C-Met expression and mechanical activation of satellite cells on cultured muscle fibers

Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0-6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.