Skeletal muscle atrophy is of great operational concern to NASA 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 productive crew member time. Therefore, a countermeasure designed to either 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 his/her 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 is that both rats and humans share similar sensorimotor/proprioceptive pathways and neuromuscular activation responses to DFS.

Project Rationale

The overall aim of this proposal is to build on the information obtained in our rat DFS model, to investigate 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, gene expressions studies 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 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. As such, experimental techniques which focus on the expression of actual proteins, rather than DNA/RNA, in atrophied muscle are by definition directly observing biochemical changes that are modulated during the mechanical unloading response, rather than providing a genetic marker to those proteins that may or may not be modulated.

The second limitation noted above, namely the inclusion of tissue obtained from a rescue experiment, is a function of the development and availability of such rescue measures. The approach that will be used 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.

Experimental Tasks

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 generate from tissues of interest. This technology has been widely used to identify potential serum biomarkers of particular disease states, as well as being 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. This 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.

Facilities

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 Postdoctoral 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 for sample preparation.

Desired Academic Background/Work Experience

The candidate for this ISSO Postdoctoral Position will be a recent Ph.D. graduate with a strong back-ground in biochemistry/cell biology with an emphasis in protein biochemistry/proteomics. The applicant should also have direct research experience in protein analysis/protein interactions as evidenced by either publications in peer-reviewed journals or Ph.D. dissertation research. Appropriate training will be provided in SELDI-TOFMS analysis techniques.

Professional Biographies of Investigators

Mark S. F. Clarke, Ph.D., Associate Professor
Department of Health and Human Performance
University Of Houston

Dr. Clarke received both his M.I. BIOL (Pharmacology) and Ph.D. (Cell Biology/Biochemistry) from Manchester Metropolitan University in the United Kingdom. His post-doctoral training included post-doctoral fellow positions in the Departments of Cell Biology and Anatomy at Harvard Medical School (1991-92) and the Medical College of Georgia (1992-95). He then served as National Research Council Resident Research Fellow at NASA-Johnson Space Center in the Biomedical Operations Branch (1995-98) before becoming a Staff Scientist with Universities Space Research Association in the Division of Space Life Sciences (1998-2002). Dr. Clarke recently joined the faculty at the University of Houston as an Associate Professor in order to establish the multi-disciplinary Laboratory of Integrated Physiology which serves as the research focus for the Department of Health and Human Performance at UH. His research has focused on applying an integrated approach to understanding the physiological de-adaptation of the musculo-skeletal system during space flight using a wide variety of ground-based analogues, including human bed-rest models, rat unloading models and various tissue culture models. During that time, he has developed a variety of novel technologies compatible with the microgravity environment in order to allow real-time biomedical monitoring of crew members. Dr. Clarke has published over 20 articles in peer-reviewed journals, five books chapters, received four U.S. patents (with an additional four pending) and been awarded four NASA Space Awards for Technical Innovation. He serves as an academic advisor to five Ph.D. graduate students and one M.Sc. student in HHP at the University of Houston.

Daniel L. Feeback, Ph.D.
Head, Muscle Research Laboratory and STS Sortie Mission Scientist
Human Adaptations and Countermeasures Office
Lyndon B. Johnson Space Center
NASA-Johnson Space Center Principal Investigator

Dr. Feeback received a B.S. in Medical Technology from Missouri Western State and completed his Medical Technology Internship at North Kansas City Memorial Hospital. He received a Ph.D. in Pathology and Laboratory Medicine from the University of Oklahoma Health Sciences Center in Oklahoma City. After receiving his doctorate, he joined the faculty of the University of Oklahoma College of Medicine as an Assistant Professor in the Department of Pathology, as well as Adjunct Assistant Professor in the Colleges of Dentistry, Pharmacy and Allied Health. Currently, he is Adjunct Associate Professor at Rice University in the Department of Biochemistry, Institute of Biosciences and Bioengineering and lectures at the University of California at Berkeley, University of Houston, and the University of Texas Medical Branch in Galveston. Dr. Feeback is a board-certified Clinical Laboratory Scientist and a member of numerous professional and scientific societies, including the American Society for Clinical Pathology, American Association of Anatomists, Histochemical Society, New York Academy of Sciences, and Society for Experimental Biology and Medicine, and is a founding member of the Society for Experimental Neuropathology. His research interests center on alterations in skeletal muscle structure and function associated with decreased mechanical loading such as occurs in association with space flight. A variety of model systems including human bed-rest, murine hindlimb suspension, and skeletal muscle tissue culture have been used in the study of muscle atrophy in the contest of unloading with the particular goal of discovering novel approaches to counter muscle loss under unloading conditions. Dr. Feeback has published over 75 scientific papers in his areas of research interest, three books and four book chapters and is co-inventor on four U.S. patents.