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University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2003 • 113-114
Subtractive Proteomic Profiling of Control, Atrophied, and Protected Rat Skeletal Muscle by Dynamic Foot Stimulation (DFS)
SKELETAL MUSCLE ATROPHY IS OF great concern to NASA in its daily operations due to the potential impact upon crew physical performance. To date, the single most effective muscle atrophy countermeasure that can be easily deployed on-orbit is exercise. However, the projected amount of time—as high as three hours per day—required to perform daily prescribed exercise countermeasures on the International Space Station (ISS) will be a significant drain on the productive time of crew members. Therefore, a countermeasure designed to enhance the effect of existing exercise countermeasures or one that provides an intermittent level of muscle stimulation/contraction in an unobtrusive fashion to the crew member throughout the daily routine may prove of great value in maintaining muscle mass and function during the extended periods of low or microgravity that will be encountered during the planned Lunar and Martian exploration missions.
Previous work funded, in part by, the Institute for Space Operations (ISSO) in both human and rat models has shown that mechanical stimulation of the soles of the feet (a.k.a. Dynamic Foot Stimulation–DFS) increases neuromuscular activation in the lower limb musculature of humans and, more importantly, attenuates development of muscle atrophy in the lower limb musculature of hind-limb suspended (HLS) rats, a well-accepted ground analogue of space flight-induced muscle atrophy (see Preliminary Studies Section). Based on the concept that neuromuscular activation promotes a eutrophic state in skeletal muscle tissue (e.g., maintenance of muscle mass and fiber type, maintenance of neuromuscular junction size and complexity, promotion of synaptic efficiency), we have recently demonstrated that DFS applied to the sole of the foot during HLS results in a marked attenuation of muscle atrophy in rats. These results indicate that DFS induces trophic effects in skeletal muscle capable of overcoming the atrophic effects of unloading. Most importantly, with regard to the transferability of our observations in a rodent model to astronauts, we find that both rats and humans share similar sensorimotor/proprioceptive pathways and neuromuscular activation responses to DFS.
The overall aim of this project is to build on the information obtained in our rat DFS model by investigating the underlying cellular and biochemical mechanisms involved in the muscle atrophy response to mechanical unloading. To date, experimental studies aimed at understanding the biochemical and molecular events responsible for muscle atrophy have primarily been carried out using gene chip arrays that compare gene expression profiles between control and atrophied tissue. Although an extremely powerful analytical technique, studies in gene expressions suffer from two basic limitations: (1) Not all gene expression changes between experimental conditions are directly attributable to the experimental variable of interest (e.g., mechanical unloading). (2) Even if a gene expression change is detected, this phenomenon does not necessarily mean that there is concomitant change in either the expression or activity of the gene product, the true biological effector molecule in living systems, namely the protein encoded for by that gene. Experimental techniques which focus on the expression of actual proteins, rather than DNA/RNA in atrophied muscle, by definition directly observe biochemical changes modulated during the mechanical unloading response, rather than provide a genetic marker to those proteins that may or may not be modulated.
The second limitation noted above, specifically, the inclusion of tissue obtained from a rescue experiment, is a function of the development and availability of such rescue measures. The approach in this project is to compare protein expression profiles in skeletal muscle from control, HLS (i.e., atrophied) and DFS-treated (i.e., atrophy prevention) animals. By performing a "subtractive" proteomics approach to the experimental problem, only those proteins which are modulated by mechanical unloading and that respond in turn to the rescue measure (i.e., DFS) will be detected.
Discovery Phase (Year 1) This task will utilize a technology known as surface enhanced laser desorption/ionization (SELDI) time of flight mass spectroscopy (TOFMS) that allows a comprehensive protein expression profile to be generated from tissues of interest. This technology has been widely used to identify potential serum biomarkers of particular disease states. It has also been employed to detect protein changes in different tissues. The UH Principal Investigator has direct experimental experience in utilizing this technology to compare protein expression profiles in a number of different tissues and model systems.
Identification Phase (Year 2) This task will focus on identifying those protein changes detected in the discovery phase. Discovery can be achieved by a number of different approaches. The simplest approach is comparison of the approximate iso-electric point (pI) value and highly accurate molecular mass value of the protein obtained using SELDI-TOFMS analysis to existing protein databases, such as the SWISS protein data base which contain (pI, Mr) values for a wide number of previously reported proteins. The second approach is to perform protein identification by means of tryptic digestion of the protein followed by peptide mapping using SELDI-TOFMS analysis of the peptides. The peptide map is then compared to an NIH-provided peptide map database for previously reported proteins.
The Laboratory for Integrated Physiology (LIP), housed in the Department of Health and Human Performance at the University of Houston has a fully functional biochemistry laboratory in which the ISSO Post-Doctoral Aerospace Fellow will carry out specific elements of this study including biochemical analysis. The Muscle Research Laboratory at NASA-Johnson Space Center is also a fully equipped biochemistry laboratory. Core facilities at NASA-JSC available for use in this project are a fully staffed animal facility (currently the home of the DFS-HLS rat model of muscle atrophy) and a proteomics core facility that is equipped with a SELDI-TOFMS analysis system and robotic work station to ease the task of preparing samples.
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Institute for Space Systems Operations - Y2003 Annual Report
Copyright © 2004
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