High-Resolution Measurements of Subsarcomere Calcium-Binding Distributions from EPXMA Images

Abstract

The distribution of calcium binding to thin filaments in vertebrate striated muscle provides clues about the role of calcium in regulation of contraction. A number of studies have suggested that the extent of Ca binding to the thin filament regulatory protein, troponin, is enhanced by the attachment of crossbridges. Electron probe X-ray microanalysis (EPXMA) has provided direct evidence of this enhancement by showing that more Ca is found in the overlap than in the nonoverlap region of sarcomeres in skinned rabbit psoas muscle in rigor. In that study, characterization of the spatial extent of this enhancement was limited by the pixel width of bands (4 pixels) within which Ca counts were averaged at each position across the sarcomere. In order to improve the resolution with which data are extracted from the image we have developed an automated routine for averaging calcium counts from successive columns of pixels across each half sarcomere in the image.