PubMed Health⌕ Search

Biomedical subjects

R J Podolsky

Publications and source records attributed to R J Podolsky.

At least 19 recordsLinked to original sources
PubMed1991

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength.

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium ("weak-binding") state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.

Animals↗
PubMed1990

Single skinned muscle fibers in Duchenne muscular dystrophy generate normal force.

We measured the intrinsic mechanical properties and protein content of single skinned muscle fibers obtained from patients who had Duchenne muscular dystrophy. To check for possible nonspecific changes caused by muscle disease per se, we also studied the properties of muscle fibers obtained from patients exhibiting severe muscle weakness due to polymyositis. Relative to control fibers obtained from 4 patients with normal or nonmyopathic muscle, we found no significant changes in the ability of muscle fibers from the patients with Duchenne muscular dystrophy or polymyositis to generate active tension in response to calcium or resting tension in response to stretch. In addition, we found no significant changes in the concentrations of the major contractile proteins myosin and actin, of the elastic protein titin, or of the structural proteins nebulin and alpha-actinin. In contrast, immunocytochemical studies showed that dystrophin was absent in the biopsy specimens from the patients with Duchenne muscular dystrophy, but localized at the cell membrane in all of the other muscle biopsy specimens used in this study. These results indicate that myofibrils assemble and function normally in Duchenne muscular dystrophy. Therefore, the absence of dystrophin, which is the primary biochemical defect in this disease, leads to clinical weakness by causing the breakdown of muscle fibers that were once capable of generating normal force, while the surviving fibers exhibit normal contractility.

Adult↗
PubMed1989

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

Actins↗
PubMed1989

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle.

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I-band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.

Actin Cytoskeleton↗
PubMed1988

Thick filament movement and isometric tension in activated skeletal muscle.

Thick filaments can move from the center of the sarcomere to the Z-disc while the isometric tension remains stable in skinned rabbit psoas fibers activated for several minutes (Horowits and Podolsky, 1987). Using the active and resting tension-length relations and the force-velocity relation, we calculated the time course and mechanical consequences of thick filament movement in the presence and absence of the elastic titin filaments, which link the ends of the thick filaments to the Z-discs and give rise to the resting tension. The calculated time course of thick filament movement exhibits a lag phase, during which the velocity and extent of movement are extremely small. This lag phase is dependent only on the properties of the cross-bridges and the initial position of the thick filament. The time course of thick filament movement in skinned rabbit psoas fibers at 7 degrees C is well fit assuming a small initial thick filament displacement away from the center of the sarcomere; this leads to a lag of approximately 80 s before any significant thick filament movement occurs. In the model incorporating titin filaments, this lag is followed by a phase of slow, steady motion during which isometric tension is stable. The model excluding titin filaments predicts a phase of acceleration accompanied by a 50% decrease in tension. The observed time course of movement and tension are consistent with the model incorporating titin filaments. The long lag phase suggests that in vivo, significant movement of thick filaments is unlikely to occur during a single contraction. Therefore, the primary physiological function of titin filaments may be to keep the thick filaments centered during passive stretch and to prevent sarcomere asymmetry from accumulating over several contractions by recentering the thick filaments each time the muscle is relaxed.

Animals↗
PubMed1988

Force generating mechanisms in striated muscle.

Force generation in muscle fibers is generally believed to arise from a structural change in the actomyosin crossbridge. The structure of the low ionic strength crossbridge has been found to be significantly different from that of the rigor crossbridge, which raises the possibility that contractile force may be generated by a transition between these two actomyosin configurations. Another possibility for force generation is based on data suggesting that the length of the subfragment 2 moiety of the myosin molecule may shorten when it moves away from the backbone of the thick filament. Experiments bearing these potential force generating processes are reviewed.

Actins↗
PubMed1987

The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments.

Electron microscopy was used to study the positional stability of thick filaments in isometrically contracting skinned rabbit psoas muscle as a function of sarcomere length at 7 degrees C. After calcium activation at a sarcomere length of 2.6 micron, where resting stiffness is low, sarcomeres become nonuniform in length. The dispersion in sarcomere length is complete by the time maximum tension is reached. A-bands generally move from their central position and continue moving toward one of the Z-discs after tension has reached a plateau at its maximum level. The lengths of the thick and thin filaments remain constant during this movement. The extent of A-band movement during contraction depends on the final length of the individual sarcomere. After prolonged activation, all sarcomeres between 1.9 and 2.5 micron long exhibit A-bands that are adjacent to a Z-disc, with no intervening I-band. Sarcomeres 2.6 or 2.7 micron long exhibit a partial movement of A-bands. At longer sarcomere lengths, where the resting stiffness exceeds the slope of the active tension-length relation, the A-bands remain perfectly centered during contraction. Sarcomere symmetry and length uniformity are restored upon relaxation. These results indicate that the central position of the thick filaments in the resting sarcomere becomes unstable upon activation. In addition, they provide evidence that the elastic titin filaments, which join thick filaments to Z-discs, produce almost all of the resting tension in skinned rabbit psoas fibers and act to resist the movement of thick filaments away from the center of the sarcomere during contraction.

Actin Cytoskeleton↗
PubMed1986

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.

Actin Cytoskeleton↗
PubMed1985

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.

Animals↗
PubMed1985

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.

Forearm↗
PubMed1984

A stopped-flow investigation of calcium ion binding by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid.

The kinetics of calcium ion complexation by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) were investigated using the stopped-flow technique. This study was performed within the pH range 5.8 to 8.4. The reaction was found to be first order in EGTA and complex order in calcium, with an observed second-order rate constant (pH 7, T = 25 degrees C, ionic strength = 0.1 M) of about 1.5 X 10(6) M-1 s-1. The rate constant was independent of hydrogen ion concentration between pH 5.8 and 7.3; above pH 7.3 it increased in magnitude with increasing pH, and was 2.0 X 10(8) M-1 s-1 at pH 8.4. The rate constant at 16 and 38 degrees C (pH 6.8) was found to be 0.9 and 7 X 10(6) M-1 s-1, respectively. These data imply that calcium ion buffering by EGTA will require times on the order of milliseconds when EGTA is present in millimolar concentrations.

Binding Sites↗
PubMed1984

Structural studies of muscle during force development in various states.

Structural studies concerned with force generating mechanisms in striated muscle fibers in different states are described. The first study deals with fibers in the "rigor" state, where ATP is absent and all the myosin heads form cross-bridges with the actin-containing filaments. In this state large axial forces are developed when ionic strength is reduced to very low levels. At the same time, the fiber expands radially, as indicated by both X-ray diffraction and light microscopy. Comparison of the latter two measurements indicates that force is developed in part because of differences in the lateral expandability of different parts of the sarcomere. Thus, under these conditions, force appears to be modulated by factors that operate at the filament rather than the cross-bridge level. The second study deals with the location of the myosin heads in the relaxed state, and the mass movement that takes place when the fiber is physiologically activated. By using the intense X-ray source at the Stanford Synchrotron Radiation Laboratory, five equatorial reflections were recorded for both the relaxed and the activated state, and a spatial resolution of 100 A was obtained. Analysis of the data indicates that (1) in the resting state, myosin heads protrude out from the thick filaments and extend toward the thin filaments, and (2) upon activation, significant loss of mass occurs only in the region peripheral to the thick filament backbone through a movement that has a pronounced azimuthal component. The latter movement can be taken as the overall shift in myosin subfragment 1 during the cross-bridge cycle.

Actins↗
PubMed1984

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.

Actins↗
PubMed1984

X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers.

Biochemical data, stiffness measurements, and equatorial x-ray diffraction patterns provide evidence that actomyosin cross-bridges form in relaxed skinned rabbit fibers at low ionic strength (20 mM). In the present study we examined the structure of these cross-bridges by using two-dimensional x-ray diffraction. In contrast to rigor cross-bridges, which significantly weaken the myosin-based reflections characteristic of relaxed fibers at 120 mM ionic strength (notably the 86-A and 108-A layer lines and the 72-A and 143-A meridionals), the formation of low ionic strength cross-bridges produced only small changes in these reflections. In addition, these cross-bridges did not produce the additional intensity on the 59-A actin-based layer line near the meridian that is associated with rigor cross-bridges. However, the formation of low ionic strength cross-bridges caused the 215-A meridional reflection to decrease in intensity, as is also the case when rigor cross-bridges are formed. These observations show that the structure of the low ionic strength cross-bridge is significantly different from that of the rigor cross-bridge, and they raise the possibility that contractile force may be generated by a transition between these two actomyosin configurations.

Actomyosin↗
PubMed1982

Cross-bridge properties in the rigor state.

The inability of applied strain to affect the equatorial diffraction pattern of the rigor fiber indicates that the attachment angle of S1 to the actin filament is extremely stable. The effects of lattice spacing on fiber length (at constant force) and force (at constant length) indicate that the S1 moiety is connected to the surface of the myosin filament by a link that is between 3 and 12 nm in length, which is considerably less than 60 nm length that has been assumed previously.

Animals↗
PubMed1981

X-ray diffraction of strained muscle fibers in rigor.

The effect of strain on the equatorial x-ray diffraction pattern of glycerinated rabbit psoas fibers was studied in the rigor (ATP free) state. Strains between 30 and 100 A per half sarcomere, measured directly by laser diffraction, did not change the intensity ratio, (10)/ . (11). Because the intensity ratio depends on the distribution of mass within the myofilament lattice, the negative result indicates that strain does not change the angle of attachment of the subfragment 1 (S1) moiety of the myosin molecule to the actin filament. The effect of strain on the ordering of the actin filaments also was considered and judged to be negligible.

Actomyosin↗
PubMed1981

Isotonic contraction of skinned muscle fibers on a slow time base: effects of ionic strength and calcium.

The force development by calcium-activated skinned frog skeletal muscle fibers and the motion on a slow time base after a quick decrease in load were studied at 0-1 degrees C as a function of the ionic strength and the degree of activation. The ionic strength was varied between 50 and 190 mM by adding appropriate concentrations of KCl to the bathing solution. Under these conditions, the fibers could be maximally activated for several cycles at low ionic strength without developing residual tension. We found that the steady isometric force in fully activated fibers linearly decreased when the KCl concentration was increased from 0 to 140 mM. The steady isotonic motion at a given relative load in fully activated fibers was almost the same at KCl concentration greater than or equal to 50 mM. In 0 and 20 mM KCl, the isotonic velocity decreased continuously for more than 300 ms. At a given relative load, the initial velocity of the motion in 0 and 20 mM KCl was about 0.6 and 0.9 times, respectively, that in 140 mM KCl. The initial velocity decreased further when residual tension developed; this observation provides additional evidence that residual tension may reflect the presence of an internal load. The effect of calcium on the motion was examined at 70 mM KCl. In this solution, the motion during the velocity transient at a given relative load appeared to be the same at different levels of activation. The speed of the subsequent motion was almost steady at high calcium levels but decreased continuously in low calcium levels. These results support the idea that at low ionic strength the response of the fiber to calcium is switch-like, but that other factors also affect the contraction mechanism under these conditions.

Animals↗