Quantitative electrophoretic analysis of myosin heavy chains in single muscle fibers

To better understand the molecular basis of the large variation in mechanic al properties of different fiber types, there has been an intense effort to relate the mechanical and energetic properties measured in skinned single fibers to those of their constituent cross bridges. There is a significant technical obstacle, however, in estimating the number of cross bridges in a single fiber. In this study, we have developed a procedure for extraction and quantification of myosin heavy chains (MHCs) that permits the routine a nd direct measurement of the myosin content in single muscle fibers. To val idate this method, we also compared MHC concentration measured in single fi bers with the MHC concentration in whole fast-twitch (psoas and gracilis) a nd slow-twitch (soleus) muscles of rabbit. We found that the MHC concentrat ion in intact psoas (184 muM) was larger than that in soleus (144 muM), as would be expected from their differing mitochondrial content and volume of myofibrils. We obtained excellent agreement between MHC concentration measu red at the single fiber level with that measured at the whole muscle level. This not only verifies the efficacy of our procedure but also shows that t he difference in concentration at the whole muscle level simply reflects th e concentration differences in the constituent fiber types. This new proced ure should be of considerable help in future attempts to determine kinetic differences in cross bridges from different fiber types.