Tim Leonard

Regulation of muscle force in the absence of actin-myosin-based cross-bridge interaction

For the past half century, the sliding filament-based cross-bridge theory has been the cornerstone of our understanding of how muscles contract. According to this theory, active force can only occur if there is overlap between the contractile filaments, actin and myosin. Otherwise, forces are thought to be caused by passive structural elements and are assumed to vary solely because of the length of the muscle. We observed increases in muscle force by a factor of 3 to 4 above the purely passive forces for activated and stretched myofibrils in the absence of actin-myosin overlap. We show that this dramatic increase in force is crucially dependent on the presence of the structural protein titin, cannot be explained with calcium activation, and is regulated by actin-myosin-based cross-bridge forces before stretching. We conclude from these observations that titin is a strong regulator of muscle force and propose that this regulation is based on cross-bridge force-dependent titin-actin interactions. These results suggest a mechanism for stability of sarcomeres on the inherently unstable descending limb of the force-length relationship, and they further provide an explanation for the protection of muscles against stretch-induced muscle injuries.

Reflex responses associated with activator treatment

BACKGROUNDnPrevious studies have demonstrated the existence era reflex response, measurable by surface electromyography (sEMG), after manually delivered spinal manipulative therapy (SMT). This reflex response has been characterized as consistent, reproducible within individual subjects, and nonlocal because it extends beyond the site of manipulation. However, the nature and magnitude of possible reflex responses in the paraspinal and proximal limb muscles elicited by nonmanual SMT, such as with an adjusting instrument, remain unknown.nnnOBJECTIVEnTo characterize the reflex responses associated with SMT by using sEMG to record the responses of 16 muscles before, during, and after treatment.nnnSTUDY DESIGNnThe eleetromyographic responses of 16 para-spinal and proximal limb muscles in 9 healthy, asymptomatic male volunteers were measured simultaneously by sEMG before, during, and after chiropractic SMT.nnnMETHODSnSMT thrusts were delivered to 9 asymptomatic volunteers at 6 bilateral sites (C3/4, T2/3, T6/8, T11/12, L2-4, and s1). Reflex responses were measured from 16 muscles with bipolar sEMG electrodes and collected at 2000 Hz per channel with data acquisition software.nnnRESULTSnApproximately 68% of the SMT thrusts resulted in a detectable reflex response. The cervical spine resulted in a detectable response of 50%, thoracic spine 59%, lumbar spine 83%, and sacroiliac joints 94%. Treatments delivered to the thoracic spine elicited the largest peak-to-peak amplitude sEMG responses, whereas the lumbar spine demonstrated the most heterogeneous responses. When a reflex response was observed, it always occurred close to the treatment site ipsilaterally and was detected in muscles that had either their origin or insertion at the vertebral level that was adjusted.nnnCONCLUSIONSnBased on the local nature, magnitude, and characteristic shape of all reflex responses observed, we hypothesized that they were likely generated by a single proprioceptor. Furthermore, the temporal properties of this reflex response suggest that they originated from the muscle spindles. In contrast to previous observations on reflex responses after manual SMT, these treatments elicited reflex responses that varied between subjects but were consistent within an individual and were local in nature. We conclude that SMT delivered in this manner results in a reflex response that is both quantitatively and qualitatively different from a manual SMT.

Coordination of medial gastrocnemius and soleus forces during cat locomotion

SUMMARY We studied force-sharing behavior between the cat medial gastrocnemius (MG) and soleus (SOL) muscles by direct measurement of the muscle forces and electromyographic activities (EMGs), muscle lengths, speeds of contraction, joint kinematics and kinetics, for a variety of locomotor conditions. Previous studies suggested that the modulation of MG force and activation is associated with movement demands, while SOL force and activation remain nearly constant. However, no systematic, quantitative analysis has been done to evaluate the degree of (possible) modulation of SOL force and activation across a range of vastly different locomotor conditions. In the present study, we investigated the effects of speed and intensity of locomotion on the modulation of SOL force and EMG activity, based on quantitative, statistical analyses. We also investigated the hypothesis that MG forces are primarily associated with MG activation for changing movement demands, while SOL forces are primarily associated with the contractile conditions, rather than activation. Seven cats were trained to walk, trot and gallop at different speeds on a motor-driven treadmill, and to walk up and down different slopes on a walkway. Statistical analysis suggested that SOL activation (EMG activity) significantly increased with increasing speeds and intensities of locomotion, while SOL forces remained constant in these situations. MG forces and EMG activities, however, both increased with increasing speeds and intensities of locomotion. We conclude from these results that SOL is not maximally activated at slow walking, as suggested in the literature, and that its force remains nearly constant for a range of locomotor conditions despite changes in EMG activity. Therefore, SOL forces appear to be affected substantially by the changing contractile conditions associated with changing movement demands. In contrast, MG peak forces correlated well with EMG activities, suggesting that MG forces are primarily associated with activation while its contractile conditions play a minor role for the movement conditions tested here.

Titin force is enhanced in actively stretched skeletal muscle

The sliding filament theory of muscle contraction is widely accepted as the means by which muscles generate force during activation. Within the constraints of this theory, isometric, steady-state force produced during muscle activation is proportional to the amount of filament overlap. Previous studies from our laboratory demonstrated enhanced titin-based force in myofibrils that were actively stretched to lengths which exceeded filament overlap. This observation cannot be explained by the sliding filament theory. The aim of the present study was to further investigate the enhanced state of titin during active stretch. Specifically, we confirm that this enhanced state of force is observed in a mouse model and quantify the contribution of calcium to this force. Titin-based force was increased by up to four times that of passive force during active stretch of isolated myofibrils. Enhanced titin-based force has now been demonstrated in two distinct animal models, suggesting that modulation of titin-based force during active stretch is an inherent property of skeletal muscle. Our results also demonstrated that 15% of the enhanced state of titin can be attributed to direct calcium effects on the protein, presumably a stiffening of the protein upon calcium binding to the E-rich region of the PEVK segment and selected Ig domain segments. We suggest that the remaining unexplained 85% of this extra force results from titin binding to the thin filament. With this enhanced force confirmed in the mouse model, future studies will aim to elucidate the proposed titin–thin filament interaction in actively stretched sarcomeres.

Effects of cyclic changes in muscle length on force production in in-situ cat soleus

Muscle shortening and stretch are associated with force depression and force enhancement, respectively. Previously, we have investigated the effect of combined dynamic contractions (i.e. a single shortening-stretch and stretch-shortening cycle) on force production (Herzog and Leonard, 2000). In order to investigate the relationship between force depression and force enhancement systematically, we studied the effects of a single as well as multiple stretch-shortening and shortening-stretch cycles on the ascending limb of the force-length relationship. Furthermore, by systematically varying the speed and magnitude of stretch preceding shortening and the speed and magnitude of shortening preceding stretch, we investigated the influence of these varying contractile conditions on force depression and force enhancement, respectively. Test contractions were performed on cat soleus (n=6) by electrical stimulation using four conceptually different protocols containing a single or repeated stretch-shortening and shortening-stretch cycles. The results of this study showed that: (1) force depression was not influenced by stretch preceding shortening independent of the speed and amount of stretch; (2) force enhancement was influenced in a dose-dependent manner by the amount of shortening preceding stretch but was not affected by the speed of shortening; (3) repeated stretch-shortening (shortening-stretch) cycles showed cumulative effects; (4) the number of shortening steps over a given distance did not influence the amount of force depression. The findings of this study support the idea that the mechanism of force depression associated with muscle shortening is different from that of force enhancement associated with muscle stretch. Furthermore, they support and extend our previous findings that stretch-shortening and shortening-stretch cycles are not commutative.

Molecular mechanisms of muscle force regulation: a role for titin?

Muscle contraction and force regulation is thought to occur exclusively through the interaction of the contractile proteins actin and myosin and in accordance with the assumptions underlying the cross-bridge theory. Here, we demonstrate that a third protein, titin, plays a major role in muscle force regulation, particularly for eccentric contractions and at long muscle and sarcomere lengths.

Does residual force enhancement increase with increasing stretch magnitudes

It is generally accepted that force enhancement in skeletal muscles increases with increasing stretch magnitudes. However, this property has not been tested across supra-physiological stretch magnitudes and different muscle lengths, thus it is not known whether this is a generic property of skeletal muscle, or merely a property that holds for small stretch magnitudes within the physiological range. Six cat soleus muscles were actively stretched with magnitudes varying from 3 to 24 mm at three different parts of the force-length relationship to test the hypothesis that force enhancement increases with increasing stretch magnitude, independent of muscle length. Residual force enhancement increased consistently with stretch amplitudes on the descending limb of the force-length relationship up to a threshold value, after which it reached a plateau. Force enhancement did not increase with stretch amplitude on the ascending limb of the force-length relationship. Passive force enhancement was observed for all test conditions, and paralleled the behavior of the residual force enhancement. Force enhancement increased with stretch magnitude when stretching occurred at lengths where there was natural passive force within the muscle. These results suggest that force enhancement does not increase unconditionally with increasing stretch magnitude, as is generally accepted, and that increasing force enhancement with stretch appears to be tightly linked to that part of the force-length relationship where there is naturally occurring passive force.

Are titin properties reflected in single myofibrils

Titin is a structural protein in muscle that spans the half sarcomere from Z-band to M-line. Although there are selected studies on titins mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces occur. Three repeat stretch-shortening cycles with magnitudes between 1.0 and 3.0μm/sarcomere were performed at a speed of 0.1μm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with 2,3 butanedionemonoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5μm that was associated with an average force of 68±5nN/mm. This inflection point was thought to reflect the onset of Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties observed here are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.

Reply from Walter Herzog (on behalf of the authors) and Tim Leonard

We would like to thank the authors of the letter to the editor for bringing up some interesting points of discussion, and we will attempt in the following to address some of the more important issues. The general conclusion of the authors of the letter is that nothing new was provided in the Topical Review (Herzog et al. 2006), that many of the cited papers suffer from limits of accuracy and additional obvious problems, and that the whole collection of observations is readily explained by non-uniformities of half-sarcomere lengthening. In the Topical Review we challenge the view that sarcomere length non-uniformity can readily explain residual force enhancement observed in muscles, single fibres and myofibril preparations. Our arguments centre primarily on the observations that force enhancement is observed on the ascending part of the force–length relationship, can exceed the forces observed for purely isometric contractions on the plateau of the force–length relationship, and occurs in the presence of sarcomere clamping in single fibres (e.g. Edman et al. 1982). Furthermore, we review literature and show recent evidence that sarcomere and half-sarcomere lengths are stable on the descending limb of the force–length relationship (e.g. Rassier et al. 2003; Telley et al. 2006), and demonstrate that in single myofibrils, individual (half-) sarcomeres show force enhancement and (half-) sarcomere lengths are non-uniform prior to and after stretch, and if anything, seem to be more uniform following a stretch compared to before, in agreement with suggestions by Edman et al. (1982) and Telley et al. (2006). Regarding some specific comments, in the section on ‘Excess tension’ the authors of the letter question the results that ‘tension after stretch can . . . exceed the tension in a fixed-end contraction at the initial length . . .’ However, as pointed out in the Topical Review, we define residual force enhancement as the increase in steady-state, isometric force following active muscle (fibre, myofibril) stretching compared to the force for a purely isometric contraction at the same length. Therefore, our comparisons are not made with respect to the initial length, but with respect to the length after the stretch. This is the accepted definition for residual force enhancement (Edman et al. 1982), as it does not make sense to expect isometric forces to be the same at different muscle length. The second point of controversy surrounds whether there is force enhancement on the ascending part of the force–length relationship. Such force enhancement has been observed frequently by groups other than ours (e.g. Abbott & Aubert, 1952; Cook & McDonagh, 1995; and De Ruiter et al. 2000 for whole muscle preparations; and Sugi, 1972 for fibre bundles). In fact, force enhancement on the ascending limb of the force–length relationship has also been observed by proponents of the sarcomere length non-uniformity theory. For example, Fig. 1 was scanned and redrawn from Morgan et al. (2000 – their Fig. 3) where it was stated that they did not observe force enhancement on the ascending part of the force–length curve, while in actuality it was present. For the longest stretches, they observed higher forces on 10 out of 11 conditions (one was the same force with and without stretch), the average force enhancement on the ascending limb of the force–length relationship (estimated from their figure) was 5.7%, and the maximum force enhancement at the plateau was about 15%. Our published values on the same preparation are similar to those shown by Morgan et al. (2000), except that our interpretation was that these results (which were observed consistently) are not caused by noise or inaccuracies in measurements, but are real. With a resolution of our force measurement system of 0.02 N (or about 0.1% of the total soleus force in a cat), we can easily resolve force enhancement of 5–15%. The average force enhancement above plateau force shown in Fig. 3 of our Topical Review was about 6% for 10 fibres. However, the stretch conditions (a 10% stretch from the ascending limb onto the plateau) shown in that figure do not maximize force enhancement. Nevertheless, force enhancement was observed in all 10 fibres used in that experiment (Peterson et al. 2004). Therefore, suggesting that our results might only show force enhancement beyond optimum length, and that force enhancement was ‘insignificantly above optimum tension’ is, at least from a statistical point of view, incorrect. Needless to say, all our equipments can resolve force differences of much smaller than 1%, and therefore consistent force enhancement of 5% or 10% above the plateau forces cannot be dismissed with arguments based on limits of accuracy. Regarding the points on internal movements, we completely agree with the authors of the letter: it is very difficult, if not impossible to know if sarcomere lengths have reached a steady-state, or if some residual internal sarcomere length redistribution occurs when the measurements of force enhancement are taken. Moreover, complete data on sarcomere length redistributions in whole muscle contractions are not available, and records of sarcomere length measurements in single fibres must be viewed and interpreted carefully, for they represent average sarcomere lengths for thousands of sarcomeres when a laser diffraction or segment clamp approach is used. More importantly, a sarcomere in a single fibre is not arranged strictly in series with other sarcomeres but has parallel connections (for example through desmin at the z-bands), and thus, the force measured at the end of the fibre cannot be related in any simple way to individual sarcomere lengths. Arguably, the only preparation in which sarcomere dynamics and force can be studied is a single myofibril, as in this preparation sarcomeres are arranged mechanically in series, and therefore, the forces measured at the end of the myofibril also represent the instantaneous sarcomere forces. In myofibrils (half-) sarcomere length ratios differ prior to and following stretching of myofibrils on the descending limb of the force–length relationship, and there is distinct force enhancement in this preparation, which exceeds the isometric plateau forces. In Fig. 2, we show an active myofibril with six sarcomeres stretched from an initial average sarcomere length of 2.7 μm to a final average length of 3.1 μm. The following observations regarding the sarcomere length non-uniformity theory seem particularly important. First, there is clear force enhancement; second, sarcomere lengths do not become non-uniform

MECHANISM OF ELECTRICALLY ELICITED MUSCLE VIBRATIONS IN THE IN SITU CAT SOLEUS MUSCLE

Ifmusce vibrationsare really produced by the contraction of unfusedmotor units, the following predictions can be madeabout electrically stimulated musce: (1) there shouldbe no vibratory signals at rest or when the musce istetanically stimulated; (2) for stimulation frequenciesproducing unfused contractions, a vibratory signalshould be measured for each electrical stimulus;(3) when increasing the stimulation frequencies from4 Hz to 20 Hz, the amplitude of the vibratory signalsshould decrease while the frequency content shouldincrease to reflect that the musce becomes increasxadingly stiff, and to reflect that fewer MUs contract inan unfused manner; and (4) the magnitude and thefrequency content of the vibratory signals should dexadcrease with decreasing numbers of activated MUs toreflect that nonactive units are passive1y moved bythe active MUs. The purpose of this study was to testthe above hypothesis by testing these four specificpredictions.