Dr. Larry L. Lowe, Department of Biological and Physical Sciences, Benedict College, and Dr. James A. Carson, Department of Exercise Science, University of South Carolina, Columbia, have been awarded $75,000 from the EPSCoR/BRIN Collaborative Research Program (CRP) to work on a project entitled “Aging Skeletal Muscle’s Decreased Regenerative Capacity: Inflammation’s Critical Role.”  The overall goal of this project is to determine if aging alters the inflammatory response that follows overload-induced muscle injury, and if the altered response causes decreased muscle regeneration in aged skeletal muscle.  Drs. Lowe and Carson plan to reach this goal by conducting studies to (1) determine the effect of aging on circulating pro-inflammatory cytokines in rats that are undergoing skeletal muscle regeneration caused by induced overload injury; (2) determine the effect of aging on local pro-inflammatory cytokine production within skeletal muscle induced to regenerate from overload-induced injury; and (3) determine if anabolic steroid administration alters systemic and local pro-inflammatory cytokine induction in animals’ skeletal muscle regeneration that results from overload injury.   As Principal Investigator, Dr. Lowe, whose research centers on skeletal muscle biochemistry and molecular biology, with an emphasis on muscular loading, is directing the project and serving as mentor to Co-Principal Investigator Dr. Carson.  Dr. Carson, whose research involves the production of overload-induced hypertrophy in animal models, with particular emphasis on the effects of aging, will help ensure successful administration of treatments and collection of samples for analysis during the CRP project.

 

Although skeletal muscle is an extremely adaptive tissue, its regenerative capacity is reduced with aging.   Understanding how and why older skeletal muscle does not regenerate as fully as younger muscle would help care givers make more informed decisions regarding the prescription of environmental interventions like changes in diet and exercise, and pharmaceutical interventions like the administration of anabolic steroids or other substances.  This knowledge would, therefore, lead to programs and therapies that could result in greater muscle regeneration or growth in older people.  This is significant, given that muscle loss is linked to many of the “symptoms” of old age, including frailty, obesity, and the resultant decreased quality of life.

 

Studies of elderly people clearly demonstrate that the ability to increase muscle mass through resistance exercise is maintained as we age. However, the extent to which muscle repair and regeneration after overload-induced injury is altered in elderly humans is less clear.  Evidence strongly suggests that decreases in muscle regenerative capacity caused by age are not intrinsic to the muscle itself, but rather dependent on the aging individual as a whole.  These facts suggest that signaling stimuli that target muscles may be deficient in the aged individual, and therefore aged muscle regenerative capacity could be restored or improved if provided the appropriate stimulus.

 

Functionally overloading skeletal muscle results in injury and inflammation prior to muscular growth and regeneration. The inflammatory response, which is necessary to initiate growth and healing, involves chemoattractants and cytokines that recruit and activate leukocytes to remove injured tissue and release growth factors to activate latent satellite cells.  This response is altered in aged animals, as plasma cytokines are elevated and leukocyte phagocytic activity to infectious challenges is reduced.  It is not known if aging also alters the inflammatory response to functional overload-induced skeletal muscle injury.  If so, this alteration could contribute to decreased muscle regeneration in aged individuals.

 

Research conducted by Drs. Lowe and Carson during this CRP project centers on the role age plays in determining inflammatory response that follows an overload induced muscle injury.  CRP project studies are designed to also determine if this inflammatory response, which is expected to be different in older skeletal muscle that in younger muscle, contributes to decreased muscle regeneration in aged muscle.  Drs. Lowe and Carson hypothesize that plasma cytokine concentrations will increase during muscle regeneration induced by functional overload, and that the increase will be greater in the older rats.  They further hypothesize that aging will cause an increased pro-inflammatory cytokine induction in the regenerating muscle, that the corresponding molecular RNA (mRNA) concentrations will increase, and that the increase will again be greater in the older rats.  Finally, Drs. Lowe and Carson expect that the administration of the anabolic steroid will decrease both the elevated cytokine concentrations and the pro-inflammatory cytokine induction in the aged animals.

 

To meet the CRP research goals, Drs. Lowe and Carson are conducting studies on male Fisher Brown Norway rats, half approximately 5 months old, and half approximately 25 months old.  Experiments involve the surgical removal of part of the gastrocnemius to produce functional overload, resulting in rapid and large increase in muscle mass, on the remaining calve muscles, the soleus and plantaris; the administration of a testosterone derivative (nandrolone decanoate) that has been shown to have an anabolic effect on rat skeletal muscle to test subjects, and sesame seed oil to controls; blood collection and analysis to determine plasma cytokine protein concentrations; protein extraction to determine protein concentration; Western Blot analysis; and total RNA isolation, cDNA synthesis and RT-PCR. 

 

Dr. Carson’s “Integrative Muscle Biology Laboratory,” located in the USC Arnold School of Public Health’s Department of Exercise Science, is conducting the surgeries on the rats required for the project, and is completing the initial preparation work for protein analysis and imaging.  USC Exercise Science graduate students Tyrone Washington and Joe McClung are working full-time on the CRP project to assist Dr. Carson with the surgeries, tissue harvesting, and sample and data analysis.  Mr. Washington and Mr. McClung are also using CRP project studies to teach other USC graduate and USC and Benedict undergraduate students about relevant research methods and techniques.  USC graduate students working in Dr. Carson’s lab have also presented their research results to undergraduates on the Benedict College campus.

 

Benedict College is a 4-year HBCU (Historically Black College and University), whose Department of Biological and Physical Sciences offers majors in Biology, Chemistry, Environmental Health Science, Physics, and Physics-Engineering.  In recent years Benedict has encouraged its faculty and undergraduate students to participate in research by recruiting new faculty, enhancing research space, and purchasing equipment.   Dr. Lowe’s laboratory, located in the newly renovated science building, Alumni Hall, is equipped with state-of-the-art instruments that are being used to complete mRNA and gene expression studies associated with the CRP project.  Studies conducted in Dr. Lowe’s lab include the use of ELISA analysis to determine circulating cytokine levels, and the use of semi-quantitative PCR analysis to determine cytokine mRNA levels.   

 

Dr. Lowe recently became Chair of Benedict’s Department of Biological and Physical Sciences, and has been involved with several programs that are designed to promote research in the sciences.  He is currently the Benedict College Campus Director of the South Carolina Space Grant Consortium. Composed of eleven institutions of higher education in South Carolina, the Consortium supports NASA-related research grants and scholarship and research awards to South Carolina colleges and universities.  The objective of the National Space Grant Program is to support faculty and students who intend to pursue further study or careers in science, technology, and engineering. Four Benedict undergraduates have received research awards under the supervision of Dr. Lowe since the College became a member institution in March 1996. All four former students are currently attending graduate or professional schools in science and health careers.

 

Dr. Lowe continues his mentorship of undergraduates while working on the CRP project.  Benedict undergraduate Diana Registe (biology major) and Maria Velez Quinones (biology major – Universidad Metropolitana of Puerto Rico) began full-time work on the project this past Summer, and will also work full-time during the Summer of 2003.  Zennette Lee (Benedict College undergraduate biology major and South Carolina Space Grant recipient) is also involved in the project under Dr. Lowe’s supervision.  These undergraduates are working under the direct supervision of Dr. Lowe and in collaboration with Dr. Carson, and have to date assisted with lactate dehydrogenase (LDH) zymogram analysis and messenger RNA (mRNA) differential display PCR experiments involving overload-induced injured muscles provided by Dr. Carson’s lab.  Students learned about the biochemistry and molecular biology relationships to the overall project goals and objectives.  Students also gain the experience of working with state-of-the art equipment including the Stratagene Eagle Eye II Still Video DNA Imaging and Sequencing System, the Beckman Coulter LS 6500 Multipurpose Scintillation Counter, the Futura 2000 K Automatic X-Ray Film Processor and the Perkin-Elmer 2400 Thermal Cycler.   Dr. Lowe has also assigned individual subprojects involving CRP project research to both students.  Diana Regiate’s project involves bacteria transformation and phenotypic changes due to the influence of unique cDNAs generated from total RNA extracted from skeletal muscles exposed to the atrophy of  simulated microgravity.  Zennette Lee’s  and Maria V. Quinone’s  projects involves the isolation and cloning of  unique cDNAs generated by means of mRNA differential display PCR from total RNA extracted skeletal muscles exposed to overload-induced injury.   Students will present the results of their findings at state and national conferences, including the South Carolina Academy of Sciences Annual Meeting and the 60^th Joint Annual Meeting of Beta Kappa Chi/National Institutes of Science/Brookhaven Semester Program to be held in New York March 2003.

 

To date, Drs. Lowe and Carson have made substantial progress in completing the research outlined in their CRP proposal.  They have conducted biochemical analysis studies on 40 rats (20 old and 20 young), and have characterized muscle response at the onset of functional overload.  Drs. Lowe and Carson are currently examining how this response differs in old and young muscle.  They have determined that markers of cell proliferation and differentiation are altered in aged muscle, and that many of the markers induced during overload in young muscle are not induced in old muscle.  They have also determined that the satellite cells are attenuated with age.  Currently, they are initiating immunohistochemical studies to try to link the inflammatory cytokine response to satellite cell proliferation differentiation.  Drs. Lowe and Carson will also begin conducting immunohistochemical studies on a new group of rats to determine if the testosterone derivative, which does have an effect on markers of differentiation, also has an effect on the inflammatory response.

 

Three recent articles by Drs. Lowe and/or Carson feature studies relating to the CRP project.  Two of these articles, (1) “Androgen receptor expression in aged rat hindlimb muscle after functional overload with nandrolone decanoate administration.”  J. Appl. Physiol. in press 2002.  Lee, W.J., J. McClung, G.A. Hand, and J. A. Carson; and (2) “Steroid Receptor Concentration in aged rat hindlimb muscle: The effect of anabolic steroid administration.” J. Appl. Physiol. 93:242-250, 2002. Carson, JA, WJ Lee, J McClung, and GA Hand, document preliminary studies on aged rat muscle that were used to write the CRP proposal.  In addition, a portion of the research conducted during the CRP project period is featured in an article that will be published in the American Journal of Physiology.  The article, entitled “RhoA Induction By Functional Overload And Nandrolone Decanoate Administration In Rat Skeletal Muscle," by Joseph M. McDlung, Won J. Lee, Raymond W. Thompson, and Drs. Lowe and Carson, details studies that demonstrate that RhoA protein expression, mRNA abundance, and activity are increased by the combination of functional overload and anabolic steroid treatments in rat plantaris muscle. 

 

Drs. Lowe and Carson’s project meets the primary objectives of the EPSCoR/BRIN CRP program, as it involves collaboration between a minority four-year college and a research institution to obtain significant data that can lead to extramural funding.  Data collected during the CRP project period will lay the groundwork for future studies, which will focus on regulatory mechanisms integrating androgen and cytokine signaling.  These studies would potentially include the use of pharmaceutical drugs and transgenic mouse models to quantify and manipulate the regulatory point of macrophage infiltration in injured skeletal muscle.  Drs. Lowe and Carson plan to submit an application during the Summer or Fall 2003 to the National Institutes of Health’s National Institute on Aging in response to the program announcement (PA) “Precursor Cells In Skeletal Muscle Repair And Hypertrophy, ” which funds studies to “to isolate, characterize and identify precursor cells required for normal growth and repair of injured, aged, or diseased muscle.” (Please see http://grants1.nih.gov/grants/guide/pa-files/PA-02-136.html for the full PA).