MOLECULAR MECHANISM OF CONTRACTILE REGULATION IN MUSCLE

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In all muscles studied, contraction is regulated by changes in intracellular calcium. In vertebrate striated muscles, the molecular mechanism involves calcium binding to the TnC subunit of the regulatory protein troponin located on the thin filament. Complete calcium regulation requires the concerted action of the two other troponin subunits (TnT and TnI), and interaction with tropomyosin and actin. In cells lacking surface membranes (so-called "skinned" fibers) from mammalian skeletal and cardiac muscle, TnC can be partially removed, rendering the fiber relaxed and calcium-insensitive. Fibers can then be repleted with TnC permitting restoration of calcium control. Repletion with genetically engineered TnC has been very useful in probing the molecular regions of TnC involved in regulation. Unfortunately, similar depletion/repletion experiments with skinned fibers to study TnT and TnI have been completely unsuccessful. Recently, however, we discovered that TnT (and a 75 kD protein of unknown function) could be extracted from skinned fast muscle fibers of the lobster, rendering the fibers fully activated even in the absence of calcium. Fibers could be relaxed by addition of a mixture of isolated lobster proteins rich in lobster TnT and the 75 kD protein. This represents the only skinned fiber preparation where TnT extraction and repletion has been successful. The overall goal of this project is to establish lobster skinned muscle fibers as a model system for the study of striated muscle contraction. We wish to capitalize on this unique preparation to probe the function of TnT in the same fashion as the Tnc experiments outlined above. In addition, we hope to extract (and then replete) TnI from skinned lobster fibers using a recently described proteinase isolated from lobster muscle which appears to be specific for the regulatory proteins. We have also discovered that contraction of skinned fibers from lobster fast muscle is remarkably insensitive to acidosis (i.e. decreased pH), as compared with skinned fibers from mammalian skeletal or cardiac muscle. In the mammal, acidosis is thought to play a major role in the decreased muscle contraction associated with fatigue and hypoxia, although the molecular basis for this influence is poorly understood. Through comparative studies of mammalian and lobster muscle, we hope to elucidate which steps of the contractile process are affected by changes in pH.