BACKGROUND
aging in skeletal muscle is characterized by loss of motor neurons, variations in size and type of muscle fibers, infiltration of fat and connective tissue, and a overall decrease in muscle mass   <cit> .
sarcopenia, the leading cause of frailty and disability in the elderly, has also been linked to other age-associated pathology, such as osteoporosis and impaired thermoregulation  <cit> .
several biological processes, such as an increase in inflammatory cytokines and a decrease in sex and growth hormones with age  <cit> , have been suggested as contributing factors to the aging of skeletal muscle.
at the cellular level, older muscle is associated with decreased protein synthesis  <cit> , an increase in dna, protein and lipid oxidation  <cit> , and the accumulation of mitochondrial abnormalities  <cit> .
several studies have employed microarray technology to uncover changes in gene expression that accompany aging in skeletal muscle of mice  <cit> , rats  <cit> , monkeys  <cit>  and humans  <cit> .
when taken as a whole, results obtained from these studies suggest that the expression of genes indicative of cellular damage, such as those involved in the stress or inflammatory response, increase with age, while expression of metabolic and biosynthetic genes decrease with aging.
we had previously investigated age-related changes in gene expression of mouse gastrocnemius tissue using high density oligonucleotide arrays  <cit> , but our study was limited by low sample number , an arbitrary fold change criteria  with no statistical analysis to determine differentially expressed genes, and a limited survey of genes expressed in the mouse genome .
we therefore decided to expand our initial investigation using a larger sample size , a stringent statistical criteria to determine differentially expressed genes , and a greater representation of the mouse genome .
our current microarray study of young and old c57bl/6nhsd mice supports previous findings on specific transcript classes differentially expressed with aging and also suggests that increased p <dig> activity in skeletal muscle of older animals may contribute to aging phenotypes.
caloric restriction, which extends the life span of this strain of mice by approximately 40%, prevents the majority of the changes in gene expression observed in the older mice under the control diet.
RESULTS
general
we used an empirical bayes  hierarchical modeling approach called ebarrays to identify  <dig> genes differentially expressed  in old mouse gastrocnemius tissue .
there are a number of advantages to our statistical approach.
the main ones are the ability to combine information across genes and the dual nature of posterior probabilities.
the posterior probabilities quantify evidence in favor of differential expression; however, unlike classic p-values, they can also be used to measure evidence in favor of equivalent expression.
measurements taken from gastrocnemius tissue from a group of old mice on caloric restriction , a regimen previously shown to increase lifespan and retard aging parameters in mice, was used as a negative control to aging.
there were  <dig> genes with higher expression in the young animals and  <dig> genes with greater expression in the old.
as expected, cr completely or partially reversed 87% of the genes with age-associated changes in expression .
in agreement with previous microarray studies done on skeletal muscle of mice, monkeys and humans, we also found that genes associated with metabolic functions, particularly energy pathways, were higher in young control animals .
in contrast, genes indicative of cellular damage, such as stress response and apoptotic genes, had higher expression levels in the muscle tissue of older animals .
among the biological classes of transcripts with an age effect, there were several gene families whose mrna levels were significantly increased or decreased with age .
there were  <dig> procollagen genes  that were altered with aging;  <dig> of these showed higher levels in the young muscle  while only one procollagen gene was increased with aging .
the keratins, important cytoskeletal structural proteins, were represented by  <dig> genes  all of which displayed increased transcript levels in older muscle.
three members  of the ccaat/enhancer-binding protein  family of transcription factors had increased levels of transcripts in the older mice.
the encoded cebp alpha, beta and delta proteins are important in the regulation of genes involved in immune and inflammatory responses, cell cycle arrest and body weight homeostasis .
because many genes are represented more than once on the moe430a affymetrix microarray, several genes had multiple hits on our list of genes with an age effect in skeletal muscle.
among those genes with four or more independent listings were: fibromodulin , transferrin receptor , immunoglobulin kappa chain variable  <dig> , and myristoylated alanine rich protein kinase c substrate  .
transcript and protein levels of p53-related genes altered with aging
given that skeletal muscle is largely a post-mitotic tissue, we were surprised to find that a large number of genes involved in the regulation of the cell cycle had increased mrna levels with age .
two gene products that are known to play a critical role in initiating cell cycle arrest following dna damage, cyclin-dependent kinase inhibitor 1a  and growth arrest and dna-damage-inducible, alpha , were both increased significantly in the skeletal muscle of the aged mice.
expression levels for p <dig> and gadd45a were  <dig>  and  <dig>  fold higher respectively in older mouse compared to the younger animals.
an age-related increase in p <dig> has previously been observed in muscle of both male and female humans   <cit>  and monkeys   <cit> , while the expression of gadd45a has been shown to be elevated in mouse gastrocnemius muscle   <cit>  and both male and female human muscle   <cit> .
because the expression of these two genes is typically mediated by the action of p <dig>  we decided to systematically examine our list of genes differentially expressed between young and old to determine if there were any other transcripts that are also connected to p <dig> activity.
as shown in table  <dig>  the transcripts that are activated by p <dig>  or whose gene products are known to bind to p <dig>  increase significantly with age, while the expression of genes that are known to inhibit p <dig> activity decline in old muscle.
the average cr prevention on these p53-related genes was 77%.
to confirm the age-related changes in expression observed in p <dig> and gadd45a using microarrays, we probed young, old and old cr mice using rt-pcr .
we also examined the expression levels of the p <dig> gene itself, along with the expression levels of another important tumor suppressor, p <dig>  both p <dig> and p <dig> transcripts were considered absent  in all animals according to the affymetrix software, but we were able to detect expression using the more sensitive method of rt-pcr.
as is shown in figure 1a, transcript levels for these genes increased significantly  with age.
furthermore, old animals undergoing cr demonstrated reduced levels for these genes compared to age-matched mice fed normal diets .
to examine how the expression of p <dig>  p <dig>  gadd45a and p <dig> is altered over the course of the adult mouse life span, we used rt-pcr to measure transcript levels for these genes in gastrocnemius muscle at  <dig> month intervals from  <dig> to  <dig> months of age .
with the exception of gadd45a , the increase in expression of these genes does not show a linear pattern with age.
p <dig> and gadd45a expression increases significantly only in the last third of the lifespan, whereas expression of p <dig> and its transcriptional target p <dig> occur as early as  <dig> months of age.
to determine whether the p53-mediated transcriptional program observed in older mouse skeletal muscle translated into higher levels of p53-associated proteins being expressed, we probed young, old and old cr muscle for p53-transcriptional targets p <dig> and gadd45a.
western immunoblot confirmed a significant increase in p <dig> protein  and gadd45a  in older mouse skeletal muscle .
levels for these proteins were reduced in the old cr mice as compared to old mice on the control diet .
an attempt was made with several antibodies to measure p <dig> protein levels  in gn muscle, but the signal from the western blots was either absent or too low to accurately quantitate p <dig> in any age group .
transcript levels of p <dig>  p <dig> and gadd45a in mouse models of increased oxidative stress
to test whether the changes in expression of the p53-related genes could be attributed to cumulative oxidative damage with age, we compared transcript levels for p <dig>  p <dig>  gadd45a and p <dig> in c57bl/ <dig> mice with a heterozygous knockout for either glutathione peroxidase  <dig>   or mnsod   mutations to age-matched wild-type mice.
gpx4+/-mice have approximately half the amount of mrna and protein levels for this essential se-dependent antioxidant protein as compared to wild type mice  <cit> .
mouse embryonic fibroblasts  derived from mice heterozygous for the gpx <dig> gene show increased evidence of lipid and dna oxidation, and are sensitive to gamma irradiation  <cit> .
sod2+/- mice show a ~50% reduction in mnsod protein content and mnsod activity in gatrocnemius tissue  <cit>  and have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine  in both nuclear and mitochondrial dna in all surveyed tissues compared to wild type mice  <cit> .
rt-pcr analysis of p <dig>  p <dig>  gadd45a and p <dig>  in gpx4+/- mice showed a significant increase  in p <dig> expression compared to the age-matched wild-type mice.
we did observe a ~ <dig> fold increase in p <dig>  although it was not statistically significant .
transcript levels of these genes were not significantly different between sod2+/- mice and age-matched controls .
thus, constitutive oxidative damage does not always trigger the expression of the genes examined.
possibly, different types of dna damage in gpx4-/+ and sod2+/- mice explain the observed differences.
increased p <dig> levels in older muscle is associated with increased expression of genes involved in p53-mediated apoptosis
a critical function of p <dig> is the induction of apoptosis in cells that have undergone dna damage or exhibit uncontrolled cellular growth  <cit> .
to investigate whether increased p <dig> activity with age is associated with transcription profiles indicative of apoptosis in old gastrocnemius muscle, we examined mrna levels of four genes known to be key transducers of p53-mediated apoptosis: bcl- <dig> binding component  <dig>   <cit> , phorbol-12-myristate-13-acetate-induced protein  <dig>   <cit> , tumor necrosis factor receptor superfamily, member 10b   <cit>  and bcl-2-related ovarian killer protein   <cit> .
we observed a significant increase in mrna levels with age for these genes in mouse gastrocnemius muscle and a significant decrease in levels due to caloric restriction in old, age-matched controls .
the expression levels for all of these p53-mediators of apoptosis were also examined in the gastrocnemius muscle over the course of the mouse adult lifespan , and increased levels of transcript were usually observed in the first half of the lifespan.
we also compared the levels for these proapoptotic genes in the gastrocnemius tissue of gpx4+/- and the sod+/- mice to age-matched wild type mice using rt-pcr.
only the levels for tnfrsf10b were considered significantly higher  in the gpx4+/- mice while expression of these genes was not significantly different in the sod2+/- mice .
these findings indicate that p53-dependent mediators of apoptosis are regulated with age and that the mechanism of activation is independent of endogenous levels of oxidative stress.
increase in mrna levels of p <dig> and p53-associated genes in multiple tissues of old c57bl6/j mice
to determine whether the p53-mediated transcriptional program we observe in older skeletal muscle may also occur in other tissues, we used rt-pcr to measure mrna levels of p53-associated genes in heart, lung, liver, kidney, brain and intestine of young, old, and old cr mice .
transcript levels for p <dig>  p <dig>  p <dig> and pmaip <dig> demonstrated increased levels in multiple tissue types in the old mice, while an increase in transcript levels for gadd45a, bbc <dig>  tnfrsf10b and bok was only observed in old skeletal muscle.
these findings suggest that activation of a p53-mediated transcriptional program may be a common feature of aging in multiple tissues, but the pattern of expression of p53-dependent genes is tissue specific.
discussion and
CONCLUSIONS
our initial investigation of muscle aging in mice revealed many differences in the transcriptional patterns between young and old mice  <cit> .
in general, transcript levels for genes that are associated with cellular damage were elevated in older muscle, while transcript levels for genes involved in energy metabolism were reduced with age.
the current study further demonstrates that the intrinsic transcriptional patterns are altered with aging in mouse skeletal muscle.
as with other microarray studies done on older mammalian muscle  <cit> , we have also found a disproportionate number of metabolic genes, such as those involved in lipid and carbohydrate metabolism, decrease in transcription levels with age.
the age-related decrease in expression of these energy-associated classes likely reflects the reduction in mitochondria observed in older gastrocnemius muscle.
for example, a 23–25% reduction in mtdna copy number and a corresponding 17–22% reduction in the expression of mitochondrial dna-encoded cytochrome c oxidase  subunits i and iii was previously reported in old rat gastrocnemius tissue  <cit> .
in our study, out of  <dig> transcripts that are associated with the mitochondria, the expression of  <dig> of these probe sets are significantly reduced with age  .
conversely, genes associated with the stress and immune response, such as those involved in the induction of apoptosis or the response to dna damage, have elevated mrna levels in the older animals.
the age-associated increase in genes that are activated during cellular stress and injury is probably in response to a wide variety of biological damage known to accumulate in older muscle over the course of the lifespan  <cit> .
we also observed that the expressions of a large number of genes involved in the regulation of the cell cycle were increased with age in mouse skeletal muscle.
a closer examination of these cell cycle genes revealed a pattern of expression indicative of increased p <dig> activity in older mouse muscle .
welle et al.
also found transcriptional evidence of increased p <dig> activity with age in the skeletal muscle of young  and old  women  <cit> .
in their study, genes that regulate or enhance p <dig> activity  or that are induced by p <dig>  demonstrate increased expression in older female muscle.
based on this evidence, welle and coworkers suggested that future studies should examine the possibility that p <dig> levels or activity are increased in older muscle.
one possible mechanism for the observed increase in p <dig> activity is elevated levels of dna damage with age.
the concentration of 8-oxo- <dig> deoxyguanosine , a byproduct of dna oxidation, has been observed to increase with age in a variety of tissues, including skeletal muscle, in rats and various strains of mice   <cit> .
we tested this hypothesis by examining the levels of p <dig> mrna and related transcripts in transgenic mice with half the wild type levels of antioxidants gpx <dig> or sod <dig>  these mice have elevated levels of oxidative dna damage yet failed to show any significant increase in expression of p <dig> and gadd45a, two well studied p53-induced genes .
the gpx4+/- mice did display a significant increase in transcript levels for p <dig> as compared to age-matched controls, but no change was observed for the sod2+/- mice.
possibly, different types of dna damage impact the expression of these genes differently.
another possibility is that the dna damage signaling that is the trigger for activation of p <dig> in old muscle originates from chromosomal damage, such as double-strand breaks or telomere shortening, rather than from oxidative alterations.
we also note that there are two likely possibilities for the cellular origin of the alteration in expression of cell cycle regulators.
one is that these alterations in gene expression reflect a decline in proliferation of a muscle-associated cell such as the satellite cell.
it has recently been reported that hematopoietic stem cell numbers are higher in old mice with reduced p <dig> activity  and fail to increase with age in old mice with hypermorphic p <dig> activity  as compared to their wild type counterparts  <cit> .
a second possibility is that the expression of these genes reflects a general p53-mediated dna damage response associated with age-related dna modifications in myocytes.
many recent studies have linked tumor suppressor activity, in particular the tumor suppressor p <dig>  to organismal aging  <cit> .
p16ink4a, a product of the ink4a/arf  locus also increases in expression with age in most tissues as shown in previous studies  <cit>  and also in the current analysis.
this increase in p16ink4a expression with age has recently been shown to contribute to age-dependent decline in regenerative potential of pancreatic islet regenerative capacity  <cit> , decreases in forebrain progenitors and neurogenesis  <cit>  and the decrease in hematopoietic stem cell repopulation defects  <cit> .
however, we note that it is possible that only rare cells in tissues express p16ink4a with aging.
under these conditions, a large effect of increased ros in a specific biologically relevant muscle compartment  could be missed by studying unfractionated muscle as we did in our analysis.
thus, the induction of a cell senescence and apoptotic program with age may underlie key features of the aging process.
given our finding that mrna levels for p53-associated genes were elevated in multiple tissues of old mice , our data suggests that increased p <dig> activity is not limited to aging muscle but is also an overall consequence of organismal aging.
increased p <dig> mrna levels is observed in the grey and white matter of the cortex, cerebellum, and medulla oblongata of old  verses young  rat brains and an increase in p <dig> protein levels is observed in the grey matter of old rats  <cit> .
we did not find a significant difference in p <dig> gene expression between young and old brains, but our analysis was performed on whole brain homogenates and therefore any age-effect on p <dig> expression within the different regions of the brain might have been missed.
further support for the role of increased p <dig> activity contributing to aging process comes from transgenic studies in mice that express overactive p <dig> and also demonstrate accelerated aging  <cit> .
mice with augmented p <dig> function develop typical age-associated phenotypes such as delayed wound healing, reduced hair growth and reduced subcutaneous adipose tissue much earlier than their p <dig> wild type counterparts.
conversely, the long-lived p66shc-/- mouse, shown to have a 30% extended life span, exhibits impaired p <dig> apoptotic signaling  <cit> .
our study demonstrates that the majority of the p53-related transcriptional changes observed in old skeletal muscle are either completely or partially reversed through caloric restriction, a proven method to extended longevity.
based on the evidence for the involvement of the p <dig> pathway in organismal aging, it follows that p <dig> and p <dig> transcriptional targets have the potential to be good biomarkers of aging in multiple tissue types.
it has been well documented that there is a significant decrease in muscle mass with age, yet little is known as to the actual mechanism for this loss of tissue.
it has been hypothesized that the decrease in muscle fiber can be attributed to increased apoptosis with age.
in fact, an increase in biological markers of apoptosis, such as dna fragmentation and proapoptotic caspase-type proteins, are increased with age in human  <cit>  and rat skeletal muscle  <cit> .
however, the mechanism for this age-associated apoptosis is unknown.
it has been suggested that increased oxidative damage to the mitochondria, along with sarcoplasmic reticulum stress in older skeletal muscle could initiate caspase activation and lead to the apoptotic pathway that ultimately results in the destruction of the aged myocyte  <cit> .
moreover, dirks and leeuwenburgh also observed a significant reduction in dna fragmentation  and caspase levels in old  rats on lifelong caloric restriction when compared to their age-matched, ad libitum-fed counterparts.
the authors suggest the reduction of apoptosis in response to caloric restriction is due to reduced mitochondrial oxidant production and nuclear oxidative dna damage in skeletal muscle  <cit> .
our results do not support this hypothesis.
although young gpx4+/- mice did show increased levels of expression, compared to wild-type young mice, of p53-associated mediator of apoptosis tnfrsf10b, little or no change was observed for bbc <dig> and bok .
the sod2+/- mice, on the other hand, demonstrated almost identical levels of expression for all of the surveyed p <dig> mediators of apoptosis to the age-matched, wild type controls.
it is possible that increased oxidative damage may play a role in p53-mediated apoptosis in older muscle, but our data suggests that it may not be the sole causative effect.
old calorie restricted mice displayed a significant decrease in expression levels of all four of the p53-mediators of apoptosis compared to the normally fed old mice.
it is clear that caloric restriction has an inhibitory effect on age-associated apoptosis of skeletal muscle, but this prevention may not be completely explained by the reduction of endogenous reactive oxygen species and the resulting decrease in oxidative dna damage in these animals.
the role of p <dig> in the aging process of skeletal muscle is likely to be a complex process involving many different cellular effectors.
various proteins and pathways interact with p <dig> to modulate its activity.
furthermore, there are numerous p53-transcriptional targets, each with their own associated modulators that may influence muscle aging.
as evidence of this complexity, we show that the expression profiles for p53-regulated genes are not identical to that of p <dig>  as well as to each other, over the course of the mouse adult lifespan in gastrocnemius muscle .
future studies will concentrate on determining which specific components of the p <dig> pathway play a functional role in muscle aging, and organismal aging in general.

 aging in skeletal muscle is characterized by loss of motor neurons, variations in size and type of muscle fibers, infiltration of fat and connective tissue, and a overall decrease in muscle mass. sarcopenia, the leading cause of frailty and disability in the elderly, has also been linked to other age-associated pathology, such as osteoporosis and impaired thermoregulation. several biological processes, such as an increase in inflammatory cytokines and a decrease in sex and growth hormones with age, have been suggested as contributing factors to the aging of skeletal muscle. at the cellular level, older muscle is associated with decreased protein synthesis, an increase in dna, protein and lipid oxidation, and the accumulation of mitochondrial abnormalities. several studies have employed microarray technology to uncover changes in gene expression that accompany aging in skeletal muscle of mice, rats, monkeys and humans. when taken as a whole, results obtained from these studies suggest that the expression of genes indicative of cellular damage, such as those involved in the stress or inflammatory response, increase with age, while expression of metabolic and biosynthetic genes decrease with aging. we had previously investigated age-related changes in gene expression of mouse gastrocnemius tissue using high density oligonucleotide arrays, but our study was limited by low sample number, an arbitrary fold change criteria with no statistical analysis to determine differentially expressed genes, and a limited survey of genes expressed in the mouse genome. we therefore decided to expand our initial investigation using a larger sample size, a stringent statistical criteria to determine differentially expressed genes, and a greater representation of the mouse genome. our current microarray study of young and old c57bl/6nhsd mice supports previous findings on specific transcript classes differentially expressed with aging and also suggests that increased p activity in skeletal muscle of older animals may contribute to aging phenotypes. caloric restriction, which extends the life span of this strain of mice by approximately 40%, prevents the majority of the changes in gene expression observed in the older mice under the control diet.  general we used an empirical bayes hierarchical modeling approach called ebarrays to identify genes differentially expressed in old mouse gastrocnemius tissue. there are a number of advantages to our statistical approach. the main ones are the ability to combine information across genes and the dual nature of posterior probabilities. the posterior probabilities quantify evidence in favor of differential expression; however, unlike classic p-values, they can also be used to measure evidence in favor of equivalent expression. measurements taken from gastrocnemius tissue from a group of old mice on caloric restriction, a regimen previously shown to increase lifespan and retard aging parameters in mice, was used as a negative control to aging. there were genes with higher expression in the young animals and genes with greater expression in the old. as expected, cr completely or partially reversed 87% of the genes with age-associated changes in expression. in agreement with previous microarray studies done on skeletal muscle of mice, monkeys and humans, we also found that genes associated with metabolic functions, particularly energy pathways, were higher in young control animals. in contrast, genes indicative of cellular damage, such as stress response and apoptotic genes, had higher expression levels in the muscle tissue of older animals. among the biological classes of transcripts with an age effect, there were several gene families whose mrna levels were significantly increased or decreased with age. there were procollagen genes that were altered with aging; of these showed higher levels in the young muscle while only one procollagen gene was increased with aging. the keratins, important cytoskeletal structural proteins, were represented by genes all of which displayed increased transcript levels in older muscle. three members of the ccaat/enhancer-binding protein family of transcription factors had increased levels of transcripts in the older mice. the encoded cebp alpha, beta and delta proteins are important in the regulation of genes involved in immune and inflammatory responses, cell cycle arrest and body weight homeostasis. because many genes are represented more than once on the moe430a affymetrix microarray, several genes had multiple hits on our list of genes with an age effect in skeletal muscle. among those genes with four or more independent listings were: fibromodulin, transferrin receptor, immunoglobulin kappa chain variable, and myristoylated alanine rich protein kinase c substrate. transcript and protein levels of p53-related genes altered with aging given that skeletal muscle is largely a post-mitotic tissue, we were surprised to find that a large number of genes involved in the regulation of the cell cycle had increased mrna levels with age. two gene products that are known to play a critical role in initiating cell cycle arrest following dna damage, cyclin-dependent kinase inhibitor 1a and growth arrest and dna-damage-inducible, alpha, were both increased significantly in the skeletal muscle of the aged mice. expression levels for p and gadd45a were and fold higher respectively in older mouse compared to the younger animals. an age-related increase in p has previously been observed in muscle of both male and female humans and monkeys, while the expression of gadd45a has been shown to be elevated in mouse gastrocnemius muscle and both male and female human muscle. because the expression of these two genes is typically mediated by the action of p we decided to systematically examine our list of genes differentially expressed between young and old to determine if there were any other transcripts that are also connected to p activity. as shown in table the transcripts that are activated by p or whose gene products are known to bind to p increase significantly with age, while the expression of genes that are known to inhibit p activity decline in old muscle. the average cr prevention on these p53-related genes was 77%. to confirm the age-related changes in expression observed in p and gadd45a using microarrays, we probed young, old and old cr mice using rt-pcr. we also examined the expression levels of the p gene itself, along with the expression levels of another important tumor suppressor, p both p and p transcripts were considered absent in all animals according to the affymetrix software, but we were able to detect expression using the more sensitive method of rt-pcr. as is shown in figure 1a, transcript levels for these genes increased significantly with age. furthermore, old animals undergoing cr demonstrated reduced levels for these genes compared to age-matched mice fed normal diets. to examine how the expression of p p gadd45a and p is altered over the course of the adult mouse life span, we used rt-pcr to measure transcript levels for these genes in gastrocnemius muscle at month intervals from to months of age. with the exception of gadd45a, the increase in expression of these genes does not show a linear pattern with age. p and gadd45a expression increases significantly only in the last third of the lifespan, whereas expression of p and its transcriptional target p occur as early as months of age. to determine whether the p53-mediated transcriptional program observed in older mouse skeletal muscle translated into higher levels of p53-associated proteins being expressed, we probed young, old and old cr muscle for p53-transcriptional targets p and gadd45a. western immunoblot confirmed a significant increase in p protein and gadd45a in older mouse skeletal muscle. levels for these proteins were reduced in the old cr mice as compared to old mice on the control diet. an attempt was made with several antibodies to measure p protein levels in gn muscle, but the signal from the western blots was either absent or too low to accurately quantitate p in any age group. transcript levels of p p and gadd45a in mouse models of increased oxidative stress to test whether the changes in expression of the p53-related genes could be attributed to cumulative oxidative damage with age, we compared transcript levels for p p gadd45a and p in c57bl/ mice with a heterozygous knockout for either glutathione peroxidase or mnsod mutations to age-matched wild-type mice. gpx4+/-mice have approximately half the amount of mrna and protein levels for this essential se-dependent antioxidant protein as compared to wild type mice. mouse embryonic fibroblasts derived from mice heterozygous for the gpx gene show increased evidence of lipid and dna oxidation, and are sensitive to gamma irradiation. sod2+/- mice show a ~50% reduction in mnsod protein content and mnsod activity in gatrocnemius tissue and have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine in both nuclear and mitochondrial dna in all surveyed tissues compared to wild type mice. rt-pcr analysis of p p gadd45a and p in gpx4+/- mice showed a significant increase in p expression compared to the age-matched wild-type mice. we did observe a ~ fold increase in p although it was not statistically significant. transcript levels of these genes were not significantly different between sod2+/- mice and age-matched controls. thus, constitutive oxidative damage does not always trigger the expression of the genes examined. possibly, different types of dna damage in gpx4-/+ and sod2+/- mice explain the observed differences. increased p levels in older muscle is associated with increased expression of genes involved in p53-mediated apoptosis a critical function of p is the induction of apoptosis in cells that have undergone dna damage or exhibit uncontrolled cellular growth. to investigate whether increased p activity with age is associated with transcription profiles indicative of apoptosis in old gastrocnemius muscle, we examined mrna levels of four genes known to be key transducers of p53-mediated apoptosis: bcl- binding component, phorbol-12-myristate-13-acetate-induced protein, tumor necrosis factor receptor superfamily, member 10b and bcl-2-related ovarian killer protein. we observed a significant increase in mrna levels with age for these genes in mouse gastrocnemius muscle and a significant decrease in levels due to caloric restriction in old, age-matched controls. the expression levels for all of these p53-mediators of apoptosis were also examined in the gastrocnemius muscle over the course of the mouse adult lifespan, and increased levels of transcript were usually observed in the first half of the lifespan. we also compared the levels for these proapoptotic genes in the gastrocnemius tissue of gpx4+/- and the sod+/- mice to age-matched wild type mice using rt-pcr. only the levels for tnfrsf10b were considered significantly higher in the gpx4+/- mice while expression of these genes was not significantly different in the sod2+/- mice. these findings indicate that p53-dependent mediators of apoptosis are regulated with age and that the mechanism of activation is independent of endogenous levels of oxidative stress. increase in mrna levels of p and p53-associated genes in multiple tissues of old c57bl6/j mice to determine whether the p53-mediated transcriptional program we observe in older skeletal muscle may also occur in other tissues, we used rt-pcr to measure mrna levels of p53-associated genes in heart, lung, liver, kidney, brain and intestine of young, old, and old cr mice. transcript levels for p p p and pmaip demonstrated increased levels in multiple tissue types in the old mice, while an increase in transcript levels for gadd45a, bbc tnfrsf10b and bok was only observed in old skeletal muscle. these findings suggest that activation of a p53-mediated transcriptional program may be a common feature of aging in multiple tissues, but the pattern of expression of p53-dependent genes is tissue specific. discussion and  our initial investigation of muscle aging in mice revealed many differences in the transcriptional patterns between young and old mice. in general, transcript levels for genes that are associated with cellular damage were elevated in older muscle, while transcript levels for genes involved in energy metabolism were reduced with age. the current study further demonstrates that the intrinsic transcriptional patterns are altered with aging in mouse skeletal muscle. as with other microarray studies done on older mammalian muscle, we have also found a disproportionate number of metabolic genes, such as those involved in lipid and carbohydrate metabolism, decrease in transcription levels with age. the age-related decrease in expression of these energy-associated classes likely reflects the reduction in mitochondria observed in older gastrocnemius muscle. for example, a 23–25% reduction in mtdna copy number and a corresponding 17–22% reduction in the expression of mitochondrial dna-encoded cytochrome c oxidase subunits i and iii was previously reported in old rat gastrocnemius tissue. in our study, out of transcripts that are associated with the mitochondria, the expression of of these probe sets are significantly reduced with age. conversely, genes associated with the stress and immune response, such as those involved in the induction of apoptosis or the response to dna damage, have elevated mrna levels in the older animals. the age-associated increase in genes that are activated during cellular stress and injury is probably in response to a wide variety of biological damage known to accumulate in older muscle over the course of the lifespan. we also observed that the expressions of a large number of genes involved in the regulation of the cell cycle were increased with age in mouse skeletal muscle. a closer examination of these cell cycle genes revealed a pattern of expression indicative of increased p activity in older mouse muscle. welle et al. also found transcriptional evidence of increased p activity with age in the skeletal muscle of young and old women. in their study, genes that regulate or enhance p activity or that are induced by p demonstrate increased expression in older female muscle. based on this evidence, welle and coworkers suggested that future studies should examine the possibility that p levels or activity are increased in older muscle. one possible mechanism for the observed increase in p activity is elevated levels of dna damage with age. the concentration of 8-oxo- deoxyguanosine, a byproduct of dna oxidation, has been observed to increase with age in a variety of tissues, including skeletal muscle, in rats and various strains of mice. we tested this hypothesis by examining the levels of p mrna and related transcripts in transgenic mice with half the wild type levels of antioxidants gpx or sod these mice have elevated levels of oxidative dna damage yet failed to show any significant increase in expression of p and gadd45a, two well studied p53-induced genes. the gpx4+/- mice did display a significant increase in transcript levels for p as compared to age-matched controls, but no change was observed for the sod2+/- mice. possibly, different types of dna damage impact the expression of these genes differently. another possibility is that the dna damage signaling that is the trigger for activation of p in old muscle originates from chromosomal damage, such as double-strand breaks or telomere shortening, rather than from oxidative alterations. we also note that there are two likely possibilities for the cellular origin of the alteration in expression of cell cycle regulators. one is that these alterations in gene expression reflect a decline in proliferation of a muscle-associated cell such as the satellite cell. it has recently been reported that hematopoietic stem cell numbers are higher in old mice with reduced p activity and fail to increase with age in old mice with hypermorphic p activity as compared to their wild type counterparts. a second possibility is that the expression of these genes reflects a general p53-mediated dna damage response associated with age-related dna modifications in myocytes. many recent studies have linked tumor suppressor activity, in particular the tumor suppressor p to organismal aging. p16ink4a, a product of the ink4a/arf locus also increases in expression with age in most tissues as shown in previous studies and also in the current analysis. this increase in p16ink4a expression with age has recently been shown to contribute to age-dependent decline in regenerative potential of pancreatic islet regenerative capacity, decreases in forebrain progenitors and neurogenesis and the decrease in hematopoietic stem cell repopulation defects. however, we note that it is possible that only rare cells in tissues express p16ink4a with aging. under these conditions, a large effect of increased ros in a specific biologically relevant muscle compartment could be missed by studying unfractionated muscle as we did in our analysis. thus, the induction of a cell senescence and apoptotic program with age may underlie key features of the aging process. given our finding that mrna levels for p53-associated genes were elevated in multiple tissues of old mice, our data suggests that increased p activity is not limited to aging muscle but is also an overall consequence of organismal aging. increased p mrna levels is observed in the grey and white matter of the cortex, cerebellum, and medulla oblongata of old verses young rat brains and an increase in p protein levels is observed in the grey matter of old rats. we did not find a significant difference in p gene expression between young and old brains, but our analysis was performed on whole brain homogenates and therefore any age-effect on p expression within the different regions of the brain might have been missed. further support for the role of increased p activity contributing to aging process comes from transgenic studies in mice that express overactive p and also demonstrate accelerated aging. mice with augmented p function develop typical age-associated phenotypes such as delayed wound healing, reduced hair growth and reduced subcutaneous adipose tissue much earlier than their p wild type counterparts. conversely, the long-lived p66shc-/- mouse, shown to have a 30% extended life span, exhibits impaired p apoptotic signaling. our study demonstrates that the majority of the p53-related transcriptional changes observed in old skeletal muscle are either completely or partially reversed through caloric restriction, a proven method to extended longevity. based on the evidence for the involvement of the p pathway in organismal aging, it follows that p and p transcriptional targets have the potential to be good biomarkers of aging in multiple tissue types. it has been well documented that there is a significant decrease in muscle mass with age, yet little is known as to the actual mechanism for this loss of tissue. it has been hypothesized that the decrease in muscle fiber can be attributed to increased apoptosis with age. in fact, an increase in biological markers of apoptosis, such as dna fragmentation and proapoptotic caspase-type proteins, are increased with age in human and rat skeletal muscle. however, the mechanism for this age-associated apoptosis is unknown. it has been suggested that increased oxidative damage to the mitochondria, along with sarcoplasmic reticulum stress in older skeletal muscle could initiate caspase activation and lead to the apoptotic pathway that ultimately results in the destruction of the aged myocyte. moreover, dirks and leeuwenburgh also observed a significant reduction in dna fragmentation and caspase levels in old rats on lifelong caloric restriction when compared to their age-matched, ad libitum-fed counterparts. the authors suggest the reduction of apoptosis in response to caloric restriction is due to reduced mitochondrial oxidant production and nuclear oxidative dna damage in skeletal muscle. our results do not support this hypothesis. although young gpx4+/- mice did show increased levels of expression, compared to wild-type young mice, of p53-associated mediator of apoptosis tnfrsf10b, little or no change was observed for bbc and bok. the sod2+/- mice, on the other hand, demonstrated almost identical levels of expression for all of the surveyed p mediators of apoptosis to the age-matched, wild type controls. it is possible that increased oxidative damage may play a role in p53-mediated apoptosis in older muscle, but our data suggests that it may not be the sole causative effect. old calorie restricted mice displayed a significant decrease in expression levels of all four of the p53-mediators of apoptosis compared to the normally fed old mice. it is clear that caloric restriction has an inhibitory effect on age-associated apoptosis of skeletal muscle, but this prevention may not be completely explained by the reduction of endogenous reactive oxygen species and the resulting decrease in oxidative dna damage in these animals. the role of p in the aging process of skeletal muscle is likely to be a complex process involving many different cellular effectors. various proteins and pathways interact with p to modulate its activity. furthermore, there are numerous p53-transcriptional targets, each with their own associated modulators that may influence muscle aging. as evidence of this complexity, we show that the expression profiles for p53-regulated genes are not identical to that of p as well as to each other, over the course of the mouse adult lifespan in gastrocnemius muscle. future studies will concentrate on determining which specific components of the p pathway play a functional role in muscle aging, and organismal aging in general. 
these observations support a role for p53-mediated transcriptional program in mammalian aging and suggest that mechanisms other than reactive oxygen species are involved in the age-related transcriptional activation of p targets. age-related induction of p53-related genes was observed in multiple tissues, but was not observed in young sod2+/- and gpx4+/- mice, suggesting that oxidative stress does not induce the expression of these genes. western blot analysis confirmed that protein levels for both p and gadd45a, two established transcriptional targets of p were higher in the older muscle tissue. expression levels for these proapoptotic genes increased significantly with age, while cr significantly lowered expression levels for these genes as compared to control fed old mice. caloric restriction completely or partially reversed 87% of the changes in expression.
 aging has been associated with widespread changes at the gene expression level in multiple mammalian tissues. we have used high density oligonucleotide arrays and novel statistical methods to identify specific transcriptional classes that may uncover biological processes that play a central role in mammalian aging.  we identified transcripts that are differentially expressed in young and old mouse skeletal muscle. caloric restriction completely or partially reversed 87% of the changes in expression. examination of individual genes revealed a transcriptional profile indicative of increased p activity in the older muscle. to determine whether the increase in p activity is associated with transcriptional activation of apoptotic targets, we performed rt-pcr on four well known mediators of p53-induced apoptosis: puma, noxa, tnfrsf10b and bok. expression levels for these proapoptotic genes increased significantly with age, while cr significantly lowered expression levels for these genes as compared to control fed old mice. age-related induction of p53-related genes was observed in multiple tissues, but was not observed in young sod2+/- and gpx4+/- mice, suggesting that oxidative stress does not induce the expression of these genes. western blot analysis confirmed that protein levels for both p and gadd45a, two established transcriptional targets of p were higher in the older muscle tissue.  these observations support a role for p53-mediated transcriptional program in mammalian aging and suggest that mechanisms other than reactive oxygen species are involved in the age-related transcriptional activation of p targets. 