OPTICAL DEPOLARIZATION CHANGES ON THE DIFFRACTION PATTERN IN THE TRANSITION OF SKINNED MUSCLE FIBERS FROM RELAXED TO RIGOR STATE Y. YEH, M. E. CORCORAN, R. J. BASKIN, AND R. L. LIEBER Department of Applied Science and Zoology Department, University of California, Davis, California 95616 ABSTRACT Light diffraction spectra from single or small bundles of skinned striated muscle fibers show large changes in polarization properties when muscles are placed into rigor. The technique of combining optical diffraction and ellipsometry measurements has previously been shown by Yeh and Pinsky to be a sensitive probe of periodic anisotropic regions of the fiber. In the present work, using this method, the observed spectrum shows marked decrease in the measured phase angle, 6, as the fiber approaches the rigor state. The degree of phase angle change is a function of sarcomere length: Maximum overlap of - 2.3 um gives the most change in 6 a A.R-R - 350 decrease for a bundle of three fibers. At a sarcomere length of 2.9 um this A.R.R value is only 100. At a nonoverlapping length of -3.8 Mm, 6 does not vary at all upon the removal of ATP. The rigor state was confirmed by stiffness measurements made after small-amplitude (0.75%), quick length changes. Upon re-relaxation, the stiffness of the skinned fiber decreased to the value of the resting state (4 mM ATP) and the phase angle 6 returned to its original value. A model based on either anisotropic subunit-2 (S-2) movements or other cross-bridge-related structural anisotropy (form birefringence) changes during the relaxed-rigor transition is suggested. INTRODUCTION Muscle contraction at the sarcomere level involves the conversion of chemical energy into mechanical energy. The mechanical movement leads to actual shortening of the sarcomere length. The sliding filament theory (1, 2) for sarcomere shortening states that the thick myosin fila- ments slide past the relatively thin f-actin filaments. The point where mechanical force generation affects the sliding motion is generally thought to be at the site where the actomyosin complex is formed (3). Because electron micro- scopic (4) and small-angle x-ray diffraction (5) data support the existence of a myosin moiety (S-1) that shifts its location from the thick filament during the resting state to the actin filament upon contraction, cross-bridge move- ment by the heavy meromyosin (HMM) parts of the thick filament toward the thin filament have been postulated. Motion of the S- I element has been the subject of investi- gation in several experiments using fluorescent (6) and phosphorescent (7) tags, electron spin labels (8), as well as x-ray scattering using a synchrotron source (9). It is difficult to monitor and assess the importance of the Dr. Lieber's current address is the Division of Orthopedics, Veterans Administration Hospital, University of California, San Diego, CA 92161. BIOPHYS. J. e Biophysical Society * 0008-3495/83/12/343/09 Volume 44 December 1983 343-351 possible dynamics of the S-2 moiety of the myosin rod whose behavior in solution shows rather large angular orientation capabilities (10). In the present experiment, the technique of optical ellipsometry (11) has been used to measure changes in the optical polarization signal of the diffraction patterns from single skeletal fibers or bundles of three fibers. In a previous publication (12), we reported on the ellipsometry measurements of diffraction patterns upon passive stretch of an intact fiber. This technique provides more spatial selectivity of anisotropic elements in that only those elements with sarcomere periodicity will exhibit the diffraction pattern. In this work, these studies have been extended to measure changes in optical polariza- tion of the diffraction pattern when a fiber goes from the relaxed state to the rigor state, where all the available cross-bridges are attached. We have been able to correlate the changes in the ellipsometry measurement with changes in both the diffraction intensity and the mechanical stiff- ness of the fiber to consistently put a single fiber into rigor and then release the rigor condition upon reintroducing chilled 4 mM ATP to the solution. In almost all cases reported here, the ellipsometry measurements are revers- ible, as are diffraction intensity and stiffness. From these results, we shall discuss the source of the changes in the depolarization signal when the fiber undergoes the relaxed-rigor transition. $1.00 343