Our long term goal is to determine the molecular mechanisms underlying the diminished skeletal muscle performance with age. We hypothesize that age-dependent damage of specific amino acid residues in key skeletal muscle proteins impacts skeletal muscle performance by altering protein structure and function. In support of this hypothesis, our previous investigations clearly demonstrate that the decline in specific force is due, in part, to changes in the structure of myosin resulting from site-specific post-translational modifications. However, changes in myosin do not explain the total age-related muscle dysfunction. Our previous findings indicate an age-induced alteration in calcium sensitivity, strongly implicating Troponin C. There is a significant reduction in shortening velocity in type II fibers that cannot be explained by myosin isoform switching, suggesting that the regulatory proteins, myosin light chains, are involved. This proposal has two aims:
AIM 1. Determine the role of Troponin C in age-related skeletal muscle dysfunction (reduction in calcium sensitivity). We predict that specific amino acids are modified in Troponin C with age, rendering the protein less sensitive to calcium.
AIM 2. Determine the role of myosin light chains (MLC1f and MLC3f) in age- related skeletal muscle dysfunction (reduction in contraction velocity). In order to test mechanistically how these two proteins contribute to muscle dysfunction we will use three approaches. First, we will purify Troponin C and myosin light chains from different aged rats and test their physiological interactions. Second we will identify the in vivo type of post-translational chemical modification and the specific amino acid site of modification using proteomic technology. Third, we will use Recombinant Adenovirus technology to over- express specific myosin light chain isoforms in different aged rats to test mechanistically how alterations in protein expression contribute directly to the slowing of contraction. The Fischer 344 rat will be our aging model concentrating on three age groups (adult, old and aged) representing the lifespan of the animal. This proposed research brings together a powerful research team and a combination of techniques, including physiology, biochemistry, proteomics, and molecular biology to test fundamental questions regarding novel roles for Troponin C and myosin light chains in aging. Once we are armed with the type of chemical modification and the specific site of modification, we will be able to test if these in vivo modifications are responsible for altered protein structure/function with age. Narrative During aging there are many opportunities for appropriately transcribed peptides and proteins to become structurally altered. Previous studies show that accumulation of altered proteins, due to post-translational modifications, is correlated with a loss of function. Therefore, it is critical to identify the alterations of specific proteins during aging and to define their roles in age-related muscle dysfunction. Presently, the role of age- related post-translational modifications of specific protein amino acids on protein structure and protein function in key skeletal muscle proteins is unknown. This proposal investigates two key skeletal muscle proteins, Troponin C and myosin light chains, which are candidates to explain age-related muscle dysfunction.
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