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.     

Force produced by isolated sarcomeres and half-sarcomeres after an imposed stretch

When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres (n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5-10% of initial SL, at a speed of 0.3 μm·s(-1)·SL(-1)) after which they were maintained isometric for at least 5 s before deactivation. Single sarcomeres showed two patterns: 31 sarcomeres showed a small level of force enhancement after stretch (10.46 ± 0.78%), and 28 sarcomeres did not show force enhancement (-0.54 ± 0.17%). In these preparations, there was not a strong correlation between the force enhancement and half-sarcomere length nonuniformities. When three or more sarcomeres arranged in series were stretched, force enhancement was always observed, and it increased linearly with the degree of half-sarcomere length nonuniformities. The results show that the residual force enhancement has two mechanisms: 1) stretch-induced changes in sarcomeric structure(s); we suggest that titin is responsible for this component, and 2) stretch-induced nonuniformities of half-sarcomere lengths, which significantly increases the level of force enhancement.     

Pre-power stroke cross bridges contribute to force during stretch of skeletal muscle myofibrils

When activated skeletal muscle is stretched, force increases in two phases. This study tested the hypothesis that the increase in stretch force during the first phase is produced by pre-power stroke cross bridges. Myofibrils were activated in sarcomere lengths (SLs) between 2.2 and 2.5 microm, and stretched by approximately 5-15 per cent SL. When stretch was performed at 1 microms-1SL-1, the transition between the two phases occurred at a critical stretch (SLc) of 8.4+/-0.85 nm half-sarcomere (hs)-1 and the force (critical force; Fc) was 1.62+/-0.24 times the isometric force (n=23). At stretches performed at a similar velocity (1 microms-1SL-1), 2,3-butanedione monoxime (BDM; 1 mM) that biases cross bridges into pre-power stroke states decreased the isometric force to 21.45+/-9.22 per cent, but increased the relative Fc to 2.35+/-0.34 times the isometric force and increased the SLc to 14.6+/-0.6 nm hs-1 (n=23), suggesting that pre-power stroke cross bridges are largely responsible for stretch forces.     

Synchronous behavior of spontaneous oscillations of sarcomeres in skeletal myofibrils under isotonic conditions.

An isotonic control system for studying dynamic properties of single myofibrils was developed to evaluate the change of sarcomere lengths in glycerinated skeletal myofibrils under conditions of spontaneous oscillatory contraction (SPOC) in the presence of inorganic phosphate and a high ADP-to-ATP ratio. Sarcomere length oscillated spontaneously with a peak-to-peak amplitude of about 0.5 microns under isotonic conditions in which the external loads were maintained constant at values between 1.5 x 10(4) and 3.5 x 10(4) N/m2. The shortening and yielding of sarcomeres occurred in concert, in contrast to the previously reported conditions (isomeric or auxotonic) under which the myofibrillar tension is allowed to oscillate. This synchronous SPOC appears to be at a higher level of synchrony than in the organized state of SPOC previously observed under auxotonic conditions. The period of sarcomere length oscillation did not largely depend on external load. The active tension under SPOC conditions increased as the sarcomere length increased from 2.1 to 3.2 microns, although it was still smaller than the tension under normal Ca2+ contraction (which is on the order of 10(5) N/m2). The synchronous SPOC implies that there is a mechanism for transmitting information between sarcomeres such that the state of activation of sarcomeres is affected by the state of adjacent sarcomeres. We conclude that the change of myofibrillar tension is not responsible for the SPOC of each sarcomere but that it affects the level of synchrony of sarcomere oscillations.     

Force enhancement and relaxation rates after stretch of activated muscle fibres

The residual force enhancement following muscle stretch might be associated with an increase in the proportion of attached cross-bridges, as supported by stiffness measurements. In this case, it could be caused by an increase in the attachment or a decrease in the detachment rate of cross-bridges, or a combination of the two. The purpose of this study was to investigate if the stretch-induced force enhancement is related to cross-bridge attachment/detachment kinetics. Single muscle fibres dissected from the lumbrical muscle of frog were place at a length approximately 20% longer than the plateau of the force-length relationship; they were maximally activated, and after full isometric force was reached, ramp stretches were imposed with amplitudes of 5 and 10% fibre length, at a speed of 40% fibre length s(-1). Experiments were performed in Ringer's solution, and with the addition of 2, 5 and 10 nM of 2,3-butanedione monoxime (BDM), a drug that places cross-bridges in a pre-power-stroke, state, inhibiting force production. The total force following stretch was higher than the corresponding force measured after isometric contraction at the corresponding length. This residual force enhancement was accompanied by an increase relaxation time. BDM, which decreases force production during isometric contractions, considerably increased the relative levels of force enhancement. BDM also increased relaxation times after stretch, beyond the levels observed during reference contractions in Ringer's solution, and beyond isometric control tests at the corresponding BDM concentrations. Together, these results support the idea that force enhancement is caused, at least in part, by a decrease in cross-bridge detachment rates, as manifested by the increased relaxation times following fibre stretch.     

Force kinetics and individual sarcomere dynamics in cardiac myofibrils after rapid ca(2+) changes

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).     

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).     

Quantal sarcomere-length changes in relaxed single myofibrils.

We carried out experiments on single isolated myofibrils in which thin filaments had been functionally removed, leaving the connecting (titin) filaments as the sole agent taking up the length change. With technical advances that gave sub-nanometer detectability we examined the time course of single sarcomere-length change when the myofibril was ramp-released or ramp-stretched by a motor. The sarcomere-length change was stepwise. Step sizes followed a consistent pattern: the smallest was approximately 2.3 nm, and others were integer multiples of that value. The approximately 2.3-nm step quantum is the smallest consistent biomechanical event ever demonstrated. Although the length change must involve the connecting filament, the size of the quantum is an order of magnitude smaller than anticipated from folding of Ig- or fibronectin-like domains, implying either that folding occurs in sub-domain units or that other mechanisms are involved.     

Residual force enhancement after stretch of contracting frog single muscle fibers

Single fibers from the tibialis anterior muscle of Rana temporaria at 0.8-3.8 degrees C were subjected to long tetani lasting up to 8 s. Stretch of the fiber early in the tetanus caused an enhancement of force above the isometric control level which decayed only slowly and stayed higher throughout the contraction. This residual enhancement was uninfluenced by velocity of stretch and occurred only on the descending limb of the length-tension curve. The absolute magnitude of the effect increased with sarcomere length to a maximum at approximately 2.9 micrometers and then declined. The phenomenon was further characterized by its dependence on the amplitude of stretch. The final force level reached after stretch was usually higher than the isometric force level corresponding to the starting length of the stretch. The possibility that the phenomenon was caused by nonuniformity of sarcomere length along the fiber was examined by (a) laser diffraction studies that showed sarcomere stretch at all locations and (b) studies of 9-10 segments of approximately 0.6-0.7 mm along the entire fiber, which all elongated during stretch. Length-clamped segments showed residual force enhancement after stretch when compared with the tetanus produced by the same segment held at the short length as well as at the long length. It is concluded that residual force enhancement after stretch is a property shown by all individual segments along the fiber.     

Stepwise dynamics of connecting filaments measured in single myofibrillar sarcomeres.

Single relaxed myofibrils of bumblebee flight muscle were subjected to motor-imposed ramp-length changes. The image of the striations was projected onto a linear photodiode array, and sarcomere length was computed as the spacing between centroids of contiguous A-bands. Centroid position was determined by integrating the respective A-band intensity peak and computing the location at which the area on one side was equal to the other. The resulting trace of centroid to centroid span versus time was stepwise, with periods of rapid shortening alternating with periods of pause. An alternative nondiscrete sensor gave similar steps. If thick filament length remains constant, stepwise sarcomere length changes imply that length changes in the connecting filament must be stepwise. Thus, shortening of the connecting filament occurs as a sequence of discrete events rather than as a continuous event.     

The mechanical response of active human muscle during and after stretch

Five subjects contracted forearm supinator muscles which were stretched after development of maximal isometric torque. The ratio of torque at the end of stretch over isometric torque at that position was calculated as excess torque. Excess torque increased with stretch velocity and decreased with stretch amplitude, and it was not dependent upon final muscle length. The rate of decay of torque following stretch could not be shown to depend upon stretch variables. The absence of significant changes in myoelectric activity suggested that with high initial forces, reflex activity did not account for the observed changes. Time-constants of decay (0.15 s to 1.8 s) were much greater than time-constants of rise (approx. 0.07 s) of isometric torque at the same muscle length. This indicates that interaction of series elastic and contractile elements is not the sole cause of prolonged torque following stretch. It is concluded that stretch temporarily enhances the intrinsic contractile properties of a group of human muscles in a manner similar to, but quantitatively different from that seen in isolated muscle preparations.     

Measurement of nucleotide release kinetics in single skeletal muscle myofibrils during isometric and isovelocity contractions using fluorescence microscopy.

Rabbit psoas muscle myofibrils, in the presence of the fluorescent nucleotide analog 2'(3')-O-[N-[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP), showed selective fluorescence staining of the A-band with a reduced fluorescence at the M-line. Addition of Cy3-EDA-ATP to a myofibril in the presence of Ca2+ caused auxotonic shortening against a compliant glass microneedle. These results indicate that Cy3-EDA-ATP is a substrate for myosin in the myofibril system. The kinetics of nucleotide release from a single myofibril, held isometrically between two needles, were measured by the displacement of prebound Cy3-EDA-ATP on flash photolysis of caged ATP. The A-band fluorescence of the myofibril decayed exponentially with a rate constant of 0.3 s(-1) at 8 degrees C, an order of magnitude faster than that for isolated thick filaments in the absence of actin. When a myofibril was imposed to shorten with a constant velocity by a piezoelectric actuator, the nucleotide displacement rate constant initially increased to 0.7 s(-1) with increasing shortening velocity and then declined with a further increase in shortening velocity. These results demonstrate that the displacement rates of Cy3-EDA-nucleotides bound to the cross-bridges in the contracting myofibril reflect a process that shows strain dependence.     

EEG Dynamics in Women during Pregnancy and after Delivery

Clinical and spectral analyses of EEG recorded in the first trimester of pregnancy and six to nine months postpartum were performed in 11 women. It was shown that, in all examined women, the spectral power of virtually all rhythms of baseline postpartum EEG was uniquely decreased compared to the EEG during pregnancy in all derivations. To a lesser extent, this was characteristic of the ₂ rhythm. Changes in the postpartum EEG were most manifest during hyperventilation, predominantly, in the form of burst and paroxysmal activity. The women were divided into groups with and without postpartum paroxysmal activity. It was observed that abnormal pregnancy was more frequent in women with burst EEG activity in the nopregnant state. This index can be used as a test to reveal the group of risk of unfavorable course of pregnancy.     

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.     

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.     

Dynamics of structural changes in rat liver after single dose of gamma-irradiation

Histological changes and alterations in biophysical and biochemical parameters in liver of gamma-irradiate rats have been investigated. The gamma-irradiation of the whole body of rats with a single dose of 1 Gy did not cause any impairments of beam structure of rat liver, but resulted in the lymphocytic infiltrations of portal tracts which were not accompanied by formation of spotty areas of necrosis in adjacent areas of lever parenchyma. gamma-Irradiation stimulated proliferation of the hepatocytes and induced time-dependent mitochondrial structure lesions. Post-irradiation changes in cell cytoplasm appeared as disordering in reticulum-endothelial system, among them enlarging and fragmentation of its cisterns, cytoplasmic vacuolization, enhancement of the number of lysosomes and of the lipid inclusion contents. These facts revealed the mobilization of the additional energy resources for recovery of metabolic processes in rat liver. Post-irradiation increase of the level of the hepatocyte membrane lipid peroxidation products preceded liver morphological alterations. The membrane lipid microviscosity decreased in 1 and 3 days after irradiation. As a result of damages of hepatocyte membrane, the activity of the alanin- and asparagin-aminotransferases in blood serum increased 6 hours after. We can conclude that the whole body single gamma-irradiation with a dose of 1 Gy leads to the reversible but significant damages to the rat liver cell membrane structures. These damages might be the reason of radiation-induced liver morphological alterations.     

Efficacy of static stretching and proprioceptive neuromuscular facilitation stretch on hamstrings length after a single session

A number of studies have investigated the efficacy of several repetitions of proprioceptive neuromuscular facilitation stretching (PNF) and static stretching (SS). However, there is limited research comparing the effects of a single bout of these stretching maneuvers. The aim of this study was to compare the effectiveness of a single bout of a therapist-applied 30-second SS vs. a single bout of therapist-applied 6-second hamstring (agonist) contract PNF. Forty-five healthy subjects between the ages of 21 and 35 were randomly allocated to 1 of the 2 stretching groups or a control group, in which no stretching was received. The flexibility of the hamstring was determined by a range of passive knee extension, measured using a universal goniometer, with the subject in the supine position and the hip at 90° flexion, before and after intervention. A significant increase in knee extension was found for both intervention groups after a single stretch (SS group = 7.53°, p < 0.01 and PNF group = 11.80°, p < 0.01). Both interventions resulted in a significantly greater increase in knee extension when compared to the control group (p < 0.01). The PNF group demonstrated significantly greater gains in knee extension compared to the SS group (mean difference 4.27°, p < 0.01). It can be concluded that a therapist applied SS or PNF results in a significant increase in hamstring flexibility. A hamstring (agonist) contract PNF is more effective than an SS in a single stretching session. These findings are important to physiotherapists or trainers working in clinical and sporting environments. Where in the past therapists may have spent time conducting multiple repetitions of a PNF and an SS, a single bout of either technique may be considered just as effective. A key component of the study methodology was the exclusion of a warm-up period before stretching. Therefore, the findings of efficacy of a single PNF are of particular relevance in sporting environments and busy clinical settings where time may be limited.     

Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibers

Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly with nearly all myofibrils, and this was evident by light and electron microscopy. A few of the fibrils that reacted strongly with 12C5 reacted weakly with 5B4. These observations demonstrate that an epitope reacting with 12C5 is more abundant in some myofibrils than in others within the same cell. Three categories of myofibrils can be identified by their relative proportions of embryonic and neonatal forms of myosin: in nearly all fibrils, a neonatal isoform predominates; in a minority population, embryonic and neonatal isoforms are both abundant; and in a few fibrils, an embryonic isoform predominates. It is concluded that there are distinct populations of myofibrils in which specific isoforms are segregated within an individual cell.