Marta Nocella

Crossbridge properties during force enhancement by slow stretching in single intact frog muscle fibres.

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, Pc) and the sarcomere length needed to reach Pc (critical length, Lc) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that Pc was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, Pc was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 ± 0.17 nm per half‐sarcomere (nm hs−1) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, Lc reduced to 8.19 ± 0.039 nm hs−1. Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.

Force enhancement after stretch in mammalian muscle fiber: no evidence of cross-bridge involvement

Stretching of activated skeletal muscles induces a force increase above the isometric level persisting after stretch, known as residual force enhancement (RFE). RFE has been extensively studied; nevertheless, its mechanism remains debated. Unlike previous RFE studies, here the excess of force after stretch, termed static tension (ST), was investigated with fast stretches (amplitude: 3-4% sarcomere length; duration: 0.6 ms) applied at low tension during the tetanus rise in fiber bundles from flexor digitorum brevis (FDB) mouse muscle at 30°C. ST was measured at sarcomere length between 2.6 and 4.4 μm in normal and N-benzyl-p-toluene sulphonamide (BTS)-added (10 μM) Tyrode solution. The results showed that ST has the same characteristics and it is equivalent to RFE. ST increased with sarcomere length, reached a peak at 3.5 μm, and decreased to zero at ∼4.5 μm. At 4 μm, where active force was zero, ST was still 50% of maximum. BTS reduced force by ∼75% but had almost no effect on ST. Following stimulation, ST developed earlier than force, with a time course similar to internal Ca(2+) concentration: it was present 1 ms after the stimulus, at zero active force, and peaked at ∼3-ms delay. At 2.7 μm, activation increased the passive sarcomere stiffness by a factor of ∼7 compared with the relaxed state All our data indicate that ST, or RFE, is independent of the cross-bridge presence and it is due to the Ca(2+)-induced stiffening of a sarcomeric structure identifiable with titin.

Non-crossbridge forces in activated striated muscles: a titin dependent mechanism of regulation?

When skeletal muscles are stretched during activation in the absence of myosin-actin interactions, the force increases significantly. The force remains elevated throughout the activation period. The mechanism behind this non-crossbridge force, referred to as static tension, is unknown and generates debate in the literature. It has been suggested that the static tension is caused by Ca2+-induced changes in the properties of titin molecules that happens during activation and stretch, but a comprehensive evaluation of such possibility is still lacking. This paper reviews the general characteristics of the static tension, and evaluates the proposed mechanism by which titin may change the force upon stretch. Evidence is presented suggesting that an increase in intracellular Ca2+ concentration leads to Ca2+ binding to the PEVK region of titin. Such binding increases titin stiffness, which increases the overall sarcomere stiffness and causes the static tension. If this form of Ca2+-induced increase in titin stiffness is confirmed in future studies, it may have large implications for understating of the basic mechanisms of muscle contraction.

Effect of temperature on cross-bridge properties in intact frog muscle fibers

It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5 degrees C and 14 degrees C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, P(c)) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach P(c) (critical length, L(c)). Our results show that P(c) increased linearly with the force developed at both temperatures, but the P(c)/P ratio was considerably smaller at 14 degrees C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length L(c) was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14 degrees C are more strained. The increased cross-bridge strain accounts for the greater average force developed.

Force decline during fatigue is due to both a decrease in the force per individual cross‐bridge and the number of cross‐bridges

Non‐technical summary  Prolonged muscle activity leads to a reduction of mechanical power and force output which is commonly indicated as muscular fatigue. The development of fatigue during repetitive stimulation of a skeletal muscle consists of an initial phase during which force decreases by 10–15%. This is followed by a second phase where force remains almost constant and finally a phase during which force drops precipitously to low levels. We show here that the initial fall in force is due to a reduction of the force generated by the individual molecular force generator, the cross‐bridge, whereas in subsequent phases the force decrease is caused by a reduction in the number of molecular force generators. These results increase our understanding of muscular fatigue mechanisms.

Non-crossbridge calcium-dependent stiffness in slow and fast skeletal fibres from mouse muscle

We showed previously that force development in frog and FDB mouse skeletal muscle fibres is preceded by an increase of fibre stiffness occurring well before crossbridge attachment and force generation. This stiffness increase, referred to as static stiffness, is due to a Ca2+-dependent stiffening of a non-crossbridge sarcomere structure which we suggested could be attributed to the titin filaments. To investigate further the role of titin in static stiffness, we measured static stiffness properties at 24 and 35°C in soleus and EDL mouse muscle fibres which are known to express different titin isoforms. We found that static stiffness was present in both soleus and EDL fibres, however, its value was about five times greater in EDL than in soleus fibres. The rate of development of static stiffness on stimulation increased with temperature and was slightly faster in EDL than in soleus in agreement with previously published data on the time course of the intracellular Ca2+ transients in these muscles. The present results show that the presence of a non-crossbridge Ca2+-dependent stiffening of the muscle fibre is a physiological general characteristic of skeletal muscle. Static stiffness depends on fibre type, being greater and developing faster in fast than in slow fibres. Our observations are consistent with the idea that titin stiffening on contraction improves the sarcomere structure stability. Such an action in fact seems to be more important in EDL fast fibre than in soleus slow fibres.

Non-crossbridge stiffness in active muscle fibres

ABSTRACT Stretching of an activated skeletal muscle induces a transient tension increase followed by a period during which the tension remains elevated well above the isometric level at an almost constant value. This excess of tension in response to stretching has been called ‘static tension’ and attributed to an increase in fibre stiffness above the resting value, named ‘static stiffness’. This observation was originally made, by our group, in frog intact muscle fibres and has been confirmed more recently, by us, in mammalian intact fibres. Following stimulation, fibre stiffness starts to increase during the latent period well before crossbridge force generation and it is present throughout the whole contraction in both single twitches and tetani. Static stiffness is dependent on sarcomere length in a different way from crossbridge force and is independent of stretching amplitude and velocity. Static stiffness follows a time course which is distinct from that of active force and very similar to the myoplasmic calcium concentration time course. We therefore hypothesize that static stiffness is due to a calcium-dependent stiffening of a non-crossbridge sarcomere structure, such as the titin filament. According to this hypothesis, titin, in addition to its well-recognized role in determining the muscle passive tension, could have a role during muscle activity. Summary: This review summarizes the current knowledge regarding static stiffness, from its identification by our group, to the present, and evaluates the role of titin as the structure possibly responsible for this non-crossbridge stiffness.

Reversal of the Myosin Power Stroke Induced by Fast Stretching of Intact Skeletal Muscle Fibers

Force generation and movement in skeletal muscle result from a cyclical interaction of overlapping myosin and actin filaments that permits the free energy of ATP hydrolysis to be converted into mechanical work. The rapid force recovery that occurs after a step release imposed on a muscle is thought to result from a synchronized tilting of myosin lever arms toward a position of lower free energy (the power stroke). We investigated the power stroke mechanism in intact muscle fibers of Rana esculenta using a fast stretch to detach forcibly cross-bridges. Stretches were applied either with or without a conditioning step release. Cross-bridge rupture tension was not significantly influenced by the release, whereas sarcomere elongation at the rupture point increased immediately after the release and returned to the prerelease condition within 15-20 ms, following a slower time course compared to the recovery of tension. These observations suggest that the rupture force of a bridge is unaltered by a conditioning release, but rupture must first be preceded by a power stroke reversal, which restores the prepower stroke state. The sarcomere extension at the rupture point indicates both the extent of this power stroke reversal and the time course of strained bridge replenishment.

Phosphate increase during fatigue affects crossbridge kinetics in intact mouse muscle at physiological temperature

Actomyosin ATP hydrolysis occurring during muscle contraction releases inorganic phosphate [Pi] in the myoplasm. High [Pi] reduces force and affects force kinetics in skinned muscle fibres at low temperature. These effects decrease at high temperature, raising the question of their importance under physiological conditions. This study provides the first analysis of the effects of Pi on muscle performance in intact mammalian fibres at physiological temperature. Myoplasmic [Pi] was raised by fatiguing the fibres with a series of tetanic contractions. [Pi] increase reduces muscular force mainly by decreasing the force of the single molecular motor, the crossbridge, and alters the crossbridge response to fast length perturbation indicating faster kinetics. These results are in agreement with schemes of actomyosin ATPase and the crossbridge cycle including a low‐ or no‐force state and show that fibre length changes perturb the Pi‐sensitive force generation of the crossbridge cycle.

Effect of Temperature on Crossbridge Force Changes during Fatigue and Recovery in Intact Mouse Muscle Fibers

Repetitive or prolonged muscle contractions induce muscular fatigue, defined as the inability of the muscle to maintain the initial tension or power output. In the present experiments, made on intact fiber bundles from FDB mouse, fatigue and recovery from fatigue were investigated at 24°C and 35°C. Force and stiffness were measured during tetani elicited every 90 s during the pre-fatigue control phase and recovery and every 1.5 s during the fatiguing phase made of 105 consecutive tetani. The results showed that force decline could be split in an initial phase followed by a later one. Loss of force during the first phase was smaller and slower at 35°C than at 24°C, whereas force decline during the later phase was greater at 35°C so that total force depression at the end of fatigue was the same at both temperatures. The initial force decline occurred without great reduction of fiber stiffness and was attributed to a decrease of the average force per attached crossbridge. Force decline during the later phase was accompanied by a proportional stiffness decrease and was attributed to a decrease of the number of attached crossbridge. Similarly to fatigue, at both 24 and 35°C, force recovery occurred in two phases: the first associated with the recovery of the average force per attached crossbridge and the second due to the recovery of the pre-fatigue attached crossbridge number. These changes, symmetrical to those occurring during fatigue, are consistent with the idea that, i) initial phase is due to the direct fast inhibitory effect of [Pi]i increase during fatigue on crossbridge force; ii) the second phase is due to the delayed reduction of Ca2+ release and /or reduction of the Ca2+ sensitivity of the myofibrils due to high [Pi]i.