INTRODUCTION

TOP ABSTRACT INTRODUCTION FUNDAMENTALS OF MUSCLE... CELLULAR MECHANISMS OF... CONCLUSIONS AND FUTURE... REFERENCES

Since mitochondria are a major source of cellular ATP, we should not be surprised to learn that the number of them per cell, as well as their intracellular locations, varies with the type of cell and with its metabolic state (41).

Robert Dyson's words, taken from his 1975 undergraduate textbook, had only recently been established. Certainly, tissue differences in mitochondrial content were well known, but it was only a few years earlier that mitochondrial size, number, and/or volume was demonstrated to increase within a given tissue (i.e., skeletal muscle) in response to a physiological stimulus (i.e., endurance training; Refs. 57, 75, 87, 95). This finding became relevant to exercise physiologists interested in the cellular basis of endurance performance, and it was of practical importance for the athletes involved in training programs. In addition, clinical relevance could be derived from the fact that mitochondrial content could also be reduced during periods of prolonged muscle disuse, such as limb immobilization (16) or muscle denervation (180). However, the broader impact of changes in mitochondrial content within a tissue to other fields of cell biology were not as apparent as they are today. Discussions of mitochondrial function, gene expression, and synthesis are now a regular feature of cell biology laboratories. The term "mitochondrial biogenesis," implying the cellular processes involved in the synthesis and degradation of the organelle, is now used with more regularity and apparent relevance. Mitochondria are now recognized not only as "powerhouses of the cell," for which they are famous, but as potential loci of disease with broad clinical relevance (104, 176).

Our understanding of mitochondrial function has progressed rapidly in recent years for a number of reasons. First, the remarkable advances in molecular biology involving recombinant DNA techniques, quantitative analyses of DNA and RNA, the use of animal and cell culture models (189), and the advent of gene transfer technology (193) have all permitted researchers to probe more deeply into cellular and molecular events involved in mitochondrial function. These are complemented by advancements in microscopy techniques, which permit improved resolution of organelle structure and, when combined with fluorescent detection methodology, measurement of ion movements in various compartments of living cells (50). The result of this is that we now appreciate new roles for mitochondria, not only as the major ATP-providing organelle but also as mediators of apoptotic events leading to cell death, as housing for a separate, semi-autonomous genome [i.e., mitochondrial DNA (mtDNA)] and as a site of cellular signal transduction events, which may help coordinate nuclear and mitochondrial gene expression.

Mitochondrial biogenesis can be a dramatic event, particularly when it is invoked in a previously low-oxidative, white skeletal muscle. Because of this, it can also serve as a cellular model of organelle assembly within eukaryotic cells. In response to exercise, the conversion of phenotype of a white muscle to one with a visibly red appearance is brought about by additional heme synthesis and its incorporation into mitochondrial cytochromes as well as into myoglobin. As discussed below, the large increase in mitochondrial content observed can have a profound impact on cellular metabolism during the stress of exercise. This review will focus on current issues in mitochondrial biogenesis, with emphasis on skeletal muscle and the role of contractile activity (i.e., exercise). A number of other reviews have appeared on this topic (44, 76, 81, 180).

	FUNDAMENTALS OF MUSCLE MITOCHONDRIAL BIOGENESIS

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Mitochondrial Biogenesis Can Be Produced by a Number of Physiological Conditions

Mitochondrial Content Has a Short Half-Life, and Mitochondrial Phenotype Can Change

Measurements of representative protein markers such as cytochrome c or cytochrome-c oxidase (COX) activity have allowed exercise physiologists to recognize that, in response to a constant exercise stimulus, ~6 wk of endurance training is required to reach a new, higher steady-state mitochondrial content. This time course is dependent on the fiber type being recruited, as well as the duration, frequency, and intensity of the exercise bouts (39, 65, 72, 83). Mitochondrial adaptations will not occur in skeletal muscle cells, which are not recruited during the exercise bout, consistent with the idea that the stimulus for biogenesis originates within the contracting muscle, independent of humoral influences. A corollary of this is that endurance training programs must be geared, in part, toward the recruitment of fast-fatiguable motor units containing type IIb (fast-twitch white) fibers if any mitochondrial adaptation is expected in those fibers. Interestingly, resistance training, which does indeed recruit fast-fatiguable motor units, does not lead to a mitochondrial adaptation. Because the very high intensity and low duration of most resistance training regimens represent such a strong stimulus for the synthesis of myofibrillar proteins leading to muscle hypertrophy, the mitochondrial content within enlarged muscle fibers may even be "diluted" within the cell (113, 114). This increases the diffusion distance of oxygen and substrates and does not represent a favorable adaptation with respect to endurance performance.

The approximate 6-wk time period required to achieve a new steady-state mitochondrial content in response to endurance training clearly does not reflect the early molecular events that ultimately lead to the measurable morphological changes. Indeed, changes in mitochondrial protein content can be visibly apparent at much earlier time points. Mitochondrial proteins turn over with a half-life of ~1 wk after the onset of a new level of muscle contractile activity (15, 70, 73, 170). This means that a continuous exercise stimulus is required to maintain mitochondrial content at an elevated level following a training period; otherwise, loss of oxidative capacity and endurance performance will ensue. It is interesting to note that mitochondrial phospholipid content changes occur with an even shorter half-life (~4 days; Refs. 167 and 180), suggesting that the assembly and/or degradation of the organelle could be initiated by changes in phospholipid composition. In the absence of normal rates of cytochrome synthesis and incorporation into the inner membrane during iron deficiency, imposed contractile activity appears to lead to continued membrane lipid synthesis, producing an increased mitochondrial volume with a markedly reduced and abnormal protein content (64). These data indicate that phospholipid and protein synthesis are not necessarily coupled during contractile activity-induced mitochondrial biogenesis. The data also suggest that the ratio of lipid to protein within mitochondria must change somewhat during the transition to either higher or lower mitochondrial contents. This change appears to be subtle as a result of training (57) or chronic stimulation (141) and in most cases appears not to affect rates of oxygen consumption per unit volume of mitochondria (85, 86). However, it may be much more evident under steady-state conditions in tissues possessing widely divergent mitochondrial contents, such as heart and white skeletal muscle (180).

Mitochondrial Biogenesis Leads to Changes in Cellular Metabolism and Muscle Performance During Endurance Exercise

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Top: adenine nucleotide metabolism during acute exercise. From the onset until the termination of contractions, free ADP (ADPf) is formed by the myosin ATPase reaction. ADPf combines with phosphocreatine (CrP) in the creatine (Cr) phosphokinase (CPK) reaction, resulting in reduced CrP levels and restoration of ATP. ADPf also stimulates glycolysis and mitochondrial respiration. Two molecules of ADPf can be converted to ATP and AMP in the myokinase reaction. In fast-twitch muscle fibers, AMP is metabolized to inosine monophosphate (IMP) and ammonia (NH3) by AMP deaminase (reaction 1). In slow-twitch muscle fibers, the metabolism of AMP is predominantly in the direction of adenosine, via the 5'-nucleotidase reaction (reaction 2). The increase in AMP and the drop in CrP activate AMP kinase kinase, resulting in the phosphorylation and activation (+) of AMP kinase (AMPK). Bottom: schematic of the improved sensitivity of mitochondrial respiration to ADPf brought about by training or other models of chronic contractile activity. The greater mitochondrial content means that a lower ADPf (25 vs. 50 nmol/g) is required to achieve the same level of oxygen consumption (O2) per gram of muscle (data taken from Ref. 40). This reduces the consumption of CrP, attenuates the rate of glycolysis and lactic acid production, reduces the formation of AMP and NH3, improves the fraction of energy obtained aerobically, and decreases the activation of AMPK. It also means that each mitochondrial electron transport chain operates at a lower fraction of its maximal rate to produce the same O2 as before the adaptation took place.

The increased mitochondrial content brought about by endurance training not only leads to a reduced lactate production but also enhances the disposal of lactate. Recently, lactate dehydrogenase has been localized within the organelle (20), and lactate transport into mitochondria can occur via the mitochondrial monocarboxylate transporter MCT1 (19). A higher mitochondrial content as a result of chronic exercise will therefore improve lactate oxidation, resulting in lower rates of release from muscle (38). In addition, the higher the mitochondrial content per gram of muscle, the lower the rate of respiration required per mitochondrion for any given workload (Fig. 1, bottom). This has been predicted to reduce the level of potentially damaging reactive oxygen species (ROS; Ref. 36), even at constant rate of oxygen consumption per gram of muscle. The combination of reduced ROS production, along with higher mitochondrial ROS protective enzymes (61), supports the idea that an additional benefit of chronic contractile activity, other than increased oxidative capacity, is an attenuated potential for ROS-mediated protein, lipid, or DNA damage (159). Conversely, the lower the mitochondrial content, as is evident in some tissues as a consequence of aging (118), the greater the tendency for ROS-induced damage to become evident, particularly in its effect on mtDNA. This highlights one potentially important reason for maintaining muscle mitochondrial content with a program of regular physical activity during the aging process.

Mitochondrial Biogenesis Requires the Cooperation of the Nuclear and Mitochondrial Genomes

View larger version (84K): [in this window] [in a new window]   Fig. 2.   Overall synopsis of mitochondrial biogenesis in a muscle cell. Signals originating at the neuromuscular junction (NMJ) include propagated action potentials and the release of trophic substances, which interact with the postsynaptic membrane. Electrical activity in the sarcolemma is coupled to the release of calcium from the sarcoplasmic reticulum (SR). Calcium acts as a second messenger to activate phosphatases and/or kinases, which are ultimately translocated to the nucleus to affect the activation of transcription factors and which influence the expression of nuclear genes encoding mitochondrial proteins. mRNA produced by transcription is translated into protein in the cytosol, which can either be translocated back to the nucleus (transcription factor) or chaperoned to the protein import machinery and taken up by the organelle. Within mitochondria it may act as a single protein subunit or be combined with other subunits to form a multisubunit holoenzyme (e.g., cytochrome c oxidase). Some subunits of the holoenzyme may be derived from the mitochondrial genome (mtDNA), which also undergoes transcription and translation to synthesize a limited number (13) of proteins that are essential components of the electron transport chain.

Where does the cooperation between the genomes come in? First, these thirteen components comprise only a small fraction of the total respiratory chain proteins. Some act as single protein subunits, but many are combined with nuclear-encoded proteins to form multisubunit holoenzymes, like COX or NADH dehydrogenase (Fig. 2). The function of these holoenzymes is clearly impaired if contributions from either genome is absent (74). Second, it is known that mtDNA transcription and replication require the import of nuclear gene products, which act as polymerases or transcription factors (see Expression of mtDNA, below). Thus a longstanding question has been related to how the two genomes are regulated, or coordinated, in response to a stimulus leading to mitochondrial biogenesis. Williams et al. (190, 192) were the first to show that chronic contractile activity led to increases in mRNA levels encoding both nuclear and mitochondrial gene products. Subsequently, this was demonstrated for subunit mRNAs belonging to the same COX holoenzyme (82). Because COX contains 10 nuclear-encoded and 3 mitochondrial-encoded subunits, this enzyme is a useful model for studying the interactions of the two genomes. The mRNA expression of these subunits is also coordinated across a variety of tissues possessing a wide range of mitochondrial contents (54, 78). In addition, some evidence for a coordinated regulation of the two genomes was found during the mitochondrial biogenesis induced by cardiac hypertrophy (182), as well as in human muscle when trained and untrained individuals were compared (137) . Given the diverse promoter regions of nuclear genes encoding mitochondrial proteins (108, 124), as well as the sequences of the mtDNA promoters, it is not surprising that this coordination can be disrupted. Evidence for this has been presented in cases of thyroid hormone treatment (80, 183), suggesting that a coordination of gene expression responses leading to strict stoichiometric relationships is not absolutely necessary for the formation of a functional organelle.

The Mitochondrial Reticulum Exists in Distinct Cellular Regions, Where It Possesses Different Biochemical Features

The results of these biochemical studies were derived from mitochondria obtained using cell fractionation and differential centrifugation techniques. Although there is little doubt that the isolated fractions are reasonably pure, it is also true that mitochondria in muscle cells do not exist as discrete "populations." Strong evidence points to a structure that more closely resembles a continuous mitochondrial membrane system (i.e., a reticulum or network; Refs. 10, 96). The presence of a reticular structure that interacts with organelles such as the cytoskeleton or the endoplasmic reticulum is also apparent in other cell types (50). This type of structure might have considerable advantage for the transport and metabolism of lipid precursors or for the involvement of mitochondria in calcium homeostasis. However, a reticular structure does not preclude the possibility that regional differences in membrane protein composition exist, as mitochondria in different locations may adapt to reflect diverse metabolic demands in distinct cellular regions (96). Differences in membrane protein composition within a continuous membrane unit are found in terminal fractions of sarcoplasmic reticulum or in subsynaptic plasma membrane regions (27), and these are retained during subcellular fractionation. It is also likely that species differences exist with respect to the extensiveness of a mitochondrial reticulum. There is some evidence of a partial mitochondrial reticulum in human muscle possessing a relatively low mitochondrial volume (2-8%; Refs. 83, 111). As the mitochondrial content increases, so does the likelihood of a reticular structure as the possibility of mitochondrial fusion increases (13). Evidence for this has been provided in horse muscle fibers in which a reticulum becomes more prominent in fibers with the highest mitochondrial content (94) and in rat (97) and rabbit (151) muscle during mitochondrial biogenesis induced by contractile activity.

	CELLULAR MECHANISMS OF MITOCHONDRIAL BIOGENESIS

TOP ABSTRACT INTRODUCTION FUNDAMENTALS OF MUSCLE... CELLULAR MECHANISMS OF... CONCLUSIONS AND FUTURE... REFERENCES

The initiation of mitochondrial biogenesis in muscle cells begins with putative signals brought about by muscle contraction. The magnitude of the signal(s), up to a point, is undoubtedly related to the intensity and duration of the contractile effort. This signaling can potentially lead to 1) the activation or inhibition of transcription factors, which will affect the rate of transcription, 2) the activation or inhibition of mRNA stability factors, which mediate changes in the rate of mRNA degradation, 3) alterations in translational efficiency, 4) the posttranslational modification of proteins, 5) changes in the kinetics of mitochondrial protein import, and/or 6) alterations in the rate of folding or assembly of proteins into multisubunit complexes. Additionally, the signal(s) could be transmitted directly to the mitochondria to initiate the replication or transcription of mtDNA or have an effect on mtRNA translation or holoenzyme assembly.

Initial Signaling Events

Calcium. On its release from the sarcoplasmic reticulum, calcium permits actin and myosin interaction in muscle cells. It is also well recognized as an important second messenger in a variety of cell types (35), including muscle (102, 148, 177). Increases in the concentration of cytosolic calcium levels can activate a number of kinases [e.g., Ca²⁺/calmodulin kinase II, protein kinase C (PKC)] and phosphatases (e.g., calcineurin), which ultimately translocate their signals to the nucleus to alter the rate of gene transcription (Fig. 2). Because of the tremendous fluctuations in cytosolic calcium levels during contraction and relaxation in both skeletal and cardiac muscle, attention has turned to the role of calcium in mediating muscle adaptation. Most interesting recently was the discovery that artificially induced changes in muscle calcium levels using a calcium ionophore could reversibly alter myosin isoform expression toward that of a slow phenotype, accompanied by an increase in mitochondrial enzyme activity (102). Subsequently, it was shown that fiber-type specific differences in calcium levels attained during normal motor unit recruitment leads to the differential activation of calcineurin. Once active, calcineurin dephosphorylates the transcription factor nuclear factor of activated T cells and activates myocyte enhancer factor-2, which then bind to specific regions in the upstream regions of genes that are ultimately expressed in slow- but not fast-twitch muscle types (24, 196). This appears to be an essential determinant of the fast- and slow-twitch fiber composition of a muscle, but the involvement of calcineurin in regulating the activity of genes involved in mitochondrial biogenesis has not yet been investigated. However, it is clear that other calcium-responsive events have an impact on mitochondrial activity and gene expression. First, increases in cytosolic calcium are known to be matched within the organelle and directly influence the rate of mitochondrial respiration (93). This occurs via the activation of dehydrogenases, which require calcium for full activity (116). The change in mitochondrial calcium causes increases in ATP levels within both the cytosol and the mitochondria, which persist long after the mitochondrial calcium elevation has declined (92). Second, calcium-mediated changes in inner membrane phosphorylation events have been reported (8). These occur within the physiological range of calcium concentrations and appear to involve the phosphorylation of subunit c of the F₀F₁-ATPase. Although the significance of these two findings is not yet established with respect to mitochondrial biogenesis, they may form part of an intramitochondrial signaling cascade (see Downstream Phosphorylation Events below). Third, decreases in calcium concentration within the medium surrounding cultured myotubes result in parallel changes in mitochondrial enzyme activities (156). Fourth, calcium-mediated increases in the expression of cytochrome c, an important component of the electron transport chain, have been documented (52). With the use of the calcium ionophore A-23187 to artificially increase intracellular calcium levels in L6 muscle cells, time- and concentration-dependent increases in cytochrome c mRNA levels have been reported, which were matched by increases in transcriptional activation (52). This increase in transcription was mediated, in part, by the activation of calcium-sensitive PKC isoforms, and it was removed, also in part, by inhibition of a mitogen-activated protein kinase (MAP kinase) pathway. There was no apparent involvement of calcium/calmodulin kinase II or IV in mediating the response. These data implicate both PKC and MAP kinase signaling in cytochrome c expression in muscle. Finally, studies of cells with experimentally induced mtDNA depletion have supported the conclusion that some calcium signaling events in muscle are propagated as a direct result of energy supply-demand imbalances. In mtDNA depletion, the synthesis of ATP is reduced because of a defective respiratory chain resulting from inadequate levels of mitochondrial gene products (14). This leads to an increase in cytosolic calcium concentration, presumably because the energy-dependent processes of calcium extrusion and uptake are impaired. An upregulation of a number of proteins related to calcium release and responsiveness (e.g., NFATc, calcineurin, ryanodine receptor-1), as well as nuclear genes encoding mitochondrial proteins, like COX subunit Vb, was observed (14). The interpretations of these data are that imbalances between cellular ATP demand and mitochondrial ATP supply, leading to alterations in calcium homeostasis, can trigger the induction of signal transduction pathways, leading to the phosphorylation or dephosphorylation of transcription and/or stability factors. Thus this may be indicative of the signaling events that take place during contractile activity. However, it also appears that an increase in calcium cannot, by itself, lead to an augmentation in overall mitochondrial biogenesis. Subsequent studies using A-23187 have shown that, although a number of nuclear genes encoding mitochondrial proteins [e.g., malate dehydrogenase (MDH), the -subunit of the F₀F₁-ATPase] are increased along with cytochrome c, a number of others that would be critical to mitochondrial biogenesis are not. For example, mRNAs encoding COX subunits (IV, Vb, and VIc) are unchanged, whereas those encoding glutamate dehydrogenase and mitochondrially encoded COX subunits II and III are decreased (Freyssenet D, Irrcher I, Connor MK, Di Carlo M, and Hood DA, unpublished observations). Thus calcium likely forms only part of a broader series of positive and negative signals that mediate changes in the synthesis of mitochondrial components.

ATP turnover. For at least 25 years, it has been hypothesized that disturbances in energy metabolism, leading to ATP depletion, an alteration in energy charge, or a change in the phosphorylation potential could initiate a compensatory response, ultimately leading to an increase in mitochondrial content (140). However, moderate-intensity exercise training can lead to mitochondrial biogenesis in the absence of marked changes in cellular ATP levels. Thus, although conditions of ATP depletion are known to lead to a higher mitochondrial content, it is likely that an increased rate of ATP turnover (i.e., increased rates of ATP synthesis and degradation) is sufficient to provoke mitochondrial biogenesis. These ideas have received considerable experimental support. First, experiments conducted using the drug -guanidinoproprionic acid to deplete the high-energy phosphate compounds phosphocreatine and ATP illustrated an upregulation of mitochondrial enzymes (162) and cytochrome c mRNA (103) in muscle. Second, as a result of accelerated ATP turnover, exercise increases the level of AMP and decreases the level of phosphocreatine in muscle, resulting in an activation of 2-AMPK (Fig. 1; Ref. 194). This activation can be simulated by administration of the drug 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside to animals. After 4 wk of treatment, an increase in some (e.g., citrate synthase, succinate dehydrogenase, cytochrome c) but not all [e.g., carnitine palmitoyltransferase (CPT), hydroxy-acyl-CoA dehydrogenase] mitochondrial enzymes was observed (187). This suggests that AMPK activation may be partially involved in signaling the mitochondrial adaptation to contractile activity. Third, the hypothesis has received support from recent studies of mitochondrial uncoupling. Uncoupling refers to the dissipation of the electrochemical gradient across the inner membrane, which diminishes ATP production by disabling the activity of the F₁F₀-ATPase. Subsequently, electron transport and oxygen consumption are accelerated because control exerted by ADP is lost, and ATP utilization exceeds ATP synthesis. In this respect, the condition is analogous to intense exercise. Uncoupling can be achieved by using specific drugs in vitro, or by the overexpression of uncoupling protein (UCP). Within 6 h of uncoupling, an induction of the transcription factor nuclear respiratory factor-1 (NRF-1) was observed (109). NRF-1 has been widely implicated in the transcriptional activation of multiple genes involved in mitochondrial biogenesis (153). Subsequent to the induction of NRF-1, an increase in the expression of NRF-1 target genes, including -aminolevulinic acid synthase (ALAs), the rate-limiting enzyme in the synthesis of heme, was noted (109). As the prosthetic group in all mitochondrial cytochromes, heme is a vital component of the respiratory chain. Thus it appears that the increase in mitochondrial respiration, or the deficit between cellular ATP demand and mitochondrial ATP supply, provides a stimulus for the sequential induction of a variety of genes involved in the biogenesis of the organelle.

Downstream Phosphorylation Events

A novel series of phosphorylation events also occurs at the level of the mitochondria, the implications of which remain to be fully established. It has been known for many years that certain subunits of the multisubunit complexes pyruvate dehydrogenase and branched-chain ketoacid dehydrogenase are reversibly phosphorylated. This can change the activation of the entire complex. Recently, other mitochondrial proteins, with more remote functions, have been found to be phosphorylated. These include COX subunit IV (164) and the 18-kDa subunit of complex I. The mechanism involved appears to be via the newly established presence of a cAMP-dependent protein kinase A located in the inner mitochondrial membrane (131). Interestingly, phosphorylation of the 18-kDa subunit activates mitochondrial respiration via an increase in complex I activity (152), whereas phosphorylation of subunit IV reduces respiration by inhibiting COX activity (12). This inhibition can be reversed by calcium-mediated activation of a protein phosphatase. It is also worth noting that calcium cannot only activate phosphatases but also has been shown to modulate the phosphorylation of subunit c of the F₀F₁-ATPase (8). The dominant influence leading to stimulation or inhibition of respiration may be determined by the presence of A-kinase anchoring proteins (AKAPs), which define the precise localizations of cAMP-dependent protein kinase A in various organelles (88). These data illustrate that important intracellular signaling pathways involving both calcium and cAMP extend beyond the cytosol and nucleus and into the mitochondria, where they certainly affect mitochondrial function and enzyme activity. Their roles in mitochondrial biogenesis have yet to be determined.

Transcription of Nuclear Genes

A coordinated increase in the transcription of nuclear and mitochondrial genes would be achieved if a universal activator of the relevant genes was identified. In this respect, NRF-1 sites exist in the promoters of many nuclear genes, including mitochondrial transcription factor A (Tfam), as well as ALAs, the rate-limiting enzyme of heme synthesis. However, NRF-1 sites are not universally found. For example, not all of the 10 nuclear-encoded subunits of COX have NRF-1 sites in their upstream regulatory regions (108). Thus it cannot be expected that NRF-1 could coordinately transactivate these genes to produce a functional COX holoenzyme with stoichiometric increases in the synthesis of each subunit. However, the recent discovery of a coactivator of PPAR- termed PGC-1 has revealed it to be a potential regulatory candidate (197). Overexpression of PGC-1 was shown to induce morphologically measurable indexes of mitochondrial content, as well as the mRNA expression of a number of nuclear genes in muscle cells, including NRF-1, NRF-2, Tfam, UCP-2, nuclear- and mitochondrial-encoded COX subunits, and cytochrome c. The increase in Tfam expression, mediated by transcriptional activation of the Tfam promoter, led to an increase in mtDNA. This effect was probably a result of PGC-1 coactivation of NRF-1 on the Tfam promoter. Further evidence provided by loss-of-function experiments suggested that NRF-1 was necessary for the effect of PGC-1 on mitochondrial biogenesis. Subsequent work by Kelly and co-workers (173) showed that PGC-1 was capable of coactivating a second protein, PPAR-, a receptor known to regulate the expression of enzymes involved in fatty acid oxidation such as medium-chain acyl CoA dehydrogenase (MCAD) and CPT-1. Using cardiac cells isolated during the perinatal period, they showed coincident increases in the mRNA expression of PGC-1 with the expression of MCAD, CPT-1, and PPAR- (107). Furthermore, forced overexpression of PGC-1 led to increased expression of nuclear and mitochondrial genes located in the matrix and respiratory chain. This was accompanied by increased mitochondrial number and higher rates of coupled cellular respiration (107). Thus PGC-1 has emerged as a critically important protein in determining mitochondrial biogenesis and function. It is interesting to note that one of its binding partners, PPAR-, is increased in muscle subject to chronic contractile activity (34), whereas another, PPAR-2, is located in the mitochondrial matrix (21). Further work will be required to determine the role that PGC-1 plays in regulating contractile activity-induced mitochondrial biogenesis in muscle.

mRNA stability

In Hep G2 cells in culture, the half-life of nuclear transcripts encoding mitochondrial proteins has been reported to range from 10 to 28 h (25). Remarkably, inhibition of mitochondrial protein synthesis led to a widespread increase in nuclear-encoded mRNA stability averaging approximately sevenfold. No effect was observed on those transcripts that were mitochondrially encoded (25). This suggests that a reduction in mitochondrial protein synthesis, which disrupts the assembly of the respiratory chain, could lead to an enhanced activity of RNA binding proteins that protect nuclear transcripts from degradation and results in increased mRNA levels. Interestingly, this is also observed in cells with respiratory impairment brought about by experimentally induced mtDNA depletion, in which a marked upregulation of mRNAs derived from multiple nuclear genes with products localized to the mitochondria (110, 178) is observed. In addition, patients harboring extensive mtDNA mutations exhibit large, compensatory increases in nuclear-derived transcript levels (68). The common feature of these diverse conditions is probably the reduced energy status of the cell or possibly the increase in ROS, which are produced when the electron transport chain is inhibited (18). The data suggest that deleterious modifications in respiratory chain structure, leading to reduced function (i.e., decreased ATP supply) relative to normal, represent a signal for the upregulation of nuclear-encoded gene products. This upregulation, often measured as mRNA levels on Northern blots, may largely be a product of increased mRNA stability. The data lend further support to the idea that a metabolically derived signal related to ATP turnover is involved in mediating mitochondrial biogenesis (see ATP turnover, above). It is also worth noting that the identity of the RNA binding proteins that confer stability or instability have remained largely unknown in most tissues, and even less is understood about mRNA stability in skeletal muscle or the role of contractile activity in modifying this process.

Example of a Nuclear Gene Encoding a Mitochondrial Protein: Cytochrome c

View larger version (90K): [in this window] [in a new window]   Fig. 3.   Partial description of cytochrome c expression in muscle during contractile activity. The schematic is derived from data published on both cardiac and skeletal muscle cells. A putative signal (see text) stimulates the transcription of nuclear respiratory factor-1 (NRF-1) and/or specificity protein 1 (Sp1). The mRNAs derived from these genes are exported from the nucleus to the cytosol via nuclear pores. These mRNAs are translated into protein in the cytosol, which are then translocated back into the nucleus. Contractile activity is known to increase the expression and DNA binding of both NRF-1 and Sp1, thereby enhancing the transcription of the cytochrome c (cyto c) gene. Many other nuclear genes encoding mitochondrial proteins are responsive to transcriptional activation by NRF-1 and Sp1, suggesting that these transcription factors are very important for mitochondrial biogenesis. C, COOH terminus; N, NH2 terminus.

To gain an understanding of the regulation of cytochrome c expression resulting from contractile activity, electrical stimulation of neonatal cardiac myocytes (198, 199) was employed. The increase in cytochrome c mRNA observed after 48 h of stimulation was preceded by increases in the mRNA levels of NRF-1 (1-12 h) and c-Fos and c-Jun (0.25-3 h). Increases in cytochrome c transcriptional activation were observed initially at ~12 h of stimulation. The stimulation effect was markedly attenuated if the NRF-1 site or the CRE were mutated. Both basal transcription and the magnitude of the stimulation response were reduced if the Sp1 site was mutated (199). Subsequently, it was shown that c-Jun, rather than CRE binding protein (CREB), was the major protein binding to the CRE that conferred transcriptional activation during electrical stimulation (198). These data illustrate the importance of c-Jun, NRF-1, and Sp1 in the transcriptional activation of cytochrome c, at least in heart cells (Fig. 3). Using skeletal muscle C2C12 cells, we have reported that contractile activity increases cytochrome c transcription, and that this is accompanied by an increase in Sp1 protein level and DNA binding after 4 days of stimulation (29). Additional studies in vivo have also indicated that cytochrome c expression can be regulated by mRNA stability and that this may be the initial event involved. Freyssenet et al. (51) employed a direct plasmid DNA injection technique to introduce the cytochrome c promoter, along with a chloramphenicol acetyltransferase reporter construct, into tibialis anterior muscles of animals undergoing unilateral chronic electrical stimulation (3 h/day) for 1-7 days. With the use of this technique along with an in vitro mRNA decay assay, time-dependent changes in the transcriptional activity and mRNA stability could be discerned. The results indicated that the increase in cytochrome c mRNA observed was initially due to an induced increase in mRNA stability between 2 and 4 days and that this was followed by transcriptional activation at 5 days of chronic stimulation. It was suspected that changes in mRNA stability might be involved in the regulation of cytochrome c mRNA because Yan et al. (200) had previously shown that the 3' end of cytochrome c mRNA bound less of a yet unidentified destabilizing protein as a result of 9-13 days of chronic stimulation. This suggested that contractile activity led to the induction of an inhibitory factor responsible for decreasing the interaction between a destabilizing protein and the cytochrome c mRNA, thereby permitting its upregulation. These data emphasize the importance of recognizing mRNA stability as an important contributor to changes in gene expression resulting from contractile activity in muscle.

Mitochondrial Protein Import

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Left: mitochondrial transcription factor A (Tfam) is a nuclear-encoded transcription factor that is synthesized in the cytosol as a larger, "precursor" protein with a positively charged NH2-terminal presequence (blue). It must interact with the protein import machinery to enter the organelle. Once inside the matrix, mature Tfam will bind within the D-loop region of the circular (not shown) mtDNA on the heavy-strand (HSP) and light-strand promoters (LSP) and stimulate the transcription and replication of mtDNA. Right: enlarged view of the components of the protein import machinery. A typical matrix-destined precursor like Tfam is unfolded and directed to the import machinery by a cytosolic chaperone, either cytosolic 70-kDa heat shock protein (cHSP70) or mitochondrial import stimulating factor (MSF). On interaction with the translocase of the outer membrane (Tom complex), it is correctly oriented by interacting with the inner membrane phospholipid cardiolipin (not shown) before being transferred to the translocase of the inner membrane (Tim complex). The matrix chaperone mtHSP70 pulls in the precursor, and the signal sequence is cleaved by the mitochondrial processing peptidase (MPP). Subsequently, the mature protein is refolded by matrix chaperonins HSP60 and Cpn10. ATP is required at multiple steps during the import process. The number within each import machinery component refers to its size in kDa.

Two other elements are required for correct import of precursor proteins into the matrix. These are 1) the presence of an inner membrane potential (, negative inside) across the inner membrane to help pull the positively charged presequence into the matrix and 2) the availability of ATP both in the cytosol and in the matrix (168). Uncoupling agents that dissipate reduce protein import, whereas ATP depletion prevents the unfolding of the precursor in the cytosol and/or the action of mtHSP70 in the matrix (55). Thus reductions in cellular ATP levels such as that produced by severe contractile activity or defects in ATP production as might be encountered in cells with mtDNA mutations could affect the rate of import into mitochondria.

After its arrival in the matrix, the NH₂-terminal signal sequence is cleaved by a mitochondrial processing peptidase (MPP) to form the mature protein. It is then refolded into its active conformation by a mitochondrial chaperonin system consisting in part of 60-kDa heat shock protein (HSP60) and 10-kDa chaperonin (Cpn10).

The vast majority of work that defines the components of the protein import machinery, as well as their cellular function, has been done in Saccharomyces cerevisiae and Neurospora crassa. This is now being extended to mammalian cells. For example, the kinetics of matrix precursor protein that import into skeletal muscle SS and IMF mitochondrial fractions, the ATP and cardiolipin dependence of the process, and the relationship to mitochondrial respiration have all been recently defined (168). IMF mitochondria import precursor proteins more rapidly than SS mitochondria, and there is a direct relationship between the capacity for mitochondrial respiration (and thus ATP production) and the rate of protein import (168). It has also been shown that a number of protein import machinery components are induced in response to chronic contractile activity. These include the chaperones MSF, cytosolic HSP70 (cHSP70), mtHSP70, HSP60, Cpn10, as well as the import receptor Tom20 (125, 129, 166). Coincident with these increases are contractile activity-induced increases in the rate of import into the matrix but not into the outer membrane (166). This differential effect on protein targeting to mitochondrial compartments provides an example of how contractile activity can result in an altered mitochondrial protein stoichiometry. The accelerated rate of protein import into the matrix can be reproduced in cardiac mitochondria obtained from animals treated with thyroid hormone (33). Thus the effect is not a unique response to contractile activity but appears to be common to stimuli that increase mitochondrial biogenesis.

To more easily define the role of specific components of the import pathway in determining the kinetics of import, measurement of import in intact cells can be employed. When C2C12 cells were incubated with [³⁵S]methionine and the import of radiolabeled MDH into mitochondria was measured, a greater rate of import was found during the progress of mitochondrial biogenesis occurring coincident with muscle differentiation (63). As expected, thyroid hormone accelerated the rate of import and induced the expression of Tom20. To evaluate the role of Tom20 alone in mediating the accelerated import rate, forced overexpression of Tom20 in these cells using a mammalian expression construct was used. Parallel increases in the rate of import and the magnitude of overexpression were observed. Conversely, inhibition of Tom20 expression using specific antisense oligonucleotides led to equivalent decreases in MDH import (63). These data suggest that the import of matrix-destined proteins is controlled, at least in part, by the expression of Tom20.

The protein import pathway represents an example of intracellular trafficking that is important for organelle biogenesis, and it may, under some conditions, determine the increase in mitochondrial content as a result of chronic exercise. For this to be the case, it must be shown that it is inducible and that it operates at a rate that limits the overall pathway under some conditions (i.e., chronic exercise). If the import rate was slow enough to limit mitochondrial biogenesis, then a pool of precursor proteins in the cell cytosol would be measurable. In the absence of such a pool, the assumption is that newly synthesized precursor proteins are rapidly taken up by mitochondria, and the kinetics do not limit the synthesis of the organelle as a whole. This has yet to be rigorously tested in a cellular system in which any other fates of the precursor (i.e., cytosolic degradation) are blocked. It is possible that the import of proteins might become limiting under conditions of chronic contractile activity if upstream steps (i.e., transcription, translation) are accelerated such that a saturating abundance of precursor proteins are presented to the import machinery. In any event, the physiological value of the observed contractile activity-induced increases in mitochondrial protein import is that mitochondria are more sensitive to changes in precursor protein concentration, a situation that would be advantageous for mitochondrial biogenesis at any given upstream production rate of cytosolic precursor proteins.

Progress in the area of protein import will advance substantially as additional mammalian homologues of the import machinery are identified. Recently, the first disease that can solely be attributed to a mutation in a protein component of the import machinery has been identified. A mutation in deafness dystonia protein (DPP) results in a neurodegenerative disorder characterized by muscle dystonia, sensorineural deafness, and blindness. DPP has been shown to be a mitochondrial protein that closely resembles Tim8p, a protein of the intermembrane space involved in the import process (99). In addition, mutations in the import receptor Tom70 have been shown to produce mtDNA rearrangements in the fungus Podospora anserina, presumably because of defective import of a component involved in mtDNA maintenance (91). The recent cloning of Tom22 (150), as well as members of the Tim machinery (11, 89, 175), will be of help in elucidating the functional roles of individual import machinery components in the import process and the relevance of import in mitochondrially based diseases and in organelle biogenesis.

Expression of mtDNA

mtDNA can replicate independently of nuclear DNA, and it is present in high copy number (10³-10⁴ copies) in virtually all cells of the body (111). Its replication requires the presence of DNA polymerase- (Pol-), a single-stranded binding protein (SSB), which facilitates Pol- activity, Tfam, which initiates transcription and generates primers to permit DNA replication, and a mitochondrial RNA-processing endonuclease (RNase MRP), which cleaves the nascent transcript to form the primer for replication (26).

Using the chronic stimulation model to induce mitochondrial biogenesis, Williams (188) showed that mitochondrial transcript levels correlated well with variations in mtDNA, as well as with the proportion of triplex DNA structure in the D-loop region (5). A close relationship between mtDNA and mtRNA was also found when muscles from trained and untrained individuals were compared (137) and in patients with a significant mtDNA deletion (67). These data suggest that the replication of mtDNA into multiple copies, or its specific conformation within the controlling D-loop region, regulates the level of mitochondrial gene expression in skeletal muscle cells. However, studies in muscle and other tissues suggest that this is not a universal finding. A variety of conditions appear to exist in which the copy number of mtDNA is not matched with the change in the level of transcripts or with changes in oxidative capacity (163, 181). Arguments can be made that, because both replication and transcription appear to be controlled by the action of Tfam and because replication relies on the formation of a mRNA primer, transcription may be the more important process of the two (105, 181). Whether control via mtDNA copy number is specific to contractile activity-induced mitochondrial biogenesis or whether differences in the results can be explained by variations in experimental approaches or measurement devices remains to be determined. In any event, the increase in mtDNA copy number observed as a result of chronic contractile activity is accompanied by the augmented expression of SSB (158), the RNA subunit of RNAse MRP (128), and Tfam (60), but not Pol- (158). These data suggest that Pol- is sufficiently abundant and nonlimiting in the transcription of mtDNA.

Tfam has been extensively studied as the most important mammalian transcription factor for mtDNA. The level of Tfam correlates well with mtDNA abundance, and its loss, either in patients with mitochondrial myopathies (105, 136) or produced by experimental disruption of the Tfam gene (106), results in partial or total depletion of mtDNA. Homozygous Tfam knockout animals die before embryonic day 10 and are characterized by abnormal mitochondria and abolished oxidative phosphorylation (106). Animals possessing heart and muscle-specific disruptions of Tfam exhibit modestly reduced levels of mtDNA and mtRNA but little change in the activities of respiratory chain enzymes in skeletal muscle, possibly because of a low mRNA turnover in this tissue (30, 179). However, these animals displayed characteristics found in the mitochondrial disease Kearns-Sayre syndrome, evident by dilated cardiomyopathy, abnormal mitochondrial morphology and proliferation, and a progressive atrioventricular conduction block (179). These data provide evidence for the importance of Tfam in normal cardiac function, perhaps because the heart possesses the highest amount of mitochondria of all tissues. Furthermore, they illustrate the benefit of developing animal models to improve our understanding of the function of specific proteins and their role in mitochondrially based diseases (104, 176).

In skeletal muscle, Tfam expression and function is modified by contractile activity. During imposed chronic stimulation (3 h/day) ranging from 1 to 7 days in duration, Gordon et al. (60) recently showed an initial increase in Tfam mRNA which took place by 4 days. This was followed by accelerated import into mitochondria (5 days), increased mitochondrial Tfam protein content, and concomitantly higher DNA binding (7 days). This latter result was accompanied by parallel increases in COX III mRNA and COX enzyme activity (60). These data suggest that Tfam expression is well correlated with alterations in mitochondrial transcriptional activation and oxidative capacity. Thus the cellular events that limit Tfam expression may also act to restrain mitochondrial biogenesis as a whole. Because the increase in Tfam mRNA was the first noticeable event, it is likely that transcription of the Tfam gene plays an important role. However, given the remarkably slow import rate of Tfam into mitochondria compared with other matrix proteins (60), it also seems reasonable that accelerations in import rate, coincident with augmented Tfam expression as a result of contractile activity, compliment each other in facilitating the overall rate of mtDNA replication and transcription.

A second transcription factor is now established as important in regulating mtDNA. Apart from Tfam, a 43-kDa protein known as p43 has recently been described that binds both thyroid hormone (T₃) as well as DNA sequences in the mtDNA D-loop control region (22, 195). This is an important finding because it helps to explain the direct action of T₃ on mitochondrial transcription in in vitro experiments using isolated mitochondria (22, 45). p43 was also shown to markedly influence muscle differentiation by enhancing mitochondrial enzyme activity and, as a result, the expression of the myogenic regulatory factor myogenin (147). If mitochondrial biogenesis in response to contractile activity is truly independent of hormonal (i.e., T₃) influences (see above), then p43 should play no role in the adaptation observed, and the only relevant transcription factor is likely Tfam. Indeed, it would be of interest to determine whether p43 has any ligand-independent (i.e., T₃-independent) influences or whether it exerts an additive effect with Tfam in determining the expression of mtDNA in muscle cells.

Mitochondrial Protein Synthesis

Recovery

	CONCLUSIONS AND FUTURE DIRECTIONS

TOP ABSTRACT INTRODUCTION FUNDAMENTALS OF MUSCLE... CELLULAR MECHANISMS OF... CONCLUSIONS AND FUTURE... REFERENCES

Contractile activity initiates a series of physiological and biochemical events that lead to mitochondrial biogenesis. Those events that are now established are shown in Fig. 5, an illustration that is meant to briefly highlight recent work, to describe the approximate time course of the changes involved, and to provide a working model for expansion as our detailed knowledge improves. Note that the resulting alteration in muscle phenotype leads to a metabolic adaptation that reduces the "stress signal" imposed by an acute exercise bout of the same intensity and duration, acting in a negative feedback fashion. Thus the signal to produce further mitochondrial biogenesis is presumably reduced in magnitude once the adaptation has taken place.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Exercise (i.e., contractile activity) produces time-dependent physiological, biochemical, and molecular changes within the contracting muscle cells (see the text for details). The magnitude of these changes is dependent on the intensity and duration of the aerobic exercise stimulus. This sequence of events produces mitochondrial biogenesis, in which the phenotype is altered from a low-oxidative white muscle to a higher oxidative red muscle. This change attenuates a number of metabolic responses to exercise (e.g., lactate production and removal) and results in improved endurance performance.

Interest in mitochondrial function, biogenesis, and gene expression in muscle and nonmuscle cells has never been higher. This is because mitochondria provide an excellent adaptive model for the study of organelle biogenesis in mammalian cells and because mitochondria are now known to be implicated in a wide variety of diseases. Some avenues of future research that are likely to be rewarding include the following, enumerated below.

1) Understanding the relationship between the capacity for oxidative phosphorylation, the formation of ROS, its effects on mtDNA mutations, and its consequences can be studied. This work would have implications for improving our understanding of the decline in oxidative capacity in aging muscles. It would also permit us to forecast the benefit of regular exercise and the potential of maintaining oxidative capacity above a muscle-specific threshold, below which mtDNA damage and organ dysfunction ensue (i.e., mitochondrial myopathy; Ref. 176).

2) The role of chronic contractile activity in modifying the process of apoptosis can be evaluated. It is now fully established that mitochondria have an important role in triggering some forms of programmed cell death in response to stress (62). A number of pro-apoptotic (e.g., Bax, cytochrome c, apoptosis-inducing factor) and anti-apoptotic (e.g., Bcl-2) proteins reside within the organelle. The function and expression of those proteins in muscle cells are worthy of investigation. One of the most fascinating challenges for those interested in mitochondrial biogenesis will be to determine the positive or negative role played by regular exercise in the modification of apoptosis, with consequences relevant to understanding the regulation of myonuclear domains (3).

3) The interactions of important participants (Tfam, PGC-1, AMPK, NRF-1) involved in mitochondrial biogenesis during a carefully planned time course of contractile activity, followed by recovery, within the initial stages of organelle biogenesis can be fully investigated. In addition, expansion of our knowledge with respect to the effects of contractile activity on the transcription, mRNA stability, or posttranslational modification of proteins other than cytochrome c is required.