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One mechanism that may influence the quality of skeletal muscle proteins, and explain the age-related decline in contractility, is protein damage. Advanced glycation end-products (AGE) in vivo are useful biomarkers of damage. In this study, comparison of extensor digitorum longus (EDL) muscles from young (8 months), old (33 months), and very old (36 months) Fischer 344 Brown Norway F1 (F344BNF1) hybrid rats shows that muscles from the very old rats have a significantly higher percentage of myofibers that immunolabel intracellularly for AGE-antibody 6D12 compared to the younger age group. The AGE-modified proteins, determined in the semimembranosus muscles from young (9 months) and old (27 months) F344 rats, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry include creatine kinase, carbonic anhydrase III, beta-enolase, actin, and voltage-dependent anion-selective channel 1. Moreover, there is a significant increase in AGE modification of beta-enolase with age. These results identify a common subset of proteins that contain AGE and suggest that metabolic proteins are targets for glycation with aging.

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On the basis of the accelerated age-related effects in type II muscle, we hypothesized that with aging the semimembranosus (type II) muscle would accumulate a greater amount of oxidized proteins compared to proteins in the soleus (type I) muscle. In this study, 3-nitrotyrosine (3-NT) was used as a stable marker of protein oxidative damage. The presence of 3-NT was evaluated in muscles from young adult, old, and very old Fischer 344 rats to provide an indication of the time course of muscle protein oxidative damage. A significant age-associated increase in nitrotyrosine-modified proteins was observed. The modified proteins identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry include the sarcoplasmic reticulum Ca(+2)-ATPase, aconitase, beta-enolase, triosephosphate isomerase, and carbonic anhydrase III. These proteins, involved in metabolism and calcium homeostasis, exhibited an age-dependent increase in 3-NT content in both muscles. However, significant levels of 3-NT modification were present at an earlier age in the semimembranosus muscle.

While the age-related loss in muscle mass partially explains the decline in strength, other yet undefined mechanisms contribute. This study investigates whether changes in myosin-actin stoichiometry and oxidative modification could help explain the decrement in muscle strength with aging. Protein expression and oxidation were evaluated in myosin and actin isolated from the soleus and semimembranosus muscles from young adult, old, and very old Fischer 344 rats. In the soleus muscle, actin and myosin content did not change with aging. In the semimembranosus, actin content was stable, but myosin exhibited decreased content in muscles from very old rats, resulting in a decrease in the myosin-to-actin ratio. 3-Nitrotyrosine and 4-hydroxy-2-nonenal were used as markers of protein oxidative damage. Although myosin and actin are modified with 3-nitrotyrosine and 4-hydroxy-2-nonenal, the extent of chemical modification does not increase with age. The results suggest that the decline in force production with age is not due to the accumulation of these two specific markers of protein oxidation on the myofibrillar proteins. Additionally, age-dependent changes in myofibrillar stoichiometry do not contribute to the decline in force production in the soleus, but may play a role in the semimembranosus with advanced age. Keywords 3-nitrotyrosine; 4-hydroxynonenal; skeletal muscle; proteomics Aging is accompanied by a general decline in muscle strength (3, 36). The decrease in muscle strength is explained, at least in part, by a decrease in muscle mass. However, strength declines more than would be expected from the reduced muscle or fiber size, thus suggesting other mechanisms are involved (3, 4, 35, 37). One possibility is there are defects in the myofibrillar proteins responsible for force generation. This idea is supported by the age-related loss in force measured in permeabilized fibers, where muscle membranes are experimentally removed and force generation is entirely dependent on the interaction of myofibrillar proteins (35, 37, 39). With aging, a disruption in the interaction of myosin and actin is noted in several studies. Electron paramagnetic resonance spectroscopy was used to measure the percentage of myosin that is in optimal interaction with actin to maximize force generation, referred to as the strong binding structural state, vs. when myosin and actin are weakly bound and thus not producing force. In aged muscle, there was a reduction in the fraction of myosin heads in the strong-binding structural state, such that there are fewer myosin-actin interactions capable of generating force (25). In addition, a significant age-related inhibition of myosin ATPase, critical for generating force, was reported from investigations of isolated proteins (myosin and actin) from young and old animals (32). Thus mechanisms that decrease or interrupt the interaction of myosin and actin are likely to explain the age-related reduction in force-generating capacity. This study investigates two potential mechanisms that could contribute...