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COMPARATIVE NUTRITION PAPERS MINI

REVIEW

MITOCH~NDRIAL ADAPTATIONS TO CHRONIC USE: EFFECT OF IRON DEFICIENCY

MUSCLE

DAVID A. HOD,* ROGERKELTON and MARYL. NK%IIO Department of Physical Education, York University, 4700 KeeIe St, North York, Ontario, Canada, M3J lP3 (Received 23 April 1991)

Abstract-I. The effects of chronic muscle use on mit~hon~al structure, enxymes and gene expression is reviewed. The role of iron deficiency in modulating this adaptation is discussed. 2. Chronic muscle use and disuse alter mitochondrial composition and alIe& mitochondrial subpopulations differentially. This has implications for an understanding of organelle assembly. 3. Iron deficiency decreases mitochondrial functional mass within muscle by reducing the level of heme and non-heme iron-containing components. This alters the metabolic response during exercise. and results in a reduced endurance performance. 4. Both iron deficiency and chronic muscle use represent contrasting experimental models for the study of mitochondrial function and biogenesis.

use provides an excellent model for the study of mitochond~~ biogenesis and its physiolo~c~ consequences. Recent work has begun to explore the underlying cellular mechanisms through which mitochondria are assembled. As a starting point, clues can be obtained from morphological studies.

INTRODUCTION

Muscle performance in endurance-type exercise events is dependent on three factors: (1) adequate muscle blood flow, (2) the mitochondrial content of the exercising muscle, and (3) the economy of ATP turnover, determined by the presence of specific myosin and sarcoplasmic reticulum ATPase isoforms (Holloszy, 1988; Laughlin and Armstrong, 1985; Rall, 1985; Terjung and Hood, 1986). These important factors are adaptable in response to chronic muscle use, performed either in the form of regular, locomotory exercise, or ex~~mentally induced by chronic, indirect muscle stimulation via the motor nerve. The adaptations are of physiological benefit in improving the capacity of the muscle for aerobic metabolism, and therefore prolonging submaximal exercise performance. The present paper will provide a review of the mit~hond~al adaptations in muscle subject to chronic muscle use, and will discuss the role of iron deficiency and its metabolic and cellular consequences. M~~HGNDRIAL

ADA~ATIONS MUSCLE USE

TO CHRONIC

Historically, endurance training has provided a good model for the study of alterations in the mitochondrial content of muscle. Holloszy (1967) first demonstrated that endurance training of sufficient intensity and duration provided an adequate stimulus for the synthesis of mitochondria within skeletal muscle. Since that pioneering work, a large number of studies (cf. later) have verified that chronic muscle *To whom correspondence

should be addressed.

~~~~chondr~~ structure and populations Mitochondria are present in skeletal muscle in a variety of shapes and sizes. They exist in two distinct areas of the cell: under the sarcolemma (subsarcolemmal, or SS mit~hond~a) and between the myofibrils (inte~yofib~llar, or IMF ~t~hond~a). Some work has illustrated these as distinct populations (Ogata and Yamasaki, 1985) while other investigators have suggested that mitochondria exist as a continuous reticulum-like structure (Bakeeva et al., 1978; Kirkwood et al., 1986). The work of Kayar et al. (1988) suggests that both situations may exist. Computer reconstructions of electron micrographs of serial sections were obtained from horse muscle fibers possessing inherently high and low mitochondrial contents. The complexity and variety of mitochondrial structure was evident from the appearance of small, oval structures, as well as those resembling mitochondrial columns. In high mitochondrial content fibers, structures resembling a network of interconnected columns were also apparent. These data are strongly suggestive of the pattern of mitochondrial growth and expansion during adaptation to chronic muscle use. Furthermore, the data confirm the hypothesis that muscle mitochondria are not stagnant structures, but are in a dynamic state of budding, branching and expansion during adaptive increases in ~t~hond~al volume (Kirkwood et al., 1987).

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The studies of Kayar et al. (1988) also illustrated the presence of SS and IMF mitochondria. However, it was evident that this geographical distinction between the two populations became less well defined in high mitochondrial content fibers. Thus, during adaptive stages of biogenesis, mitochondrial structure and volume probably expand to infiltrate adjacent cell areas. None the less, sufficient evidence appears to exist suggesting differences in the properties of these two populations. Subsarcolemmal mitochondria adapt more readily, and to a greater degree, during mitochondrial synthesis induced by chronic muscle use (Hoppeler et al., 1973, 1987; Kreiger et al., 1980). The opposite trend occurs during muscle disuse: SS mitochondrial content decreases more than does the IMF mitochondrial content (Desplanches et al., 1990; Kreiger et al., 1980). The fact that IMF and SS mitochondria are differentially affected by ischemia (Duan and Karmazyn, 1989) and myopathies (Hoppel et al., 1982) is further suggestive that they have functionally different roles in muscle. However, this remains to be proven. In cardiac muscle, biochemical studies have indicated that isolated IMF and SS mitochondria possess enzymatic and respiratory properties which distinguish them from one another (Palmer et al., 1977, 1985). This is less well documented in skeletal muscle, although some evidence exists supporting differences in rates of state 3 respiration, as well as in enzyme activities (Federico et al., 1987; Kreiger et al., 1980). Preliminary results indicate that the underlying basis for functionally distinct mitochondrial populations may be differing capacities for mitochondrial protein synthesis (Cogswell and Hood, unpublished observations). Models of mitochondrial biogenesis in muscle

A number of studies have reported an increase in the size, number and volume of mitochondria in response to endurance training (Gollnick and King, 1969; Hoppeler, 1986; Morgan et al., 1971). These mitochondria appear normal under the electron microscope (Terjung et al., 1972) and exhibit excellent respiratory control and tightly coupled oxidative phosphorylation (Holloszy, 1967). The rates of oxidative phosphorylation and electron transport, as well as the activities of enzymes providing reducing equivalents for oxidation, can be increased up to 2-fold, depending on the intensity and duration of the training protocol employed (cf. Holloszy and Booth, 1976; Saltin and Gollnick, 1983; Terjung and Hood, 1986 for reviews). It has been emphasized that the complexity of mitochondrial morphology is markedly increased during biogenesis of the organelle induced by chronic muscle use (Salmons et al., 1978). In order to evaluate this, as well as the molecular events underlying these morphological changes, a model which results in more rapid and extensive mitochondrial biogenesis than found with endurance training would be advantageous. Chronic, indirect, low frequency (10 Hz) stimulation of the motor nerve (Salmons and Vrbova, 1969) is a suitable model, since the mitochondrial changes are large (2-6 fold, Hood et al., 1989; Reichmann et al., 1985). With 24 hr of continuous stimulation at 10 Hz, a 2.5fold increase in mitochondrial enzyme activities can be achieved in 5-10 days

(Takahashi and Hood, unpublished observations). This represents a remarkable increase in the rate of mitochondrial biogenesis when compared to endurance training, which typically requires 6-12 weeks to elicit 1%2-fold increases in mitochondrial enzyme activities (Dudley et al., 1982; Holloszy, 1967; Hood and Terjung, 1987). Thus, mechanisms of mitochondrial biogenesis are more easily studied in the chronic stimulation model. The imposed 10 Hz stimulation pattern represents an immediate increase in workload for the muscle from the onset of the protocol. This results in well characterized metabolic and contractile changes within the muscle (Green et al., 1990; Hood and Parent, 1991) and does not invoke whole body temperature or hormonal reactions found during normal endurance training. Thus, adaptations observed are most easily ascribed to muscle use (Pette, 1986). Species -speciJic responses

The adaptive responses of oxidative enzymes within muscle to chronic stimulation appear to be species-specific. This has been documented with regard to mitochondrial enzymes, as well as the transition of lactate dehydrogenase from the M- to H-subunit type of isoform. Greatest changes are observed in the rabbit, intermediate in the rat and guinea pig, and lowest in the mouse (Simoneau et al., 1990). These differential responses may be due to the initial levels of mitochondrial enzymes found in these species. The magnitude of the adaptation to a standard workload appears to be inversely related to the enzyme level initially present. The phrase “adaptive range” has been coined to describe the species-specific alterations which occur (Simoneau and Pette, 1988), since the imposed 10 Hz workload does not lead to equalization of the enzyme activities in each species. Mitochondrial gene expression

Mechanisms of mitochondrial adaptations have begun to be investigated at the level of gene expression. Williams et al. (1986) first illustrated the use of the chronic stimulation model to study the expression of nuclear and mitochondrial genes at the mRNA level. Twenty-one days of chronic stimulation of the rabbit tibialis anterior muscle led to a 5-fold enhancement of cytochrome b mRNA, as well as a 4-fold increase in mitochondrial DNA. Subsequently, Williams et al. (1987) demonstrated 1.9- and 5.9-fold increases in cytochrome c oxidase subunit VIc and cytochrome b mRNA levels, respectively, under similar stimulation conditions. These data illustrated an uncoordinated response between a mitochondrial gene product (cytochrome b) and a nuclear gene product (subunit VIc) during the mitochondrial biogenesis induced by chronic muscle use. This was recently confirmed by Annex et al. (1991) who have reported a very rapid (3 days) increase in nuclearencoded citrate synthase mRNA levels in response to chronic stimulation in the rabbit. The increase occurred prior to any change in mitochondrial gene products, suggesting that molecular signals induced by chronic muscle use initially affect nuclear gene expression. This is a reasonable hypothesis, since many of the proteins required for mitochondrial gene

Mitochondrial adaptations in muscle expression are nuclear gene products (Fox, 1986). However, when two proteins which combine to form the same holoenzyme were examined, a coordinated response was observed. Hood et al. (1989) confirmed the results of Williams et al. (1987) with respect to increases in cytochrome c oxidase subunit VIc mRNA levels in the rat, and illustrated that these changes were coordinated with the level of the mitochondrially-encoded subunit III mRNA from 3 to 35 days of chronic stimulation. This coordinated expression was also demonstrated under steady state conditions between selected rat tissues possessing a 13-fold range of mitochondrial cytochrome c oxidase activity (Hood, 1990). These studies also illustrated that pre-translational regulation of the expression of the nuclear and mitochondrial genomes could not account for all of the phenotypic changes observed. Thus, some evidence for translational or post-translational control was demonstrated (Hood et al., 1989; Seedorf et al., 1986; Williams et al., 1987). An intriguing possibility is that this post-translational regulation may occur at the level of holoenzyme mitochondrial protein import, or assembly. Compositional alterations in mitochondria: implications for orgunelle assembly Endurance training studies indicated that, following a 612 week period, the composition of mitochondria with respect to protein : lipid ratios, as well as the specific activities of several mitochondrial membrane and matrix enzymes, remain unaltered (Davies et al., 1981). Comparative studies have indicated that many mitochondrial enzymes exist as a “constant proportion group” across a wide range of oxidative capacities (Pette and Diilken, 1975). This appears to be also true during an adaptive response imposed by cross-innervation or chronic stimulation (Pette and Dtilken, 1975; Reichmann et al., 1985). In addition, it is evident that the rate of turnover of mitochondrial inner membrane components of the respiratory chain is approximately equal (5-8 days; Aschenbrenner et al., 1970; Booth and Holloszy, 1977; Druyan et al., 1969; Terjung, 1979) and that these components respond in a uniform way (Zfold) during the mitochondrial biogenesis induced by endurance training (Holloszy, 1967). In addition, morphological changes in mitochondrial volume density closely parallel alterations in enzyme activities belonging to this constant proportion group (Kirkwood et al., 1987; Reichmann et al., 1985). This information suggests that mitochondria are assembled in a coordinated fashion with respect to lipid and protein components, However, a class of subtle but well documented changes occur in mitochondrial composition as a result of muscle use. A significant increase (Gollnick and King, 1969; Salmons et al., 1978) or no change (Reichmann et al., 1985) in the surface density of the inner membrane following a period of chronic muscle use has been reported. An approximate 20% decline in the surface density of the outer membrane has been observed following 28 days of chronic stimulation (Reichmann et al., 1985). This may be related to the shorter half-life of outer membrane proteins relative to those proteins in the inner membrane (Druyan et al., 1969). It is also evident that some enzymes of

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the inner membrane (a-glycerophosphate dehydrogenase (GPDH), fl-hydroxybutyrate dehydrogenase (HBDH)) behave differently from those in the “constant proportion group” such as citrate synthase and succinate dehydrogenase. GPDH decreased in response to chronic muscle use (Holloszy and Oscai, 1969; Pette, 1984), while HBDH increased far more than those associated with the Krebs cycle and respiratory chain (Pette, 1984). Selected matrix enzymes (glutamate dehydrogenase, malate dehydrogenase, cr-ketoglutarate dehydrogenase) increased only 35-50% as much as the respiratory chain proteins following 12 weeks of endurance training (Holloszy et al., 1970). Enzymes of the intermembrane space (adenylate kinase) or associated with the outer membrane (creatine phosphokinase) were not found to increase following a training period (Oscai and Holloszy, 1971). Thus, adenylate kinase and creatine phosphokinase have reduced specific activities when expressed per mg mitochondrial protein, because of the increased amount of mitochondrial protein (Holloszy, 1967). These data provide support for alterations in mitochondrial composition in response to chronic muscle use, and illustrate that the expression of genes encoding proteins which are associated with mitochondria do not all behave uniformly. Some evidence also exists supporting the stepwise assembly of mitochondria. During development, liver mitochondria exhibit an increase in density during the latter stages of gestation (Aprille, 1986). This indicates that the insertion of protein occurs into a pre-existing lipid bilayer. This has also been demonstrated in yeast during the transition from an anaerobic to an aerobic environment (Aithal and Tustanoff, 1975), and in developing liver mitochondria where the formation of the phospholipid cardiolipin has been shown to precede that of cytochrome aa, (Hallman and Kankare, 1971). During the cell cycle of a leukemic cell line, Leprat et al. (1990) illustrated that membrane synthesis was followed by a time lag prior to the development of its functional organization. Finally, during the process of mitochondrial degradation induced by chronic denervation, the cardiolipin content of muscle decreased significantly by 5 days, and was subsequently followed by declines inner in mitochondrial membrane enzymes cytochrome c oxidase and succinate dehydrogenase by 8-14 days (Wicks and Hood, 1991). Taken together, these data are supportive of the stepwise accretion and removal of mitochondrial components during organelle turnover, and suggest that initiating events involve the synthesis or degradation of mitochondrial phospholipids. Physiological implications of mitochondriul biogenesis Work in recent years has led to a much more thorough understanding of the physiological implications of an enhanced mitochondrial content, and increased mitochondrial enzymes in muscle. As classic Michaelis-Menten kinetics would predict, a lower substrate concentration is required to achieve the same biological effect in a tissue with a greater enzyme content (Gollnick and Saltin, 1982; Holloszy et al., 1971). For example, in 1 g of muscle a lower free ADP (ADP,) concentration would be required to

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stimulate the same rate of oxygen consumption in trained vs untrained muscle. This has been elegantly demonstrated experimentally (Dudley et al., 1987). Since ADP, is a potent regulator of flux through phosphofructokinase (Newsholme and Start, 1973), and it activates AMP deaminase (Wheeler and Lowenstein, 1979), the physiological implications of this adaptation include a reduced production of lactate, IMP and ammonia in the cell (Constable et al., 1987; Dudley et al., 1987). This, along with the concomitant increase in enzymatic capacity for fatty acid utilization (Molt et al., 1971), result in a diminished glycogen utilization during a given submaximal work bout (Hollosxy, 1988). These adaptations form the basis for the improved endurance performance which is observed. EFFRCB

OF IRON DEFICIENCY ON MUSCLE MITOCWONDRIA

Iron deficiency has recently been adopted as a method for the investigation of the functional role of iron-containing proteins in blood (i.e. hemoglobin) and in muscle (i.e. myoglobin), particularly in mitochondria (i.e. cytochromes, iron-sulfur proteins). The treatment can be induced in weanling experimental animals by feeding them a diet which is reduced from the normal 50 mg iron/kg feed, to one containing 2-6mg iron/kg feed for 4-8 weeks. Not surprisingly, skeletal muscle mitochondria obtained from iron deficient animals demonstrate morphological changes. The cristae are sparse (Cartier et al., 1986), broken (Johnson et al., 1990), deformed and swollen (Harlan and Williams, 1988). Iron deficiency resulted in a 28% expansion of mitochondrial volume density, and mitochondria appeared to develop a more cylindrical shape (Johnson et al., 1990). In addition, mitochondria were more closely associated with an increased number of lipid droplets. Subsarcolemmal mitochondria illustrated the most dramatic alteration in structure in response to iron deficiency. These were more fragmented, larger, vacuolar structures in comparison to the inter-myofibrillar mitochondria (Johnson et al., 1990). This may be a direct consequence of the lack of iron, or may be secondary to a reduced synthesis of heme for incorporation into the apocytochromes (cf. later). These constituents of the respiratory chain appear not to be incorporated into the membrane in the absence of heme attachment (Cartier et al., 1986), and since they have structural (Tzagoloff, 1982) as well as functional roles in the inner membrane, lack of protein incorporation results in large morphological alterations. As outlined below, the protein stoichiometry which normally exists is no longer present in mitochondria isolated from iron deficient animals, resulting in mitochondria of grossly abnormal composition (Cartier et al., 1986; Ohira et al., 1987; Willis et al., 1987). Mitochondrial respiration is decreased by irondeficiency to variable degrees depending on the extent and severity of the condition. The magnitude of the decrease is greatest in red, oxidative muscle @O-60%), while fast-twitch white muscle is less affected (30-35%; McLane et al., 1981). This is undoubtedly the result of the accumulated effect of

iron deficiency on individual mitochondrial proteins. The heme iron-containing respiratory chain proteins cytochrome c and cytochrome c oxidase (Cartier ef al., 1986; Davies et al., 1982; Hagler et al., 1981; Harlan and Williams, 1988; McKay et al., 1983; h&Lane et al., 1981; Ohira et al., 1987; Willis et al., 1987) are diminished to 3060% of control values in animals raised on iron deficient diets. The non-heme iron-containing enzymes succinate dehydrogenase and NADH dehydrogenase are decreased even more markedly during iron deficiency, with values ranging from 930% of those in control muscle (Ackrell et al., 1984; Cartier et al., 1986; Davies et al., 1982; Maguire et al., 1982; McKay et al., 1983; Ohira et al., 1987). The non-heme iron-sulfur protein content is also diminished by approximately 4060% (Maguire et al., 1982). The effect on these proteins is greater in highly oxidative red muscle, than in low oxidative white muscle fiber sections (McKay et al., 1983). A variable response has been observed with respect to non-iron containing constituents of the inner membrane. Carnitine palmitoyltransferase, /Ihydroxybutyrate dehydrogenase (Cartier et al., 1986) and the adenine nucleotide translocator (Willis and Dallman, 1989) are not diminished during iron deficiency. However, ubiquinone and the F,-ATPase are reduced to 63 and 87% of control activities, respectively (Cartier et al., 1986). This effect may reflect a role for iron in the regulation of gene expression or assembly of these proteins. The response of matrix enzymes to iron deficiency depends on the severity and duration of the condition. Cartier et al. (1986) originally reported no effect of 8-10 weeks of a 6mg iron/kg diet on non-iron containing matrix enzymes. Subsequently, studies using more severe iron-deficiency treatments found increases in citrate synthase (CS), isocitrate dehydrogenase (ICD) and ketoacid CoA transferase in slow-twitch red and fast-twitch red muscle types (Ohira et al., 1987). In contrast, fast-twitch white muscle exhibited decreases in CS activity (Harlan and Williams, 1988; Ohira et al., 1987). It was postulated that the increases observed in these non-iron containing matrix enzymes were induced by the recruitment of the red fibers during posture and weight bearing. It this were true, then an exaggerated response should be observed following endurance training. Indeed, Willis et al. (1987) found greater increases in CS and ICD in trained, iron-deficient muscle. These data illustrate that the ratios apparent in the “constant proportion group” of enzymes (Pette and Dolken, 1975) may be forcibly disrupted during iron deficient conditions (Cartier et al., 1986). In addition, the adaptations observed in non-iron containing enzymes may serve to maintain ATP synthesis in the face of reduced 0, delivery and oxidative capacity (Johnson et al., 1990). Physiological and metabolic implications of iron dejiciency constituents The changes in mitochondrial documented above have profound implications for organelle function, as well as the metabolic response of the muscle to a given exercise workload. The respiratory capacity of muscle is markedly reduced, as is whole muscle V02 during contractions (McLane

Mitochondrial adaptations in muscle et al., 1981). This decreased oxidative capacity results in an adaptive shift in energy metabolism during exercise. A reduction in functional mitochondrial mass implies that a greater concentration of ADP, is required to sustain a given rate of oxygen consumption in iron deficient muscle (Dudley et al., 1987; Willis and Dallman, 1989). This effect contrasts directly with the role of chronic muscle use in enhancing mitochondrial enzyme concentration per gram of muscle mass. Thus, a greater ADP, content will serve to accelerate glycolytic flux, and will increase the free AMP (AMP,) content via the myokinase reaction. In turn, this will activate AMP deaminase, since the AMP, concentration is lower than the K, of AMP deaminase for its substrate (Tullson and Terjung, 1990), resulting in a greater rate of NH, and IMP production in iron deficient muscle. In addition, iron deficiency results in elevated blood glucose levels at rest and during endurance exercise (Davies et al., 1984), an increased lactate production (Davies et al., 1984; McLane et al., 1981; Willis et al., 1988), and a greater rate of glucose turnover during exercise (Henderson et al., 1986). These data suggest a greater reliance on gluconeogenesis in iron deficient animals (Henderson et al., 1986) to support both the accelerated glucose catabolism, and the increased substrate (i.e. pyruvate, lactate, alanine) delivery to the liver during exercise. Indeed, liver enzymes of amino acid metabolism are increased in iron deficiency (Azevedo et al., 1989). This shift away from lipid metabolism and towards more limited supplies of carbohydrate fuels is likely an important contributor to the reduced endurance performance observed in iron deficient animals (McLane et al., 1981; Perkkio et al., 1985). When the reduced mitochondrial oxidative capacity of the muscle is restored during iron repletion, this is closely associated with the amelioration of endurance performance (Davies et al., 1982). Implications of iron dejciency for heme metabolism Iron is an integral component of several mitochondrial proteins either by direct association with the molecule, as in Fe-S proteins of the respiratory chain, or through its association with heme, the prosthetic group of the cytochromes. Heme is involved in regulating the structure and function of hemoproteins, and it has been hypothesized that the synthesis of heme may be a limiting factor in mitochondrial assembly (Holloszy and Winder, 1979). Therefore, iron deficiency can have profound indirect effects on mitochondrial assembly through its association with heme. The effect of iron deficiency on the cellular heme content is unknown, but it is reasonable to assume that it is reduced, given the wealth of data (cf. earlier) which illustrate that iron deficiency decreases heme-containing proteins. However, no data are available to support this assumption. Heme synthesis and degradation Eight enzymes are involved in heme biosynthesis and three enzymes are associated with heme catabolism (Sassa, 1990). Delta-aminolevulinate synthase (ALA-S) catalyses the first step in heme synthesis, the condensation of glycine and succinylCoA to form delta-aminolevulinic acid (ALA). In

601

liver (Granick and Urata, 1963), and probably most other tissues (May et al., 1986; Sedman et al., 1982), ALA-S activity limits the rate of heme biosynthesis, and the reaction is a critical point of regulation. The synthesis of ALA-S is regulated at several stages from the transcription of the gene in the nucleus, to its tinal location in the mitochondria. ALA-S mRNA levels are subject to end-product feedback inhibition. Evidence for this is that hemin administration inhibits ALA-S gene transcription in rat liver (Yamamoto et al., 1988) and heme appears to negatively regulate ALA-S gene transcription in most other tissues as well (Srivastava et al., 1988). The cellular concentration of heme in vivo is sufficient to inhibit ALA-S gene transcription under basal conditions in chick embryo hepatocytes (May et al., 1986). A reduction of heme levels in rat liver derepresses the gene and increases ALA-S mRNA (Srivastava et al., 1989). The half-lives of the ALA-S protein and mRNA are short, on the order of 35-120min (May et al., 1986) and 20min (Yamamoto et al., 1988), respectively. Thus, heme availability can rapidly regulate its own synthesis through altered gene expression (Granick and Reale, 1978). Heme also appears to influence ALA-S synthesis via a post-translational mechanism, by inhibiting the removal of the pre-sequence from pre-ALA-S, and increasing its rate of turnover in the cytosol (Ades et al., 1983). This appears to be due to heme-mediated blocking of pre-ALA-S transport from the cytosol into the mitochondria (Hayashi et al., 1980). The final step in the heme synthetic pathway is the one responsible for the incorporation of iron into the porphyrin ring, catalyzed by heme synthase. The V_ of this enzyme is approximately 24-fold greater than that of ALA-S (Granick and Reale, 1978). Therefore, in the presence of sufficient substrate (i.e. iron), heme synthase activity is not rate-limiting for heme biosynthesis. However, it is conceivable that this enzyme could become a more dominant site of regulation during iron deficiency if cellular iron levels are severely reduced. Heme oxygenase (HO) catalyses the release of iron from the heme molecule, and is the major site of regulation in heme degradation. Expression of the HO gene is positively regulated by heme concentration. Alam and Smith (1989) showed that heme caused an increase in HO mRNA in hepatoma cells via transcriptional regulation. Role of heme in mitochondriul assembly Approximately 90% of mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and subsequently targeted and assembled in the proper compartment. If the final protein contains heme, heme attachment to the apoprotein is an important part of the assembly process. This is catalysed by heme lyase, an enzyme of the intermembrane space. The importance of heme attachment for protein (e.g. cytochrome) import was demonstrated in a study employing heme lyase deficient yeast mutants (Dumont et al., 1988). Mitochondria isolated from these mutants had negligible heme lyase activities, and were unable to import newly synthesized apocytochrome c. The import inhibition

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was specific for cytochrome c, since the import of the j-subunit of F,-ATPase was unaffected. Heme also appears to be required to maintain the stability of the protein, since apocytochrome c not imported into mitochondria exhibited increased rates of degradation (Dumont et al., 1988). Assembly of the respiratory chain enzyme cytochrome c oxidase (CYTOX) is also regulated by heme. CYTOX is a multisubunit inner membrane protein comprised of gene products from both the nuclear and the mitochondrial genomes. In yeast, heme has been shown to positively regulate the transcription of three of the nuclear-encoded subunits, IV, Va, and VI (Trawick et al., 1989). This was demonstrated by an increase in mRNA levels when heme-deficient cells were grown in the presence of heme precursors. Saltzgaber-Miiller and Schatz (1978) have further demonstrated that heme-deficient yeast grown in the absence of heme produced measurable quantities of only three of the seven CYTOX subunits. They showed that heme plays a role in the assembly of the subunits, since the residual subunits were not attached to each other when heme was not available. Reattie (1971) examined the relationship between heme synthesis and mitochondrial protein synthesis in animals with experimentally-induced porphyria. She found that the incorporation of radioactive precursors into heme was followed by increased levels of protein synthesis. When the increased synthesis of heme was abolished, mitochondrial protein synthesis was prevented, as was the induction of cytochromes aa, and b. Thus, the synthesis of heme appears to be closely related to mitochondrial protein synthesis. Taken together, this information suggests that heme plays a fundamental role in the assembly and function of mitochondria. Therefore, it is not surprising that iron deficiency has such marked and drastic effects on mitochondrial morphology, as noted above. Under contrasting conditions of mitochondrial biogenesis as induced by chronic contractile activity, the search for regulatory events which underlie this biogenesis has naturally led to the investigation of the heme pathway. Work has focused on ALA-S activity because of its rate-limiting role. Acute exercise has been shown to increase ALA-S activity, with a peak attained 6 hr post-exercise in cardiac muscle of untrained rats (Abraham and Tejung, 1978). In skeletal muscle, a peak in ALA-S activity was observed 17 hr post-exercise, and occurred in the absence of a change in cytochrome c content (Holloszy and Winder, 1979). Following a period of endurance training, an acute exercise bout no longer resulted in an increased ALA-S activity. It was hypothesized that following training the adaptive phase within the muscle was complete, and the exercise stress no longer posed a stimulus for mitochondrial biogenesis (Abraham and Terjung, 1978). ALA-S gene expression was also evaluated in an overloaded skeletal muscle model (Essig et al., 1990). These authors demonstrated a 4-fold increase in enzyme activity following 7 days of overload, but this was only accompanied by a 1.5-fold increase in ALA-S mRNA. Thus, post-transcriptional regulation must be important in increasing ALA-S activity during chronic muscle use. In this case, the increase

HOOD et al.

in ALA-S activity also preceded the increase in cytochrome c oxidase activity. These data suggest that, in muscle, ALA-S activity is controlled by additional factors other than changes in the free heme pool, as determined by cytochrome formation (Essig et al., 1990). The studies described above demonstrate the profound effect which heme has on the composition and assembly of mitochondrial proteins. A reduction in heme levels as a result of iron deficiency affects gene transcription, mitochondrial protein import and stability, as well as mitochondrial assembly. This results in a reduced mitochondrial functional capacity, which may, in part, be attenuated by chronic muscle use. EFFECT OF CHRONIC MUSCLE USE ON MITOCHONDRIA DURING IRON DEFICIENCY

Loss of functional mitochondrial mass following iron deficiency, coupled with the associated anemia and decreases in myoglobin stores, lead to a marked reduction in endurance performance. Endurance training has been shown to improve performance in iron deficient animals, and this is accompanied by alterations which appear not to be restricted solely to skeletal muscle. Perkkio et al. (1985) found that mild endurance training of iron-deficient animals, which was not sufficient in intensity to elicit changes in normal animal muscle, resulted in a lesser anemic condition, as well as 34% and 30-60% increases in muscle cytochrome c and cytochrome c oxidase activities, respectively. A 6-fold improvement in endurance performance accompanied these changes. Willis et al. (1987; 1988) demonstrated 15-58% increases in Krebs cycle enzymes, resulting in ratios of Krebs cycle enzymes:respiratory proteins which were maintained, or augmented, following a similar training protocol. It was suggested that this adaptation could serve to sustain a greater electron pressure in the face of a reduced phosphorylation potential during the stimulation of mitochondrial respiration (Willis et al., 1987). Chronic stimulation of iron deficient rabbit muscle elicited responses similar to those described above; 54 and 99% increases in cytochrome c and cytochrome c oxidase were observed (Harlan and Williams, 1988), while much larger increases (172%) were observed in citrate synthase, a protein which does not require iron for its synthesis. It appeared that muscles which were chronically used (heart, soleus, stimulated tibialis anterior) were less affected by the iron deficiency treatment, perhaps by more effectively competing for the limited iron stores available (Harlan and Williams, 1988). Cardiac muscle hypertrophy has also been demonstrated in iron deficient animals (Willis et al., 1988). Skeletal muscle appears to be quite capable of adapting to chronic exercise under iron deficient conditions, in attempting to compensate for the lack of iron-containing proteins. The marked amelioration in endurance performance is likely due, not only to adaptations within skeletal muscle, but also to beneficial changes in liver and cardiac function. However, the adaptations observed with respect to enzymes, morphology and endurance performance

Mitochondrial adaptations in muscle still fall short of compensating for the large effect which iron deficiency exerts (Harlan and Williams, 1988; Willis et al., 1988). The use of iron deficiency and chronic muscle use, either alone or in combination, provide interesting models for probing mitochondrial structure, biochemical adaptations, alterations in gene expression, as well as their physiological consequences. Future studies devoted to the interaction of heme metabolism with mitochondrial biogenesis in skeletal muscle appear warranted. REFERENCES

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