Long Term Functional and Morphological Characterization of Muscle Cell Cultures

Skeletal muscle is the mammalian body's largest protein resevoir, serving the critical role of mobilizing proteins during acute conditions, such as trauma and starvation, and chronic conditions, such as AIDS, cancer, and muscular dystrophy. When proteins are mobilized in excess of physiological needs, muscle wasting ensues. With advances in the treatment of the primary effects of these conditions, muscle wasting, which formerly may have been of secondary concern, has become an impedement to full recovery from these conditions. With recognition of the growing importance of muscle wasting to clinical rehabilitation, the need to elucidate the mechanistic underpinnings of muscle wasting has become more acute. Previously, whole body and organ culture have been the choice models for investigation of protein turnover in skeletal muscle. These models have been useful in showing that myofibrillar proteins are under physiological regulation by hormones and cytokines. However, these models are valid for only several hours, contain many inherent couplings and cell types, and it is difficult to separate protein synthesis from degradation, all of which make elucidation of complex proteolytic pathways difficult. Cell culture can be a very powerful system for investigation of cellular metabolic pathways, offering more precise control of the cell culture micro-environment and the ability to measure synthesis and degradation independently. However, caution must be taken to insure that the muscle specific components and degradative pathways which are regulated in adult muscle are present in the cell culture system. This study was undertaken to address these concerns and emphasize the need for thorough biochemical analysis before muscle specific protein turnover may be investigated in cell culture. The L6 cell line was used as a model cell culture system in this study. Using creatine phosphokinase (CPK) and DNA, media conditions were optimized to produce maximal differentiation in the short-term. Next, using CPK, tropomyosin, and ax-actinin, long-term studies were undertaken to evaluate the suitability of the L6 cell culture system for protein turnover experiments. Finally, static mechanical tension was applied to the cultured L6 cells as a potential method of augmenting of muscle specific function. Long-term biochemical analysis of CPK showed that differentiated muscle cells were quasistable in the optimized media for a period of 7 to 14 days depending on the stringency of the requirements. However, immunoflourescence staining during this period revealed that muscle specific tropomyosin degraded heterogeneously across the cultures before organizing into myofibrils. Work with short-term passive mechanical stretch showed that CPK levels increased two-fold over controls, suggesting that, after appropriate optimization, passive tension may be a viable method for maintaining muscle specific function during protein turnover studies. Thesis Supervisor: Dr. Mehmet Toner Title: Assistant Professor of Surgery