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G H Pollack

Publications and source records attributed to G H Pollack.

At least 37 records · Page 2Linked to original sources
PubMed1996

Phase transitions and the molecular mechanism of contraction.

In this paper, the rotating cross-bridge mechanism for muscle contraction is discussed and much contradictory evidence is put forward. As an alternative, a model is given in which the motor of muscle contraction is placed in the myosin-rod hinge and/or in the actin filament. No definite choice for one of the proposed models can be made yet, although it is clear that some kind of phase transition plays an important role in the mechanism.

Amino Acid Sequence↗
PubMed1996

Limits of titin extension in single cardiac myofibrils.

Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from approximately 2-5 microns. Myofibril stretch up to sarcomere lengths of approximately 3 microns resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of approximately 2.7 microns. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond approximately 3 microns sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 microns. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.

Animals↗
PubMed1995

Actin filaments in honeybee-flight muscle move collectively.

To investigate the pattern of actin-filament translation in the intact myofibrillar matrix, we carried out electron micrographic experiments on the "rigor-stretch" model of insect-flight muscle. In this model, thin filaments are mechanically severed from their connections to the Z-line and may then slide freely over the myosin filaments when activated. The model is similar to the in vitro motility assay in that untethered actin filaments slide over myosin, but here the natural filament lattice is retained: sliding takes place through the lattice of thick filaments. We find, in this model, that while the extent of thin filament translation is variable from sarcomere to sarcomere, filaments never translate far enough to enter the opposite I-band. Unlike the in vitro motility assay, where the actin filament translates over the entire thick filament even with "incorrectly" polarized cross-bridges as the sole driver, in this intact filament-lattice model, cross-bridges are apparently unable to move filaments in both directions. We also find that the pattern of filament translation is collective. Although the extent of translation may vary among sarcomeres, in any given half-sarcomere all actin filaments translate by the same degree. Further, the extent of translation is the same in both halves of a given sarcomere. In rare instances where the extent of translation exhibited a transverse gradient across the myofibrillar half-sarcomere, the gradient was similar on both sides of the sarcomere. Filament translation within the sarcomere is thus collective. Some mechanism ensures that nearby but distinctly separated actin filaments move together and that cooperative-like behavior therefore extends to the supramolecular level.

Actin Cytoskeleton↗
PubMed1995

Visualization of the transverse cytoskeletal network in insect-flight muscle by scanning-electron microscopy.

Located at the level of the Z-line, the transverse cytoskeletal network of insect-flight muscle interconnects adjacent myofibrils with one another, and interconnects peripheral myofibrils with the cell membrane. This network has been presumed to keep myofibrils in register, or to distribute tension laterally among myofibrils. In this study, we used scanning-electron microscopy to reveal details of the three-dimensional arrangement of this network. The network is seen to interconnect longitudinal elements of the cytoskeletal network which surround each myofibril. The arrangement is not unlike that seen in vertebrate skeletal muscle. Interestingly, the transverse network makes contact with cell components such as dense bodies and mitochondria. Such contacts imply potential roles over and above those noted above. The network may be involved not only in mechanical function, but possibly also in intracellular communication.

Animals↗
PubMed1995

Persisting in vitro actin motility at nanomolar adenosine triphosphate levels: comparison of skeletal and cardiac myosins.

We have previously demonstrated in vitro actin movement at nanomolar adenosine triphosphate (ATP) levels using heavy meromyosin from skeletal muscle. In the present work we tested whether the motility at nonomolar ATP-concentrations could be supported by cardiac myosin as well. Actomyosin (skeletal actin and bovine ventricular myosin) was pretreated in the in vitro motility assay with 1 mM ATP; subsequently, the ATP level was reduced by multiple rigor-solution washes. By the final rigor-solution wash, the ATP concentration, monitored by the luciferin-luciferase assay, dropped to the order of 100 nM. Even at this low ATP level actin-filament movement remained in evidence. This was in marked contrast to the situation where ATP concentration was gradually increased from zero; in the latter, filament movement began only as ATP levels exceeded 1-2 microM. The difference indicates that potential energy is stored during the initial ATP treatment, and utilized later as the free ATP falls below micromolar levels. Although the velocity of cardiac myosin-supported movement was only one fourth of that of skeletal myosin, both myosins supported actin movement down to similar ATP concentrations. The similarity in response of the two myosins to ATP implies a similar degree of potential energy storage. Given the significantly different specific ATPase activities, however, it appears that the mechanism of potential energy storage and release involves factors different from those involved in the release of chemical energy by the myosin ATPase.

Actins↗
PubMed1995

Do vertebrate thick filaments contain a core? Observations using ultrathin sectioning and freeze-etching.

Transverse ultrathin sections and the freeze-fracture deep-etch method were used to study the structure of vertebrate thick filaments. We examined intact frog semitendinosus and glycerinated rabbit psoas muscle fibers. In transverse thin sections, using standard staining methods, cores were moderately well revealed. The cores were particularly distinct when sections were stained with potassium permanganate followed by differentiation with bleach. For freeze-etching, the specimens were generally fractured transversely, and occasionally obliquely, to filament axis, and were then deep-etched and rotary-shadowed. In both transverse and oblique images, thick filaments were made up of a central core, surrounded by an annular region. Since cores are characteristic of invertebrate thick filaments, it is tempting to suggest a similarity. On the other hand, the cores observed here seem simplest to interpret as being hollow, as in microtubules.

Animals↗
PubMed1994

Passive and active tension in single cardiac myofibrils.

Single myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1-2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n = 15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal myofibrils under similar conditions (Bartoo et al., 1993).

Animals↗
PubMed1993

Elastic properties of titin filaments demonstrated using a "freeze-break" technique.

A "freeze-break" technique (Trombitás, K.: Acta Biochim. Biophys. Hung. 6:419-427, 1971) and immunoelectron microscopy were used to study the elastic properties of titin filaments. Small bundles of freshly prepared rabbit psoas muscle fibers were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axis, in each of various regions along the sarcomere. The still-frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken specimens were then labelled with monoclonal anti-titin antibodies against an unique epitope in the I-band. The titin epitopes were normally positioned symmetrically about the Z-line. However, in sarcomeres broken at the A-I junction, the epitopes no longer remained symmetrical: the titin filaments in the broken half-sarcomere retracted, independently of the thin filaments, forming a dense band just near the Z-line. The retracted density apparently did not reach the Z-line; retraction stopped at the level of the so-called N1-line. In sarcomeres broken at the Z-line level, the titin filaments retracted in the opposite direction. In this case the titin epitope retracted all the way to the ends of the thick filaments. It appears then that titin molecules form elastic filaments that are independent of thin filaments in most of the I-band. Near the Z-line, however, the titin filaments either have an inelastic domain or associate firmly with the thin filaments at the N1-line level.

Actin Cytoskeleton↗
PubMed1993

Shortening-induced tension enhancement: implication for length-tension relations.

Length-tension relations come in two types--the classical type with linearly descending limb, and the "flat," higher type. The classical type, now confirmed in several laboratories, is obtained when sarcomeres are servo-controlled to maintain constant length. The flat type, also confirmed in several laboratories, is obtained in fixed-end contractions, where some sarcomeres have the opportunity to shorten at least slightly. We find that the higher tensions seen in the flat type are indeed the result of very small shortening: when isometric sarcomeres shorten to a slightly shorter (e.g., by 40 nm) length, they go on to produce considerably more isometric tension than if they had remained at the shorter length throughout contraction. We term this phenomenon "shortening-induced tension enhancement." The phenomenon accounts not only for the higher, flatter length-tension relation seen in fixed-end contractions but can explain the creep of tension that occurs in extended tetani. Thus, several issues concerning the interpretation of length-tension relations are resolved by this newly discovered phenomenon.

Animals↗
PubMed1993

Elastic properties of connecting filaments along the sarcomere.

The elasticity of the connecting filament--the filament that anchors the thick filament to the Z-line--has been investigated using rigor release, freeze-break and immunolabelling techniques. When relaxed insect flight muscle was stretched and then allowed to go into rigor, then released, the recoil forces of the connecting filaments caused sarcomeres to shorten. Thin filaments, prevented from sliding by rigor links, were found crumpled against the Z-line. Thus, rigor release experiments demonstrate the spring-like nature of the connecting filaments in insect flight muscle. In vertebrate skeletal muscle, however, the same protocol did not result in sarcomere shortening. Absence of shortening was due to either smaller stiffness of connecting filaments and/or higher stiffness of the thin filaments relative to insect flight muscle. The spring-like nature of the connecting filament was confirmed with the freeze break technique. When the frozen sarcomeres were broken along the A-I junction, the broken connecting filaments retracted to the N1-line level, independently of the thin filaments, demonstrating the basic elastic nature of these filaments. To study the elastic properties of the connecting filaments along the sarcomere, the muscle was labelled with monoclonal antibodies against a titin epitope near the N1-line, and another very near the A-I junction in the I-band. Before labelling, fibers were pre-stretched to varying extents.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1993

Filament lengths in frog semitendinosus and tibialis anterior muscle fibres.

In frog semitendinosus muscle the descending limb of the length-tension curve is shifted rightward relative to that of tibialis anterior. Both the plateau right corner and the zero-force intercept are equally shifted. To investigate the reason for this shift, we compared filament lengths in the two muscles. Single fibres were mechanically skinned, stretched to reveal filaments clearly, incubated in a solution containing one of several antibodies to enhance filament visualization, and examined by electron microscopy. We found no differences of filament length. Thick filament lengths were 1.62 and 1.61 microns, respectively. I-segment lengths were measured by two methods. With the first, filament length was the same for both muscles, 1.95 or 1.98 microns, depending on the value taken for the troponin repeat; with the second it was 1.92 and 1.94 microns, respectively, for the two muscles. These differences are insignificant. Thus, the reported differences of shape of the length-tension curve are not explainable in terms of differences of filament length.

Actin Cytoskeleton↗
PubMed1993

Elastic properties of the titin filament in the Z-line region of vertebrate striated muscle.

The characteristics of the titin filament in the vicinity of the Z-line were investigated using immunoelectron microscopy. We used monoclonal titin antibodies T-11 and T-12 on single fibres of frog skeletal muscle, and on Z-line-extracted fibres. It is well established that the I-band region of titin is elastic. We find, however, that the elastic properties are not uniform. The T-12 epitope, which binds near the Z-line at the N1-line level, hardly changes position relative to the Z-line as the sarcomere is stretched. This demonstrates the functional inextensibility of the N1-Z-line region. After extreme stretch (above 6-microns sarcomere length), this zone finally does elongate; thus, the titin molecule in this region is intrinsically elastic. The functional inextensibility seen at shorter sarcomere lengths may, therefore, be a result of binding of titin to the actin filament in the zone near the Z-line. When the Z-line was extracted, the T-12 epitope remained in the same position as in the unextracted fibres; it did not retract from the Z-line. Failure to retract implies that functional anchoring of titin is not exclusive to the Z-line, but includes some site closer to the A-band. Combined with the results of the above-mentioned stretch experiment, this result implies a likely binding of titin to the thin filament either focally at the N1 line or all along the entire N1-Z region. Thus, this region of titin is functionally stiff, but intrinsically elastic.

Animals↗
PubMed1993

Contraction-induced movements of water in single fibres of frog skeletal muscle.

Although X-ray diffraction measurements imply almost constant filament separation during isometric contraction, such constancy does not hold at the level of the isolated cell; cell cross-section increases substantially during isometric contraction. This expansion could arise from accumulation of water drawn from other fibre regions, or from water drawn into the cell from outside. To distinguish between these hypotheses, we froze single fibres of frog skeletal muscle that were jacketed by a thin layer of water. Frozen fibres were freeze-substituted, sectioned transversely, and examined in the electron microscope. In fibres frozen during contraction, we found large amounts of water just beneath the sarcolemma, less in deeper regions, and almost none in the fibre core. Such gradients were absent or diminished in fibres frozen in the relaxed state. The water was not confined to the myofibril space alone; we found large water spaces between myofibrils, particularly near mitochondria. Accumulation of water between myofibrils and around mitochondria implies that the driving force for water movement probably lies outside the filament lattice, and may therefore be osmotic. The fact that the distribution was nonuniform-highest near the sarcolemma and lowest in the core--implies that the water was likely drawn from the thin jacket surrounding the cell. Thus, the contractile cycle appears to be associated with water entry into and exit from the cell.

Animals↗
PubMed1993

Active tension generation in isolated skeletal myofibrils.

Single or double myofibrils isolated from rabbit psoas muscle were suspended between a fine needle and an optical force transducer. By using a photodiode array, the length of every sarcomere along the specimen could be measured. Relaxed specimens exhibited uniform sarcomere lengths and their passive length-tension curve was comparable to that of larger specimens. Most specimens could be activated and relaxed four to five times before active force levels began to decline; some specimens lasted for 10-15 activation cycles. Active tension (20-22 degrees C) was reproducible from contraction to contraction. The contractile response was dependent on initial sarcomere length. If initially activated at sarcomere lengths of > or = 2.7 microns, one group of sarcomeres usually shortened to sarcomere lengths of 1.8-2.0 microns, while the remaining sarcomeres were stretched to longer lengths. Myofibrils that were carefully activated at shorter initial sarcomere lengths usually contracted homogeneously. Both homogeneous and inhomogeneous contractions produced high levels of active tension. Calcium sensitivity was found to be comparable to that in larger preparations; myofibrils immersed in pCa 6.0 solution generated 30% of maximal tension, while pCa 5.5-4.5 resulted in full activation. Active tension at full overlap of thick and thin filaments ranged from 0.34 to 0.94 N mm-2 (mean of 0.59 N mm-2 +/- 0.13 SD. n = 65). Even allowing for a maximum of 20% nonmyofibrillar space in skinned or intact muscle fibres, the mean tension generated by isolated myofibrils per cross-sectional area is higher than by fibre preparations.

Animals↗
PubMed1993

An optical fiber transducer for single myofibril force measurement.

A force transducer has been developed for use in force measurement of skeletal muscle myofibrils. The transducer is suitable for measurement of passive and contractile forces in a range up to 200 micrograms, with 1 microgram resolution. It is based upon the operating principle of the deflection of an optical fiber of known compliance, sensed by the differential illumination of two phototransistors. Attractive features include ease of operation and specimen mounting, high bandwidth, adaptability for different force ranges, and simple and inexpensive construction.

Biomechanical Phenomena↗
PubMed1993

Force-length relation of isometric sarcomeres in fixed-end tetani.

The higher force observed in fixed-end tetani relative to sarcomere-isometric tetani is commonly attributed to sarcomere length inhomogeneity; sarcomeres in the end regions of the fiber shorten extensively at the expense of the central sarcomeres. By shortening, these sarcomeres supposedly attain higher force production capacity and can thus account for the extra force. However, the fibers could also contain sarcomeres that stay isometric throughout most of the tetanic force plateau. If such sarcomeres undergo slight shortening before their isometric phase, their force-length relation should be elevated (A. Horowitz, H. P. M Wussling, and G. H. Pollack. Biophys. J. 63: 3-17, 1992). These sarcomeres may therefore account for the higher force in fixed-end tetani. To test this possibility, single frog semitendinosus fibers were tetanized under fixed-end conditions. Sarcomere length change during the tetanus was measured at different locations along the fiber by optical diffraction. Fibers stretched to average sarcomere lengths between 2.2 and 3.2 microns contained sarcomeres that, except for some initial shortening during the early part of the tetanus, remained isometric. These sarcomeres were located between the ends and the central region of the fibers. Their force-length relation was higher than the linear force-length relation based on sarcomere length clamps by an average of 14% between sarcomere lengths of 2.4-3.2 microns. Thus slight (1-5%) shortening may explain the relatively higher fixed-end force-length relation.

Animals↗