A non-cross-bridge stiffness in activated frog muscle fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14 degrees C at approximately 2.1 microm sarcomere length on fibers bathed in Ringer's solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed "static tension," and the ratio between the force and the accompanying sarcomere length change was termed "static stiffness." The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1-2.8 microm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.     

Stiffness and fraction of Myosin motors responsible for active force in permeabilized muscle fibers from rabbit psoas

The stiffness of the single myosin motor (epsilon) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T0) modulated by changing either [Ca2+] or temperature. The hs compliance is 43.0+/-0.8 nm MPa-1 in isometric contraction at saturating [Ca2+], whereas in rigor it is 28.2+/-1.1 nm MPa-1. The equivalent compliance of myofilaments is 21.0+/-3.3 nm MPa-1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5+/-1.7 kPa nm-1 in isometric contraction at saturating [Ca2+] (e0), and in rigor (er) it rises to 138.9+/-21.2 kPa nm-1. Epsilon, calculated from er and the lattice molecular dimensions, is 1.21+/-0.18 pN nm-1. epsilon estimated, using a thermodynamic approach, from the relation of T0 at saturating [Ca2+] versus the reciprocal of absolute temperature is 1.25+/-0.14 pN nm-1, similar to that estimated for fibers in rigor. Consequently, the ratio e0/er (0.33+/-0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e0 and er increase by approximately 40%. Epsilon becomes approximately 1.7 pN nm-1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.     

Stiffness and force in activated frog skeletal muscle fibers.

Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs.(ABSTRACT TRUNCATED AT 400 WORDS)     

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements.In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pattern indicated a specific mitochondrial enzyme defect, which was confirmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two patients with combined complex I and IV deficiency). In the 6 patients with spinal muscular atrophy (SMA) oxidation rates were found to be decreased to 23 ± 5% of controls. The normalized respiration pattern was comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxidation rates (42 ± 5%) were detected. In addition a low RCI (1.2) indicated a loose coupling of oxidative phosphorylation in the mitochondria of these patients.It is concluded that investigation of mitochondrial function in saponin skinned muscle fibers using high resolution respirometry in combination with multiple substrate titration offers a valuable tool for evaluation of mitochondrial alterations in muscle biopsies of children suffering from neuromuscular disorders. (Mol Cell Biochem 174: 71–78, 1997)     

Glibenclamide increases post-fatigue tension in slow skeletal muscle fibers of the chicken

In contrast to fast-twitch skeletal muscle fibers of the chicken, slow-twitch fibers are fatigue-resistant. In fast fibers, the fatigue process has been related to K_ATP channels. In the present study, we investigated the action of glibenclamide (an anti-diabetic sulphonylurea that acts on K_ATP channels) on fatigued slow skeletal muscle, studying twitch and tetanus tension after inducing the muscle to fatigue by continuous electrical stimulation. Our results showed that glibenclamide (150 μM) increased post-fatigue twitch tension by about 25% with respect to the fatigued condition (P < 0.05). In addition, glibenclamide (150 μM) increased post-fatigue tetanic tension (83.61 ± 15.7% in peak tension, and 85.0 ± 19.0% in tension-time integral, P = 0.02, and 0.04, respectively; n = 3). Moreover, after exposing the muscle to a condition that inhibits mitochondrial ATP formation in order to activate K_ATP channels with cyanide (10 mM), tension also diminished, but in the presence of glibenclamide the effect produced by cyanide was abolished. To determine a possible increase in intracellular calcium concentration, the effects of glibenclamide on caffeine-evoked contractures were explored. After muscle pre-incubation with glibenclamide (150 μM), tension of caffeine-evoked contractures increased (6.5 ± 1.5% in maximal tension, and 5.9 ± 3.8% in tension-time integral, P < 0.05). These results suggest a possible role of K_ATP channels in the fatigue process, since glibenclamide increases twitch and tetanus tension in fatigued slow muscle of the chicken and during metabolic inhibition, possibly by increasing intracellular calcium.     

Length dependence of active force production in skeletal muscle.

The sliding filament and cross-bridge theories of muscle contraction provide discrete predictions of the tetanic force-length relationship of skeletal muscle that have been tested experimentally. The active force generated by a maximally activated single fiber (with sarcomere length control) is maximal when the filament overlap is optimized and is proportionally decreased when overlap is diminished. The force-length relationship is a static property of skeletal muscle and, therefore, it does not predict the consequences of dynamic contractions. Changes in sarcomere length during muscle contraction result in modulation of the active force that is not necessarily predicted by the cross-bridge theory. The results of in vivo studies of the force-length relationship suggest that muscles that operate on the ascending limb of the force-length relationship typically function in stretch-shortening cycle contractions, and muscles that operate on the descending limb typically function in shorten-stretch cycle contractions. The joint moments produced by a muscle depend on the moment arm and the sarcomere length of the muscle. Moment arm magnitude also affects the excursion (length change) of a muscle for a given change in joint angle, and the number of sarcomeres arranged in series within a muscle fiber determines the sarcomere length change associated with a given excursion.     

Tension recovery in permeabilized rat soleus muscle fibers after rapid shortening and restretch

Permeabilized rat soleus muscle fibers were subjected to rapid shortening/restretch protocols (20% muscle length, 20 ms duration) in solutions with pCa values ranging from 6.5 to 4.5. Force redeveloped after each restretch but temporarily exceeded the steady-state isometric tension reaching a maximum value approximately 2.5 s after relengthening. The relative size of the overshoot was <5% in pCa 6.5 and pCa 4.5 solutions but equaled 17% +/- 4% at pCa 6.0 (approximately half-maximal Ca2+ activation). Muscle stiffness was estimated during pCa 6.0 activations by imposing length steps at different time intervals after repeated shortening/restretch perturbations. Relative stiffness and relative tension were correlated (p < 0.001) during recovery, suggesting that tension overshoots reflect a temporary increase in the number of attached cross-bridges. Rates of tension recovery (k(tr)) correlated (p < 0.001) with the relative residual force prevailing immediately after restretch. Force also recovered to the isometric value more quickly at 5.7 < or = pCa < or = 5.9 than at pCa 4.5 (ANOVA, p < 0.05). These results show that k(tr) measurements underestimate the rate of isometric force development during submaximal Ca2+ activations and suggest that the rate of tension recovery is limited primarily by the availability of actin binding sites.     

Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].     

Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers

Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction, Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.     

Effects of shortening on stretch-induced force enhancement in single skeletal muscle fibers

The main purpose of this study was to evaluate the effects of shortening on the stretch-induced force enhancement in single muscle fibers, and indirectly test the hypothesis that force enhancement may be associated with the engagement of a passive element upon activation. Fibers were placed on the descending limb of the force-length relationship, and stretch and shortening contractions were performed. Fibers underwent two sets of shortening-stretch cycles. First, fibers were shortened by a fixed amplitude and speed (10% fiber length, and at 40% fiber length/s), and then were stretched (10% fiber length, and at 40% fiber length/s) immediately following shortening, or 500 or 1000 ms following the shortening. Second, fibers were shortened by varying amounts (5%, 10% and 15% fiber length) and at a constant speed (40% fiber length/s) immediately preceding a given fiber stretch (10% fiber length, and at 40% fiber length/s). When stretching was immediately preceded by shortening, force enhancement was decreased proportionally with the shortening magnitude. When intervals were introduced between shortening and stretch, the effects of shortening on the stretch-induced force enhancement became less prominent. We concluded that, in contrast to published suggestions, shortening affects the stretch-induced force enhancement in an amplitude-dependent manner in single fibers, as it does in whole muscles, but this effect is diminished by increasing the time period between the shortening and stretch phases.     

Stretch-induced,steady-state force enhancement in single skeletal muscle fibers exceeds the isometric force at optimum fiber length

Stretch-induced force enhancement has been observed in a variety of muscle preparations and on structural levels ranging from single fibers to in vivo human muscles. It is a well-accepted property of skeletal muscle. However, the mechanism causing force enhancement has not been elucidated, although the sarcomere-length non-uniformity theory has received wide support. The purpose of this paper was to re-investigate stretch-induced force enhancement in frog single fibers by testing specific hypotheses arising from the sarcomere-length non-uniformity theory. Single fibers dissected from frog tibialis anterior (TA) and lumbricals (n=12 and 22, respectively) were mounted in an experimental chamber with physiological Ringer's solution (pH=7.5) between a force transducer and a servomotor length controller. The tetantic force-length relationship was determined. Isometric reference forces were determined at optimum length (corresponding to the maximal, active, isometric force), and at the initial and final lengths of the stretch experiments. Stretch experiments were performed on the descending limb of the force-length relationship after maximal tetanic force was reached. Stretches of 2.5-10% (TA) and 5-15% lumbricals of fiber length were performed at 0.1-1.5 fiber lengths/s. The stretch-induced, steady-state, active isometric force was always equal or greater than the purely isometric force at the muscle length from which the stretch was initiated. Moreover, for stretches of 5% fiber length or greater, and initiated near the optimum length of the fiber, the stretch-enhanced active force always exceeded the maximal active isometric force at optimum length. Finally, we observed a stretch-induced enhancement of passive force. We conclude from these results that the sarcomere length non-uniformity theory alone cannot explain the observed force enhancement, and that part of the force enhancement is associated with a passive force that is substantially greater after active compared to passive muscle stretch.     

Effect of temperature on residual force enhancement in single skeletal muscle fibers

It is well accepted that the steady-state isometric force following active stretching of a muscle is greater than the steady-state isometric force obtained in a purely isometric contraction at the same length. This property of skeletal muscle has been called residual force enhancement (FE). Despite decades of research the mechanisms responsible for FE have remained largely unknown. Based on previous studies showing increases in FE in fibers in which cross-bridges were biased towards weakly bound states, we hypothesized that FE might be associated with a stretch-induced facilitation of transitioning from weakly to strongly bound cross-bridges. In order to test this hypothesis, single fibers (n=11) from the lumbrical muscles of frog (Rana pipiens) were used to determine FE at temperatures of 7 and 20 degrees C. At the cold temperature, cross-bridges are biased towards weakly bound states, therefore we expected FE to be greater at 7 degrees C compared to 20 degrees C. The average FE was significantly greater at 7 degrees C (11.5+/-1.1%) than at 20 degrees C (7.8+/-1.0%), as expected. The enhancement of force/stiffness was also significantly greater at the low (13.3+/-1.4%) compared to the high temperature (5.6+/-1.7%), indicating an increased conversion from weakly to strongly bound cross-bridges at the low temperature. We conclude from the results of this study that muscle preparations that are biased towards weakly bound cross-bridge states show increased FE for given stretch conditions, thereby supporting the idea that FE might be caused, in part, by a stretch-induced facilitation of the conversion of weakly to strongly bound cross-bridges.     

A K(ATP) channel deficiency affects resting tension, not contractile force, during fatigue in skeletal muscle

Theobjective of this study was to determine how an ATP-sensitiveK⁺ (K_ATP) channel deficiency affects thecontractile and fatigue characteristics of extensor digitorum longus(EDL) and soleus muscle of 2- to 3-mo-old and 1-yr-old mice.K_ATP channel-deficient mice were obtained by disrupting theKir6.2 gene that encodes for the protein forming the pore ofthe channel. At 2-3 mo of age, the force-frequency curve, the twitch,and the tetanic force of EDL and soleus muscle of K_ATPchannel-deficient mice were not significantly different from those inwild-type mice. However, the tetanic force and maximum rate of forcedevelopment decreased with aging to a greater extent in EDL and soleusmuscle of K_ATP channel-deficient mice (24-40%) thanin muscle of wild-type mice (7-17%). During fatigue, theK_ATP channel deficiency had no effect on the decrease intetanic force in EDL and soleus muscle, whereas it caused asignificantly greater increase in resting tension when compared withmuscle of wild-type mice. The recovery of tetanic force after fatiguewas not affected by the deficiency in 2- to 3-mo-old mice, whereas in1-yr-old mice, force recovery was significantly less in muscle ofK_ATP channel-deficient than wild-type mice. It is suggestedthat the major function of the K_ATP channel during fatigueis to reduce the development of a resting tension and not to contributeto the decrease in force. It is also suggested that theK_ATP channel plays an important role in protecting muscle function in older mice.

     

Trifluoperazine inhibition of contraction in permeabilized skeletal, cardiac and smooth muscles

To gain insights into the mechanism of the central helix of calmodulin and troponin-C in the Ca2(+)-regulation of force development in striated and smooth muscles, the present study was made of the TFP induced inhibition of contraction, and of the uptake of these proteins by skinned fibers. Calmodulin was four-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac muscles despite the differences in their troponin-C isoforms. Also, the results were comparable between fast-twitch fiber, when calmodulin was exchanged for troponin-C to act on TnI, and smooth muscle, where calmodulin acts on myosin light chain kinase. These findings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sites in the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFP-independent domain in troponin-C that binds TnI.     

Ultrastructure of skeletal muscle fibers in monkeys after space flight

It is known that exposure of humans and animals to microgravity causes reduction in the cross-sected area of muscle fibers and muscle atrophy. These changes also involve ultrastructural alterations in muscle fibers. Therefore primates, that are physiologically close to humans, are to be examined to help a better understanding of the nature of these ultrastructural changes is muscles and muscle fibers. Although failed to find any relevant published data on the quantitative aspects of ultrastructural changes in muscle fibers of space-flown primates we believe that it is important to examine these aspects. The postflight study of monkey's m. soleus, and m. vastus lateralis did not reveal any significant changes in volume density of the myofibrillar apparatus. Mitochondria of m. soleus showed a distinct reduction in volume density, being more obvious in the subsarcolemmal zone than in the central one. Mitochondria of m. vastus lateralis showed a decrease (P > 0.05) in volume density. Following the flight, m. soleus and m. vastus lateralis of the monkeys showed a significant increase in the mean area of myofibrils, and a trend towards a decrease in the number of myofibrils per 100 micron 2. Besides, m. soleus showed a significant increase in the mean area of mitochondria, and a trend towards a decrease in the number of mitochondria per 100 micron 2. In m. vastus lateralis of the monkeys after space flight the number opf mitochondria tended to decrease and the mean area showed differential changes. It can be postulated that these phenomena may be associated with a reduction in the diffusion surface of mitochondria resulting from the diminished myofibrillar volume.     

Nicorandil improves post-fatigue tension in slow skeletal muscle fibers by modulating glutathione redox state

Fatigue is a phenomenon in which force reduction has been linked to impairment of several biochemical processes. In skeletal muscle, the ATP-sensitive potassium channels (K_ATP) are actively involved in myoprotection against metabolic stress. They are present in sarcolemma and mitochondria (mitoK_ATP channels). K⁺ channel openers like nicorandil has been recognized for their ability to protect skeletal muscle from ischemia-reperfusion injury, however, the effects of nicorandil on fatigue in slow skeletal muscle fibers has not been explored, being the aim of this study. Nicorandil (10 μM), improved the muscle function reversing fatigue as increased post-fatigue tension in the peak and total tension significantly with respect to the fatigued condition. However, this beneficial effect was prevented by the mitoK_ATP channel blocker 5-hydroxydecanoate (5-HD, 500 μM) and by the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 1 mM), but not by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM). Nicorandil also decreased lipid peroxidation and maintained both reduced glutathione (GSH) levels and an elevated GSH/GSSG ratio, whereas total glutathione (TGSH) remained unaltered during post-fatigue tension. In addition, NO production, measured through nitrite concentrations was significantly increased with nicorandil during post-fatigue tension; this increase remained unaltered in the presence of nicorandil plus L-NAME, nonetheless, this effect was reversed with nicorandil plus MPG. Hence, these results suggest that nicorandil improves the muscle function reversing fatigue in slow skeletal muscle fibers of chicken through its effects not only as a mitoK_ATP channel opener but also as NO donor and as an antioxidant.     

Changes in force and stiffness during stretch of skeletal muscle fibers, effects of hypertonicity.

Slow stretch ramps (velocity: 0.17 fiber lengths s-1) were imposed during fused tetanic contractions of intact muscle fibers of the frog (1.4-3.0 degrees C; sarcomere length: 2.12-2.21 microns). Instantaneous force-extension relations were derived both under isometric conditions and during slow stretch by applying fast (0.2 ms) length steps to the fiber. An increase in tonicity (98 mM sucrose added to control Ringer solution) led to significant reduction of the maximum isometric tension but at the same time to marked increase in the force enhancement during slow stretch. The maximum force level reached during the stretch was affected very little. Experiments on relaxed fibers showed that recruitment of passive parallel elastic components were of no relevance for these effects. Hypertonicity slightly increased the instantaneous stiffness of the active fiber both in the presence and in the absence of stretch. The total extension of the undamped fiber elasticity was considerably reduced by increased tonicity under isometric conditions but was only slightly affected during slow stretch. The change in length of the undamped cross-bride elasticity upon stretch was thus greater in the hypertonic than in the normotonic solution suggesting a greater increase in force per cross-bridge in the hypertonic medium. The contractile effects are consistent with the assumptions that hypertonicity reduces the capability of the individual cross-bridge to produce active force and, furthermore, that hypertonicity has only minor effects on the number of attached cross-bridges and the maximum load-bearing capacity of the individual bridge.