The deterioration of the musculoskeletal system with age that leads to sarcopenia and osteoporosis is a multi-factorial reality that has significant clinical limitations for muscle strength, mobility and loss of functional independence in frail older adults. Murine models may provide a window into the environmental and genetic factors that contribute to the process. This grant will test two independent yet converging hypothesis: one model based on lineage regulation that focuses on a common progenitor cell with the ability to develop into the myogenic, osteogenic or adipogenic lineages, and a second model in which invading adipocytes adversely influence the immunological and contractile status of mature muscle tissue. The first model envisions that age-related changes in gene activity result in a disruption of lineage progression from its primary or default direction (bone and muscle) to a secondary alternative direction (fat). To test this model, GFP-reporter constructs that mark specific levels of cellular differentiation (Col3.6, bone;AP2, fat;myoD, muscle;smooth muscle actin.SMA, myofibroblast) will be introduced in to C57BI/6 (affected) and 129 P3/J (resistant) mice and the animals will be aged to 10 and 24 months. By combining reporter constructs with complementary colors, the ability of the lineage to progress from a progenitor to fully mature bone or muscle cells, or the branch point of the progenitor to select the primary or adipocytic lineage will be assessed in a primary cell culture model. The tempo and extent of differentiation will be assess in real time using repetitive fluorescence imaging of the same culture well, and the proportion of cells at a defined levels of development will be obtained by FAC analysis. In vitro analysis will suggest levels of differentiation that are particularly sensitive to genetic and age-related factors and the potential genetic mediators of the effect will be examined by microarray analysis of the FAC isolated cell populations. In vivo validation of the culture results will be performed by progenitor transplantation experiments into young and aged hosts, and by a BrdU labeling study that assesses lineage choices of the endogenous progenitor cells. To test the second model of in-situ tissue disruption by adipocytes, the two experimental groups will be phenotyped for appearance of GFP positive fat cells in bone and muscle and for a spatial correlation of adipocytes with disorganized microstructure within muscle fiber cells. The tools, techniques and molecular concepts that are developed in this grant will provide a foundation for future QTL studies in mice designed to map genetic factors that contribute to age-related changes in skeletal health.
