Application of angle-resolved fluorescence depolarization in muscle research

Angle-resolved fluorescence depolarization (AFD) experiments have been used for over a decade in studies of fluorescent molecules in macroscopically aligned systems such as lipid bilayers and stretched polymer films. The importance of this technique lies in the fact that it affords the determination of both the second- and the fourth-rank order parameters of the orientational distribution of the probe molecules in the sample. Here we apply the technique to the study of the orientational distribution of crossbridges in muscle fibers. This orientational distribution is particularly relevant in muscle research, as crossbridge rotation is commonly regarded to be the driving mechanism in force development. An unfortunate consequence of the fact that the crossbridges have an average orientation of approximately 45o relative to the fiber axis is that the values of the second-rank order parameter 〈P2〉 of the crossbridge distribution are close to 0. Therefore, knowledge of 〈P4〉 is essential for a reliable reconstruction of the form of the distribution function. AFD of dyelabeled muscle was measured under rigor and relaxation conditions. The results indicate that no significant changes in depolarization take place upon a transition from the rigor to the relaxed state in the muscle and seem not to support the rotating crossbridge model, which postulates a clear change of orientation of the crossbridges.