The overall long term goal of this research is to understand the mechanisms by which elevation in myoplasmic free calcium regulates contraction of vertebrate striated muscle and in particular, to explore the ways in which calcium binding to myofilaments may modulate and be modulated by the contractile event. The hypothesis to be examined proposes that calcium binding to the thin filament is enhanced by the attachment of cycling crossbridges to actin in both cardiac and skeletal muscle. This proposed phenomenon has been cited as a molecular basis for the Frank- Starling relation, and may underlie other features of the contractile event in striated muscle. This hypothesis will be tested by mapping the distribution of calcium along the sarcomere using quantitative electron probe X-ray microanalysis (EPXMA) and elemental imaging of freeze dried cryosections of glycerinated rabbit psoas muscles, frog semitendinosis, and rabbit and frog ventricular trabeculae. Fibers will be frozen in solutions with or without MgATP and with varying levels of free calcium. EPXMA of fibers following partial extractions of TnC and myosin regulatory light chains would be used to identify the source of any observed enhancement in calcium binding in the overlap region. Orthovanidate or N- phenylmaleimide will be used to determine characteristics of the attachments with lead to this phenomenon. The spatial extent of any calcium enhancement will be examined to determine whether the thin filament behaves as a single unit with respect to calcium binding, or whether other models predicting a shorter range of cooperativity are favored. Also to be examined is whether there are intrinsic variations in calcium affinity along the thin filament, independent of interactions between actin and myosin. These studies will provide insight into the fundamental mechanisms which modulate activation of force in skeletal and cardiac muscle.