Life-long calorie restriction in Fischer 344 rats attenuates age-related loss in skeletal muscle-specific force and reduces extracellular space.

The decline in muscle function is associated with an age-related decrease in muscle mass and an age-related decline in strength. However, decreased strength is not solely due to decreased muscle mass. The age-related decline in muscle-specific force (force/muscle cross-sectional area), a measure of intrinsic muscle function, also contributes to age-related strength decline, and the mechanisms by which this occurs are only partially known. Moreover, changes in the extracellular space could have a profound effect on skeletal muscle function. Life-long calorie restriction in rodents has shown to be a powerful anti-aging intervention. In this study, we examine whether calorie restriction is able to attenuate the loss of muscle function and elevations in extracellular space associated with aging. We hypothesize that calorie restriction attenuates the age-associated decline in specific force and increases in extracellular space. Measurements of in vitro contractile properties of the extensor digitorum longus (type II) and soleus (type I) muscles from 12-mo and 26- to 28-mo-old ad libitum-fed, as well as 27- to 28-mo-old life-long calorie-restricted male Fischer 344 rats, were performed. We found that calorie restriction attenuated the age-associated decline in muscle mass-to-body mass ratio (mg/g) and strength-to-body mass ratio (N/kg) in the extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle (P > 0.05). Importantly, muscle-specific force (N/cm2) in the extensor digitorum longus, but not in the soleus muscle, of the old calorie-restricted rats was equal to that of the young 12-mo-old animals. Moreover, the age-associated increase in extracellular space was reduced in the fast-twitch extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle with calorie restriction. We also found a significant correlation between the extracellular space and the muscle-specific force in the extensor digitorum longus (r = -0.58; P < 0.05) but not in the soleus muscle (r = -0.38; P > 0.05). Hence, this study shows a loss of muscle function with age and suggests that long-term calorie restriction is an effective intervention against the loss of muscle function with age.     

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: effects of caloric restriction.

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.     

Rapid onset of gene expression in lung, supportive of formation of alveolar septa, induced by refeeding mice after calorie restriction

Alveolar regenerative gene expression is unidentified partly because its onset, after a regenerative stimulus, is unknown. Toward addressing this void, we used a mouse model in which calorie restriction produces alveolar loss, and ad libitum access to food after calorie restriction induces alveolar regeneration. We selected four processes (cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion) that would be required to convert a flat segment of alveolar wall into a septum that increases gas-exchange surface area. Global gene expression supportive of processes required to form a septum was present within 3 h of allowing calorie-restricted mice food ad libitum. One hour after providing calorie-restricted mice food ad libitum, RNA-level expression supportive of cell replication was present with little evidence of expression supportive of angiogenesis, extracellular matrix remodeling, or guided cell motion. Cell replication was more directly assayed by measuring DNA synthesis in lung. This measurement was made 3 h after allowing calorie-restricted mice food ad libitum because translation may be delayed. Ad libitum food intake, following calorie restriction, elevated DNA synthesis. Thus RNA expression 1 h after allowing calorie-restricted mice food ad libitum supported increased cell replication; measurements at 3 h revealed increased DNA synthesis and RNA expression, supportive of the three other processes required to form a septum. These findings identify the first hour after providing calorie-restricted mice ad libitum access to food as the onset of gene expression in this model that supports processes needed for alveolar regeneration.     

Age-related alterations in expression of apoptosis regulatory proteins and heat shock proteins in rat skeletal muscle

Aging of skeletal muscle is often accompanied by muscle atrophy and it appears that apoptosis plays an important role in this process. The detailed mechanism(s) is not completely understood, however. In this study, we examined expression of the apoptosis regulatory proteins as well as the heat shock proteins, which have been shown to modulate the apoptotic process in certain cell types, in order to more completely elucidate apoptotic signaling in aged skeletal muscle. To more specifically identify alterations that are likely to be the result of aging, we compared 16-month-old middle-aged (MD) and 29-month-old senescent (SE) male Fischer 344 x Brown Norway rats in our study. Our results show that the degree of DNA laddering was higher in SE compared to MD rats. Using total tissue homogenates we examined the level of expression of several apoptosis-related proteins in two categories: mitochondria-associated proteins and caspases. Of the mitochondria-associated proteins, the levels of p53 showed a significant increase in SE compared to MD rats. There was also a significant increase in the expression of Bax, Bcl-2 and Apaf-1 in SE rats over that of MD rats; cytochrome c and AIF levels remained unchanged, however. Regarding the caspases, there were increases in the levels of pro-caspases-12 and -7 and cleaved caspase-9, although the levels of pro- and cleaved caspase-3 as well as cleaved caspase-12 remained unchanged. Furthermore, our results showed significant increases in HSP27, HSP60, and the inducible HSP70. These data show that in rat skeletal muscle increased apoptosis occurs between middle-age and senescence, indicating an aging-related increase in apoptosis in skeletal muscle. The involvement of different apoptotic pathways in the aging process is suggested by the selective alterations in the apoptosis regulatory proteins. The increased expression of the HSPs suggests a relationship between HSPs and the aging-related apoptotic process.     

Iron accumulation with age, oxidative stress and functional decline

Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength) in male Fischer 344 X Brown Norway rats fed ad libitum (AL) or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age) at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR) rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.     

Caloric restriction optimizes the proteasome pathway with aging in rat plantaris muscle: implications for sarcopenia

To gain insight into the significance of alterations in the proteasome pathway for sarcopenia and its attenuation by calorie restriction, we examined protein oxidation and components of the proteasome pathway in plantaris muscle in 8-, 30-, and 35-mo-old ad libitum-fed (AL) rats; and in 8-, 35-, and 40-mo-old calorie-restricted (CR) rats. We hypothesized that CR rats would exhibit a lesser accumulation of protein carbonyls with aging and that this would be associated with a better maintenance of skeletal muscle proteasome activity and function with aging. Consistent with this view, whereas AL rats had a significant increase in protein carbonylation with aging, there was no such increase in CR rats. Protein levels of the ubiquitin ligases MuRF1 and MAFbx increased similarly with aging in both AL and CR rats. On the other hand, chymotrypsin-like activity of the proteasome increased with aging more gradually in CR rats, and this increase was paralleled by increases in the expression of the C2 subunit in both groups, suggesting that differences in activity were not related to differences in proteasome function with aging. Interestingly, the plot of muscle mass vs. proteasome activity showed that the oldest animals in both diets had a lower muscle mass than would be predicted by their proteasome activity, suggesting that other factors explain the acceleration of sarcopenia at advanced age. Since calorie restriction better protects skeletal muscle function than muscle mass with aging (Hepple RT, Baker DJ, Kaczor JJ, Krause DJ, FASEB J 19: 1320-1322, 2005), and our current results show that this protection of function is associated with a prevention of oxidative protein damage accumulation, we suggest that calorie restriction optimizes the proteasome pathway to preserve skeletal muscle function at the expense of modest muscle atrophy.     

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle

Apoptosis and autophagy are critical in normal skeletal muscle homeostasis; however, dysregulation can lead to muscle atrophy and dysfunction. Lipotoxicity and/or lipid accumulation may promote apoptosis, as well as directly or indirectly influence autophagic signaling. Therefore, the purpose of this study was to examine the effect of a 16-week high-fat diet on morphological, apoptotic, and autophagic indices in oxidative and glycolytic skeletal muscle of female rats. High-fat feeding resulted in increased fat pad mass, altered glucose tolerance, and lower muscle pAKT levels, as well as lipid accumulation and reactive oxygen species generation in soleus muscle; however, muscle weights, fiber type-specific cross-sectional area, and fiber type distribution were not affected. Moreover, DNA fragmentation and LC3 lipidation as well as several apoptotic (ARC, Bax, Bid, tBid, Hsp70, pBcl-2) and autophagic (ATG7, ATG4B, Beclin 1, BNIP3, p70 s6k, cathepsin activity) indices were not altered in soleus or plantaris following high-fat diet. Interestingly, soleus muscle displayed small increases in caspase-3, caspase-8, and caspase-9 activity, as well as higher ATG12-5 and p62 protein, while both soleus and plantaris muscle showed dramatically reduced Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) levels. In conclusion, this work demonstrates that 16 weeks of high-fat feeding does not affect tissue morphology or induce a global autophagic or apoptotic phenotype in skeletal muscle of female rats. However, high-fat feeding selectively influenced a number of apoptotic and autophagic indices which could have implications during periods of enhanced muscle stress.     

Response of XIAP, ARC, and FLIP apoptotic suppressors to 8 wk of treadmill running in rat heart and skeletal muscle.

Although it has been demonstrated that exercise training has an antiapoptotic effect on postmitotic myocytes, the mechanisms responsible for this effect are still largely unclear. Because the antiapoptotic effect of exercise training in postmitotic myocytes could be possibly mediated by the upregulation of apoptotic suppressors, this study examined the effect of endurance training on endogenous apoptotic suppressors including X-chromosome-linked inhibitor of apoptosis protein (XIAP), apoptosis repressor with caspases recruitment domain protein (ARC), and FADD-like inhibitor protein (FLIP) in skeletal and cardiac muscles. Eight adult Sprague-Dawley rats were trained 5 days weekly for 8 wk on treadmill, and eight sedentary rats served as controls. Soleus and ventricle muscles were dissected 2 days after the last training session. The mRNA content of XIAP, ARC, and FLIP was estimated by RT-PCR with ribosomal 18S RNA used as an internal control. The protein expression of XIAP, ARC, FLIP(S), and FLIP(alpha) was assessed by Western immunoblot. After training, mRNA content of ARC and FLIP was not different between the control and trained animals, whereas XIAP mRNA content was elevated by 22 and 14% in the trained soleus and cardiac muscles, respectively, relative to the control samples. No difference was found in the protein content of FLIP(S) and FLIP(alpha) between control and trained muscles, whereas XIAP and ARC protein content was increased by 18 and 38%, respectively, in the soleus muscle of trained animals. Furthermore, negative relationships were found between XIAP and apoptotic DNA fragmentation as well as ARC and caspase-3 activity. These findings are consistent with the hypothesis that the modulation of apoptotic suppressors is involved in training-induced attenuation of apoptosis in skeletal and cardiac muscles.     

Age-related differences in apoptosis with disuse atrophy in soleus muscle

Muscle atrophy is associated with a loss of muscle fiber nuclei, most likely through apoptosis. We investigated age-related differences in the extent of apoptosis in soleus muscle of young (6 mo) and old (32 mo) male Fischer 344 x Brown Norway rats subjected to acute disuse atrophy induced by 14 days of hindlimb suspension (HS). HS-induced atrophy (reduction in muscle weight and cross-sectional area) was associated with loss of myofiber nuclei in soleus muscle of young, but not old, rats. This resulted in a significant decrease in the myonuclear domain (cross-sectional area per nucleus) in young and old rats, with changes being more pronounced in old animals. Levels of apoptosis (TdT-mediated dUTP nick end labeling and DNA fragmentation) were higher in soleus muscles of old control rats than young animals. Levels were significantly increased with HS in young and old rats, with the greatest changes in old animals. Caspase-3 activity in soleus muscle tended to be increased with age, but changes were not statistically significant (P=0.052). However, with HS, caspase-3 activity significantly increased in young, but not old, rats. Immunohistochemistry showed that the proapoptotic endonuclease G (EndoG, a mitochondrion-specific nuclease) was localized in the subsarcolemmal mitochondria in control muscles, and translocation to the nucleus occurred in old, but not young, control animals. There was no difference between EndoG total protein content in young and old control rats, but EndoG increased almost fivefold in soleus muscle of old, but not young, rats after HS. These results show that deregulation of myonuclear number occurs in old skeletal muscle and that the pathways involved in apoptosis are distinct in young and old muscles. Apoptosis in skeletal muscle is partly mediated by the subsarcolemmal mitochondria through EndoG translocation to the nucleus in response to HS.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Age-dependent upregulation of p53 and cytochrome c release and susceptibility to apoptosis in skeletal muscle fiber of aged rats: role of carnitine and lipoic acid

Mitochondrial dysfunction has been implicated in the regulation of myofiber loss during aging, possibly by apoptotic pathways. However, the mitochondrial-mediated pathway of apoptosis by cytochrome c in skeletal muscle remains ambiguous. To understand this, we have studied the upstream and downstream events of cytochrome c release, and assessed the efficacy of carnitine and lipoic acid cosupplementation. The results show that elevated levels of cytosolic cytochrome c activate apoptosis in aged rats, and was confirmed further by in vitro caspase-3 assay. Interestingly, the exogenous addition of cytochrome c results in a much higher increase of caspase-3 activity in aged treated rats than age-matched control rats, strongly suggesting that cytochrome c is a limiting factor for caspase-3 activation in the cytosol. Carnitine and lipoic acid supplement decreased apoptosis in aged rats by maintaining mitochondrial membrane integrity and thereby preventing further loss of cytochrome c in vivo. Furthermore, the upregulation of p53 observed in aged rats is attributed to the loss of outer mitochondrial membrane integrity and subsequent release of cytochrome c through BH3-only proteins. In conclusion, the p53-dependent activation of the mitochondrial-cytochrome c pathway of apoptosis in the present study suggests the existence of cross talk between mitochondria and nucleus. However, the exact molecular mechanism remains to be explored. Oral supplements of carnitine and lipoic acid play an antiapoptotic role in aged rat skeletal muscle by protecting mitochondrial membrane integrity.     

Combined effects of short-term calorie restriction and exercise on insulin action in normal rats

The present study examined the effect of combination of short-term calorie restriction (CR) and moderate exercise on insulin action in normal rats. Rats were divided randomly into 4 groups: ad libitum, sedentary (A-Sed); calorie restriction, sedentary (CR-Sed); ad libitum, exercise (A-Ex); and calorie restriction, exercise (CR-Ex). Rats in the exercise groups were run on a rodent treadmill. Rats in the CR groups were fed every alternate day. Oral glucose tolerance test (OGTT) showed improvements in both CR-Sed and A-Ex groups compared with the A-Sed group; no further improvement in glucose tolerance was observed in the CR-Ex group. In contrast, glucose infusion rates (GIRs) determined by the hyperinsulinemic-euglycemic clamp method indicated that the GIR of the CR and exercise combination was significantly better than that of the sole intervention of CR or exercise. There was no difference in the levels of fasting glucose, insulin, or high-molecular weight forms of adiponectin among the 4 groups. Protein expression of GLUT-4 in the skeletal muscle increased by exercise, but not by CR. Our findings indicate that the combination of exercise and CR may be effective in enhancing insulin sensitivity at the skeletal muscle in normal subjects.     

The effect of aging and dietary restriction on DNA repair

DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.     

24-hour changes in ACTH, corticosterone, growth hormone, and leptin levels in young male rats subjected to calorie restriction

Calorie restriction of young male rats increases plasma prolactin, decreases luteinizing hormone (LH) and testosterone, and disrupts their 24 h secretory pattern. To study whether this could be the consequence of stress, we examined the 24 h variations of plasma adrenocorticotropic hormone (ACTH) corticosterone, growth hormone (GH), leptin, and adrenal corticosterone. Rats were submitted to a calorie restriction equivalent to a 66% of usual intake for 4 weeks, starting on day 35 of life. Controls were kept in individual cages and allowed to eat a normal calorie regimen. Significantly lower ACTH levels were detected in calorie-restricted rats. Plasma corticosterone levels during the light phase of the daily cycle were significantly higher in calorie-restricted rats. Time-of-day variation in plasma ACTH and corticosterone levels attained significance in calorie-restricted rats only, with a maximum toward the end of the resting phase. The daily pattern of adrenal gland corticosterone mirrored that of circulating corticosterone; however, calorie restriction reduced its levels. Plasma ACTH and corticosterone correlated significantly in controls only. Calorie restriction decreased plasma GH and leptin, and it distorted 24 h rhythmicity. In a second study, plasma ACTH and corticosterone levels were measured in group-caged rats, isolated control rats, and calorie-restricted rats during the light phase of the daily cycle. Plasma ACTH of calorie-restricted rats was lower, and plasma corticosterone was higher, compared with isolated or group-caged controls. The changes in the secretory pattern of hormones hereby reported may be part of the neuroendocrine and metabolic mechanisms evolved to maximize survival during periods of food shortage.     

Attenuation of bleomycin-induced Hprt mutant frequency in female and male rats by calorie restriction

Calorie restriction modulates spontaneous and chemically induced tumors and increases maximal life span in experimental animals; however, the mechanism by which calorie restriction exerts its ameliorating effects is not fully elucidated, although reduced levels of reactive oxygen species (ROS) by calorie restriction has generated much interest. In the present study, we have determined whether or not calorie restriction would affect the mutagenic response in rats treated with bleomycin (BLM) a radiomimetic drug that is associated with DNA damage by a free radical mechanism. Fourteen weeks after weaning, the rats were divided into two groups; ad libitum (AL)-fed and 40% calorie restriction. Both AL and calorie-restricted animals were injected with 2.5, 5.0 and 10.0 mg BLM/kg, or with phosphate-buffered saline (PBS), and they were killed 4 weeks post drug treatment. Lymphocytes from the spleens were seeded in 96-well microtiter plates to determine mutant frequency in the hypoxantine guanine phosphoribosyl transferase (Hprt) gene. The mutant frequency in the BLM-treated rats was higher in AL males (P=0.001), and AL females (P=0.0174) than in their calorie-restricted counterparts. The difference in mutagenic response relative to AL males and AL females appeared unrelated to a low percent cloning efficiency seen in the males, since the mean absolute number of Hprt mutant clones was higher in the AL males compared to the females. A reduction in animal weight by calorie restriction was significant in both sexes (P<0.001), but the dose effect appeared non-significant. The results indicate that calorie intake of 60% reduced the mutagenic response of BLM, a compound known to induce oxidative DNA damage, and suggest a possible decrease in ROS as a function of calorie restriction.     

Modulation of age-induced apoptotic signaling and cellular remodeling by exercise and calorie restriction in skeletal muscle

Aging is inevitably associated with a progressive loss of muscle mass and strength, a condition also known as sarcopenia of aging. Although the precise mechanisms underlying this syndrome have not been completely elucidated, recent studies point toward several key cellular mechanisms that could contribute to age-associated muscle loss. Among these, mitochondrial dysfunction and deregulation of apoptotic signaling have emerged as critical players in the onset and progression of sarcopenia. Interestingly, calorie restriction, a well-known antiaging intervention, and, more recently, exercise training have been shown to beneficially affect both mitochondrial function and apoptotic signaling in skeletal muscle from young and old animals. Preliminary observations also indicate that even a small (8%) reduction in food intake may still provide protective effects against sarcopenia and cellular remodeling in aging skeletal muscle, with the advantage of being more applicable to human subjects than the traditional 30-40% restriction regimen. The most recent evidence on the relevance of skeletal muscle apoptosis to sarcopenia, as well as its modulation by calorie restriction and exercise, is reviewed.     

Effect of burn injury on apoptosis and expression of apoptosis-related genes/proteins in skeletal muscles of rats

The purpose of this study was to investigate the occurrence and possible mechanisms of apoptosis in skeletal muscles after burn injury. After a 40% body surface area burn to rats, TA muscles were examined for apoptosis at varying times by TEM, TUNEL and cell death ELISA assay. Thermal injury was found to induce apoptosis in skeletal muscle on the first day and maximal apoptosis appeared 4 days post-injury. Apoptotic ligands in serum assessed by ELISA revealed rapidly increase of TNF-α and subsequent increase of sFasL to sFas ratio after burn injury. It implied TNF-α induced apoptosis in early stage and FasL induced apoptosis in later stage after burn injury. Apoptosis-related genes/proteins in skeletal muscles examined by real-time PCR array and Western blotting showed pro-apoptotic genes/proteins, including Tnfrsf1a, Tnfrsf1b and Tnfsf6 in TNF ligand and receptor family, Bax and Bid in Bcl-2 family, caspase-3 and caspase-6 in caspase family, Dapk1, FADD and Cidea in death and CIDE domain family, Apaf-1 in CARD family, and Gadd45a were up-regulated, while anti-apoptotic gene Bnip1 was down-regulated compared with that of time-matched controls. In addition, increment of caspase-3, caspase-8 and caspase-9 activity provided further evidence for their role in apoptosis in skeletal muscle. Significant increase in expression in pro-apoptotic genes/proteins and activity of caspases suggested that death receptor-mediated signaling pathways and other apoptotic related pathways participated in apoptosis in skeletal muscle after burn injury. However, it was found that some anti-apoptotic genes such as Bcl2l1, Mcl-1, Nol-3, Il-10 and Prok2 were also up-regulated, which might imply the co-existence of protective response of the body after burns. In conclusion, the data suggest that apoptosis and pro-apoptotic signaling are enhanced in muscles of burned rats. To further elucidate the underlying apoptotic mechanisms mediating the atrophic response is important in establishing potential therapeutic interventions that could prevent and/or reduce skeletal muscle wasting and preserve its physiological function.