Richard J. Podolsky

X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths.

Equatorial x-ray diffraction patterns from single skinned rabbit psoas fibers were studied at various ionic strengths to obtain structural information regarding cross-bridge formation in relaxed muscle fibers. At ionic strengths between 20 and 50 mM, the intensity of the 11 reflection, I11, of the relaxed state was close to that of the rigor state, whereas the intensity of the 10 reflection, I10, was approximately twice that of rigor reflection. Calculations by two-dimensional Fourier synthesis indicated that substantial extra mass was associated with the thin filaments under these conditions. With increasing ionic strength between 20 and 100 mM, I10 increased and I11 decreased in an approximately linear way, indicating net transfer of mass away from the thin filaments towards the thick filaments. These results provided evidence that cross-bridges were formed in a relaxed fiber at low ionic strengths, and that the number of cross-bridges decreased as ionic strength was raised. Above mu = 100 mM, I10 and I11 both decreased, indicating the onset of increasing disorder within the filament lattice.

Length-Force Relation of Calcium Activated Muscle Fibers

Calcium activated skinned frog muscle fibers develop a large relative force at a sarcomere length of 1.0 micrometer. Since the normal myofilament lattice is perturbed at this length, regularity of the lattice does not appear to be an important factor in the contraction mechanism.

Distribution of mass in relaxed frog skeletal muscle and its redistribution upon activation.

Five orders of equatorial reflection were recorded from both relaxed and fully activated intact frog sartorius muscle using synchrotron x-ray radiation. Electron density maps of the myofilament lattice in axial projection were calculated from the integrated intensities by Fourier synthesis, using all possible phase combinations. These maps were evaluated systematically in terms of their compatibility with electron microscopically and biochemically derived properties of the lattice structure and with the minimum wavelength principle. For the relaxed state, one phase combination emerged as most consistent with these constraints: it shows a thick filament with a compact core surrounded by an annular shell of density. The distribution of mass suggests that the S-2 moiety of the myosin molecule is an integral part of the thick-filament backbone and the S-1 moiety makes up the shell and is tilted or slewed around the backbone. For the active state, there are two feasible maps, which differ according to whether or not the activation process is associated with phase inversion in two of the reflections. Both maps represent patterns of redistribution of mass upon activation in which the thick-filament backbone is practically unaffected and there is movement of density from the annular shell towards the thin filaments. In addition to this outward radial flux of density from the thick-filament periphery, the pattern of net mass transfer involves a pronounced azimuthal component in both cases. The total net mass transfer is equivalent to approximately 20% (no phase change) or approximately 40% (with phase change) of the S-1 mass. From the observed systematic increase in peak widths of the higher orders, the size of the crystalline domain in the myofilament lattice in the relaxed sartorius is estimated to be greater than 650 nm and the variations in myofilament lattice spacing among different myofibrils to be about +/- 3%. Furthermore, in the activated state, the equilibrium positions of the myofilaments are no longer well ordered, but are distributed statistically about the lattice points with a standard deviation of approximately 3 nm.

Characterization of a non-indexible equatorial x-ray reflection from frog sartorius muscle

Abstract X-ray equatorial reflections from frog sartorius muscle were studied using a position sensitive detector. A weak reflection appeared between the 10 and 11 peaks which did not index on the hexagonal filament lattice. This reflection, first reported by Elliott et al . (1967), was further characterized. The spacing of the reflection varied in direct proportion to that of the 10 peaks for sarcomere lengths between 2·0 μm and 3·0 μm. Its intensity appeared relatively insensitive to length changes. Optical diffraction patterns from electron micrographs of oblique sections through muscle gave ratios for the spacings of the myosin filaments and the Z -disc lattice that correlated very closely with the X-ray results. It is suggested that the Z -disc structure is the major source of this nonindexible reflection.

Equatorial x-ray intensities and isometric force levels in frog sartorius muscle.

Abstract Isometric force levels, ranging between 0 and 100% of maximal force P 0 at 2 to 3 °C, were elicited in frog sartorius muscle by means of rapidly cooling a Ringer solution containing 1·25 to 2·0 m m -caffeine. Equatorial X-ray diffraction patterns were obtained in the resting state and during contraction. The ratio of the intensities I 11 I 10 increased with force almost linearly, with a slight upward curvature. The individual intensities for the contracting state were normalized relative to both the intensity of the undiffracted beam and the intensity of each reflection in the resting state. These normalized intensities were found to vary in a reciprocal way: I 10 decreased while I 11 increased throughout the range of forces studied. The gradual change in I 11 I 10 with force level indicates that this ratio is a sensitive measure of the number of cross-bridges in the isometric state. A two-state model in which myosin projections are either in a resting or attached state and in which force is proportional to the fraction of projections in the attached state was applied to the experimental data of the individual reflections. I 10 deviates from this model in a way that suggests that formation of the first few cross-bridges may decrease the regularity of the remaining unattached myosin projections.

A laser diffraction method for measuring muscle sarcomere length in vivo for application to tendon transfers.

A technique that uses laser light diffraction to measure muscle sarcomere length allows direct determination of optimal muscle length during tendon transfers. Forearm muscle sarcomere length with the hand in the position of function is 2.4, and muscle length corresponds directly to sarcomere length. We have used these observations to restore optimal muscle length during tendon transfers. Standard high radial nerve tendon transfers in six fresh cadaver forearms demonstrated the efficacy of the laser diffraction method in accurately measuring sarcomere length. In two clinical trials with the laser, standard high radial nerve palsy tendon transfers were performed. In each case the clinical tendency was to overpull the muscle during the transfer. With the laser it was possible to identify excessive muscle stretch and restore optimal muscle length.

Ordering of the myofilament lattice in muscle fibers

The effect of pH on the muscle filament lattice in skinned rabbit psoas fibers was studied by X-ray diffraction. In relaxed fibers, the intensity of the 11 equatorial reflection, I11, remained constant between pH 7.0 and pH 6.0 and fell markedly when the pH was decreased to 5.5. The intensity of the 10 reflection was almost constant over this pH range. These results indicate that the thick-filament lattice is more stable than that of the thin filaments, and that the thin filaments are positioned within the thick-filament lattice by a charge-dependent force. In rigor fibers, the decrease in I11 over this pH range was much smaller, which shows that the thin filament lattice can also be stabilized by the presence of actomyosin crossbridges. These conclusions were confirmed by electron microscopy. Thus, the thin filaments can be positioned in the trigonal positions of the thick-filament lattice by two different mechanisms, one electrostatic and the other steric.

Muscle activation: the current status

I was pleased to have been asked to organize a session in this Symposium for a number of reasons, some sentimental and some scientific. One of the sentimental ones is that Kacy Cole was one of my advisors in graduate school at the University of Chicago and I am grateful to him for the guidance he provided at that time. Kacy left Chicago before 1 finished my thesis work to become Technical Director of the Naval Medical Research Institute in Bethesda, but we met again when I went there to take a postdoctoral fellowship with Manuel Morales, who introduced me to muscle cells. Kacy “welcomed me aboard” when I arrived, and I began a stay that lasted a number of happy years.

Crossbridge Rotation in EDC-Crosslinked Striated Muscle Fibers

The rotatability of the strong- and weak-binding myosin heads was tested by stretching glycerinated rabbit psoas fibers after crosslinking the heads to actin by using a carbodiimide EDC. The equatorial 1,1 reflection intensity (I1,1) decreased by approximately 10% upon 1% stretch in the presence of various ligands (ATP, ATP-gamma-S, pyrophosphate and AMPPNP). As the action of ligands to dissociate actomyosin increased, the relaxation of tension response to stretch and the I1,1 decrease were accelerated. This result is best explained if the ligand converts the crosslinked head to a weak-binding state, in which the head is rotatable because of its acquired elasticity. Conversely, the weak-to-strong transition was induced in the crosslinked system by removing a ligand (ATP-gamma-S) from myosin. Force was produced upon weak-to-strong transition and was accounted for by the increased stiffness of each crosslinked myosin head. However, the comparison of stress-strain curves for the weak- and strong-binding myosin showed that the equilibrium angle of myosin attachment was unchanged, making it unlikely that the weak-to-strong transition is the sole mechanism for active contraction. The calcium-activated force of the same crosslinked fibers showed several features in marked contrast to the force produced by the weak-to-strong transition. This leads to a possibility that the active force is supported by a third class of intermediate which is distinct not only from the weak-binding but also from the strong-binding intermediates in a classical sense.

Two‐dimensional photon counter for x‐ray imaging

This paper characterizes an imaging x‐ray detector formed by coupling a gadolinium oxysulphide phosphor to the input of an optical imaging photon detector. The device is small, light, easy to use, and features a direct digital readout. It exhibits fairly high efficiency (40%–80%) and high resolution (160 μm‐width point spread function) near the center of its 40‐mm active area. There is a small amount of pincushion distortion which seems to be associated with a loss in resolution toward the edge of the active area. The device has very low noise and can be used at x‐ray fluxes down to about 0.1 x‐ray/mm2/s without loss of accuracy but it is count rate limited at 105 x rays/s over the active area and so is not usable in high‐flux situations such as are often found at synchrotrons. We have used the device to record good diffraction patterns from striated rabbit muscle in 30 min on a rotating anode x‐ray generator: less than one tenth the time needed under similar conditions when using film.