An increase in force after stretch of diaphragm fibers and myofibrils is accompanied by an increase in sarcomere length nonuniformities and Ca2+ sensitivity.

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca2+ sensitivity as shown by a change in the force-pCa2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.

Impaired Intracellular Ca2+ Dynamics, M-Band and Sarcomere Fragility in Skeletal Muscles of Obscurin KO Mice.

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.

The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms.

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca(2+) activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca(2+)-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 µm for the psoas, 2.68 and 4.21 µm for the soleus, and 1.51 and 2.86 µm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca(2+)-dependent change in titin properties and not associated with changes in titin-actin interactions.

Profiling Carbonylated Proteins in Heart and Skeletal Muscle Mitochondria from Trained and Untrained Mice.

Understanding the relationship between physical exercise, reactive oxygen species, and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Heart and skeletal muscles have a high density of mitochondria with robust energetic demands, and mitochondria plasticity has an important role in both the cardiovascular system and skeletal muscle responses. The aim of this study was to investigate the influence of regular physical activity on the oxidation profiles of mitochondrial proteins from heart and tibialis anterior muscles. To this end, we used the mouse as animal model. Mice were divided into two groups: untrained and regularly trained. The carbonylated protein pattern was studied by two-dimensional gel electrophoresis followed by Western blot with anti-dinitrophenyl hydrazone antibodies. Mass spectrometry analysis allowed the identification of several different protein oxidation sites, including methionine, cysteine, proline, and leucine residues. A large number of oxidized proteins were found in both untrained and trained animals. Moreover, mitochondria from skeletal muscles and heart showed almost the same carbonylation pattern. Interestingly, exercise training seems to increase the carbonylation level mainly of mitochondrial proteins from skeletal muscle.

Non-crossbridge stiffness in active muscle fibres.

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.

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 Ca(2+)-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 Ca(2+) concentration leads to Ca(2+) 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 Ca(2+)-induced increase in titin stiffness is confirmed in future studies, it may have large implications for understating of the basic mechanisms of muscle contraction.

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.

Mechanism of force enhancement during stretching of skeletal muscle fibres investigated by high time-resolved stiffness measurements.

Stretching of active muscles leads to a great enhancement of the force developed without increased ATP consumption. The mechanism of force enhancement is still debated and it is not clear if it is due to increased crossbridge strain or to a stretch-induced increase in crossbridge number. The present study, performed on single fibres from tibialis anterior or interosseus muscles of the frog at 5 °C, was aimed at clarifying this point. A striation follower device was used to measure sarcomere length changes. Force was measured during the application of stretches (0.15-3.9 ms duration, 3-7.8 nm per half-sarcomere amplitude) to activated fibres. Small 4 kHz sinusoidal length oscillations, superimposed on the stretches, were used to calculate fibre stiffness with high time resolution. Stiffness increased during the stretch then subsequently decayed, all in parallel with tension. Likewise, during quick releases, stiffness decreased during the release then subsequently recovered in parallel with tension. Comparison of tension and stiffness both during the tetanus rise and also during stretches which doubled tension, imposed on the tetanus rise, indicated that stretch-induced crossbridge recruitment was only about 11 %, suggesting that force enhancement by stretching is mainly due to an increase of individual crossbridge force, whereas crossbridge recruitment plays only a minor role. The accompanying stiffness changes can be explained by non-linearity of myofilament compliance.

The muscle shortening maneuver in individuals with stroke: a consideration-of-concept randomized pilot trial.

BACKGROUND AND PURPOSE: The Muscle Shortening Maneuver (MSM) is derived from Feldman's λ model of motor control, and seems to induce a more balanced agonist- antagonist-muscular action. The hypothesized mechanism of action is a modulation of the Tonic Stretch Reflex Threshold (TSRT). We designed a pilot, randomized trial aimed to explore the mechanisms of action of the technique. An ancillary objective was to research the implementation of the MSM as a stroke rehabilitation intervention. METHODS: A sample of 10 participants with chronic stroke was enrolled and randomly assigned to MSM (n, 5) or conventional physical therapy (CPT) (n, 5) treatments. The TSRTs were assessed by the Montreal Spasticity Measure device. A selection of clinical and instrumental outcome measures was taken to investigate function and activity levels. Data were collected at baseline, end-of-treatment, and one month after the end-of-treatment. RESULTS: No adverse events were observed. In both between- and within-group post-treatment assessments, in the affected ankle the MSM group showed decreased TSRTs of the plantar flexor, increased strength of the dorsiflexor and active range of motion; also, the time needed to perform the Timed Up and Go test decreased. No changes were evident across assessments in the CPT group. DISCUSSION AND CONCLUSIONS: The MSM seems able to modulate the TSRTs in individuals with stroke. Although with the limitations due to the pilot design, the variation in participants' responses appear to be promising. Many methodological issues have to be clarified and specified conceiving the progression toward a confirmatory trial.

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 Ca(2+)-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 Ca(2+) transients in these muscles. The present results show that the presence of a non-crossbridge Ca(2+)-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.

The Muscle Shortening Maneuver: a noninvasive approach to the treatment of peroneal nerve injury. A case report.

BACKGROUND: The treatment of peripheral nerve injuries is a debated topic. The Muscle Shortening Maneuver (MSM), a physiotherapy approach, is noninvasive and free of side effects; it consists of a muscle shortening and a solicitation in traction applied simultaneously. OBJECTIVE: The focus of this report is to describe the effects of the MSM combined with walking retraining in a patient with incomplete injury of the peroneal nerve. DESCRIPTION: The patient was a 17-year-old man, who underwent osteotomy surgery of the proximal two-thirds of the fibula, due to an Ewing sarcoma that caused a partial injury of the left peroneal nerve. Our assessment plan of the left ankle movement ability comprised range of movement, muscle strength, and surface electromyography (EMG); and a gait analysis was conducted by using an iPhone application. MSM and walking retraining were administered twice and once a week, respectively, for 4 weeks. OUTCOMES: The active range of movement substantially improved in dorsiflexion (≥15°), whereas slightly decreased in plantar flexion (-5°). Aside from the tibialis anterior, an increase in muscle strength was detected. Surface EMG showed an increased activation, particularly in the peroneus longus. A decrease in gait speed and step length was recorded from the gait analysis, with a better bilateral symmetry. CONCLUSIONS: Positive outcomes were reported without evidence of risk or adverse events for the participant.

Shoulder impingement syndrome in water polo players: muscle shortening manoeuvre controls pain intensity, recovers function and normalizes sonographic parameters.

PURPOSE: To evaluate the effects of muscle shortening manoeuvre (MSM) by sonography (US) in professional water polo players with shoulder impingement syndrome (SIS). METHODS: Twenty-four professional water polo players (mean age: 22.13 ± 3.34) with SIS were assigned to one of 2 different treatment interventions: Group (1) MSM: a series of fast accelerations in the upward direction was applied to the upper limb that's connected to a spring through a metal plate with a ring. The ring was linked to a pulley system that was submitted to forces acting in the opposite direction (added mass). Group (2) Simple traction: the series of fast accelerations were performed without the springs. Pain intensity, Yocum and Hawkins tests for SIS, Neer's impingement sign, range of motion, muscle strength and shoulder US were assessed. The examination was performed before, immediately after and 30 days after each treatment to study the US width of subacromial-subdeltoid bursa (SSB), thickness of supraspinatus (ST), long biceps tendons (LBT); hypoechoic halo of surrounding the long biceps (LBH) and subscapular tendons (STH); width of acromio-clavicular joint capsule (ACJ) and the distance between bone heads (ACD). Impingement sign (IS) was evaluated by dynamic examination. RESULTS: Immediately after treatment with MSM, pain was much reduced (p = 0.002); Yocum and Hawkins tests were decreased (p = 0.008, p = 0.031); Neer's impingement sign was negative; range of motion and muscle strength were increased. US showed that the following parameters were significantly reduced: SSB (p = 0.001), LBT (p = 0.014), LBH (p = 0.014), SSH (p = 0.002), ACJ (p = 0.004), ACD (p = 0.001). IS was no more detected. After 30 days, the improvement of clinical and US findings was maintained. In the control group, after simple traction, no clinical amelioration of US parameters was found immediately after the procedure. CONCLUSION: These data show that MSM could be significantly and rapidly effective against pain and the loss of function due to shoulder impingement in water polo players.

THE MUSCLE SHORTENING MANOEUVRE: APPLICABILITY AND PRELIMINARY EVALUATION IN CHILDREN WITH HEMIPLEGIC CEREBRAL PALSY: A RETROSPECTIVE ANALYSIS.

INTRODUCTION: Physiotherapy plays a key role in cerebral palsy rehabilitation, through addressing body function/structure deficits, minimizing activity limitations, and encouraging participation. The muscle shortening manoeuvre is an innovative therapeutic technique, characterized by the ability to induce changes in muscle strength in a short time. OBJECTIVE: To describe the applicability and estimate the effect of the muscle shortening manoeuvre applied to improve motor weakness and joint excursion of the ankle in children with hemiplegic cerebral palsy. METHODS: Nine children with hemiplegic cerebral palsy received 3 intervention sessions in one week. Muscle strength, passive and active range of motion were assessed before, during and after the training, and at 1-week follow-up. RESULTS: The children experienced an immediate increase in muscle strength and joint excursion of the ankle; the improvements were still present at follow-up after 7 days. CONCLUSION: The muscle shortening manoeuvre may be an effective intervention to induce an immediate increase in muscle strength and range of motion of the ankle in children affected by hemiplegia due to cerebral palsy, thus promoting better physical functioning.

Precision in drawing and tracing tasks: Different measures for different aspects of fine motor control.

Drawing and tracing tasks, by being relatively easy to execute and evaluate, have been incorporated in many paradigms used to study motor control. While these tasks are helpful when examining various aspects relative to the performance, the relationship in proficiency between these tasks was not evaluated to our knowledge. Seeing that drawing is thought to be an internally cued and tracing an externally cued task, differences in performances are to be expected. In this study, a quantitative evaluation of the precision of circle drawing and tracing, and spiral tracing was made on 150 healthy subjects. Our results show that, while precision is correlated when repeating drawing circles, tracing spirals, or tracing circles as well as between tracing spirals and tracing circles; there is no correlation when subjects performed drawing circles and tracing spirals or between drawing and tracing of circles. These results suggest that this lack of correlation is task dependent and not shape dependent. We suggest that the evaluation of fine motor control should include both a tracing and a drawing task, taking in consideration the precision in each task. We believe that this approach could help not only to evaluate fine motor control more accurately, but also to identify subjects who are more reliant on either internal or external cueing and to what extent.

S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.

To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.

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 Ca(2+) release and /or reduction of the Ca(2+) sensitivity of the myofibrils due to high [Pi]i.

Effects of solution tonicity on crossbridge properties and myosin lever arm disposition in intact frog muscle fibres.

The aims of this study were to investigate the effects of solution tonicity on muscle properties, and to verify their consistence with the lever arm theory of force generation. Experiments were made in single muscle fibres and in fibre bundles from the frog, using both fast stretches and time-resolved X-ray diffraction, in isotonic Ringer solution (1T), hypertonic (1.4T) and hypotonic (0.8T) solutions. Fast stretches (0.4-0.6 ms duration and 16-25 nm per half-sarcomere (nm hs(-1)) amplitude) were applied at various tensions during the force development in isometric tetani. Force increased during the stretch up to a peak (critical tension, Pc) at which it started to fall, in spite of continued stretching. In all solutions, Pc was proportional to the initial isometric tension developed. For a given isometric tension, Pc increased with solution tonicity and occurred at a precise sarcomere elongation (critical length, Lc) which also increased with tonicity. M3 meridional layer line intensity (I M3) was measured during the application of sinusoidal length oscillations (1 kHz frequency, and about 2% fibre length amplitude) at tetanus plateau. I M3 changed during the length oscillations in a sinusoidal manner in phase opposition to length changes, but a double peak distortion occurred at the peak of the release phase. The presence of the distortion, which decreased with tonicity, allowed calculation of the mean position of the myosin head (S1) during the oscillation cycle. In agreement with the lever arm theory, both X-ray diffraction and mechanical data show that solution tonicity affects S1 mean position and consequently crossbridge individual extension and force, with no effect on crossbridge number. The force needed to break the single crossbridge was insensitive to solution tonicity suggesting a non-ionic nature of the actomyosin bond.

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.