they are organized into a complex, heterarchical network that is currently insufficiently understood. Untangling the complexity of such a network will likely provide clinicians with novel and highly effective therapeutics to counter the muscle loss associated with aging and disuse. In this review, we summarize the current knowledge on the mechanisms whereby mitochondrial dysfunction intervenes in the pathogenesis of sarcopenia and disuse atrophy, and highlight the prospect of targeting specific processes to treat these conditions.

The transcriptional coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been identified as an important mediator of mitochondrial biogenesis based on its ability to interact with transcription factors that activate nuclear genes encoding mitochondrial proteins. The induction of PGC-1α protein expression under conditions that provoke mitochondrial biogenesis, such as contractile activity or thyroid hormone (T 3 ) treatment, is not fully characterized. Thus we related PGC-1α protein expression to cytochrome c oxidase (COX) activity in 1) tissues of varying oxidative capacities, 2) tissues from animals treated with T 3 , and 3) skeletal muscle subject to contractile activity both in cell culture and in vivo. Our results demonstrate a strong positive correlation ( r = 0.74; P < 0.05) between changes in PGC-1α and COX activity, used as an index of mitochondrial adaptations. The highest constitutive levels of PGC-1α were found in the heart, whereas the lowest were measured in fast-twitch white muscle and liver. T 3 increased PGC-1α content similarly in both fast- and slow-twitch muscle, as well as in the liver, but not in heart. T 3 also induced early (6 h) increases in AMP-activated protein kinase (AMPKα) activity, as well as later (5 day) increases in p38 MAP kinase activity in slow-twitch, but not in fast-twitch, muscle. Contractile activity provoked early increases in PGC-1α, coincident with increases in mitochondrial transcription factor A (Tfam), and nuclear respiratory factor-1 (NRF-1) protein expression, suggesting that PGC-1α is physiologically important in coordinating the expression of the nuclear and mitochondrial genomes. Ca 2+ ionophore treatment of muscle cells led to an approximately threefold increase in PGC-1α protein, and contractile activity induced rapid and marked increases in both p38 MAP kinase and AMPKα activities. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) treatment of muscle cells also led to parallel increases in AMPKα activity and PGC-1α protein levels. These data are consistent with observations that indicate that increases in PGC-1α protein are affected by Ca 2+ signaling mechanisms, AMPKα activity, as well as posttranslational phosphorylation events that increase PGC-1α protein stability. Our data support a role for PGC-1α in the physiological regulation of mitochondrial content in a variety of tissues and suggest that increases in PGC-1α expression form part of a unifying pathway that promotes both T 3 - and contractile activity-induced mitochondrial adaptations.

We investigated the role of PPAR gamma coactivator 1alpha (PGC-1alpha) in muscle dysfunction in Huntington's disease (HD). We observed reduced PGC-1alpha and target genes expression in muscle of HD transgenic mice. We produced chronic energy deprivation in HD mice by administering the catabolic stressor beta-guanidinopropionic acid (GPA), a creatine analogue that reduces ATP levels, activates AMP-activated protein kinase (AMPK), which in turn activates PGC-1alpha. Treatment with GPA resulted in increased expression of AMPK, PGC-1alpha target genes, genes for oxidative phosphorylation, electron transport chain and mitochondrial biogenesis, increased oxidative muscle fibers, numbers of mitochondria and motor performance in wild-type, but not in HD mice. In muscle biopsies from HD patients, there was decreased PGC-1alpha, PGC-1beta and oxidative fibers. Oxygen consumption, PGC-1alpha, NRF1 and response to GPA were significantly reduced in myoblasts from HD patients. Knockdown of mutant huntingtin resulted in increased PGC-1alpha expression in HD myoblast. Lastly, adenoviral-mediated delivery of PGC-1alpha resulted increased expression of PGC-1alpha and markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice. These findings show that impaired function of PGC-1alpha plays a critical role in muscle dysfunction in HD, and that treatment with agents to enhance PGC-1alpha function could exert therapeutic benefits. Furthermore, muscle may provide a readily accessible tissue in which to monitor therapeutic interventions.

Chronic muscle disuse induced by denervation reduces mitochondrial content and produces muscle atrophy. To investigate the molecular mechanisms responsible for these adaptations, we assessed 1) mitochondrial biogenesis- and apoptosis-related proteins and 2) apoptotic susceptibility and cell death following denervation. Rats were subjected to 5, 7, 14, 21, or 42 days of unilateral denervation of the sciatic or peroneal nerve. Muscle mass and mitochondrial content were reduced by 40–65% after 21 and 42 days of denervation. Denervation-induced decrements in mitochondrial content occurred along with 60% and 70% reductions in transcription factor A (Tfam) and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, respectively. After 42 days of denervation, Bax was elevated by 115% and Bcl-2 was decreased by 89%, producing a 16-fold increase in the Bax-to-Bcl-2 ratio. Mitochondrial reactive oxygen species production was markedly elevated by 5- to 7.5-fold in subsarcolemmal mitochondria after 7, 14, and 21 days of denervation, whereas reactive oxygen species production in intermyofibrillar (IMF) mitochondria was reduced by 40–50%. Subsarcolemmal and IMF mitochondrial levels of MnSOD were also reduced by 40–50% after 14–21 days of denervation. The maximal rate of IMF mitochondrial pore opening ( V max ) was elevated by 25–35%, and time to V max was reduced by 20–25% after 14 and 21 days, indicating increased apoptotic susceptibility. Myonuclear decay, assessed by DNA fragmentation, was elevated at 7–21 days of denervation. Our data indicate that PGC-1α and Tfam are important factors that likely contribute to the reduced mitochondrial content after chronic disuse. In addition, our results illustrate that, despite the reduced mitochondrial content, denervated muscle has greater mitochondrial apoptotic susceptibility, which coincided with elevated apoptosis, and these processes may contribute to denervation-induced muscle atrophy.

p53 is a tumor suppressor protein that also plays a role in regulating aerobic metabolism. Since skeletal muscle is a major source of whole body aerobic respiration, it is important to delineate the effects of p53 on muscle metabolism. In p53 knockout (KO) mice, we observed diminished mitochondrial content in mixed muscle and lowered peroxisome proliferator-activated receptor-γ (PPARγ) coactivator (PGC)-1α protein levels in gastrocnemius muscle. In intermyofibrillar (IMF) mitochondria, lack of p53 was associated with reduced respiration and elevated reactive oxygen species production. Permeability transition pore kinetics remained unchanged; however, IMF mitochondrial cytochrome c release was reduced and DNA fragmentation was lowered, illustrating a resistance to mitochondrially driven apoptosis in muscle of KO mice. p53-null animals displayed similar muscle strength but greater fatigability and less locomotory endurance than wild-type (WT) animals. Surprisingly, the adaptive responses in mitochondrial content to running were similar in WT and KO mice. Thus p53 may be important, but not necessary, for exercise-induced mitochondrial biogenesis. In WT animals, acute muscle contractions induced the phosphorylation of p53 in concert with increased activation of upstream kinases AMP-activated protein kinase and p38, indicating a pathway through which p53 may initiate mitochondrial biogenesis in response to contractile activity. These data illustrate a novel role for p53 in maintaining mitochondrial biogenesis, apoptosis, and performance in skeletal muscle.

Regularly performed exercise in the form of endurance training produces a well‐established adaptation in skeletal muscle termed mitochondrial biogenesis. The physiological benefit of this is an enhanced performance of muscle when subject to endurance exercise. This is not only of great advantage for athletic endeavours, but it also clearly improves the quality of life of previously sedentary individuals and those involved in injury rehabilitation. Here we review the molecular basis for mitochondrial biogenesis in muscle, from the initial signals arising in contracting muscle, to the transcription factors involved in mitochondrial and nuclear DNA transcription, as well as the post‐translational import mechanisms required for the synthesis of the organelle. We discuss specific protein components associated with reactive oxygen species production, and suggest some questions which remain unanswered with respect to the role of exercise‐induced mitochondrial biogenesis in ageing, apoptosis and disease.

Mitochondria are critical for cellular bioenergetics, and they mediate apoptosis within cells. We used whole body peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) knockout (KO) animals to investigate its role on organelle function, apoptotic signaling, and cytochrome-c oxidase activity, an indicator of mitochondrial content, in muscle and other tissues (brain, liver, and pancreas). Lack of PGC-1alpha reduced mitochondrial content in all muscles (17-44%; P < 0.05) but had no effect in brain, liver, and pancreas. However, the tissue expression of proteins involved in mitochondrial DNA maintenance [transcription factor A (Tfam)], import (Tim23), and remodeling [mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1)] did not parallel the decrease in mitochondrial content in PGC-1alpha KO animals. These proteins remained unchanged or were upregulated (P < 0.05) in the highly oxidative heart, indicating a change in mitochondrial composition. A change in muscle organelle composition was also evident from the alterations in subsarcolemmal and intermyofibrillar mitochondrial respiration, which was impaired in the absence of PGC-1alpha. However, endurance-trained KO animals did not exhibit reduced mitochondrial respiration. Mitochondrial reactive oxygen species (ROS) production was not affected by the lack of PGC-1alpha, but subsarcolemmal mitochondria from PGC-1alpha KO animals released a greater amount of cytochrome c than in WT animals following exogenous ROS treatment. Our results indicate that the lack of PGC-1alpha results in 1) a muscle type-specific suppression of mitochondrial content that depends on basal oxidative capacity, 2) an alteration in mitochondrial composition, 3) impaired mitochondrial respiratory function that can be improved by training, and 4) a greater basal protein release from subsarcolemmal mitochondria, indicating an enhanced mitochondrial apoptotic susceptibility.

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H2O2 (0, 25, 50, and 100 microM). However, IMF mitochondria were more sensitive to H2O2 and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, V(max)) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H2O2-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn2+-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy.

Substantial evidence indicates bioenergetic dysfunction and mitochondrial impairment contribute either directly and/or indirectly to the pathogenesis of numerous neurodegenerative disorders. Treatment paradigms aimed at ameliorating this cellular energy deficit and/or improving mitochondrial function in these neurodegenerative disorders may prove to be useful as a therapeutic intervention. Creatine is a molecule that is produced both endogenously, and acquired exogenously through diet, and is an extremely important molecule that participates in buffering intracellular energy stores. Once creatine is transported into cells, creatine kinase catalyzes the reversible transphosphorylation of creatine via ATP to enhance the phosphocreatine energy pool. Creatine kinase enzymes are located at strategic intracellular sites to couple areas of high energy expenditure to the efficient regeneration of ATP. Thus, the creatinekinase/phosphocreatine system plays an integral role in energy buffering and overall cellular bioenergetics. Originally, exogenous creatine supplementation was widely used only as an ergogenic aid to increase the phosphocreatine pool within muscle to bolster athletic performance. However, the potential therapeutic value of creatine supplementation has recently been investigated with respect to various neurodegenerative disorders that have been associated with bioenergetic deficits as playing a role in disease etiology and/or progression which include; Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis (ALS), and Huntington’s disease. This review discusses the contribution of mitochondria and bioenergetics to the progression of these neurodegenerative diseases and investigates the potential neuroprotective value of creatine supplementation in each of these neurological diseases. In summary, current literature suggests that exogenous creatine supplementation is most efficacious as a treatment paradigm in Huntington’s and Parkinson’s disease but appears to be less effective for ALS and Alzheimer’s disease.

Mitochondrial DNA (mtDNA) mutations lead to decrements in mitochondrial function and accelerated rates of these mutations has been linked to skeletal muscle loss (sarcopenia). The purpose of this study was to investigate the effect of mtDNA mutations on mitochondrial quality control processes in skeletal muscle from animals (young; 3-6 months and older; 8-15 months) expressing a proofreading-deficient version of mtDNA polymerase gamma (PolG). This progeroid aging model exhibits elevated mtDNA mutation rates, mitochondrial dysfunction, and a premature aging phenotype that includes sarcopenia. We found increased expression of the mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its target proteins, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam) in PolG animals compared to wild-type (WT) (P<0.05). Muscle from older PolG animals displayed higher mitochondrial fission protein 1 (Fis1) concurrent with greater induction of autophagy, as indicated by changes in Atg5 and p62 protein content (P<0.05). Additionally, levels of the Tom22 import protein were higher in PolG animals when compared to WT (P<0.05). In contrast, muscle from normally-aged animals exhibited a distinctly different expression profile compared to PolG animals. Older WT animals appeared to have higher fusion (greater Mfn1/Mfn2, and lower Fis1) and lower autophagy (Beclin-1 and p62) compared to young WT suggesting that autophagy is impaired in aging muscle. In conclusion, muscle from mtDNA mutator mice display higher mitochondrial fission and autophagy levels that likely contribute to the sarcopenic phenotype observed in premature aging and this differs from the response observed in normally-aged muscle.

Mitochondria are negatively affected by ageing leading to their inability to adapt to higher levels of oxidative stress and this ultimately contributes to the systemic loss of muscle mass and function termed sarcopenia. Since mitochondria are central mediators of muscle health, they have become highly sought-after targets of physiological and pharmacological interventions. Exercise is the only known strategy to combat sarcopenia and this is largely mediated through improvements in mitochondrial plasticity. More recently a critical role for mitochondrial turnover in preserving muscle has been postulated. Specifically, cellular pathways responsible for the regulation of mitochondrial turnover including biogenesis, dynamics and autophagy may become dysregulated during ageing resulting in the reduced clearance and accumulation of damaged organelles within the cell. When mitochondrial quality is compromised and homeostasis is not re-established, myonuclear cell death is activated and muscle atrophy ensues. In contrast, acute and chronic exercise attenuates these deficits, restoring mitochondrial turnover and promoting a healthier mitochondrial pool that leads to the preservation of muscle. Additionally, the magnitude of these exercise-induced mitochondrial adaptations is currently debated with several studies reporting a lower adaptability of old muscle relative to young, but the processes responsible for this diminished training response are unclear. Based on these observations, understanding the molecular details of how advancing age and exercise influence mitochondria in older muscle will provide invaluable insight into the development of exercise protocols that will maximize beneficial adaptations in the elderly. This information will also be imperative for future research exploring pharmacological targets of mitochondrial plasticity.

Apoptosis, or programmed cell death, is now recognized to be an important cellular event during normal development and in the progression of specific diseases. Apoptosis can be triggered by stimuli initiating outside of the cell, or within the mitochondria, leading to the activation of caspases and subsequent cell death. Although apoptosis has been widely studied in a variety of tissues over the last 5 years, skeletal muscle and heart have been relatively ignored in this regard. Research on apoptosis in cardiac muscle has recently taken on a higher profile as the recognition emerges that it may be an important contributor to specific cardiac pathologies, particularly in response to ischemia-reperfusion in which reactive oxygen species are formed. In skeletal muscle, very few studies have been done under specific physiological (e.g., exercise) and pathophysiological (e.g., dystrophies, denervation, myopathies) conditions. Skeletal muscle is unique in that it is multi-nucleated, and evidence suggests that it can undergo individual myonuclear apoptosis as well as complete cell death. This review discusses the basic cellular mechanisms of apoptosis, as well as the current evidence of this process in cardiac and skeletal muscle. The need for more work in this area is highlighted, particularly in exercise and training.

Our intent was to investigate the mechanisms driving the adaptive potential of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in young (6 mo) and senescent (36 mo) animals in response to a potent stimulus for organelle biogenesis. We employed chronic electrical stimulation (10 Hz, 3 h/day, 7 days) to induce contractile activity of skeletal muscle in 6 and 36 mo F344XBN rats. Subsequent to chronic activity, acute stimulation (1 Hz, 5 min) in situ revealed greater fatigue resistance in both age groups. However, the improvement in endurance was significantly greater in the young, compared to the old animals. Chronic muscle use also augmented SS and IMF mitochondrial volume to a greater extent in young muscle. The molecular basis for the diminished organelle expansion in aged muscle was due, in part, to the collective attenuation of the chronic stimulation-evoked increase in regulatory proteins involved in mediating mitochondrial protein import and biogenesis. Furthermore, adaptations in mitochondrial function were also blunted in old animals. However, chronic contractile activity evoked greater reductions in mitochondrially-mediated proapoptotic signaling in aged muscle. Thus, mitochondrial plasticity is retained in aged animals, however the magnitude of the changes are less compared to young animals due to attenuated molecular processes regulating organelle biogenesis.

Sirt1 is a NAD + -dependent histone deacetylase that interacts with the regulatory protein of mitochondrial biogenesis PGC-1α and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins, and PGC-1α existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days, 10 Hz; 3 h/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1α levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70–90% in soleus and plantaris muscles of running animals, whereas Sirt1 activity remained unchanged. In chronically stimulated muscle, cytochrome c was increased by 30% compared with nonstimulated muscle, whereas Sirt1 activity was increased modestly by 20–25%. In contrast, in denervated muscle, these markers of mitochondrial content were decreased by 30–50% compared with the control muscle, whereas Sirt1 activity was increased by 75–80%. Our data suggest that Sirt1 and PGC-1α expression are independently regulated and that, although Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse.

Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P<0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P<0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (approximately 30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both mitochondrial subfractions. Our data suggest the possibility that chronic contractile activity can exert a protective effect on mitochondrially mediated apoptosis in muscle.

Mitochondrial myopathy patients (MMPs) have impaired oxidative phosphorylation and exercise intolerance. Endurance training of MMPs improves exercise tolerance, but also increases mutational load. To assess the regulation of mitochondrial content in MMPs, we measured proteins involved in 1) biogenesis, 2) oxidative stress, and 3) apoptosis in MMPs and healthy controls (HCs) both before and after endurance training. Before training, MMPs had a greater mitochondrial content, along with a 1.4-fold (P < 0.05) higher expression of the biogenesis regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha). The DNA repair enzyme 8-oxoguanine DNA glycolase-1 (OGG-1), the antioxidant manganese superoxide dismutase (MnSOD), and the apoptotic proteins AIF and Bcl-2 were higher in MMPs compared with HCs. Aconitase, an enzyme sensitive to oxidative stress, was 52% lower (P < 0.05) in MMPs when calculated based on an estimate of mitochondrial volume and oxidative stress-induced protein modifications tended to be higher in MMPs compared with HCs. Endurance training (ET) induced increases in mitochondrial content in both HC subjects and MMPs, but there was no effect of training on the regulatory proteins Tfam or PGC-1alpha. In MMPs, training induced a selective reduction of OGG-1, an increase in MnSOD, and a reduction in aconitase activity. Thus, before training, MMPs exhibited an adaptive response of nuclear proteins indicative of a compensatory increase in mitochondrial content. Following training, several parallel adaptations occurred in MMPs and HCs, which may contribute to previously observed functional improvements of exercise in MMPs. However, our results indicate that muscle from MMPs may be exposed to greater levels of oxidative stress during the course of training. Further investigation is required to evaluate the long-term benefits of endurance training as a therapeutic intervention for mitochondrial myopathy patients.

Aging is associated with a loss in muscle known as sarcopenia that is partially attributed to apoptosis. In aging rodents, caloric restriction (CR) increases health and longevity by improving mitochondrial function and the polyphenol resveratrol (RSV) has been reported to have similar benefits. In the present study, we investigated the potential efficacy of using short-term (6 weeks) CR (20%), RSV (50 mg/kg/day), or combined CR + RSV (20% CR and 50 mg/kg/day RSV), initiated at late-life (27 months) to protect muscle against sarcopenia by altering mitochondrial function, biogenesis, content, and apoptotic signaling in both glycolytic white and oxidative red gastrocnemius muscle (WG and RG, respectively) of male Fischer 344 × Brown Norway rats. CR but not RSV attenuated the age-associated loss of muscle mass in both mixed gastrocnemius and soleus muscle, while combined treatment (CR + RSV) paradigms showed a protective effect in the soleus and plantaris muscle (P < 0.05). Sirt1 protein content was increased by 2.6-fold (P < 0.05) in WG but not RG muscle with RSV treatment, while CR or CR + RSV had no effect. PGC-1α levels were higher (2-fold) in the WG from CR-treated animals (P < 0.05) when compared to ad-libitum (AL) animals but no differences were observed in the RG with any treatment. Levels of the anti-apoptotic protein Bcl-2 were significantly higher (1.6-fold) in the WG muscle of RSV and CR + RSV groups compared to AL (P < 0.05) but tended to occur coincident with elevations in the pro-apoptotic protein Bax so that the apoptotic susceptibility as indicated by the Bax to Bcl-2 ratio was unchanged. There were no alterations in DNA fragmentation with any treatment in muscle from older animals. Additionally, mitochondrial respiration measured in permeabilized muscle fibers was unchanged in any treatment group and this paralleled the lack of change in cytochrome c oxidase (COX) activity. These data suggest that short-term moderate CR, RSV, or CR + RSV tended to modestly alter key mitochondrial regulatory and apoptotic signaling pathways in glycolytic muscle and this might contribute to the moderate protective effects against aging-induced muscle loss observed in this study.

Diaphragm muscle weakness in chronic heart failure (CHF) is caused by elevated oxidants and exacerbates breathing abnormalities, exercise intolerance, and dyspnea. However, the specific source of oxidants that cause diaphragm weakness is unknown. We examined whether mitochondrial reactive oxygen species (ROS) cause diaphragm weakness in CHF by testing the hypothesis that CHF animals treated with a mitochondria-targeted antioxidant have normal diaphragm function. Rats underwent CHF or sham surgery. Eight weeks after surgeries, we administered a mitochondrial-targeted antioxidant (MitoTEMPO; 1 mg·kg −1 ·day −1 ) or sterile saline (Vehicle). Left ventricular dysfunction (echocardiography) pre- and posttreatment and morphological abnormalities were consistent with the presence of CHF. CHF elicited a threefold ( P &lt; 0.05) increase in diaphragm mitochondrial H 2 O 2 emission, decreased diaphragm glutathione content by 23%, and also depressed twitch and maximal tetanic force by ∼20% in Vehicle-treated animals compared with Sham ( P &lt; 0.05 for all comparisons). Diaphragm mitochondrial H 2 O 2 emission, glutathione content, and twitch and maximal tetanic force were normal in CHF animals receiving MitoTEMPO. Neither CHF nor MitoTEMPO altered the diaphragm protein levels of antioxidant enzymes: superoxide dismutases (CuZn-SOD or MnSOD), glutathione peroxidase, and catalase. In both Vehicle and MitoTEMPO groups, CHF elicited a ∼30% increase in cytochrome c oxidase activity, whereas there were no changes in citrate synthase activity. Our data suggest that elevated mitochondrial H 2 O 2 emission causes diaphragm weakness in CHF. Moreover, changes in protein levels of antioxidant enzymes or mitochondrial content do not seem to mediate the increase in mitochondria H 2 O 2 emission in CHF and protective effects of MitoTEMPO.

Fatigue is a symptom of many diseases, but it can also manifest as a unique medical condition, such as idiopathic chronic fatigue (ICF). While the prevalence of ICF increases with age, mitochondrial content and function decline with age, which may contribute to ICF. The purpose of this study was to determine whether skeletal muscle mitochondrial dysregulation and oxidative stress is linked to ICF in older adults. Sedentary, old adults (n = 48, age 72.4 ± 5.3 years) were categorized into ICF and non-fatigued (NF) groups based on the FACIT-Fatigue questionnaire. ICF individuals had a FACIT score one standard deviation below the mean for non-anemic adults > 65 years and were excluded according to CDC diagnostic criteria for ICF. Vastus lateralis muscle biopsies were analyzed, showing reductions in mitochondrial content and suppression of mitochondrial regulatory proteins Sirt3, PGC-1α, NRF-1, and cytochrome c in ICF compared to NF. Additionally, mitochondrial morphology proteins, antioxidant enzymes, and lipid peroxidation were unchanged in ICF individuals. Our data suggests older adults with ICF have reduced skeletal muscle mitochondrial content and biogenesis signaling that cannot be accounted for by increased oxidative damage.

The importance of the mitochondrial protein import pathway, discussed relative to other steps involved in the overall biogenesis of the organelle, are reviewed.Mitochondrial biogenesis is a product of complex interactions between the nuclear and mitochondrial genomes. Signaling pathways, such as those activated by exercise, initiate the activation of transcription factors that increase the production of mRNA from nuclear and mitochondrial DNA. Nuclear gene products are translated in the cytosol as precursor proteins with inherent targeting signals. These precursor proteins interact with molecular chaperones that direct them to the import machinery of the outer membrane (Tom complex). The precursor is unfolded and transferred through the outer membrane, across the intermembrane space to the mitochondrial inner membrane translocases (Tim complex). Intramitochondrial components (mtHSP70) pull the precursor into the matrix, cleave off the targeting sequence (mitochondrial processing peptidase), and refold the protein (HSP60, cpn10) into its mature conformation. Physiological stressors such as contractile activity and thyroid hormone accelerate protein import into the mitochondria, coincident with an increase in the expression of some components of the import machinery. This is important for the overall expansion of the mitochondrial reticulum. Conversely, impairments in the import process can be a cause of mitochondrial dysfunction and disease.Efforts to further characterize the components of the import machinery, to define the role of specific machinery components on the import rate, and to examine protein import function in a variety of mitochondrial diseases are warranted.

In an effort to better characterize uncoupling protein-3 (UCP3) function in skeletal muscle, we assessed basal UCP3 protein content in rat intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial subfractions in conjunction with measurements of state 4 respiration. UCP3 content was 1.3-fold ( P &lt; 0.05) greater in IMF compared with SS mitochondria. State 4 respiration was 2.6-fold greater ( P &lt; 0.05) in the IMF subfraction than in SS mitochondria. GDP attenuated state 4 respiration by ∼40% ( P &lt; 0.05) in both subfractions. The UCP3 activator oleic acid (OA) significantly increased state 4 respiration in IMF mitochondria only. We used chronic electrical stimulation (3 h/day for 7 days) to investigate the relationship between changes in UCP3 protein expression and alterations in state 4 respiration during contractile activity-induced mitochondrial biogenesis. UCP3 content was increased by 1.9- and 2.3-fold in IMF and SS mitochondria, respectively, which exceeded the concurrent 40% ( P &lt; 0.05) increase in cytochrome- c oxidase activity. Chronic contractile activity increased state 4 respiration by 1.4-fold ( P &lt; 0.05) in IMF mitochondria, but no effect was observed in the SS subfraction. The uncoupling function of UCP3 accounted for 50–57% of the OA-induced increase in state 4 respiration in IMF mitochondria, which was independent of the induced twofold difference in UCP3 content due to chronic contractile activity. Thus modifications in UCP3 function are more important than changes in UCP3 expression in modifying state 4 respiration. This effect is evident in IMF but not SS mitochondria. We conclude that UCP3 at physiological concentrations accounts for a significant portion of state 4 respiration in both IMF and SS mitochondria, with the contribution being greater in the IMF subfraction. In addition, the contradiction between human and rat training studies with respect to UCP3 protein expression may partly be explained by the greater than twofold difference in mitochondrial UCP3 content between rat and human skeletal muscle.

In Brief Apoptosis is an essential process that plays a critical role in both tissue development and maintenance. Apoptosis has been shown to be involved in skeletal muscle atrophy resulting from chronic muscular disuse, sarcopenia, and mitochondrial myopathies. Exercise may attenuate some of the proapoptotic adaptations that occur during these conditions. This review will focus on the factors influencing mitochondrially mediated apoptosis in skeletal muscle. This paper reviews the evidence that mitochondrially-mediated apoptosis in skeletal muscle contributes to muscle atrophy/dysfunction, and that exercise may represent a therapeutic intervention to reduce muscle apoptotic susceptibility.

Near-infrared (NIR) light is a complementary therapy used to treat musculoskeletal injuries but the underlying mechanisms are unclear. Acute NIR light treatment (~ 800–950 nm; 22.8 J/cm2) induced a dose-dependent increase in mitochondrial signaling (AMPK, p38 MAPK) in differentiated muscle cells. Repeated NIR light exposure (4 days) appeared to elevate oxidative stress and increase the upstream mitochondrial regulatory proteins AMPK (3.1-fold), p38 (2.8-fold), PGC-1α (19.7%), Sirt1 (26.8%), and reduced RIP140 (23.2%), but downstream mitochondrial regulation/content (Tfam, NRF-1, Sirt3, cytochrome c, ETC subunits) was unaltered. Our data indicates that NIR light alters mitochondrial biogenesis signaling and may represent a mechanistic link to the clinical benefits.

Mitochondrial biogenesis occurs when the tissue energy demand is chronically increased to stress the ATP producing capacity of the preexisting mitochondria. In muscle, endurance training is a metabolic stress that is capable of inducing mitochondrial biogenesis, the consequence of which is improved performance during exercise. Expansion of the mitochondrial volume requires the coordinated response of the nuclear and mitochondrial genomes. During acute exercise, the initial signaling events are the perturbations in ATP turnover and calcium (Ca2+) concentrations caused by the contractile process. These alterations activate signal transduction pathways which target transcription factors involved in gene expression. Nuclear gene products are then posttranslationally imported into mitochondria. One of these, Tfam, is important for the regulation of mitochondrial DNA (mtDNA) gene expression. In muscle, a broad range of mitochondrial-specific diseases due to mutations in nuclear DNA or mtDNA exist, termed mitochondrial myopathies. These mutations result in dysfunctional mitochondrial assembly which ultimately leads to reduced ATP production. Mitochondrial myopathy patients exhibit a variety of compensatory responses which attempt to reconcile this energy deficiency, but the extent and the type of compensatory adaptations are disease-specific. Understanding the role of exercise in mediating these compensatory responses leading to mitochondrial biogenesis could help us in prescribing exercise designed to improve mitochondrial function in patients with mitochondrial myopathies. In addition, numerous other diseases (e.g., neurological disorders, cancer, diabetes, and cardiomyopathies), as well as the aging process, have etiologies or consequences attributed, in part, to mitochondrial dysfunction. Thus, insight gained by investigating the steps involved in exercise-induced mitochondrial biogenesis may help us to understand the underlying basis of these other disease states.

Unilateral, chronic low-frequency electrical stimulation (CLFS) is an experimental model that evokes numerous biochemical and physiological adaptations in skeletal muscle. These occur within a short time frame and are restricted to the stimulated muscle. The humoral effects of whole body exercise are eliminated and the nonstimulated contralaterai limb can often be used as a control muscle, if possible effects on the contralateral side are considered. CLFS induces a fast-to-slow transformation of muscle because of alterations in calcium dynamics and myofibrillar proteins, and a white-to-red transformation because of changes in mitochondrial enzymes, myoglobin, and the induction of angiogenesis. These adaptations occur in a coordinated time-dependent manner and result from altered gene expression, including transcriptional and posttranscriptional processes. CLFS techniques have also been applied to myocytes in cell culture, which provide a greater opportunity for the delivery of pharmacological agents or for the application of gene transfer methodologies. Clinical applications of the CLFS technique have been limited, but they have shown potential therapeutic value in patients in whom voluntary muscle contraction is not possible due to debilitating disease and/or injury. Thus the CLFS technique has great value for studying various aspects of muscle adaptation, and its wider scientific application to a variety of neuromuscular-based disorders in humans appears to be warranted. Key words: skeletal muscle, muscle plasticity, endurance training, mitochondrial biogenesis, fiber types

Evidence exists that mitochondrial content and/or function is reduced in muscle of aging individuals. The purposes of this study were to investigate the contribution of outer membrane protein import and assembly processes to this decline and to determine whether the assembly process could adapt to chronic contractile activity (CCA). Tom40 assembly into the translocases of the outer membrane (TOM complex) was measured in subsarcolemmal mitochondria obtained from young (6 mo old) and aged (36 mo old) Fischer 344 × Brown Norway animals. While the initial import of Tom40 did not differ between young and aged animals, its subsequent assembly into the final ∼380 kDa complex was 2.2-fold higher ( P &lt; 0.05) in mitochondria from aged compared with young animals. This was associated with a higher abundance of Tom22, a protein vital for the assembly process. CCA induced a greater initial import and subsequent assembly of Tom40 in mitochondria from young animals, resulting in a CCA-induced 75% increase ( P &lt; 0.05) in Tom40 within mitochondria. This effect of CCA was attenuated in mitochondria from old animals. These data suggest that the import and assembly of proteins into the outer membrane do not contribute to reduced mitochondrial content or function in aged animals. Indeed, the greater assembly rate in mitochondria from aged animals may be a compensatory mechanism attempting to offset any decrements in mitochondrial content or function within aged muscle. Our data also indicate the potential of CCA to contribute to increased mitochondrial biogenesis in muscle through changes in the outer membrane import and assembly pathway.

Mitochondrial dysfunction in various tissues has been associated with numerous conditions including aging. In testes, aging induces atrophy and a decline in male reproductive function but the involvement of mitochondria is not clear. The purpose of this study was to examine whether the mitochondrial profile differed with (1) aging, and (2) 10-weeks of treadmill exercise training, in the testes of young (6 month) and old (24 month) Fischer-344 (F344) animals. Old animals exhibited significant atrophy (30 % decline; P < 0.05) in testes compared to young animals. However, relative mitochondrial content was not reduced with age and this was consistent with the lack of change in the mitochondrial biogenesis regulator protein, peroxisome proliferator-activated receptor gamma coactivator 1-alpha and its downstream targets nuclear respiratory factor-1 and mitochondrial transcription factor A. No effect was observed in the pro- or anti-apoptotic proteins, Bax and Bcl-2, respectively, but age increased apoptosis inducing factor levels. Endurance training induced beneficial mitochondrial adaptations that were more prominent in old animals including greater increases in relative mtDNA content, biogenesis/remodeling (mitofusin 2), antioxidant capacity (mitochondrial superoxide dismutase) and lower levels of phosphorylated histone H2AX, an early marker of DNA damage (P < 0.05). Importantly, these exercise-induced changes were associated with an attenuation of testes atrophy in older sedentary animals (P < 0.05). Our results indicate that aging-induced atrophy in testes may not be associated with changes in relative mitochondrial content and key regulatory proteins and that exercise started in late-life elicits beneficial changes in mitochondria that may protect against age-induced testicular atrophy.

Mitochondria are dynamic and complex cellular organelles that are involved in a wide range of cellular events and are essential for tissue adaptation, survival, death, and renewal. In addition to their important role in energy metabolism making them popularly known as the cellular powerhouse in textbook definitions, mitochondria are malleable structures that are also intimately involved in controlling cellular redox status, cellular signaling, calcium homeostasis, and cell death and autophagy processes. Thus, mitochondria have emerged from simply being the powerhouse of the cell to being at the forefront of numerous research avenues. In fact, mitochondrial perturbations evoked by physiological and pathological stimuli have been shown to contribute towards the pathogenesis of many diseases and mitochondrial research now constitutes a very significant and ever-expanding research area. It is now understood that mitochondria and their associated pathways may represent areas for the development of preventive and therapeutic strategies to potentially mitigate diseases/disorders such as diabetes, obesity, neurodegeneration, and sarcopenia. In the present issue of Oxidative Medicine and Cellular Longevity devoted to mitochondria in health and disease, a variety of original research articles were published covering distinct aspects of cellular physiology and adaptation involving mitochondria. These include the role of mitochondrial and peroxisome group VIB phospholipase A2 (iPLA2g) in β-cell proliferation and redox control (Bao et al., 2013), the study of mitochondrial metabolic and structural phenotypes in liver and skeletal muscle from obese animals (Cao et al., 2013), the modulator effects of hydrogen disulfide in neuronal cells and mitochondria (Guo et al., 2013), the mechanisms by which astragaloside IV protects against oxidative stress-induced increased permeability transition pore opening in cardiac cell line (He et al., 2012), the effects of alpha-lipoic acid on mitochondrial superoxide and glucocorticoid-induced hypertension (Ong et al., 2013), and the development of in vitro approaches to study population mitochondrial genomic variations (Lin et al., 2013). In the present issue, the potential use of two-photon microscopy probes for the study of mitochondrial redox environment (Kim and Cho, 2013) and the potential of translocator protein 18 as therapeutic target and also a diagnostic tool for cardiovascular diseases (Qi et al., 2013) are also reviewed and discussed. It is our belief that the articles in this special mitochondrial issue could provide an important contribution to improve the use of mitochondrial-related models for health and disease research, as well as identifying mitochondrial pathways and associated mechanisms as important subcellular targets in the prevention and treatment of many pathological conditions. Jose  Magalhaes Paola Venditti Peter J. Adhihetty Jon Jay Ramsey Antonio Ascensao

Curcumin, a polyphenol found in the spice turmeric, is shown to have antioxidant and anti‐inflammatory properties in multiple tissues, but whether it alters mitochondrial biogenesis/apoptotic pathways is unknown. The aging process is associated with impaired mitochondrial function and elevated mitochondrial apoptotic susceptibility. Potential pharmacological and/or neutraceutical therapeutic interventions capable of improving mitochondrial function, like curcumin, have been postulated to delay this process. Thus, we investigated whether short‐term (21d) dietary curcumin supplementation (5% diet) altered mitochondrial biogenesis in muscle and brown adipose tissue (BAT) of aged mice (24 month; C57BL/6) compared to control diet mice (n=4‐6/group). While curcumin supplementation increased the mitochondrial content markers cytochrome c (14%) and COX Vb (26%) and enhanced the mitochondrial regulators Tfam (25%) and NRF‐1 (14%) in BAT, it suppressed and/or caused no change in these mitochondrial indices in muscle. In contrast, curcumin treatment evoked significant decreases (P&lt;0.05) in the pro‐apoptotic BAX protein in both BAT (20%) and muscle (55%). Our data indicate short‐term curcumin treatment in aged mice causes tissue‐specific mitochondrial biogenesis adaptations in BAT and muscle while potentially suppressing mitochondrial apoptotic susceptibility in both tissues.

Skeletal muscle aging is partly attributed to the oxidation of macromolecules caused by increased reactive oxygen species. DNA base excision repair (BER) pathways offer protection against oxidative damage through the removal and repair of DNA lesions. While the activity of these BER enzymes is proposed to be reduced with aging, the molecular details remain elusive. The purpose of this study was to investigate oxidative stress/damage, BER enzyme activities, and antioxidant capacity in skeletal muscle obtained from two sedentary but functionally‐distinct groups of elderly (&gt; 65 yr) individuals classified as high‐ and low‐functioning (HF and LF). Results showed increased oxidative damage in both HF and LF individuals compared to young, as indicated by higher levels of 3‐Nitrotyrosine (3‐NT) and 4‐Hydroxynonenal (4‐HNE) (P&lt;0.05). This increase corresponded with lower levels of 8‐oxoguanine DNA glycosylase (OGG1; P&lt;0.05) protein, the main mitochondrial oxidative DNA repair enzyme, in elderly subjects compared to young. No changes in protein content were observed for apurinic/apyrimidinic (AP) endonuclease, APE1, corresponding to similar rates of AP sites incision when protein extracts from young and elderly individuals were utilized to measure APE1 enzymatic activity. These data suggest that BER capacity may be impaired in aging muscle leading to greater susceptibility to oxidative damage. Grant Funding Source : Supported by Claude D. Pepper OAIC

Cancer afflicts numerous primary tissues and is often associated with secondary skeletal muscle atrophy and wasting, known as cachexia. Mitochondrial abnormalities contribute to muscle atrophy/dysfunction in various diseases, and experimental perturbations (i.e. exercise) augmenting mitochondrial content tend to improve muscle but whether this occurs in prostate cancer is unknown. We used an orthotopic prostate cancer model, injecting AT‐1 adenocarcinoma cells (tumor‐bearing; TB) or saline (non‐tumor bearing; NTB) into the prostate of young (5 mo) rats, and assigned a sedentary (Sed) or exercise (7 weeks; Ex) protocol (n=5‐10/group). Exercise did not alter tumor size (4.12 g TB‐Sed; 4.3 g TB‐Ex), nor was there tumor‐associated atrophy in muscles (SOL, EDL, Pl, TA). As expected, training induced mitochondrial biogenesis by increasing (P&lt;0.05) COX activity, cytochrome c, and electron transport complexes (30%, 3.3‐fold, 1.8‐fold, respectively) in NTB animals while the TB group had a significantly blunted response. Interestingly, tumor presence per se did not alter mitochondrial content/regulation but tended to reduce (~25%) mitochondrial respiration and elevate (~2.5‐fold) mitochondrial free radical production, and exercise did not evoke improvements. Our data suggests prostate cancer impairs exercise‐induced mitochondrial biogenesis and impairs mitochondrial function in skeletal muscle.

Although fatigue at old age is often attributed to specific conditions, in many cases the association between indicators of disease severity and the degree of fatigue is equivocal, suggesting that other factors influence this condition. Research has demonstrated that circadian rhythms and molecular clocks exist in skeletal muscle. Furthermore, molecular clock proteins can greatly impact maximal muscle contraction, myofiber architecture and mitochondrial volume which may in part contribute to chronic idiopathic fatigue. PURPOSE: To determine whether circadian rhythm protein expression is different in skeletal muscle between community-dwelling fatigued and non-fatigued older adults. METHODS: Sedentary, older adults (N = 47, age 72.2±5.3 yrs) were categorized into fatigued and non-fatigued groups based on the 13-item Functional Assessment of Chronic Illness Therapy (FACIT) Fatigue questionnaire. Fatigued cases were defined as having a FACIT score that was one standard deviation below the mean for non-anemic adults > 65 years (n = 20). To identify individuals with idiopathic fatigue, participants were excluded for depression, untreated sleep apnea, heart failure, pulmonary disorders, thyroid conditions, uncontrolled diabetes, morbid obesity, and substance abuse, among others. Quadriceps muscle biopsies were analyzed for total protein expression of circadian rhythm markers Brain muscle arnt-like1 (Bmal1), Period1 (Per1) and Rev-Erbα. RESULTS: The circadian rhythm proteins Per1 and Rev-Erbα trended to be lower in expression levels in fatigued compared to non-fatigued skeletal muscle (-11.7% p=0.0822, and -20.8% p=0.0989 respectively). There was no significant effect of Bmal1 between gropus (-0.8% p=0.9300). CONCLUSIONS: Our data suggests that older adults who complain of fatigue symptoms not directly linked to a disease condition may have lower molecular clock protein expression, suggesting a potential mechanism to the pathology of idiopathic chronic fatigue. Interventions such as scheduled exercise may be a viable therapeutic intervention to improve molecular clock regulation and treat older adults who report severe fatigue. Supported by University of Florida, Claude D. Pepper Center from the National Institute on Aging (Award Number P30AG028740).

Mitochondrial dysfunction in aged skeletal muscle is involved in sarcopenia and may contribute to reduced physical function in the elderly. We examined whether mitochondrial regulation differed in muscle from elderly subjects classified as high- or low-functioning (HF, LF; 70–99 yrs) according to scores on the Short Physical Performance Battery test (HF≥l11; LF≤7), when compared to young subjects (YS; 20–35 yrs). Mitochondrial respiration rates in permeabilized myofibers and cytochrome c oxidase enzyme activity (mitochondrial content marker) tended to be lower in HF and were significantly lower (p<0.05) in LF subjects when compared to YS. PGC-1α, a mitochondrial regulator, was 47% and 64% lower (p<0.05) in HF and LF subjects, respectively, compared to YS. Sirt3, a mitochondrial deacetylase, was similarly reduced (50%; p<0.05) in HF and LF subjects. The mitochondrial fusion protein, Opa1, was markedly suppressed in HF and LF (p<0.05) but no changes were evident in Mfn2, Drp1 or Fis1. Additionally, LF but not HF subjects, displayed significant abnormalities in key mitochondrial import proteins (cytosolic and mitochondrial Hsp70, and Tom22; p<0.05). Our findings indicate that mitochondrial regulatory pathways are impaired in skeletal muscle from elderly subjects (HF and LF) when compared to YS, but that this is more evident in LF subjects, and may play a role in the differential physical abilities of the elderly.

It is well established that impairments in mitochondrial function/regulation contribute to tissue decline with aging. Caloric restriction (CR) and resveratrol (RSV) treatment in rodents induces beneficial mitochondrial adaptations in tissues such as heart and skeletal muscle but whether similar benefits occur in fat is unknown. Thus, we investigated whether RSV (50 mg/kg/day; 6 weeks) and/or CR (20% reduced AL; 6 weeks) could alter mitochondrial regulation/biogenesis in aged rodent adipose tissues (visceral; VIS, epididymal; EPI, brown adipose tissue; BAT). Aged F344xBN rats (26 mo) were divided into 4 groups (n=4): ad libitum (AL), CR, RSV, RSV+CR and mitochondrial content (cyto c, COX activity, and COX I) and mitochondrial signaling/regulation (AMPK, PGC‐1α) were assessed. Expectedly, mitochondrial content (cyto c, COX activity) and PGC‐1α were significantly elevated (~2–3‐fold) in the BAT compared to EPI and VIS fat. CR and RSV tended to increase COX activity (~20–50%) in all adipose depots but showed inconsistencies with other mitochondrial content/signaling markers. Interestingly, combined CR and RSV treatment had no effect and/or suppressed COX activity in all adipose tissues. Our data indicates that short‐term CR and RSV treatment when applied independently appears to enhance mitochondrial content in adipose tissue and may contribute to improvements in health associated with these paradigms.

Laser therapy (phototherapy) is a clinical treatment used to promote tissue healing and improve muscle function in patients with musculoskeletal injury. However, the underlying cellular and molecular mechanisms responsible for the positive clinical outcomes are poorly understood. One proposed mechanism suggests specific wavelengths (710–1100‐nm) of light are preferentially absorbed by, and activate mitochondria in skeletal muscle to alter mitochondrial bioenergetics/biogenesis and potentially improve muscle regeneration post‐injury. To elucidate the underlying mechanisms, we applied varying treatment doses of phototherapy (0.42, 2.57, 8.14, 14.85, 22.80 and 29.60 J/cm2; 810–935nm; LiteCure LCT‐1000) to cultured muscle cells (C2C12 myotubes) and examined the activation of key mitochondrial biogenesis signalling proteins (AMPK, p38). Laser treatment caused phosphorylated‐AMPK (P‐AMPK) to increase in a traditional dose‐dependent manner, while phosphorylated‐p38 (P‐p38) exhibited a biphasic dose‐response. Thus, our preliminary findings indicate laser therapy evokes differential alterations in mitochondrial‐associated signaling molecules in C2C12 cells. Whether these acute phototherapy‐induced signaling changes will lead to an increase in functional mitochondria with longer‐term phototherapy in C2C12 cells has yet to be determined and is a focus of our future work.

Tumors are associated with dysregulated mitochondrial-mediated apoptosis and impaired mitochondrial metabolism, and the incidence of tumors increases with age. While exercise has been shown to reduce mitochondrial-mediated apoptosis and improve mitochondrial bioenergetics in various tissues, its effect on aged tumorigenic testes is unknown. Thus, we examined whether mitochondrial apoptotic markers and mitochondrial content differed in, 1) young control testes (YC; 6m; Fischer 344; n=4 group), 2) old control non-tumorigenic testes (OC; 22–24m; Fischer 344), 3) old tumorigenic testes (OT), 4) old tumorigenic exercise-trained (OT-E; 10 weeks treadmill training) testes. OT and OT-E testes mass were similar but significantly larger (1.5-fold) than YC and OC. Mitochondrial content was modestly reduced in OC compared to YC but was elevated similarly in OT and OT-E testes compared to YC. AIF, a pro-apoptotic factor, was not different in YC and OC testes but was significantly elevated by 60–70% in OT and OT-E testes. Bax:Bcl-2 ratio, an apoptotic index, was similar in YC and OC testes, while both OT and OT-E testes tended to be elevated compared to YC. Our findings suggest paradoxically that both mitochondrial content and mitochondrial apoptotic susceptibility were not altered by age alone, but enhanced by tumorigenesis and that exercise did not attenuate these mitochondrial abnormalities.

The mitochondrial theory of aging proposes that mitochondrial DNA mutations (mtDNA) accumulate with age and this leads to mitochondrial dysfunction and/or damage that contribute to the aging process. mtDNA mutator mice (referred to as PolG) contain a deficient version of the proofreading mtDNA polymerase gamma and consequently incur high mutation rates to mtDNA that lead to mitochondrial dysfunction. As a result of these spontaneous mutations, PolG mice have an accelerated aging phenotype, allowing their use as a model of aging. The present study investigated the effect of increased mtDNA mutations on regulators of skeletal muscle mitochondrial biogenesis and morphology in young (3–6 mo) and old (8–15 mo) PolG and wild‐type (WT) mice. In old animals, mtDNA content was significantly reduced in the PolG compared to WT. Moreover, there was an age‐dependent decrease in PGC‐1α levels exhibited in both PolG and WT animals. However, young mice with the PolG mutation contained a greater level of PGC‐1α when compared to young WT mice. Age‐dependent changes were also observed in morphology proteins, including higher levels of the fusion protein Mfn2 in both WT and PolG mice, and reduced levels of the fission protein Fis1, only in WT and not in PolG mice. These data suggest that alterations in the ratio of fusion to fission proteins may contribute to the aging phenotype that is exhibited in mtDNA mutator mice.

Chronic heart failure (CHF) is marked by myocardial dysfunction and is also associated with skeletal muscle metabolic abnormalities but the underlying cellular mechanisms are not well understood. Thus, we investigated whether CHF alters mitochondrial function/content and apoptotic susceptibility in skeletal muscle. Young Lewis rats (8 wks) were subjected to either myocardial infarction to induce CHF or, Sham (Sh) operation, and hindlimb muscles were removed 16‐weeks post‐surgery (n=6/group). Mitochondrial function tended to be impaired in CHF as indicated by a reduced respiratory control ratio (RCR‐State 3/4 respiration). CHF‐induced impairments in respiration coincided with significant reductions in cytochrome c and the mitochondrial anti‐oxidant, MnSOD, in isolated mitochondria. Additionally, oxidative damage, indicated by protein carbonylation was significantly elevated in CHF. CHF animals tended to exhibit greater mitochondrial apoptotic susceptibility (Bax:Bcl‐2) but this surprisingly did not alter the vulnerability to exogenous H2O2‐ induced pro‐apoptotic release from isolated mitochondria. Our preliminary results indicate CHF impairs mitochondrial function and/or content, increases mitochondrial apoptotic susceptibility and enhances ROS‐induced damage in muscle, and these deleterious mitochondrial‐associated adaptations likely contribute towards CHF pathogenesis.

Sarcopenia is an age‐related loss in muscle mass partially attributable to mitochondrial‐mediated apoptosis. Caloric restriction (CR) and resveratrol (RSV) treatment in rodents induces beneficial mitochondrial alterations which may serve to suppress sarcopenia. Doxorubicin (DOX) is a chemotherapeutic agent that induces cell death via mitochondria. We investigated whether RSV (50 mg/kg/day; 6 weeks) and/or CR (20% reduced AL; 6 weeks) could 1) induce mitochondrial biogenesis, 2) attenuate apoptotic susceptibility and, 3) reduce DOX‐induced toxicity, in aged rodent muscle. Aged F344xBN rats (26 mo) were split into 8 groups (n=4): ad libitum (AL), CR, RSV, RSV+CR and injected with DOX (20 mg/kg; IP) or saline prior (24h) to sacrifice. Mitochondrial content/regulation (cyto c, COX activity, PGC‐1á, SIRT3) and apoptotic susceptibility (Bax:Bcl‐2) were assessed in hindlimb muscle. Surprisingly, mitochondrial indices were unaffected by CR, RSV or CR+RSV, and DOX did not affect any group. CR+RSV reduced (50%) the Bax:Bcl2 ratio compared to AL while RSV and CR independently showed trends for reductions. DOX treatment enhanced the Bax:Bcl‐2 in AL while CR, RSV and CR+RSV tended to suppress this DOX‐induction. Our data indicates aged muscle, and DOX‐treatment, increases apoptotic susceptibility and CR+RSV treatment provides modest protection without altering mitochondrial content/regulation.

Tumors exhibit an altered mitochondrial metabolic profile and suppressed mitochondrial‐mediated apoptosis. Exercise improves mitochondrial function and reduces susceptibility to mitochondrial‐mediated apoptosis in a variety of tissues but its effect on aged tumorigenic testes is unexplored. Thus, we investigated whether key mitochondrial signaling markers, mitochondrial content, and/or apoptotic factors differed in, 1) young control testes (YC; 6m; Fischer 344; n=4 group), 2) young exercised‐trained testes (YE; 6m; 10 weeks treadmill training) 3) old control non‐tumorigenic testes (OC; 22–24m; Fischer 344), 4) old tumorigenic testes (OT), 5) old tumorigenic exercise‐trained (OT‐E) testes. OT and OT‐E testes mass were similar but significantly larger (1.5‐ fold) than YC, YE and OC. Mitochondrial content was significantly elevated by 70–80% in both OT and OT‐E testes when compared to YC, YE, and OC, and similar trends were evident in upstream mitochondrial biogenesis signaling markers LKB1, AMPK, p38 and PGC‐1á. Bax:Bcl‐2 ratio, an apoptotic index, was significantly reduced by 70–80% in both OT and OT‐E when compared to YC, YE, and OC. Our findings indicate old tumorigenic testes exhibit enhanced mitochondrial biogenesis signaling and mitochondrial content but have reduced mitochondrial apoptotic susceptibility when compared to young healthy testes and this was unaffected by exercise.

Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the huntingtin gene which has been shown to suppress mitochondrial function in various tissues. Mutant huntingtin protein alters cellular bioenergetics by interfering with the important metabolic regulator, PGC-1α, and this likely contributes to the pathogenesis of HD. PURPOSE: To examine whether metformin, a known inducer of mitochondrial biogenesis, could improve mitochondrial function in muscle and brown adipose tissue (BAT) of HD mice. METHODS: BACHD mice with the mutant huntingtin gene, and wild-type (WT) mice were given metformin (2mg/ml; n=20/group), or water, for 12 months and immunohistochemistry, histochemistry and biochemical measures were performed on gastrocnemius muscle (GM) and BAT. RESULTS: GM from HD animals tended to have less (20%) type I fibers and greater (20%) type IIA fibers compared to WT animals. Metformin treatment in HD animals increased type I fibers to levels comparable to that of WT animals, but did not alter type IIA fiber distribution. Type I and type IIA fiber size did not differ in GM from HD compared to WT, but type IIB fibers tended to be smaller in HD animals. Intramyocellular lipid (IMCL) content (assessed with Oil Red O staining) was greater in GM from HD compared to WT muscle, and metformin treatment tended to reduce lipid content in HD muscle. In BAT, lipid droplets and lipid vacuolization (Oil red O and H+E staining, respectively) were significantly greater in HD compared to WT animals. Metformin treatment restored lipid content and vacuolization in the BAT of HD animals to a level similar to non-treated WT animals. CONCLUSIONS: Our findings show that mutant huntingtin leads to a fiber-type shift in skeletal muscle that is associated with lipid accumulation in muscle and BAT, and further that metformin can partially revert these abnormalities to that of WT animals. The beneficial effects of metformin in these tissues are likely related to the upregulation of PGC-1α which is associated with improved mitochondrial function, altered fiber-type distribution and substrate oxidation, and metabolic reprogramming.

Huntington's disease (HD) is a progressive neurological disorder which affects both brain and skeletal muscle and leads to a decline in cognitive function and muscle coordination. HD is caused by an autosomal dominant mutation within the Huntingtin gene (mHtt) which has been shown to lead to mitochondrial dysfunction and increased susceptibility to cell death through both autophagy and mitochondrially-mediated apoptosis. However, the underlying mechanisms responsible for the increased cell death susceptibility in HD have yet to be fully elucidated. PURPOSE: To determine whether metformin treatment, a known inducer of mitochondrial content, can reduce signaling pathways leading to cell death in skeletal muscle of HD mice. METHODS: BACHD transgenic mice (TG), possessing the huntingtin gene mutation, and wild-type animals (WT) were provided metformin (treated) or water (non-treated) for 12 months (n=20/group). Gastrocnemius (GM) was excised and protein analysis performed. RESULTS: The pro-apoptoic protein, Bax, was 34% higher in GM of TG animals, compared to WT animals, whereas the expression of the anti-apoptotic protein Bcl-2 was unchanged. Cytochrome c protein was 20% lower in the GM of TG animals, compared to WT animals. Levels of the pro-apoptotic protein, AIF, remained unchanged in the GM of non-treated WT and TG animals. Additionally, expression of the autophagic protein, Beclin 1 did not differ in the GM of non-treated WT and TG animals. Chronic metformin treatment tended to increase cytochrome c and reduce pro-apoptotic Bax levels in the GM of TG animals to levels comparable to non-treated WT animals. CONCLUSION: These findings suggest that HD is associated with an increased susceptibility of skeletal muscle to cell death and indicate the potential of chronic metformin treatment to attenuate cell death signaling pathways and reduce the severity of HD in muscle.

Huntington's disease (HD) is an autosomal dominant disorder caused by a mutation in the huntingtin gene. HD progression is associated with mitochondrial dysfunction in neural and skeletal muscle tissue. Mutant huntingtin suppresses PGC‐1α, a transcriptional coactivator that regulates genes influencing numerous metabolic programs. The purpose was to determine whether metformin, an AMPK agonist and mitochondrial biogenesis inducer, would delay HD‐associated symptoms by improving mitochondrial content and function. BACHD mice, exhibiting a mutant form of human huntingtin, and wild‐type animals, were given metformin or water (non‐treated) for 12 months and a series of measurements were performed. Our results indicate metformin treatment provided a modest, yet significant, improvement in the behavioural and locomotory deficits in BACHD mice. Metformin treatment in BACHD mice reversed the suppressed expression of mitochondrial‐related genes (i.e. PGC‐1α, NRF‐2, cyt‐c) in soleus, but not gastrocnemius muscle, of non‐treated BACHD mice. However, metformin was incapable of reducing the striatal atrophy, or restore impaired mitochondrial respiratory function, in BACHD mice. Our findings suggest that chronic metformin treatment delays the progression of HD‐associated symptoms in HACHD mice and that the beneficial effects of metformin are fiber and tissue‐specific. This research was supported by NIH.

Mitochondrial protein import adapts to perturbations in cellular energy status by altering the rate at which nuclear‐encoded proteins are translocated into mitochondria. The purposes of this study were to determine whether the import and assembly of precursor proteins are similar in muscle from young and aged F344XBN animals, and to assess the effect of chronic contractile activity (CCA) on the import pathway in subsarcolemmal (SS) mitochondria. Import of Tom40 into the outer membrane increased (p&lt;0.05) over time in both young and aged animals, but was not different between the two groups. However, assembly of the multisubunit TOM complex containing Tom40, as assessed by Blue Native‐PAGE, was 2.2‐fold greater (p&lt;0.05) in aged, when compared to young animals. Following 7 days of CCA (3hr/day), Tom40 assembly into the TOM complex was accelerated by 75%, compared to non‐stimulated muscle in young animals. This increase due to CCA was only 65% in aged animals (p&lt;0.05). CCA also resulted in an 85% increase in the import of the matrix protein OCT in SS mitochondria from young animals, while only a 28% increase in OCT import was observed in aged animals (p&lt;0.05). Our findings suggest that aged muscle has a greater capacity to assemble Tom40 into a functional TOM complex, when compared to young animals. CCA can further accelerate the rate of import and assembly in both groups, however this effect is attenuated with age.

The intent of the present study was to investigate the adaptive potential of skeletal muscle, as well as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in young (6 mo) and senescent (36 mo) animals in response to a standard regimen of chronic contractile activity (CCA). The TA and EDL muscles of 6 and 36 mo F344XBN rats were chronically stimulated (10 Hz, 3 h/day, 7 days) to induce CCA. The contralateral limb served as a non‐stimulated control. Subsequent to CCA, acute stimulation (1 Hz, 5 min) of the TA muscle in situ revealed greater fatigue resistance in both 6 and 36 mo animals. However, the improvement in endurance was 50% greater in the young, compared to the old animals. The CCA‐mediated increase in Hsp70 was also greater in the young animals. The expression of the longevity protein SIRT1 was increased 40–50% with CCA in both age groups. CCA also induced similar 30% elevations in COX enzyme activity in young and old animals. This may be due to the CCA‐elicited increases in mitochondrial biogenesis regulatory proteins PGC‐1α and Tfam that were also comparable (40–70%) between groups. CCA reduced mitochondrial ROS production by 40–80% in both age groups, whereas mitochondrial VO 2 in the presence of succinate was increased with CCA only in the 6 mo group. Thus, muscle and mitochondria from senescent animals readily adapt to CCA, however the magnitude of the changes are less compared to young animals.

The insertion of nuclear-derived proteins into mitochondria is facilitated by the translocases of the outer membrane (TOM) complex. Tom40 is the main component of this complex. Its import and assembly has not been characterized in skeletal muscle. Thus, our purpose was to compare the import and assembly of Tom40 in muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Tom40 protein levels were not different between SS and IMF mitochondria. However, Tom40 import into the outer membrane was higher (p<0.05) in SS mitochondria at all time points (5, 10, 20 min) when compared to IMF mitochondria. Tom40 assembly was monitored using blue native electrophoresis. Tom40 first assembles into a ~250kDa complex (Intermediate I), followed by a ~120 kDa complex (Intermediate II), before its final insertion into the ~380 kDa TOM complex. In IMF mitochondria, 70–80% of Tom40 was incorporated into Intermediate I by 5 min, whereas only 40–50% was found in Intermediate I in SS mitochondria. However, the sequential incorporation of Tom40 into Intermediate II was less rapid in IMF mitochondria, and only 45% of Tom40 was incorporated into the final TOM complex by 60 min. In contrast, SS mitochondria incorporated 65–70% of Tom40 by 60 min. Thus, despite similar levels of steady state Tom40 protein content, the import and assembly of Tom40 into the TOM complex differs among muscle mitochondrial subtypes.

PPAR-gamma coactivator-1α (PGC-1α) is an important regulator of mitochondrial biogenesis. However, its effect on mitochondrial function, key regulatory proteins involved in organelle synthesis and apoptosis are not resolved in skeletal muscle, particularly in intermyofibrillar (IMF) and subsarcolemmal (SS) subfractions. Thus, we examined respiration, ROS production and protein expression in mitochondria from PGC-1α null animals. SS and IMF mitochondrial yields in muscle from PGC-1α null animals was 36% and 27% (p<0.05) lower, than in wild-type animals. Parallel decrements of 22% and 36% (p<0.05) existed in the mitochondrial markers, COX activity and cytochrome c. Despite these differences, the mtDNA transcription factor Tfam, and mitochondrial fission protein Fis1, were similar in null and wild-type animals. However, the import protein mtHSP70 was 30% lower in muscle from PGC-1α null animals. The Bax:Bcl-2 ratio, an apoptotic indicator, was unaltered but both proteins were 35% greater in muscle from PGC-1α null animals. SS mitochondria from PGC-1α null animals displayed 35% and 20% lower respiration rates and ROS production, respectively, while IMF mitochondria exhibited no differences compared to wild-type. Thus, the absence of PGC-1α does not completely reduce mitochondrial content and must evoke compensatory mechanisms to prevent the entire loss of mitochondrial function in muscle.

Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria are two morphologically distinct subfractions located in different regions of the myofiber, possessing disparate biochemical properties which may contribute to their adaptive potentials. Although mice lacking the skeletal muscle cytosolic fatty acid (FA) transporter heart-type fatty acid-binding protein (H-FABP) exhibit abnormal FA metabolism, it is unknown how SS and IMF mitochondria adapt functionally to this deficit. PURPOSE: To investigate the composition and function of SS and IMF mitochondria isolated from WT compared to H-FABP KO animals. METHODS: SS and IMF mitochondria were isolated from muscle of WT and H-FABP KO animals and cardiolipin content was assessed using flow cytometry. State 3 (active) and state 4 (passive) mitochondrial oxygen consumption (VO2) was measured. Fluorometric and flow cytometric techniques were used to measure mitochondrial reactive oxygen species (ROS) generation and membrane potential. Mitochondrial permeability transition pore (mtPTP) opening kinetics, including the maximal rate of pore opening (Vmax) and the time required to achieve Vmax, were also assessed. RESULTS: Cardiolipin content was similar between SS and IMF mitochondria isolated from WT and H-FABP KO mice. IMF mitochondrial VO2 was 1.5-and 2-fold greater compared to SS during state 4 and state 3 respiration, respectively. IMF mitochondrial ROS production, along with membrane potential, were 35-70% lower compared to the levels in SS mitochondria in both WT and H-FABP KO mice. ROS generation during SS state 3 respiration was 30% higher in H-FABP KO compared to WT animals. mtPTP Vmax was 2.7-fold higher in IMF mitochondria compared to the SS subfraction isolated from WT and H-FABP KO mice. Time to Vmax in IMF mitochondria harvested from H-FABP KO mice was 40% greater compared to WT littermates. In addition, within the H-FABP deficient genotype, time to Vmax was 66% higher for the IMF subfraction compared to SS mitochondria. CONCLUSION: These findings reveal unique adaptive strategies between SS and IMF mitochondria due to an alteration in FA handling. The data demonstrate an association between high rates of mitochondrial VO2, low membrane potential, and reduced levels of ROS production in IMF mitochondria, while the inverse appears to hold for the SS subfraction. Further, SS mitochondria upregulate ROS production as a potential signal to increase the transcription of other genes, possibly involved in FA metabolism. The IMF subfraction demonstrates a reduced susceptibility to mitochondrially-mediated apoptosis, a protective adaptation to possible lipotoxic conditions within the myocyte.

1825 Chronic muscle disuse, such as that produced by denervation, reduces mitochondrial content and produces muscle atrophy. These changes in muscle phenotype could be provoked by alterations in important regulators of mitochondrial biogenesis, such as the nuclear coactivator PGC-1α, and by increases in muscle fiber cell death via apoptosis. However, the role of PGC-1α following denervation has not been defined, and limited research has assessed apoptotic mechanisms as contributing factors to denervation-induced muscle atrophy. PURPOSE: To assess time-dependent alterations in key proteins involved in mitochondrial biogenesis (PGC-1α, cytochrome c, Tfam, cytochrome oxidase (COX) activity) and apoptosis (HSP70, Bcl-2, BAX, AIF) following chronic muscle denervation. METHODS: Male Sprague-Dawley rats (n = 20) underwent unilateral denervation (DEN) of the sciatic nerve for 5, 21, or 42 days (n = 3–4 animals/day). Denervated and contralateral (control) red gastrocnemius muscles were used for biochemical analyses. RESULTS: Denervation decreased PGC-1α expression by 45% following 5 days of DEN, which was further decreased by 51% and 65% of control at 21 and 42 days, respectively. COX activity declined with time and matched PGC-1α expression, with decreases of 35%, 55%, and 70% at 5, 21, and 42 days of DEN. These changes occurred coincident with 45% decreases in both Tfam and cytochrome c expression at 42 days of DEN. In contrast, proapoptotic BAX expression was elevated by 88% and 115% after 21 and 42 days of DEN, while coincident decreases of 79% and 89% occurred for the expression of the antiapoptotic protein Bcl-2. These changes dramatically increased the BAX:Bcl-2 ratio in denervated muscle. An increase in this ratio is indicative of a greater susceptibility to apoptosis. Additionally, 42 days of DEN resulted in increased (2.0-fold) expression of pro-apoptotic AIF, as well as increased levels (2.9-fold) of anti-apoptotic HSP70. CONCLUSIONS: These results indicate PGC-1α could be an important mediator of the decrements in mitochondrial content following periods of chronic muscle disuse. In addition, the profound shift in BAX:Bcl-2 ratio suggests that denervated muscle has a greater susceptibility to apoptosis and that this likely contributes to denervationinduced atrophy of skeletal muscle. Support: NSERC, Canada.

Caloric restriction promotes longevity in mammals. This effect is mediated by the NAD+-dependent histone deacetylase Sirt1, which is sensitive to metabolic alterations and interacts with the regulatory protein of mitochondrial biogenesis PGC-1α. Conditions of muscle use and disuse such as endurance training and denervation are known to produce alterations in mitochondrial biogenesis as well as energy metabolism. To determine whether Sirt1 is regulated under those conditions, Sirt1 deacetylase activity was evaluated in muscles of rats subjected to chronic electrical stimulation for 7 days (10 Hz; 3 hrs/day), or denervation for 14 days. Mitochondrial biogenesis was determined by measuring the protein expression of PGC-1α and cytochrome c, and COX enzyme activity. Sirt1 activity was measured using a fluorimetric deacetylation assay and its reliability was assessed as a function of sample protein concentration, incubation time, and inhibitor (nicotinamide) concentration. In chronically-stimulated extensor digitorum longus (EDL) muscle, PGC-1α, cytochrome c and COX enzyme activity were increased by 30–50% compared to non-stimulated muscle. In denervated EDL, those markers of mitochondrial biogenesis were decreased to the same extent (30–50%) compared to the control muscle. In contrast, Sirt1 activity was increased by 20% and 76% in chronically-stimulated and denervated EDL, respectively. Our data suggest that 1) SIRT1 and PGC-1α expression are independently regulated, and 2) mitochondrial biogenesis and SIRT1 function are involved in separate metabolic pathways in conditions of muscle use and disuse. Support from CIHR.

Chronic skeletal muscle disuse results in a loss of muscle mass which is partly attributable to apoptosis. This study was designed to evaluate the relationship between changes in mitochondrial function and apoptosis in chronic denervation (7, 14, 21 days) of rat tibialis anterior (TA) muscle. Denervation decreased state 3 (ADP-stimulated) respiration in subsarcolemmal (SS) mitochondria by 37 – 45 % between 7 and 21 days, whereas no changes were evident in intermyofibrillar (IMF) mitochondria. Conversely, state 4 respiration increased by 68% in IMF mitochondria with 14 days of denervation, while it remained unchanged in the SS mitochondria. This was accompanied by a 2.3-fold increase in UCP3 expression at 14 days of denervation. The expression of the anti-oxidant enzyme MnSOD declined 60–70% in SS and IMF mitochondria, whereas the production of reactive oxygen species (ROS) was elevated 5–10-fold in the SS mitochondrial subfraction. In addition, there was a 76% increase in the expression of the pro-apoptotic protein p53 at 14 days of denervation. This was matched by an increase in DNA fragmentation, assessed using TUNEL staining. An elevation in apoptotic nuclei was evident as early as 7 days, which remained similarly elevated at 14 and 21 days. Thus chronic muscle denervation: 1) induces impairments in mitochondrial function, leading to increased ROS production; and 2) increases the expression of pro-apoptotic proteins while reducing the protective effects of anti-oxidant enzymes. These data suggest that mitochondrially mediated apoptosis contributes to the muscle atrophy which is observed during muscle disuse.

1825 Chronic muscle disuse, such as that produced by denervation, reduces mitochondrial content and produces muscle atrophy. These changes in muscle phenotype could be provoked by alterations in important regulators of mitochondrial biogenesis, such as the nuclear coactivator PGC-1α, and by increases in muscle fiber cell death via apoptosis. However, the role of PGC-1α following denervation has not been defined, and limited research has assessed apoptotic mechanisms as contributing factors to denervation-induced muscle atrophy. PURPOSE: To assess time-dependent alterations in key proteins involved in mitochondrial biogenesis (PGC-1α, cytochrome c, Tfam, cytochrome oxidase (COX) activity) and apoptosis (HSP70, Bcl-2, BAX, AIF) following chronic muscle denervation. METHODS: Male Sprague-Dawley rats (n = 20) underwent unilateral denervation (DEN) of the sciatic nerve for 5, 21, or 42 days (n = 3–4 animals/day). Denervated and contralateral (control) red gastrocnemius muscles were used for biochemical analyses. RESULTS: Denervation decreased PGC-1α expression by 45% following 5 days of DEN, which was further decreased by 51% and 65% of control at 21 and 42 days, respectively. COX activity declined with time and matched PGC-1α expression, with decreases of 35%, 55%, and 70% at 5, 21, and 42 days of DEN. These changes occurred coincident with 45% decreases in both Tfam and cytochrome c expression at 42 days of DEN. In contrast, proapoptotic BAX expression was elevated by 88% and 115% after 21 and 42 days of DEN, while coincident decreases of 79% and 89% occurred for the expression of the antiapoptotic protein Bcl-2. These changes dramatically increased the BAX:Bcl-2 ratio in denervated muscle. An increase in this ratio is indicative of a greater susceptibility to apoptosis. Additionally, 42 days of DEN resulted in increased (2.0-fold) expression of pro-apoptotic AIF, as well as increased levels (2.9-fold) of anti-apoptotic HSP70. CONCLUSIONS: These results indicate PGC-1α could be an important mediator of the decrements in mitochondrial content following periods of chronic muscle disuse. In addition, the profound shift in BAX:Bcl-2 ratio suggests that denervated muscle has a greater susceptibility to apoptosis and that this likely contributes to denervationinduced atrophy of skeletal muscle. Support: NSERC, Canada.

Mitochondrial myopathy patients possessing mutations in mitochondrial DNA (mtDNA) exhibit a low exercise tolerance despite the presence of a 1.5-fold greater citrate synthase activity, indicative of mitochondrial volume. Endurance training in these patients can lead to further increases in citrate synthase, and improvements in oxidative and work capacities (Ann. Neurol. 50:133, 2001). The molecular mechanisms underlying these adaptations remain unknown. PURPOSE To assess the expression of selected nuclear-encoded mitochondrial proteins in mitochondrial myopathy patients (MMP) following endurance training. METHODS Eight MMP were trained 3–4 times/wk for 14 weeks at 70–80% max HR. The response was compared to that of healthy control subjects (C). The patients had mtDNA defects that included: 1) large-scale and multiple deletions, 2) point mutations of tRNA, and 3) point mutations in protein coding genes. The proteins measured were chosen as indices of mtDNA transcription (Tfam), protein import (mtHsp70, Tom20), apoptosis (AIF, Bcl-2) and organelle biogenesis (cytochrome c). RESULTS Cytochrome c increased by 2.6-fold with training in C, and by 1.8-fold in MMP, similar to the change observed with citrate synthase. MtHSP70 increased by 1.4-fold in C, but 1.6-fold in MMP. While C displayed training-induced increases in Tfam (1.2-fold), Tom20 (1.5-fold), Bcl-2 (1.2-fold) and AIF (1.4-fold), the response of these proteins to training in MMP was attenuated. CONCLUSION The expression of some nuclear genes encoding mitochondrial proteins, which normally respond to training, does not occur in patients with a diversity of mtDNA defects. This suggests that training induces changes in mitochondrial composition which favour an increase in oxidative phosphorylation, but that an altered mitochondrial-to-nuclear signaling mechanism exists in mtDNA-based mitochondrial disease. Supported by CIHR.

PGC-1α is a transcriptional coactivator that is an important mediator of mitochondrial biogenesis. However, it is not known whether PGC-1α protein expression increases under conditions which provoke mitochondrial biogenesis in skeletal muscle, such as contractile activity or thyroid hormone (T3) treatment. PURPOSES 1) To relate PGC-1α protein expression to cytochrome c oxidase (COX) activity in fast- and slow-twitch muscle from animals treated with T3, and in muscle subject to contractile activity; 2) to identify potential signaling pathways involved in inducing PGC-1α expression. METHODS Muscle extracts were made from animals subject to 1) chronic 10 Hz stimulation (3,5,7 and 10 days) and 2) T3-treatment (5 days), as well as from electrically-stimulated C2C12 muscle cells. Immunoblot and enzymatic analyses were used to evaluate protein expression. RESULTS T3 increased PGC-α content similarly in fast- and slow-twitch muscle. Contractile activity induced early increases in PGC-1α protein coincident with increases in Tfam and NRF-1, suggesting that PGC-1α is important in coordinating the nuclear and mitochondrial genomes. A strong correlation (r = 0.74) between changes in PGC-1α protein and COX activity was found. AMP kinase and p38 MAPK activities were increased by contractile activity. p38 MAPK is known to increase PGC-1α protein stability. T3 also increased p38 MAPK activity, but only in slow-twitch muscle. Ca2+ ionophore treatment of muscle cells also led to an induction of PGC-1α. CONCLUSIONS Our data support a role for PGC-1α in the physiological regulation of organelle biogenesis fast-and slow-twitch muscle, and suggest that increases in PGC-1α form part of a unifying pathway which promotes both T3- and contractile activity-induced mitochondrial biogenesis in muscle. These data also suggest that PGC-1α expression is mediated by Ca2+ signaling which may increase transcription, as well as fiber type-specific post-translational phosphorylation which increases protein stability. Supported by NSERC

Intermyofibrillar and subsarcolemmal mitochondria (IFM and SSM) exist in skeletal muscle, and these increase in response to chronic contractile activity (CCA). IFM and SSM contain the adenine nucleotide translocase (ANT), ATP synthase (F1ATPase), porin, and uncoupling protein-3 (UCP3) that are responsible for proton leak, resulting in state 4 respiration. PURPOSE To investigate the relationship between these proteins and state 4 respiration during CCA-induced mitochondrial biogenesis. METHODS Chronic (10-Hz) stimulation (3 h/day, 7 days) was used to induce CCA of rat muscle. Respiration was measured in SSM and IFM with the additions of glutamate (state 4), oleic acid (OA) and GDP. OA stimulates, while GDP inhibits UCP3 function. Immunoblotting was used to measure protein levels. Cytochrome c oxidase (COX) activity was used as a marker of mitochondrial biogenesis. RESULTS UCP3, F1α subunit of F1ATPase (F1α) and porin were 1.3-, 1.6- and 1.5-fold (p < .05) greater in IFM compared to SSM. CCA increased COX activity by 1.4-fold (p < .05). UCP3 content was increased by 1.9- and 2.3-fold in IFM and SSM, respectively. CCA increased F1α in SSM by 1.3-fold, but had no effect in IFM. ANT and porin were not changed by CCA. CCA increased state 4 respiration by 1.4-fold (p < .05) in IFM, but no effect was seen in the SSM. OA stimulated state 4 respiration by 2-fold in IFM, but there was no effect in SSM despite only modestly lower UCP3 levels in SSM. Inhibition of UCP3 function by GDP led to 55–70% reductions in state 4 respiration, independent of the 2-fold difference in UCP3 content due to CCA. CONCLUSIONS CCA induced changes in protein composition within IFM and SSM membranes, including an increase in UCP3 expression which exceeded the extent of mitochondrial biogenesis. Modifications in UCP3 function affect state 4 respiration, but a dissociation of UCP3 content from state 4 respiration suggests that respiration is differentially regulated in SSM and IFM, by multiple membrane components. Supported by NSERC.

Skeletal muscle contains two morphologically distinct subfractions of mitochondria which are found in separate locations in the cell. Subsarcolemmal (SS) mitochondria are located beneath the surface of the sarcolemma and intermyofibrillar (IMF) mitochondria are found intermingled within the myofibrils. SS and IMF subfractions possess different enzyme activities and respiratory rates, and they adapt differently to contractile activity. The purpose of this study was to determine if there is a differential adaptive response of IMF and SS mitochondrial respiration to chronic stimulation (1,2,3,5,7 days, 10Hz, 3hrs/d, n = 2–7/day) of rat tibialis anterior muscles. Since oxygen consumption is associated with the production of reactive oxygen species (ROS), and these are known activators of the transcription factor NF-kB, we also measured NF-kB-DNA binding using electromobility shift assays (n = 4/day). Following 1 day of stimulation, SS mitochondrial state 3 and 4 respiration rates were transiently decreased by 40–50% in stimulated, compared to control muscle. This was followed by a return to control levels of state 4 respiration, and a dramatic 63% increase in state 3 respiration above control levels between 3–5d of stimulation. SS mitochondrial state 3 respiration returned to control levels by 7d. In contrast, IMF mitochondrial state 3 and 4 respiration rates were not changed between 1–5d, but increased in parallel by 35–40% after 7d of stimulation. Electromobility shift assays revealed elevated levels of NF-kB binding above that in control muscle extracts by 40% after 1d, 130% after 5d, and 80% after 7d (n = 4) of stimulation. These data indicate chronic contractile activity induces early increases in NF-kB binding which precede changes in muscle respiratory capacity. In addition, contractile activity induces differential adaptive responses with respect to IMF and SS mitochondrial respiration rates.