Sarcomere Length Nonuniformity and Force Regulation in Myofibrils and Sarcomeres

The smallest contractile unit in striated muscles is the sarcomere. Although some of the classic features of contraction assume a uniform behavior of sarcomeres within myofibrils, the occurrence of sarcomere length nonuniformities has been well recognized for years, but it is yet not well understood. In the past years, there has been a great advance in experiments using isolated myofibrils and sarcomeres that has allowed scientists to directly evaluate sarcomere length nonuniformity. This review will focus on studies conducted with these preparations to develop the hypotheses that 1) force production in myofibrils is largely altered and regulated by intersarcomere dynamics and that 2) the mechanical work of one sarcomere in a myofibril is transmitted to other sarcomeres in series. We evaluated studies looking into myofibril activation, relaxation, and force changes produced during activation. We conclude that force production in myofibrils is largely regulated by intersarcomere dynamics, which arises from the cooperative work of the contractile and elastic elements within a myofibril.     

Diffraction rings obtained from a suspension of skeletal myofibrils by laser light illumination. Study of internal structure of sarcomeres.

Diffraction rings corresponding to the first, second, and third order were obtained by laser light illumination from a suspension of rabbit glycerinated psoas myofibrils (diameter, 1-2 microns; average length of the straight region, 44 microns; average sarcomere length, 2.2-2.6 microns) of which the optical thickness was appropriately chosen. Dispersed myofibrils were nearly randomly oriented in two dimensions, so that the effects of muscle volume were minimized; these effects usually interfere significantly with a quantitative analysis of laser optical diffraction in the fiber system. The diameters of diffraction rings represented the average sarcomere length. By using this system, we confirmed the ability of the unit cell (sarcomere) structure model to explain the intensity change of diffraction lines accompanying the dissociation from both ends of thick filaments in a high salt solution. The length of an A-band estimated from the relative intensity of diffraction rings and that directly measured on phase-contrast micrographs coincided well with each other. Also, we found that myofibrils with a long sarcomere length shorten to a slack length accompanying the decrease in overlap between thick and thin filaments produced by the dissociation of thick filaments.     

Intensity of light diffraction from striated muscle as a function of incident angle.

In a recently developed theory of light diffraction by single striated muscle fibers, we considered only the case of normal beam incidence. The present investigation represents both an experimental and theoretical extension of the previous work to arbitrary incident angle. Angle scan profiles over a 50 degrees range of incident angle (+25 degrees to -25 degrees) were obtained at different sarcomere lengths. Left and right first-order scan peak separations were found to be a function of sarcomere length (separation angle = 2 theta B), and good agreement was found between theory and experiment. Our theoretical analysis further showed that a myofibrillar population with a single common skew angle can yield an angle scan profile containing many peaks. Thus, it is not necessary to associate each peak with a different skew population. Finally, we have found that symmetry angle, theta s, also varies with sarcomere length, but not in a regular manner. Its value at a given sarcomere length is a function of a particular region of a given fiber and represents the average skew angle of all the myofibril populations illuminated. The intensity of a diffraction order line is considered to be principally the resultant of two interference phenomena. The first is a volume-grating phenomenon which results from the periodic A-I band structure of the fiber (with some contribution from Z bands and H zones). The second is Bragg reflection from skew planes, if the correct relation between incident angle and skew angle is met. This may result in intensity asymmetry between the left and right first order lines.     

Intermediate filaments connect z-discs in adult chicken muscle.

When adult chicken skeletal myofibrils are treated with a myosin-extracting solution, the Z-discs with attached actin filaments retain their linear connections with one another in the extracted myofibril. The sarcomere length increases in the extracted myofibrils from a control lenght of 2.5 micrometer up to 6 micrometer. In a sarcomere, eight to fifty 10 nm filaments can be seen in parallel array in the H-zone. The 10 nm-wide filaments do not bind heavy meromyosin and are two to four micrometers in length. These intermediate filaments are postulated to be an integral part of the sarcomere, connecting Z-bands along the length of the myofibril.     

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis. This paper is dedicated to the memory of Professor Pavel P. Rumyantsev (1927–1988), a pioneer in studies of cardiac muscle differentiation, who is a lasting inspiration to all who worked with him.     

Direct x-ray observation of a single hexagonal myofilament lattice in native myofibrils of striated muscle

A striated muscle fiber consists of thousands of myofibrils with crystalline hexagonal myofilament lattices. Because the lattices are randomly oriented, the fiber gives rise to an equatorial x-ray diffraction pattern, which is essentially a rotary-averaged "powder diffraction," carrying only information about the distance between the lattice planes. We were able to record an x-ray diffraction pattern from a single myofilament lattice, very likely originating from a single myofibril from the flight muscle of a bumblebee, by orienting the incident x-ray microbeam along the myofibrillar axis (end-on diffraction). The pattern consisted of a number of hexagonally symmetrical diffraction spots whose originating lattice planes were readily identified. This also held true for some of the weak higher order reflections. The spot-like appearance of reflections implies that the lattice order is extremely well maintained for a distance of millimeters, covering up to a thousand of approximately 2.5-microm-long sarcomeres connected in series. The results open the possibility of applying the x-ray microdiffraction technique to study many other micrometer-sized assemblies of functional biomolecules in the cell.     

Muscle diffraction theory. Relationship between diffraction subpeaks and discrete sarcomere length distributions.

A theoretical discussion is presented that describes the diffraction on monochromatic light by a three-dimensional sarcomere array having the following properties. The basic repetitive diffracting unit is the sarcomere. The contiguous arrangement of physically attached serial sarcomeres in the myofibril is contained within the model so that relative position of sarcomeres depend upon the lengths of intervening ones. Sarcomere length is described by a distribution function. This function may be discrete or continuous and contain one or more subpopulations. Two arrangements of sarcomeres are considered: (a) when sarcomeres of different lengths are arranged randomly in myofibrils the amplitude and width of mth order (m greater than or equal to 1) peaks and associated secondary diffraction maxima decrease and increase monotonically, respectively, as the standard deviation of the length distribution increases. No subpeaks are present regardless of the number of subpopulations within the distribution function. This behavior is shown to follow from the dependence of sarcomere position on the length of intervening sarcomeres. (b) When sarcomeres belonging to the same length subpopulation are arranged in serial contiguous fashion to form domains and more than one length subpopulation is present, then mth order diffraction peaks split to form subpeaks. The theoretical basis for this behavior is developed for the first time and may explain the subpeaks evident in diffraction patterns from cardiac and skeletal muscle.     

Sarcomeric pattern formation by actin cluster coalescence

Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells.     

The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes

Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin-containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s-alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha-actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.     

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).     

AN ANALYSIS OF MYOGENESIS BY THE USE OF FLUORESCENT ANTIMYOSIN

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.     

Efficiency of light diffraction by cross-striated muscle fibers under stretch and during isometric contraction.

When light is diffracted by a single frog muscle fiber the intensities I kappa of the different orders kappa (kappa = 1,2,3) strongly depend on the angle between the axis of the incident beam and the fiber axis. Maximum intensity is not obtained with perpendicular incidence (omega = 0 degree) but at angles that can be calculated for each order number and sarcomere length using Bragg's formula. In analogy to techniques developed for x-ray structure analysis of mosaic crystals we have rotated the fiber around an axis perpendicular to the fiber axis and to the incident beam axis within an angular range delta omega = +/- 35 degrees and recorded the light intensities I kappa. Diffraction efficiencies defined as E kappa = integral of I kappa d omega were studied as a function of sarcomere length and during isometric contraction. The sarcomere length dependences of the efficiencies E kappa of the first three orders show characteristic trends. E1 increases with fiber stretch, E2 has a minimum at a sarcomere length near 2.8 micrometers, and E3 has a maximum near 2.5 micrometers. These trends as well as the observed efficiency ratios are in fairly good agreement with predictions by the intensity formula developed for x-ray structure analysis. During isometric contraction, the diffraction efficiencies of the fiber decrease, with the decreases becoming greater the higher the order number. These decreases might be caused by a longitudinal displacement of myofibrils of up to 0.4 micrometers. The efficiency of light diffraction strongly depends on the tonicity of the bathing fluid. Hypertonic (3/2 x normal) solution reduces E1 to less than half, hypotonic (2/3 x normal) solution increases E1 to almost twice the value obtained in normal Ringer's solution.     

The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes

The topographical relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle-specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle alpha-actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) more SFLS were present in immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before anti-desmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.     

Uniform sarcomere shortening behavior in isolated cardiac muscle cells

We have observed the dynamics of sarcomere shortening and the diffracting action of single, functionally intact, unattached cardiac muscle cells enzymatically isolated from the ventricular tissue of adult rats. Sarcomere length was measured either (a) continuously by a light diffraction method or (b) by direct inspection of the cell's striated image as recorded on videotape or by cinemicroscopy (120--400 frames/s). At physiological levels of added CaCl2 (0.5--2.0 mM), many cells were quiescent (i.e., they did not beat spontaneously) and contracted in response to electrical stimulation (less than or equal to 1.0-ms pulse width). Sarcomere length in the quiescent, unstimulated cells (1.93 +/- 0.10 [SD] micrometers), at peak shortening (1.57 +/- 0.13 micrometers, n = 49), and the maximum velocity of sarcomere shortening and relengthening were comparable to previous observations in intact heart muscle preparations. The dispersion of light diffracted by the cell remained narrow, and individual striations remained distinct and laterally well registered throughout the shortening- relengthening cycle. In contrast, appreciable nonuniformity and internal buckling were seen at sarcomere lengths < 1.8 micrometers when the resting cell, embedded in gelatin, was longitudinally compressed These results indicate (a) that shortening and relengthening is characterized by uniform activation between myofibrils within the cardiac cell and (b) that physiologically significant relengthening forces in living heart muscle originate at the level of the cell rather than in extracellular connections. First-order diffracted light intensity, extremely variable during sarcomere shortening, was always greatest during midrelaxation preceding the onset of a very slow and uniform phase of sarcomere relengthening.     

Ultrastructure of Developing Flight Muscle in Drosophila. I. Assembly of Myofibrils

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from 1.7 to 3.2 μm, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from 4-thick filaments to 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.     

Myofilament length-dependent activation develops within 5 ms in guinea-pig myocardium

Myofilament length-dependent activation is a universal property of striated muscle, yet the molecular mechanisms that underlie this phenomenon are incompletely understood. Additionally, the rate by which sarcomere length (SL) is sensed and then transduced to form length-dependent activation is unknown. Here, using isolated guinea-pig myocardium, we employed a rapid solution-switch single myofibril technique that allows for the study of contractile action/relaxation dynamics in the virtual absence of diffusion delays. We compared contraction kinetics obtained at submaximal activation at steady-state SL with contractions observed after rapid SL ramps to that same SL just before activation. Neither the activation and relaxation kinetics nor the final submaximal force development differed significantly between the two contraction modes for SL ramps as fast as 5 ms. We conclude that the transduction of the length signal by the cardiac sarcomere to modulate thin filament activation levels occurs virtually instantaneously, possibly resulting from structural rearrangements of the contractile proteins.     

Sarcomere length determination using laser diffraction. Effect of beam and fiber diameter

An experimental and theoretical analysis is presented involving the effect of variation in fiber and beam diameter upon the determination of average sarcomere length in isolated single muscle fibers using laser light diffraction. The muscle diffraction phenomenon is simplified by first considering diffraction order position and intensity to be the result of grating and Bragg diffraction. It is the product of the intensity profiles, which results from these types of diffraction, that produces the diffracted order. These simplifying assumptions are then extended to the case of the real muscle. Based on these considerations and the theory that we recently presented, conditions are set forth under which grating information (i.e., sarcomere length) can be maximally expressed to yield accurate average sarcomere length values.     

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.     

Optical depolarization changes on the diffraction pattern in the transition of skinned muscle fibers from relaxed to rigor state

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, delta, as the fiber approaches the rigor state. The degree of phase angle change is a function of sarcomere length: Maximum overlap of approximately 2.3 microns gives the most change in delta a delta delta R-R approximately 35 degrees decrease for a bundle of three fibers. At a sarcomere length of 2.9 microns this delta delta R-R value is only 10 degrees. At a nonoverlapping length of approximately 3.8 microns, delta 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 delta 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.     

Residual force enhancement in myofibrils and sarcomeres

Residual force enhancement has been observed following active stretch of skeletal muscles and single fibres. However, there has been intense debate whether force enhancement is a sarcomeric property, or is associated with sarcomere length instability and the associated development of non-uniformities. Here, we studied force enhancement for the first time in isolated myofibrils (n=18) that, owing to the strict in series arrangement, allowed for evaluation of this property in individual sarcomeres (n=79). We found consistent force enhancement following stretch in all myofibrils and each sarcomere, and forces in the enhanced state typically exceeded the isometric forces on the plateau of the force-length relationship. Measurements were made on the plateau and the descending limb of the force-length relationship and revealed gross sarcomere length non-uniformities prior to and following active myofibril stretching, but in contrast to previous accounts, revealed that sarcomere lengths were perfectly stable under these experimental conditions. We conclude that force enhancement is a sarcomeric property that does not depend on sarcomere length instability, that force enhancement varies greatly for different sarcomeres within the same myofibril and that sarcomeres with vastly different amounts of actin-myosin overlap produce the same isometric steady-state forces. This last finding was not explained by differences in the amount of contractile proteins within sarcomeres, vastly different passive properties of individual sarcomeres or (half-) sarcomere length instabilities, suggesting that the basic mechanical properties of muscles, such as force enhancement, force depression and creep, which have traditionally been associated with sarcomere instabilities and the corresponding dynamic redistribution of sarcomere lengths, are not caused by such instabilities, but rather seem to be inherent properties of the mechanisms of contraction.