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Muscle fiber specific apoptosis and TNF- signaling in sarcopenia are attenuated by life-long calorie restriction

University of Florida, Department of Aging and Geriatric Research, College of Medicine, Biochemistry of Aging Laboratory, Gainesville, Florida, USA

¹Correspondence: University of Florida, Biochemistry of Aging Laboratory, 25 FLG, P.O. Box 118206, Gainesville, FL 32611, USA. E-mail: cleeuwen{at}ufl.edu

SPECIFIC AIMS

Loss of skeletal muscle mass and fiber number (sarcopenia) is prevalent at old age. The involvement of tumor necrosis factor- (TNF-), a catabolic cytokine found to increase with age and with the ability to signal cell survival or cell death, poses as a potential contributing factor to this condition that, so far, has been unexplored. It is not known whether life-long calorie restriction (CR) exerts some of its anti-aging capabilities by influencing the TNF- signal in vivo. We investigated apoptosis as well as inflammatory and apoptotic proteins of the TNF- signaling pathway in the soleus and superficial vastus lateralis muscles of 6-month-old, 26-month-old ad libitum-fed, and 26-month-old CR male Fischer-344 rats (CR=40% restricted compared with ad libitum). Hence, this study documents the effects of aging and life-long calorie restriction on TNF- signaling and skeletal muscle atrophy in a type I (soleus) and a type II (superficial vastus lateralis: SVL) muscle, and reports the TNF- signaling pathways demonstrated by the different muscles and the relationship between this signal and the degree of atrophy. Our results suggest that TNF- signals transmitted to specific fiber types determine the decision of selecting life or death signaling pathways and are linked to the extent of fiber loss experienced in the aging muscle; such a specific potential may constitute a major proponent in the pathogenesis of sarcopenia.

Eight 6-month-old ad libitum-fed (6AL), eight 26-month-old ad libitum-fed (26AL), and eight 26-month-old calorie restricted (26CR) male Fischer 344 rats (National Institutes of Aging Colony, Harlan Sprague Dawley, Indianapolis, IN, USA) were used. The 26CR animals had been subjected to calorie restriction starting at 3.5 months of age (10% restriction), increased to 25% restriction at 3.75 months, and maintained at 40% restriction from 4 months throughout the animal’s life (until 26 months of age). The rats were individually housed in a temperature (18–22°C) and light controlled environment with a 12 h light/dark cycle. After 1 wk of acclimation the animals were randomly killed (5/day) on consecutive days and muscles where removed for biochemical analysis.

PRINCIPAL FINDINGS

Aging was coupled with increased plasma TNF- and a greater TNF- expression in the superficial vastus lateralis muscle compared with both young ad libitum and old CR animals (Fig. 1 ). In soleus, muscle mass was not different with age, yet there was a reduction in muscle cross sectional area and fiber number in the older vs. young animals. An age-linked reduction in muscle mass, cross sectional area, and fiber number was found in the SVL; this coincided with an elevation in FADD and caspase-8 (Fig. 2 ), signaling proteins in the TNF- receptor-mediated pathway of apoptosis. Moreover, evidence of DNA fragmentation was apparent only in the SVL from the 26AL animals and not the 26CR or 6AL groups and not in the soleus muscle (data not shown). Aging also affected TNF- signaling to NF-B. Intermediary proteins (IKK, IB, and p65) key to transmission of TNF- activation of NF-B were increased with age and abrogated by CR in the soleus, but not in the SVL. NF-B activation did not differ with age or CR; however, soleus displayed 56% greater NF-B activation compared with SVL. Taken as a whole, the inflammatory and apoptotic signaling capability of TNF- appeared to be dependent on the muscle being examined. In soleus with age, we reported a greater role for the TNF- signal to be directed toward activating the transcription factor NF-B compared with that detected in the SVL. Alternatively, in the SVL, TNF- stimulated apoptotic signaling with age to a much higher extent than observed in the soleus. Moreover, TNF- stimulation of both inflammatory and apoptotic pathways were attenuated when CR was applied.

View larger version (15K): [in this window] [in a new window]   Figure 1. Immunohistochemical analysis to expose TNF- expression was performed on soleus and SVL (superficial vastus lateralis) muscle from 6- (6AL) and 26-month-old ad libitum (26AL) rats and 26-month-old calorie restricted rats (26CR) to determine whether muscle fiber type exerted an influence on the cytokines’ expression. In soleus, we found no detectable differences between the 6AL and 26AL animals (0.030±0.005 vs. 0.043±0.003 TNF- expression/fiber number/mm2; mean±SE) nor did we reveal differences when comparing 26AL with 26CR (0.043±0.003 vs. 0.03±0.01 TNF- expression/fiber number/mm2; mean±SE). There were, however, far different results found in the superficial vastus lateralis muscle. 26-month-old ad libitum animals exhibited significantly greater TNF- expression than the 6AL animals (0.069±0.02 vs. 0.039±0.003 TNF- expression/fiber number/mm2; mean±SE; *P<0.05) and compared with the 26CR animals (0.069±0.02 vs. 0.033±0.008 TNF- expression/fiber number/mm2; mean±SE; P<0.05).

View larger version (15K): [in this window] [in a new window]   Figure 2. TNF- induction of apoptosis. In view of the capability of TNF- to induce apoptosis, we investigated expression of two key proteins intrinsic to the TNF- receptor-mediated pathway of apoptosis: FADD (Fas-associated death domain) and caspase-8. Caspase-8, the activated and cleaved form of procaspase-8, belongs to the family of cysteine proteases and activates other caspases with the potential to generate programmed cell death. In soleus muscle, there were no differences between 6AL and the 26AL animals for caspase-8 protein content (cleaved caspase-8) (14557±5879 vs. 10905±4300 arbitrary units/mg protein, 6AL vs. 26AL, respectively; mean±SE); similarly, we did not observe differences when comparing the 26AL rats to the 26CR rats (10905±4300 vs. 15848±800 arbitrary units/mg protein, 26AL vs. 26CR, respectively; mean±SE). In SVL, we found a significant effect with age (7605±1296 vs. 22918±4486 arbitrary units/mg protein, 6AL vs. 26AL, respectively; mean±SE; *P=0.0478) in caspase-8 content. The dietary intervention reduced caspase-8 levels compared with the age-matched ad libitum-fed counterparts (8497±1522 vs. 22918±4486 arbitrary units/mg protein, 26CR vs. 26AL, respectively; mean±SE; P=0.0142).

CONCLUSIONS AND SIGNIFICANCE

By age 80 it is estimated that humans generally lose 30–40% of skeletal muscle fibers, particularly from muscles containing type II fibers such as the vastus lateralis. Several investigators, therefore, have attempted to elucidate the factors involved in skeletal muscle atrophy by using various experimental paradigms. Since calorie restriction has consistently been shown to increase mean and maximum life span in mammals, attenuate oxidative damage to proteins, lipids, and DNA, reduce mitochondrion dysfunction and prevent muscle contractile dysfunction and loss of muscle fibers, we investigated the ability of CR to influence TNF- signaling and the effects of life-long calorie restriction on age-driven alterations in the signaling pathways in type I and type II skeletal muscles. Moreover, relatively little is known regarding the contribution of TNF- to skeletal muscle loss with age and the possible mechanisms involved, although the inflammatory and apoptotic signaling properties of this cytokine, coupled with the levels of TNF- known to be elevated with age, may play a role during muscle aging. These data provide evidence that TNF- and TNF- receptor-mediated apoptotic events increase with age and are more prevalent in type II fiber-containing muscles (superficial vastus lateralis) than in type I fiber-containing muscles (soleus). Further, life-long calorie restriction attenuates apoptosis in aging skeletal muscle and reverses many of the age-related alterations in the TNF--mediated inflammatory and apoptotic signaling pathways. Accordingly, further examination of muscle-specific attributes from the perspective of TNF- signal transmission in an attempt to uncover why sarcopenia affects different muscles disproportionately presents an avenue worthy of additional exploration. Achieving a 10% reduction in sarcopenia prevalence in the U.S. would result in healthcare savings of $1.1 billion/year. Realizing this goal may potentially improve or generate new therapeutic interventions geared to the pathogenesis of sarcopenia and drastically curb the physical and financial burdens imposed by sarcopenia.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic diagram. Proposed overview of TNF- signaling in soleus and superficial vastus lateralis (SVL) skeletal muscles with age.

FOOTNOTES

To read the full text of this article, go to http://fasebj.org/cgi/doi/10.1096/fj.04-2870fje; doi: 10.1096/fj.04-2870fje

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