Effects of aging on regulators of muscle apoptosis in the female F344BN rat

Age-related muscle atrophy is a consequence of normal aging characterized by decreases in muscle mass and strength. The mechanism(s) underlying the loss of muscle mass with increasing age is not fully understood, however recent data has suggested that muscle cell apoptosis may be involved. Here we investigate how aging affects the regulation of muscle apoptosis in the extensor digitorum longus (EDL) and soleus muscles of young (6-month), aged (26-month), and very aged (30-month) female Fischer 344/NNiaHSD X Brown Norway / BiNia (F344BN) rats. EDL and soleus muscle mass/body weight ratios were lower in aged animals but not different between 26- and 30-months of age. Decrease in muscle mass was associated with increased TdTmediated dUTP nick-end labeling (TUNEL) positive immunoreactivity in both EDL and soleus. With advancing age the time course and magnitude of changes in Bax, Bcl-2, caspase-3, caspase-9, caspase-12 and cleavage of α-fodrin protein were regulated differently between muscles. These data demonstrated that decreases in muscle mass, and increases in muscle cell apoptosis appear to be caspase independent and differ between fiber types in the female F344BN rats with aging

Altered Regulation of Contraction-Induced Akt/mTOR/p70S6k Pathway Signaling in Skeletal Muscle of the Obese Zucker Rat

Increased muscle loading results in the phosphorylation of the 70 kDa ribosomal S6 kinase (p70S6k), and this event is strongly correlated with the degree of muscle adaptation following resistance exercise. Whether insulin resistance or the comorbidities associated with this disorder may affect the ability of skeletal muscle to activate p70S6k signaling following an exercise stimulus remains unclear. Here, we compare the contraction-induced activation of p70S6k signaling in the plantaris muscles of lean and insulin resistant obese Zucker rats following a single bout of increased contractile loading. Compared to lean animals, the basal phosphorylation of p70S6k (Thr389; 37.2% and Thr421/Ser424; 101.4%), Akt (Thr308; 25.1%), and mTOR (Ser2448; 63.0%) was higher in obese animals. Contraction increased the phosphorylation of p70S6k (Thr389), Akt (Ser473), and mTOR (Ser2448) in both models however the magnitude and kinetics of activation differed between models. These results suggest that contraction-induced activation of p70S6k signaling is altered in the muscle of the insulin resistant obese Zucker rat

Aging-Associated Dysfunction of Akt/Protein Kinase B: S-Nitrosylation and Acetaminophen Intervention

Background: Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s) underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB) is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention. Principal Findings: Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-b), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6- months. Conclusions: These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to ageassociated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction

Application of Poly(amidoamine) Dendrimers for Use in Bionanomotor Systems

The study and utilization of bionanomotors represents a rapid and progressing field of nanobiotechnology. Here, we demonstrate that poly(amidoamine) (PAMAM) dendrimers are capable of supporting heavy meromyosin dependent actin motility of similar quality to that observed using nitrocellulose, and that microcontact printing of PAMAM dendrimers can be exploited to produce tracks of active myosin motors leading to the restricted motion of actin filaments across a patterned surface. These data suggest that the use of dendrimer surfaces will increase the applicability of using protein biomolecular motors for nanotechnological applications

Differential regulation of apoptosis in slow and fast twitch muscles of aged female F344BN rats

Age-related muscle atrophy is characterized by decreases in muscle mass and is thought be mediated, at least in part, by increases in myocyte apoptosis. Recent data has demonstrated that the degree of muscle loss with aging may differ between males and females while other work has suggested that apoptosis as indicated by DNA fragmentation may be regulated differently in fast- and slow-twitch muscles. Herein, we investigate how aging affects the regulation of muscle apoptosis in the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of young (6-month), aged (26-month), and very aged (30-month) female Fischer 344/NNiaHSD × Brown Norway/BiNia (F344BN) rats. Tissue sections were stained with hydroethidium for ROS and protein extract was subjected to immunoblotting for assessing apoptotic markers. Our data suggest that decreases in muscle mass were associated with increased DNA fragmentation (TUNEL positive) and increases in reactive oxygen species (ROS) as determined by hydroethidium staining in both the EDL and soleus. Similar to our previous work using aged male animals, we observed that the time course and magnitude of changes in Bax, Bcl-2, caspase-3, caspase-9, and cleavage of α-fodrin protein were regulated differently between muscles. These data suggest that aging in the female F344BN rat is associated with decreases in muscle mass, elevations in ROS level, increased muscle cell DNA fragmentation, and alterations in cell membrane integrity and that apoptotic mechanisms may differ between fiber types

Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention.

BACKGROUND:Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s) underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB) is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention. PRINCIPAL FINDINGS:Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-beta), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6-months. CONCLUSIONS:These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction

Akt dysfunction is associated with decreases in myosin, actin and muscle fiber cross-sectional area.

<p>A. Myosin and actin protein levels in 6-, 27-, 33-month control (33C) and acetaminophen-treated (33T) rats. Data are mean±SE (n = 4–6). B. Muscle fiber cross-sectional area (µm²/fiber). Upper panel: Representative images of dystrophin-stained soleus muscle section from 6-, 27-, 33C- and 33T rats. Lower panel: Vertical box and whisker plots showing the distribution of muscle fiber cross-sectional area: median values (+), the 25th and 75th percentile (the bottom and top of box, respectively), and the minimum and maximum (the bottom and top end of whisker, respectively). Number of fibers measured for 6-, 27-, 33C- and 33T-rats were 162, 207, 350 and 318, respectively. abcd: Groups without the same letter are significantly different (P<0.05).</p

Aging-associated hyper-phosphorylation of Akt can be attenuated by acetaminophen.

<p>Akt total protein and the phosphorylation of Akt at Ser473 and Thr308 in soleus muscle from 6-, 27-, 33-month control (33C) and acetaminophen-treated (33T) F344BN rats were determined by immunoblotting. Data are mean±SE (n = 4–6). abc: Groups without the same letter are significantly different (P<0.05).</p