© 2002 New York Academy of Sciences

ANTHONY W. LINNANEa, CHUNFANG ZHANGa, NATALIA YAROVAYAa, GEORGE KOPSIDASa, SERGEY KOVALENKOa, PENNY PAPAKOSTOPOULOSa, HAYDEN EASTWOODa, STEPHEN GRAVESb and MARTIN RICHARDSONc

a Centre for Molecular Biology and Medicine, Epworth Medical Centre, Richmond, Victoria 3121, Australia b Royal Melbourne Hospital, Orthopaedic Department, Gratten Street, Parkville, Victoria 3052, Australia c Epworth Medical Centre, 173 Lennox Street, Richmond, Victoria 3121, Australia

Address for correspondence: Anthony W. Linnane, Centre for Molecular Biology and Medicine, Epworth Medical Centre, 185-187 Hoddle Street, Richmond, Victoria 3121, Australia. Voice: +61-3-9426-4200; fax: +61-3-9426-4201. email: tlinnane@cmbm.com.au

In this paper, we review two parts of our recent work on human skeletal muscle. The first part mainly describes changes occurring during aging, whereas the second part discusses the functions of coenzyme Q10 (CoQ10), particularly in relation to the aging process. During the lifetime of an individual, mtDNA undergoes a variety of mutation events and rearrangements. These mutations and their consequent bioenergenic decline, together with nuclear DNA damage, contribute to the reduced function of cells and organs, especially in postmitotic tissues. In skeletal muscle, this functional decline can be observed by means of changes with age in fiber type profile and the reduction in the number and size of the muscle fibers. In addition to the functions of coenzyme Q10 as an electron carrier in the respiratory chain and as an antioxidant, CoQ10 has been shown to regulate global gene expression in skeletal muscle. We hypothesize that this regulation is achieved via superoxide formation with H2O2 as a second messenger to the nucleus. -------------------------------------------------- INTRODUCTION
PART 1: CHANGES DURING... PART 2: EFFECTS OF... CONCLUSION REFERENCES

INTRODUCTION

This paper reviews part of our continuing studies on the human aging process, particularly as related to skeletal muscle.1-4,6,7 We have earlier proposed that damage to tissue cells, as exemplified by mitochondrial DNA in cells, is of a stochastic nature,2 whereby cells within a single tissue would suffer from various degrees of damage. During the aging process, a "damage mosaic" develops where, in different individuals, different tissues in the same individual, different cells in the same tissue, and different organelles in the same cell have undergone a range of changes. The system is a dynamic one with repair and ongoing damage in a finely tuned equilibrium, which with age tilts increasingly towards damage and cell loss. The whole process is affected by the individual's genetic makeup and environmental factors also have an impact.

PART 1: CHANGES DURING THE AGING PROCESS

Changes in Mitochondrial DNA and Consequent Decline in Bioenergy Capacity in Human TissuesIt was proposed by Linnane and colleagues1 that during one's lifetime, numerous mtDNA mutations occur and accumulate in tissues and that accumulation of such mutations makes a significant contribution to the aging process.
This idea has gained significant support. Data from a number of laboratories has confirmed that various mutations of mtDNA occur with age in postmitotic tissues (for review, see Refs. 2 and 5). Our laboratory has applied the technique of extra-long PCR (XL-PCR) to analyze the mtDNA genome extracted from human vastus lateralis muscle of individuals of different ages. We have shown that the amount of full-length mtDNA that could be amplified by XL-PCR is decreased with age and that multiple heterogenous mtDNA deletions appear in skeletal muscle of older individuals.
The decrease in the amount of full-length mtDNA and the appearance of numerous mtDNA deletions seem to be associated with a decreased activity of cytochrome c oxidase (COX), the complex IV of the electron transport chain.6 To be able to correlate the changes in mtDNA with age with the age-related decline in bioenergetic capacity of the tissue, we have performed XL-PCR on isolated single skeletal muscle fibers of predetermined COX activity.
All COX-positive muscle fibers isolated from tissues of individuals of different ages were shown to contain full-length mtDNA as well as a small number of mtDNA deletions. By contrast, muscle fibers negative for COX activity did not contain a detectable amount of full-length mtDNA but had multiple heterogeneous mtDNA species that were different in different muscle fibers of the same individual. These data lead us to the conclusion that there is a link between the amount of full-length mtDNA and the bioenergenic capacity of the tissue, measured through the activity of COX.7

Muscle Fiber Types and Muscle Aging
An important aspect of muscle physiology is the recognition that skeletal muscle consists of two major fiber groups: type I and type II. Type I fibers have greater oxidative capacity and contain more mitochondria per cell and preferentially use oxidative phosphorylation for their bioenergy metabolism. Type II fibers, which can be further subgrouped into IIa, IIb, and IIx/d fibers, have fewer mitochondria per cell and are more glycolytic with respect to energy requirements. Fiber type and, consequently, the energy metabolism of muscle fiber, are determined by the myosin heavy chain, which is expressed by the fiber. The proportion of fiber types varies among different muscles and among the same muscle of different subjects.

Fiber type composition of some muscles has been reported to change with advanced age. Early studies on aging and muscle fiber type profile have shown that type I fiber percentage increases from 39% to 66% in skeletal muscle of men aged 20 to 70.8 However, other researchers have observed much smaller changes in the percentage of type I fibers with age (from 49% to 52%) or no statistically significant changes in fiber type proportion with age.9,10 The loss of muscle mass and changes of fiber type during aging may be secondary to age-related denervation of muscle fibers.
During the aging process, preferential denervation of type II muscle fibers occurs that can lead to their atrophy. Some type II fibers may be reinnervated by type I motor neurons, which leads to the transition of type II fibers into type I fibers. As a result of these processes, the proportion of type I fibers in the elderly is higher, and the proportion of type II fibers lower, than in younger persons. It should also be noted that the aging profile is different in various muscles.
The above-mentioned phenomenon of the increase in the number of type I fibers and the decrease in the number of type II fibers with age has been reported, for example, for limb muscle including vastus lateralis. Other muscles, for example, human jaw muscles, exhibit the inverse change in fiber type profile with advanced age.11

Organ Cell Loss with Age
Another important aspect of skeletal muscle aging studies is the recognition of a gradual loss of muscle fibers throughout the life of the organism. It is considered that the muscle cell loss in humans is minimal before the age of 50 years. Thereafter, the loss accelerates and reaches about 5% per decade. The number of muscle fibers in one study has been shown to decrease by 39% in vastus lateralis muscle of old men (80+ years old) compared to young men (20-30 years old).12

Earlier studies of age-related changes in the central nervous system (CNS) have shown a similar rate of cell loss for CNS neurons. It has been reported that approximately 25% of CNS neurons are lost between the ages of 50 and 90 years, with an attrition rate of about 0.5% per year.13,14
Later studies of the age-related cell loss in CNS have shown that the cell loss is unequal in different parts of the brain, with some areas showing a slow, gradual loss of neurons with age and others not showing significant changes in neuron numbers. For brain areas exhibiting cell loss, the rate of fallout was estimated at about 10% over decades.15 For human cardiomyocytes, the cell loss between the ages of 18 and 90 years was estimated to reach 38 million cells per year in the left ventricle and 14 million cells per year in the right ventricle.16 It is also of great interest that the age-related cell loss is generally accompanied by considerable hypertrophy of remaining cells that may be necessary to compensate for the decrease in cell numbers.15,16

We suggest that cell loss is the quintessential characteristic of the aging process of postmitotic tissues. By contrast, in mitotic tissues such as liver, cells are being continually replaced over one's lifetime, so that the organ function is well maintained; thus, age-associated systemic degeneration of liver function is an uncommon phenomenon.

Dual COX/SDH Histochemistry to Follow Muscle Fiber Loss with Age As human aging is a gradual process taking place over decades, age-related changes observed at any one particular time point cannot be expected to be large relative to some earlier time.
We have used a dual COX and SDH (succinate dehydrogenase) enzyme histochemistry assay to visualize the aforementioned age-associated muscle fiber loss. The COX enzyme complex of the electron transport chain (complex IV) comprises 13 subunits; the three catalytic subunits of the COX enzyme complex are encoded by mtDNA. The loss of COX activity in some muscle fibers with age has been widely reported,17,18 and its loss coincides with nonfunctional mtDNA and mitochondrial energy loss.6,7 COX negative fibers are extremely rare in young persons, but are readily detected in skeletal muscle of persons over 50 years of age.17,18
The SDH enzyme complex (complex II) is a nuclear-encoded complex of the electron transport chain; therefore, negative SDH activity is a reflection of nuclear genome mutations.

We have used dual COX/SDH enzyme histochemistry to estimate the number of COX- and/or SDH-deficient fibers in vastus lateralis muscle of individuals of different age groups; thus, a large number of muscle fibers were examined for their COX and SDH reactivity (Table 1). The large majority of the fibers (97-99%) were COX and SDH positive. About 0-2%, however, were either of low COX activity or COX negative; more rarely and only in some patients, fibers that were both COX negative and SDH negative were detected (Fig. 1). We interpret the COX-negative fibers as severely damaged, probably progressing to death. On the other hand, the dual COX/SDH-negative fibers represent muscle cells that are either dead or in the process of dying and removal from the muscle mass as they represent cells whose mitochondrial and nuclear genomes have undergone extensive damage. These fibers exemplify the attrition rate of the tissue. Although only a low number of such fibers are seen at any one time over decades from the age of 50 to 80 years, it is suggested that such fibers could account for the overall loss of muscle mass and decrease in muscle function.

Gene Expression in Skeletal Muscle during Aging
There has long been a need for a global approach to the study of the aging process with its multiplicity of seemingly both related and unrelated random and nonrandom progression. Studies of identical twins separated early in life have demonstrated that even individuals with such closely related genomes can age very differently, emphasizing the key role played by an individual's physical and metabolic environment. It is then obvious that there will be a wide range of gene expressions in the tissues of individual subjects, some directly relevant, some irrelevant to the aging process.

About 5000-8000 particular genes can be estimated to determine an individual major cell type making up an organ such as skeletal muscle. The putative genes are largely unknown, and human gene microarrays are still in their developmental infancy. The results herein are too preliminary to comment upon except in a very generalized way, but they can serve to illustrate the direction in which future studies of the aging process will develop.

To begin the necessary studies, which will take many years to eventually detail and comprehend, we have applied microarray technology in order to study the role of changes in gene expression in muscle deterioration with age.
Eight subjects were arbitrarily assigned to four pairs, each consisting of a young subject and an old one. The ages of the individuals in the four pairs were 30/85, 56/84, 22/81, and 22/77 years. Messenger RNA (mRNA) was extracted, converted to cDNA, and labeled with the fluorescent dyes Cy-3 (for the young) and Cy-5 (for the old). The two fluorescently labeled samples (young and old) in each pair were mixed and used to hybridize a UniGEM V cDNA microarray (Incyte) containing 7000 genes and expressed sequence tags (ESTs). The hybridized array was then scanned, and the different intensities of the two fluorescent dyes were used as an indication of differential expression of particular genes.

A differential expression of a number of genes was seen in samples obtained from young and old subjects in each pair, as shown in Table 2. When the cutoff point was set at a twofold or greater difference, the number of genes showing higher expression in the young sample was 9, 14, 32, and 41 in the four pairs of samples. Eight, 7, 9, and 22 genes had higher expression in the old in the same four pairs of samples.

The differentially expressed genes were then compared between the four pairs of samples (Table 3). With 1.5-fold or greater difference as the cutoff point, only three genes changed expression consistently in all four pairs: two down-regulated and one up-regulated with age. Fifteen genes were consistent in any three pairs, and 88 genes consistent in any two pairs.

Among the genes with altered expression, the most prominent was the one encoding mitochondrial superoxide dismutase, which was down-regulated in old subjects in all four pairs, with an average 4.6-fold decrease in the old. Reactive oxygen species have been implicated in the aging process.19,20 Superoxide is particularly damaging to macromolecules, and superoxide dismutase is an essential enzyme to convert superoxide to the less toxic hydrogen peroxide, which can then be further detoxified. The downregulation of gene expression of this enzyme with age is particularly pertinent to the aging process. Indeed, overexpression of cytosolic superoxide dismutase (together with catalase) has been reported to delay the aging process in the fly Drosophila melanogaster,21 and treatment of the worm Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics increased the organism's mean lifespan.22

While our results are of a preliminary nature, they illustrate the power of these technologies for the study of the aging process. It can be readily envisioned that detailed gene expression and proteomic atlases of individual tissues of various ages will be a major future activity of many laboratories.

PART 2: EFFECTS OF CoQ10 IN SKELETAL MUSCLE OF AGED INDIVIDUALS

We have earlier reported on the re-energizing effect of CoQ10 and some of its analogues on skeletal muscle and isolated submitochondrial particles of aged rats.23-25 There are also a large number of anecdotal reports of the beneficial effects of CoQ10 for the treatment of a variety of apparently unrelated clinical syndromes, as diverse as congestive heart failure,26 muscular dystrophy,27 chronic fatigue syndrome,28 breast cancer,29 and primary biliary cirrhosis.30 It has also been suggested as an amelioration therapy, such as support for AIDS patients treated with AZT31 with improved immune function.32 However, the mechanism for these beneficial effects remains unclear.

In order to explore the wide-ranging effects of CoQ10 on human tissues, we have set up a clinical trial on the effects of CoQ10 on human vastus lateralis muscle. Human test subjects about to undergo hip replacement surgery received 300 mg CoQ10 per day for 25-30 days before surgery, whereas control subjects received placebo treatment. At the time of surgery, samples of vastus lateralis muscle were taken from the same region, and gene and protein expression patterns and muscle fiber type profiles were compared between placebo and CoQ10-treated subjects. We report here that CoQ10 regulates the expression of numerous genes and proteins. Furthermore, a dramatic change in muscle fiber types towards profiles of young people was observed in subjects treated with CoQ10. We conclude that CoQ10 can function as a gene regulator, which may account for its wide-ranging effects.

Effects of CoQ10 on Gene Expression in Skeletal Muscle of Aged Individuals
Studied by Microarray Analysis
As we have earlier reported,3 we assessed the effects of CoQ10 on gene expression in human skeletal muscle of aged individuals by gene microarray analysis. Skeletal muscle samples of three CoQ10 subjects (aged 70, 75, and 76 years) and two placebo subjects (aged 63 and 79 years) were studied. Total RNA isolated from each muscle sample obtained at surgery was used, after appropriate labeling, to hybridize a human U95A oligonucleotide array (Affymetrix) containing 12,000 annotated genes. We then compared the gene expression levels between each of the three CoQ10-treated persons and each of the two placebo subjects, generating a total of six comparisons. Many genes showed differential expression between the two samples in each comparison. Differentially expressed genes consistent in all six comparisons were then identified. At a cutoff point of 1.8-fold, there were 115 genes consistently differentially expressed in all six comparisons, with 47 genes up-regulated and 68 down-regulated in the CoQ10-treated subjects.

Examples of the up-regulated genes include the following: the glutamate receptor protein GluR5, which has a function in neuronal transmission and synapsis development;33 guanylyl cyclase, which is the receptor for nitric oxide signaling34 and is redox sensitive;35 fibroblast growth factor receptor N-SAM is essential for muscle growth and development; and a number of protein kinases that are involved in cell cycle control and cell signaling. The down-regulated genes include TTF-1 interacting peptide 20, which is important in transcription termination; and the TR3 orphan receptor, which is a steroid hormone receptor involved in apoptosis.36,37 Several transcription factors and the gene regulator hZFH helicase38 were also down-regulated, as was the major group Rhinovirus receptor, which is an adhesion molecule essential for cold virus infection.39 It is difficult to interpret these changes for a variety of reasons, and no attempt is made to do so, but the conclusion that can be reached is that CoQ10 modulates gene expression.

Effects of CoQ10 on Skeletal Muscle Proteome
In order to determine whether the regulation of gene expression by CoQ10 is also reflected at the protein level, the high-abundance proteome of vastus lateralis muscle samples from four placebo and three CoQ10-treated individual were analyzed with the aim of identifying proteins potentially regulated by CoQ10. By overlaying and matching the seven 2D gels in each of two pH ranges (pH 4-7 and pH 7-10), individual proteins could be identified and categorized into one of three groups: (a) specific proteins that only appear in the CoQ10 samples or at least three of the four placebo samples; (b) common proteins that appear in at least six of the seven samples; or (c) nonmatched proteins that appear in five or fewer samples nonspecifically between the treated and placebo samples.

Approximately 2000 proteins were visualized in each of the samples (Table 4). The results of protein matching are summarized in Table 5. Of the proteins detected, the expression of 229 proteins appear to be induced by CoQ10, whereas the expression of 236 appears to be repressed by the treatment.

Although of a preliminary nature, these results suggest that CoQ10 treatment is modulating muscle protein expression. Current work is concerned with the expansion of the number of samples in both the treated and placebo groups, and the characterization of specific proteins by MALDI-TOF mass spectroscopy.40

Change of Fiber Type Composition of Vastus Lateralis Muscle upon CoQ10 Treatment
Skeletal muscle is a highly heterogenous tissue consisting of a few different muscle fiber types that vary in their energy profile. The type of the muscle fiber is determined by the type of myosin heavy chain it expresses; some fibers express only one myosin heavy chain ("pure" fibers), whereas others may co-express two or more myosin heavy chains ("hybrid" fibers).
Myofibrillar actomyosin adenosine triphosphatase (mATPase)
histochemistry analysis is one of the most common procedures used for the delineation of muscle fiber types.41 This method is based on the observation that fast and slow myosins have different alkaline and acid stabilities. Histochemically, fast muscle fibers display high mATPase activity under alkaline conditions and low activity under acid conditions, whereas slow muscle fibers exhibit the inverse. Routine mATPase histochemistry allows delineation of the following fiber types: I, IIa, IIab, IIb, Ic, IIc, and IIac. The last three fiber types are intermediate in their staining characteristics between type I and type IIa fibers and are often referred to as C-fibers or IM (intermediate) fibers.

It is well known that aging results in a gradual loss of muscle function. This loss is due to the age-associated changes in the number and size of muscle fibers, and also to the age-related changes in the muscle fiber type composition. Type I fibers seem to be little affected by aging. Numerous studies have failed to show any significant changes in cross-sectional area of type I fibers with age,10,42,43 whereas type II fibers have been reported to decrease in size by about 25% from age 20 to 80.12 Changes in size for both type IIa and IIb muscle fibers have been documented.44

It should be emphasized that analysis of the quadriceps fiber type composition in humans is complicated because of the high variability of the muscle profile in different individuals. In addition, the proportion of fiber types varies significantly in different portions of the muscle, with a higher number of type I fibers in the medial part of the quadriceps and a lower number in the lateral part. Thus, for comparative analyses, it is important that the muscle tissue is collected from the same portion of the muscle in different subjects.

In our study, we have analyzed fiber type profiles of quadriceps muscles of 17 male subjects older than 57 years, eight receiving the placebo (mean age 65.5 ± 2.6) and nine receiving the CoQ10 supplementation (mean age 70.4 ± 2.7).
Despite large variations in fiber type composition both among placebo and CoQ10 subjects, it was clear that the placebo and CoQ10 samples were different in their fiber type composition. The number of type I fibers in vastus lateralis muscle of eight out of nine CoQ10-treated subjects was less than 50%, whereas five out of nine placebo samples showed the proportion of type I fiber type higher than 50% (Fig. 2). The difference in the number of type I fibers between placebo and CoQ10 patients was found to be statistically significant (P < 0.05). CoQ10-treated samples generally had a higher proportion of type II fibers (IIa, IIab, IIb), but the difference was statistically significant (P < 0.05) for type IIab fibers only (Fig. 3). The results obtained suggest that the patients receiving CoQ10 have an altered fiber type composition more reflective of younger muscle than the group receiving the placebo.

CONCLUSION

It is important to recall that the human organism ages to death over decades, so that deleterious aging effects on a yearly basis will be small. The number of cells of postmitotic tissues slowly decreases in late life, compensated for commonly by cellular hypertrophy and fibrous tissue expansion to maintain mass or even increase organ size. The tissues are in a dynamic equilibrium: Macromolecular damage and repair are finely balanced, with the equilibrium gradually shifting towards damage.

During a person's lifetime, mtDNA undergoes a variety of mutations and rearrangements. These mutations and the consequent bioenergenic decline, especially in postmitotic tissues, are among the factors contributing to the reduced function of cells and organs. Fiber type profiles of skeletal muscle change during aging, and the number and size of the muscle fibers are reduced. The COX/SDH double-negative fibers represent both mtDNA mutation and nuclear DNA damage in those cells, and these fibers are considered to be in the process of death and elimination.

In this paper we have asked the question whether CoQ10 can ameliorate the rate of tissue aging. The results suggest that this may indeed be possible. The data presented here indicate that CoQ10 functions as a major skeletal muscle gene regulator and modulates cellular metabolism. CoQ10, as demonstrated by microarray gene expression analysis, affects the expression of a number of genes. Proteome analysis reflects the global response of CoQ10 supplementation on the protein expression profile of the muscle tissue. In addition, skeletal muscle fiber types were shown to change as a result of CoQ10 administration to human subjects towards the muscle fiber profile of younger subjects.

How can CoQ10 have such wide-ranging effects and act as a gene regulator when it is membrane localized? The wide-ranging effects of CoQ10 may be explained by its broad-based cellular redox function. It has been proposed that CoQ10 plays a key role in manipulating the redox potential poise, thereby affecting subcellular membrane potential changes, resulting in the differential regulation of subcellular membrane activities and compartments.3 Different subcellular redox poises and their modulation would lead to wide-ranging metabolic changes. Furthermore, superoxide anions generated by reactions involving CoQ10 would reflect specific redox poise and would play a major role in cellular regulation. H2O2 produced from superoxide would act as a second messenger in the regulation of gene and proteome expression.

In deriving these concepts, the following facts were considered: It has been reported that the relative oxidation/reduction level of plastoquinone regulates chloroplast DNA transcription and the specific products of transcription. It was hypothesized that mitochondrial transcription may be similarly regulated.45 Through the Q cycle, CoQ10 is involved in determining mitochondrial membrane potential and, in turn, energy synthesis and mitochondrial substrate utilization. CoQ10 has also been shown to be an essential cofactor of the uncoupling proteins that act to downregulate mitochondrial membrane potential.46 The occurrence of a lysosomal CoQ10 oxido-reductase system, which establishes a proton gradient across the membrane, has recently been demonstrated.47 Such a system would contribute to the regulation of metabolite movement in and out of the lysosome.

Crane and colleagues have extensively reported on the properties of a CoQ10 NADH oxido-reductase enzyme complex in the plasma membrane, which again will contribute to redox potential poise and substrate movement.48 Furthermore, Crane et al.49 have made a preliminary report on a CoQ10 oxido-reductase localized in the Golgi membrane complex. Based on these observations, we have proposed that further studies may show that the CoQ10 located in other membrane systems reflects undiscovered oxido-reductase systems that will contribute to the determination of individual membrane potentials.3 CoQ10 has been shown to function as a proton/electron donor through which sulfhydryl/disulfide intraprotein crosslinks are converted, and, in part, it determines protein conformations.50

Finally, CoQ10 not only acts as an antioxidant, but also as a pro-oxidant, continually giving rise to superoxide anion, which is converted to H2O2 by superoxide dismutase. Because H2O2 has been shown to function as a mitogen and is involved in the regulation of gene expression,51,52 the regulation of gene expression by CoQ10 may be achieved using H2O2 as a second messenger.3

REFERENCES Linnane, A.W. et al. 1989. Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1: 642-645.[Medline] Kopsidas, G. et al. 2000. Tissue mitochondrial DNA change: a stochastic system. Ann. N.Y. Acad. Sci. 908: 226-243.[Abstract/Full Text] Linnane, A.W. et al. 2001. Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle. Free Radic. Res. In press. Linnane, A.W. 2000. Human skeletal muscle aging; a lifestyle determinant. Proc. Fifth Shizuoka Forum on Health and Longevity: 43-52. Shizuoka Perpetual Government, Japan. Nagley, P. & C. Zhang. 1998. Mitochondrial DNA mutations in aging. In Mitochondrial DNA Mutations in Aging, Disease and Cancer. K.K. Singh, Ed.: 205-238. Springer-Verlag and R.G. Landes. Georgetown, TX. Kovalenko, S.A. et al. 1998. Tissue-specific distribution of multiple mitochondrial DNA rearrangements during human aging. Ann. N.Y. Acad. Sci. 854: 171-182.[Abstract/Full Text] Kopsidas, G. et al. 1998. An age-associated correlation between cellular bioenergy decline and extensive mtDNA rearrangements in human skeletal muscle. Mutat. Res. 421: 27-36.[Medline] Larsson, L., B. Sjodoin & J. Karlsson. 1978. Histochemical and biochemical changes in human skeletal muscle with age in sedentary males age 22-65 years. Acta Physiol. Scand. 103: 31-39.[Medline] Lexell, J. et al. 1983. Distribution of different fibre types in human skeletal muscles: effect of aging studied in whole muscle cross-sections. Muscle Nerve 6: 588-593.[Medline] Aniansson, A. et al. 1981. Muscle morphology, enzyme activity, and muscle strength in elderly men and women. Clin. Physiol. 1: 75-86. Monemi, M. et al. 1999. Adverse changes in fibre type and myosin heavy chain compositions of human jaw muscle vs. limb muscle during ageing. Acta. Physiol. Scand. 167: 339-345. Lexell, J., C.C. Taylor & M. Sjostrom. 1988. What is the cause of the ageing atrophy? Total number, size and proportion of different fibre types studied in whole vastus lateralis muscle from 15- to 83-year old men. J. Neurol. Sci. 84: 275-294. Byrne, E. & X. Dennett. 1992. Respiratory chain failure in adult muscle fibers: relationship with ageing and possible implications for the neuronal pool. Mutat. Res. 275: 125-131.[Medline] Brooks, S.V. & J.A. Faulkner. 1994. Skeletal muscle weakness in old age: underlying mechanisms. Med. Sci. Sports Exerc. 26: 432-439.[Medline] Morrison, J.H. & P.R. Hof. 1997. Life and death of neurons in the aging brain. Science 278: 412-419. Olivetti, G. et al. 1991. Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy. Circ. Res. 68: 1560-1568. Müller-Höcker, J. 1990. Cytochrome c oxidase deficient fibers in the limb muscle and diaphragm of man without muscular disease: an age-related alteration. J. Neurol. Sci. 100: 14-21.[Medline] Nagley, P. et al. 1992. Mitochondrial DNA mutation associated with aging and degenerative disease. Ann. N.Y. Acad. Sci. 673: 92-102.[Abstract] Harman, D. 1956. Role of free radical and radiation chemistry. J. Gerontol. 11: 298-300. Harman, D. 1983. Free radical theory of aging: consequences of mitochondrial aging. Age 6: 86-94. Orr, W.C. & R.S. Sohal. 1994. Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263: 1128-1130.[Medline] Melov, S. et al. 2000. Extension of life-span with superoxide dismutase/catalase mimetics. Science 289: 1567-1569.[Abstract/Full Text] Linnane, A.W. et al. 1995. The universality of bioenergetic disease and amelioration with redox therapy. Biochim. Biophys. Acta 1271: 191-194.[Medline] Rosenfeldt, F.L. et al. 1999. Coenzyme Q10 improves the tolerance of the senescent myocardium to aerobic and ischemic stress: studies in rats and in human atrial tissue. Biofactors 9: 291-299.[Medline] Rowland, M.A. et al. 1998. Coenzyme Q10 treatment improves the tolerance of the senescent myocardium to pacing stress in the rat. Cardiovasc. Res. 40: 165-173.[Medline] Langsjoen, H. et al. 1994. Usefulness of coenzyme Q10 in clinical cardiology: a long-term study. Mol. Aspects Med. 15(Suppl.): s165-s175. Folkers, K. & R. Simonsen. 1995. Two successful double-blind trials with coenzyme Q10 (vitamin Q10) on muscular dystrophies and neurogenic atrophies. Biochim. Biophys. Acta 1271: 281-286.[Medline] Werbach, M.R. 2000. Nutritional strategies for treating chronic fatigue syndrome. Alt. Med. Rev. J. Clin. Ther. 5: 93-108. Lockwood, K. et al. 1995. Progress on therapy of breast cancer with vitamin Q10 and the regression of metastases. Biochem. Biophys. Res. Commun. 212: 172-177.[Medline] Watson, J.P. et al. 1999. Case report: oral antioxidant therapy for the treatment of primary biliary cirrhosis: a pilot study. J. Gastroenterol. Hepatol. 14: 1034-1040.[Medline] Tanner, H.A. 1992. Energy transformations in the biosynthesis of the immune system: their relevance to the progression and treatment of AIDS. Med. Hypoth. 38: 315-321.[Medline] Folkers, K., M. Morita & J. McRee Jr. 1993. The activities of coenzyme Q10 and vitamin B6 for immune responses. Biochem. Biophys. Res. Commun. 193: 88-92.[Medline] Dingledine, R. & P.J. Conn. 2000. Peripheral glutamate receptors: molecular biology and role in taste sensation. J. Nutr. 130(Suppl.): 1039S-1042S. Koesling, D. 1999. Studying the structure and regulation of soluble guanylyl cyclase. Methods 19: 485-493.[Medline] Dierks, E.A. & J.N. Burstyn. 1998. The deactivation of soluble guanylyl cyclase by redox-active agents. Arch. Biochem. Biophys. 351: 1-7.[Medline] Li, H. et al. 2000. Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 289: 1159-1164.[Abstract/Full Text] Uemura, H. & C. Chang. 1998. Antisense TR3 orphan receptor can increase prostate cancer cell viability with etoposide treatment. Endocrinology 139: 2329-2334.[Abstract/Full Text] Aubry, F., M.G. Mattei & F. Galibert. 1998. Identification of a human 17p-located cDNA encoding a protein of the Snf2-like helicase family. Eur. J. Biochem. 254: 558-564.[Abstract] Bella, J. & M.G. Rossmann. 2000. ICAM-1 receptors and cold viruses. Pharm. Acta Helv. 74: 291-297.[Medline] Walsh, B.J., M.P. Molloy & K.L Williams. 1998. The Australian Proteome Analysis Facility (APAF): assembling large scale proteomics through integration and automation. Electrophoresis 19: 1883-1890.[Medline] Brooke, M.H. & K.K. Kaiser. 1970. Muscle fibre types: how many and what kind? Arch. Neurol. 23: 369-379.[Medline] Lexell, J. & C.C. Taylor. 1991. Variability in muscle fibre areas in whole human quadriceps muscle: effects of increasing age. J. Anat. 174: 239-249.[Medline] Jakobsson, F., K. Borg & L. Edstrom. 1990. Fibre type composition, structure and cytoskeletal protein location of fibres in anterior tibialis muscle: comparison between young adults and physically active aged humans. Acta Neuropathol. 80: 459-468.[Medline] Coggan, A.R. et al. 1992. Histochemical and enzymatic comparison of the gastrocnemius muscle of young and elderly men and women. J. Gerontol. 47: B71-B76.[Medline] Pfannschmidt, T., A. Nilsson & J.F. Allen. 1999. Photosynthetic control of chloroplast gene expression. Nature 397: 625-628. van Belzen, R. et al. 1997. The iron-sulfur clusters 2 and ubisemiquinone radicals of NADH:ubiquinone oxidoreductase are involved in energy coupling in submitochondrial particles. Biochemistry 36: 886-893.[Medline] Gille L. & H. Nohl. 2000. The existence of a lysosomal redox chain and the role of ubiquinone. Arch. Biochem. Biophys. 375: 347-354.[Medline] Morre, D.J. et al. 1993. NADH oxidase activity of rat liver plasma membrane activated by guanine nucleotides. Biochem. J. 292: 647-653.[Medline] Crane, F.L. et al. 1984. Coenzyme Q in Golgi apparatus membrane redox activity and proton uptake. In Biomedical and Clinical Aspects of Coenzyme Q. Vol. 4. K. Folkers & Y. Yamamura, Eds.: 77-85. Elsevier. Amsterdam. Glockshuber, R. 1999. Where do the electrons go? Nature 401: 30-31.[Medline] Rusnak F. & T. Reiter. 2000. Sensing electrons: protein phosphatase redox regulation. Trends Biochem. Sci. 25: 527-529.[Medline] Smith, J., E. Ladi, et al. 2000. Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. Proc. Natl. Acad. Sci. USA 97: 10032-10037.[Abstract/Full Text]