Venus Joumaa

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.

Energy cost of force production is reduced after active stretch in skinned muscle fibres

Residual force enhancement has been observed consistently in skeletal muscles. Despite an abundance of experimental observations, there has been no information about the metabolic cost of the force observed after stretch. Our aim was to investigate the energy cost of force production after active stretch in skinned fibres isolated from rabbit psoas muscle, by quantifying the ATPase activity using an enzyme-coupled assay. Fibres were actively stretched from an average sarcomere length of 2.4 μm to average sarcomere lengths of 2.8 and 3.2 μm. Purely isometric reference contractions were performed at average sarcomere lengths of 2.8 and 3.2 μm. Simultaneously with the force measurements, the ATP cost per unit of force produced was measured during the last 40s of isometric contraction. Results showed that ATPase activity per unit of force was reduced by 17.2±4.1% in the isometric contractions after active stretch, compared to the purely isometric contraction at the corresponding lengths for both stretch magnitudes. Fibres stretched to an average sarcomere length of 3.2 μm showed a higher reduction in ATPase activity per unit of force compared to fibres stretched to an average sarcomere length of 2.8 μm (20.7±4.4 versus 12.4±3.2% respectively). Passive force enhancement was observed in all fibres and was correlated with the decrease in ATPase activity. No difference in stiffness was observed between reference and active stretch contractions. These results suggest that skeletal muscles become more efficient after stretch, either by increasing the amount of force produced per cross bridge or by engaging a passive element.

Force depression in single myofibrils

Force depression after active shortening has been observed in different muscle preparations. It has been assumed that force depression is caused by the development of sarcomere length nonuniformities after shortening. However, this hypothesis has never been investigated in a preparation where individual sarcomere lengths could be directly measured. Here, we investigated force depression in single myofibrils (n = 11) and tracked simultaneously the changes in individual sarcomere lengths (n = 60) before, during, and after shortening and after a purely isometric contraction performed at the final length. Shortening produced force depression in all myofibrils (mean +/- SE; 30.9 +/- 3.9%). During shortening, all sarcomeres shortened, but not by the same amount. Sarcomere lengths were nonuniform, with the same mean SD before (0.11 +/- 0.06 microm) and after shortening (0.11 +/- 0.06 microm) and after a purely isometric contraction at the final length (0.10 +/- 0.05 microm). Furthermore, greater shortening magnitudes were found for sarcomeres that were long in the initial isometric configuration. Nonuniformities of half-sarcomere lengths were also the same before (SD = 0.13 microm) and after (SD = 0.14 microm) shortening. We conclude from these results that the development of sarcomere (or half-sarcomere) length nonuniformities does not play a major role in force depression. Rather, force depression seems an intrinsic property of individual (half-) sarcomeres and muscle contraction.

New insights into force depression in skeletal muscle

SUMMARY Force depression observed following active shortening is not well understood. Previous research suggested that force depression might be associated with a stress-induced inhibition of cross-bridges in the newly formed overlap zone following shortening. Our aim was to investigate this theory in skinned fibres and determine whether there was an inhibition of the attachment of cross-bridges or a decrease in the force produced per cross-bridge. The stress-induced inhibition of cross-bridge theory gives testable predictions, including: (1) skinned fibres should show proportional force and stiffness depression, (2) force after shortening should not be lower than force before shortening, (3) stiffness following shortening should not be lower than stiffness before shortening and (4) force depression should decrease when the stress during shortening is decreased. In agreement with these predictions, force and stiffness depression were approximately proportional, and force depression decreased with decreasing stress during shortening. However, in contrast to the predictions of the stress-induced inhibition of cross-bridge theory, force after shortening from sarcomere lengths of 2.8 and 3.0 μm to a sarcomere length of 2.4 μm was smaller than force before shortening, and this was not accompanied by a corresponding decrease in stiffness. We conclude that the stress-induced inhibition of cross-bridge theory, as proposed previously, cannot be the only mechanism for force depression, but that there is an additional, stress-induced inhibition of cross-bridges in the old overlap zone. Furthermore, both mechanisms, inhibition of cross-bridge attachment and reduction of force produced per cross-bridge, contribute to force depression. Inhibition and/or reduction of force depend(s) on the amount of stress imposed on actin during the shortening phase.

Decreased force enhancement in skeletal muscle sarcomeres with a deletion in titin

ABSTRACT In the cross-bridge theory, contractile force is produced by cross-bridges that form between actin and myosin filaments. However, when a contracting muscle is stretched, its active force vastly exceeds the force that can be attributed to cross-bridges. This unexplained, enhanced force has been thought to originate in the giant protein titin, which becomes stiffer in actively compared with passively stretched sarcomeres by an unknown mechanism. We investigated this mechanism using a genetic mutation (mdm) with a small but crucial deletion in the titin protein. Myofibrils from normal and mdm mice were stretched from sarcomere lengths of 2.5 to 6.0 μm. Actively stretched myofibrils from normal mice were stiffer and generated more force than passively stretched myofibrils at all sarcomere lengths. No increase in stiffness and just a small increase in force were observed in actively compared with passively stretched mdm myofibrils. These results are in agreement with the idea that titin force enhancement stiffens and stabilizes the sarcomere during contraction and that this mechanism is lost with the mdm mutation. Summary: Force enhancement is absent in sarcomeres where amino acids in N2A and PEVK titin are deleted, indicating these specific regions are paramount in increasing titin stiffness in an active sarcomere.

An activatable molecular spring reduces muscle tearing during extreme stretching.

The purpose of this study was to determine failure stresses and failure lengths of actively and passively stretched myofibrils. As expected, myofibrils failed at average sarcomere lengths (about 6-7μm) that vastly exceeded sarcomere lengths at which actin-myosin filament overlap ceases to exist (4μm) and thus actin-myosin-based cross-bridge forces are zero at failure. Surprisingly, however, actively stretched myofibrils had much greater failure stresses and failure energies than passively stretched myofibrils, thereby providing compelling evidence for strong force production independent of actin-myosin-based cross-bridge forces. Follow-up experiments in which titin was deleted and cross-bridge formation was inhibited at high and low calcium concentrations point to titin as the regulator of this force, independent of calcium. The results of this study point to a mechanism of force production that reduces stretch-induced muscle damage at extreme length and limits injury and force loss within physiologically relevant ranges of sarcomere and muscle lengths.

The Force–Length Relationship of Mechanically Isolated Sarcomeres

The sarcomere force-length relationship is arguably the most basic property of skeletal muscle force production. It has been accepted as textbook knowledge and is in direct support of the sliding filament and cross-bridge theories of contraction. However, the sarcomere force-length relationship has never been measured directly. Here, we show results of two experiments elucidating the force-length properties of mechanically isolated sarcomeres. We demonstrate that sarcomere forces are greatly dependent on sarcomere lengths for purely isometric conditions, but can take on essentially any steady-state value depending on an individual sarcomeres contractile history. Therefore, we conclude that steady-state isometric forces in isolated sarcomeres do not only depend on sarcomere lengths (or equivalently actin-myosin overlap) but depend crucially on a sarcomeres contractile history. These results have direct implications for our understanding of the molecular mechanisms of muscle contraction.

Intermittent stretch training of rabbit plantarflexor muscles increases soleus mass and serial sarcomere number

In humans, enhanced joint range of motion is observed after static stretch training and results either from an increased stretch tolerance or from a change in the biomechanical properties of the muscle-tendon unit. We investigated the effects of an intermittent stretch training on muscle biomechanical and structural variables. The left plantarflexors muscles of seven anesthetized New Zealand (NZ) White rabbits were passively and statically stretched three times a week for 4 wk, while the corresponding right muscles were used as nonstretched contralateral controls. Before and after the stretching protocol, passive torque produced by the left plantarflexor muscles as a function of the ankle angle was measured. The left and right plantarflexor muscles were harvested from dead rabbits and used to quantify possible changes in muscle structure. Significant mass and serial sarcomere number increases were observed in the stretched soleus but not in the plantaris or medial gastrocnemius. This difference in adaptation between the plantarflexors is thought to be the result of their different fiber type composition and pennation angles. Neither titin isoform nor collagen amount was modified in the stretched compared with the control soleus muscle. Passive torque developed during ankle dorsiflexion was not modified after the stretch training on average, but was decreased in five of the seven experimental rabbits. Thus, an intermittent stretching program similar to those used in humans can produce a change in the muscle structure of NZ White rabbits, which was associated in some rabbits with a change in the biomechanical properties of the muscle-tendon unit.

Calcium sensitivity of residual force enhancement in rabbit skinned fibers

Isometric force after active stretch of muscles is higher than the purely isometric force at the corresponding length. This property is termed residual force enhancement. Active force in skeletal muscle depends on calcium attachment characteristics to the regulatory proteins. Passive force has been shown to influence calcium attachment characteristics, specifically the sarcomere length dependence of calcium sensitivity. Since one of the mechanisms proposed to explain residual force enhancement is the increase in passive force that results from engagement of titin upon activation and stretch, our aim was to test if calcium sensitivity of residual force enhancement was different from that of its corresponding purely isometric contraction and if such a difference was related to the molecular spring titin. Force-pCa curves were established in rabbit psoas skinned fibers for reference and residual force-enhanced states at a sarcomere length of 3.0 μm 1) in a titin-intact condition, 2) after treatment with trypsin to partially eliminate titin, and 3) after treatment with trypsin and osmotic compression with dextran T-500 to decrease the lattice spacing in the absence of titin. The force-pCa curves of residual force enhancement were shifted to the left compared with their corresponding controls in titin-intact fibers, indicating increased calcium sensitivity. No difference in calcium sensitivity was observed between reference and residual force-enhanced contractions in trypsin-treated and osmotically compressed trypsin-treated fibers. Furthermore, calcium sensitivity after osmotic compression was lower than that observed for residual force enhancement in titin-intact skinned fibers. These results suggest that titin-based passive force regulates the increase in calcium sensitivity of residual force enhancement by a mechanism other than reduction of the myofilament lattice spacing.

Effects of fiber type on force depression after active shortening in skeletal muscle

The aim of this study was to investigate force depression in Type I and Type II muscle fibers. Experiments were performed using skinned fibers from rabbit soleus and psoas muscles. Force depression was quantified after active fiber shortening from an average sarcomere length (SL) of 3.2µ m to an average SL of 2.6 µm at an absolute speed of 0.115f iber length/s and at a relative speed corresponding to 17% of the unloaded shortening velocity (V0) in each type of fibers. Force decay and mechanical work during shortening were also compared between fiber types. After mechanical testing, each fiber was subjected to myosin heavy chain (MHC) analysis in order to confirm its type (Type I expressing MHC I, and Type II expressing MHC IId). Type II fibers showed greater steady-state force depression after active shortening at a speed of 0.115 fiber length/s than Type I fibers (14.5±1.5% versus 7.8±1.7%). Moreover, at this absolute shortening speed, Type I fibers showed a significantly greater rate of force decay during shortening and produced less mechanical work than Type II fibers. When active shortening was performed at the same relative speed (17% V0), the difference in force depression between fiber types was abolished. These results suggest that no intrinsic differences were at the origin of the disparate force depressions observed in Type I and Type II fibers when actively shortened at the same absolute speed, but rather their distinct force-velocity relationships.