Death receptor-associated pro-apoptotic signaling in aged skeletal muscle

Abstract

Tumor necrosis factor-alpha (TNF-alpha) is elevated in the serum as a result of aging and it promotes pro-apoptotic signaling upon binding to the type I TNF receptor. It is not known if activation of this apoptotic pathway contributes to the well-documented age-associated decline in muscle mass (i.e. sarcopenia). We tested the hypothesis that skeletal muscles from aged rodents would exhibit elevations in markers involved in the extrinsic apoptotic pathway when compared to muscles from young adult rodents, thereby contributing to an increased incidence of nuclear apoptosis in these muscles. The plantaris (fast) and soleus (slow) muscles were studied in young adult (5-7 mo, n=8) and aged (33 mo, n=8) Fischer(344) x Brown Norway rats. Muscles from aged rats were significantly smaller while exhibiting a greater incidence of apoptosis. Furthermore, muscles from aged rats had higher type I TNF receptor and Fas associated death domain protein (FADD) mRNA, protein contents for FADD, BCL-2 Interacting Domain (Bid), FLICE-inhibitory protein (FLIP), and enzymatic activities of caspase-8 and caspase-3 than muscles from young adult rats. Significant correlations were observed in the plantaris muscle between caspase activity and muscle weight and the apoptotic index, while similar relationships were not found in the soleus. These data demonstrate that pro-apoptotic signaling downstream of the TNF receptor is active in aged muscles. Furthermore, our data extend the previous demonstration that type II fibers are preferentially affected by aging and support the hypothesis that type II fiber containing skeletal muscles may be more susceptible to muscle mass loses via the extrinsic apoptotic pathway.

Fig. 1

Rodent and skeletal muscle characteristics. Rodent bodyweight (A), as well as absolute (B) and normalized (C) muscles weights and incidence of apoptosis (D) was determined for plantaris and soleus muscles. Data presented as means ± SE. * Significant effect of age (p < 0.05). PL-plantaris, SOL-soleus

Fig. 2

RT-PCR analysis of muscle mRNA. The mRNA content for TNF-α (A), TNFR (B), TRADD (C), and FADD (D) was determined by RT-PCR, with PCR products normalized to the 18S gene. Representative agarose gel images following electrophoresis are displayed for each group. Data are presented as means ± SE. * Significant effect of age (p < 0.05)

Fig. 3

Western-immunoblot analyses. The protein content for the soluable TNFR (A), FLIP (B), Bid (C), and FADD (D) was determined by western immunoblot. Representative immunoblots for TNFR, FADD, BID, and FLIP are displayed for each graph. * Significant effect of age (p < 0.05). PL-plantaris, SOL-soleus

Fig. 4

Caspase enzymatic activities. The caspase activity of caspase-8 (A) and caspase-3 (B) was measured using specific fluoremetric substrates. Data are presented as means ± SE. * Significant effect of age (p < 0.05)

Fig. 5

Correlational analyses in plantaris and soleus muscles, plan-taris weight and the apoptotic index. The relationships between muscle phenotypes and apoptotic indicators were determined by calculating the Pearson correlation coefficient. The following relationships were analysed: absolute plantaris weight and the apoptotic index (A), normalized plantaris weight and the apoptotic index (B), plantaris caspase-8 activity and the apoptotic index (C), plantaris caspase-3 activity and the apoptotic index (D), absolute soleus weight and the apoptotic index (E), normalized soleus weight and the apoptotic index (F), soleus caspase-8 activity and the apoptotic index (G), soleus caspase-3 activity and the apoptotic index (sh=squ). = young adult samples, [◇ = aged samples

Fig. 6

Cross-talk between apoptotic pathways. The extrinsic apoptotic pathway, downstream of the type I TNFR, is active in aged skeletal muscle and consists of FADD, caspase-8, and caspase-3. Cleaved caspase-8 can activate BID, which can interact with Bax and promote further pro-apoptotic signaling arising from the mitochondria