Rabbit Soleus Research Articles

The Functional Consequences of Hypertrophic Cardiomyopathy Troponin C Mutations in the Regulation of Slow Skeletal Muscle Contraction: The Protective Role of Slow Skeletal Troponin I

Mutations in cardiac troponin C (cTnC) have been linked to hypertrophic cardiomyopathy (HCM) in humans. The cTnC subunit of the troponin complex is expressed in both slow skeletal and cardiac muscle. The cTnC HCM mutations A8V, C84Y and D145E increase the Ca2+-sensitivity of contraction in cardiac muscle; however, their functional consequences in slow skeletal muscle regulation are unknown. Here we investigated the Ca2+-sensitivity of TnC-extracted rabbit soleus skinned fibers reconstituted with either WT or HCM mutant cTnC. Surprisingly, the slow skeletal skinned fibers extinguished the Ca2+-sensitization typical of cardiac fibers for A8V (pCa50 = 6.01) and D145E (pCa50 = 6.05), while for C84Y (pCa50 = 6.33) the Ca2+-sensitization was maintained compared to WT (pCa50 = 6.00). To determine whether slow skeletal TnI (ssTnI) alone was responsible for the protective effect of incorporating the A8V and D145E mutants into the slow skeletal myofilaments, cardiac skinned fibers were reconstituted with a hybrid cardiac troponin complex containing cTnT, ssTnI and cTnC-WT or -HCM mutants. The presence of ssTnI in cardiac fibers partially protected against Ca2+-sensitization by the cTnC mutants in cardiac skinned fibers, with the exception of C84Y. MgATPase activity of slow or cardiac myofibrils replaced with exogenous HCM cTnC mutants were also affected. The absence of increased Ca2+-sensitivity of contraction arising from the A8V and D145E in the soleus muscle suggests that the aberrant effects of cardiomyopathic cTnCs may be tissue-specific, and ssTnI is an important partner that partially protects the slow muscle from a deleterious effect of the HCM cTnC mutants. Supported by NIH HL103840 (JRP).

Three-dimensional surface geometries of the rabbit soleus muscle during contraction: input for biomechanical modelling and its validation

There exists several numerical approaches to describe the active contractile behaviour of skeletal muscles. These models range from simple one-dimensional to more advanced three-dimensional ones; especially, three-dimensional models take up the cause of describing complex contraction modes in a realistic way. However, the validation of such concepts is challenging, as the combination of geometry, material and force characteristics is so far not available from the same muscle. To this end, we present in this study a comprehensive data set of the rabbit soleus muscle consisting of the muscles' characteristic force responses (active and passive), its three-dimensional shape during isometric, isotonic and isokinetic contraction experiments including the spatial arrangement of muscle tissue and aponeurosis-tendon complex, and the fascicle orientation throughout the whole muscle at its optimal length. In this way, an extensive data set is available giving insight into the three-dimensional geometry of the rabbit soleus muscle and, further, allowing to validate three-dimensional numerical models.

A numerical validation approach of a finite element muscle model using optical data

AbstractAim of this work is to obtain a convenient data set for the validation of a recently developed three‐dimensional constitutive muscle model. Therefore, an optical measurement technique is used to reconstruct a geometrical model of a rabbit soleus muscle. Thus, the muscle geometry and also the generated force characteristics are measured. The proposed numerical model is able to reproduce the experimental results in an adequate manner. (© 2012 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Compressive properties of passive skeletal muscle—The impact of precise sample geometry on parameter identification in inverse finite element analysis

Due to the increasing developments in modelling of biological material, adequate parameter identification techniques are urgently needed. The majority of recent contributions on passive muscle tissue identify material parameters solely by comparing characteristic, compressive stress–stretch curves from experiments and simulation. In doing so, different assumptions concerning e.g. the sample geometry or the degree of friction between the sample and the platens are required. In most cases these assumptions are grossly simplified leading to incorrect material parameters. In order to overcome such oversimplifications, in this paper a more reliable parameter identification technique is presented: we use the inverse finite element method (iFEM) to identify the optimal parameter set by comparison of the compressive stress–stretch response including the realistic geometries of the samples and the presence of friction at the compressed sample faces. Moreover, we judge the quality of the parameter identification by comparing the simulated and experimental deformed shapes of the samples. Besides this, the study includes a comprehensive set of compressive stress–stretch data on rabbit soleus muscle and the determination of static friction coefficients between muscle and PTFE.

The Functional Consequences of HCM Troponin C Mutations in the Regulation of Slow Skeletal Muscle Contraction

The cardiac troponin C (cTnC) subunit of the troponin complex is expressed in both slow skeletal and cardiac muscle. Therefore, investigating the effects of cTnC mutations associated with hypertrophic cardiomyopathy (HCM) in slow skeletal muscle may reveal new insights into the mechanisms underlying cardiomyopathies. Glycerol SDS-PAGE analysis was performed on rabbit soleus muscle in order to determine the amount of slow myosin heavy chain type I (MHC I) present and its ratio to other myosin isotypes.

The Effect of Muscle Excursion on Muscle Recovery after Tendon Repair in a Neglected Tendon Injury: A Study in Rabbit Soleus Muscles

The Effect of Muscle Excursion on Muscle Recovery after Tendon Repair in a Neglected Tendon Injury: A Study in Rabbit Soleus Muscles

VGluT2 and NMDAR1 Expression in Cells in the Inflammatory Infiltrates in Experimentally Induced Myositis: Evidence of Local Glutamate Signaling Suggests Autocrine/Paracrine Effects in an Overuse Injury Model

It is not known whether a glutamate signaling system is involved in muscle inflammation (myositis). In the present study, we examined this question in the soleus muscle in a laboratory model of myositis resulting from repetitive overuse induced by electrical stimulation and injection of pro-inflammatory substances. Sections of rabbit soleus muscle with an induced myositis, i.e., exhibiting infiltration of inflammatory cells, were examined immunohistochemically using antibodies against vesicular glutamate transporter VGluT2 and the glutamate receptor NMDAR1. In situ hybridization for demonstration of VGluT2 mRNA was also performed. Specific reactions for both VGluT2 and NMDAR1 could be observed immunohistochemically in the same cells. In situ hybridization demonstrated the occurrence of VGluT2 mRNA in the cells. Double staining showed that the VGluT2 reactions were detectable in cells marked with T cell/neutrophil marker and in cells expressing eosinophil peroxidase. These data suggest the occurrence of previously unknown glutamate-mediated autocrine/paracrine effects within the inflammatory infiltrates during the development of muscle inflammation.

The effect of muscle excursion on muscle recovery after tendon repair in a neglected tendon injury: A study in rabbit soleus muscles

We attempted to determine whether muscle excursion observed during operation can be a prognostic indicator of muscle recovery after delayed tendon repair in a rabbit soleus model. Eighteen rabbits underwent tenotomy of the soleus muscles bilaterally and were divided into three groups according to the period from tenotomy to repair. The tendons of each group were repaired 2, 4, and 6 weeks after tenotomy. The excursion of each soleus muscle was measured at the time of tenotomy (baseline), at 2, 4, 6 weeks after tenotomy, and 8 weeks after tendon repair. The amount of muscle recovery after tendon repair in terms of muscle excursion independently depended on the timing of repair and on the muscle excursion observed during repair. The regression model predicted that the muscle excursion recovered on average by 0.6% as the muscle excursion at the time of repair increased by 1% after adjusting for the timing of repair. This study suggests that measuring the muscle excursion during tendon repair may help physicians estimate the potential of muscle recovery in cases of delayed tendon repair.

The effect of muscle length and excursion on muscle contracture after tendon injury: A study in rabbit soleus muscles

If tendon repair after injury is delayed, intramuscular fibrosis occurs and finally results in irreversible muscle contracture. The purpose of this study was to determine the effect of preserving muscle length and excursion on the progression of muscle contracture after tendon injury in a rabbit soleus tenotomy model. Forty rabbits underwent tenotomy of the soleus muscles bilaterally and the tendons were managed according to the five experimental groups (N=40). In group A, the tendon was lengthened maintaining half the excursion. In group B, maximal muscle length was preserved and in group C, resting muscle length was maintained. In group D, the tendon was allowed to retract and undergo fibrosis. In group E, the tendon was partially transected and repaired. Four and 8 weeks postoperatively, soleus muscles were harvested from each hind limb and histomorphometric evaluations were performed to measure the connective tissue areas. Electrophysiological studies were carried out to measure the compound muscle action potential to assess the number of functioning muscle fibres. The results showed that maximal muscle length preservation (group B) was the most protective in preventing muscle contracture within 4 weeks of tenotomy, but this effect was gradually offset by prolonged fixation of the muscle, and 8 weeks after tenotomy, maintenance of excursion (group A) was the most protective. These observations may be helpful in the intra-operative evaluation of muscle contracture in neglected tendon ruptures, and could be applied to the management of acute tendon injuries to prevent muscle contracture when immediate anatomical reconstruction is not possible.

Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as β-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin–ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant KD) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (KAD/KD∼5 instead of KAD/KD∼50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s−1) than reported previously (138 s−1). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

When fibres go slack and cross bridges are free to run: a brilliant method to study kinetic properties of acto‐myosin interaction

When fibres go slack and cross bridges are free to run: a brilliant method to study kinetic properties of acto‐myosin interaction

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB(-/-) mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2(b/b) mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M· ADP·Pi states predominate on the cross-bridge cycle

The ATPases (+/-Ca2+) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different Ea: -Ca2+, 78 and 60 kJ/mol and +Ca2+, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132-3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the Ea data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4 degrees C. We used two methods: rapid flow quench ('cold ATP chase': titration of active sites, ATP binding kinetics, k(cat); 'Pi burst': ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free Pi; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M.ADP.Pi. We propose a reaction pathway that includes several (A)M.ADP.Pi sub-states that are either 'weak' or 'strong', depending on the mechanical condition.

Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus.

This study investigated the effects of l-thyroxine-induced hyperthyroidism on Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca(2+) pump (Ca(2+)-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca(2+)-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30-50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1 ( approximately 150%) Ca(2+) pump isoform, unaltered levels of SERCA2 Ca(2+) pump isoform, and lower levels of PLN ( approximately 50%) and delta-, beta-, and gamma-CaM kinase II (40 approximately 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca(2+) uptake and Ca(2+)-stimulated ATPase activities compared with that from euthyroid control. The V(max) of Ca(2+) uptake (in nmol Ca(2+).mg SR protein(-1).min(-1): euthyroid, 818 +/- 73; hyperthyroid, 1,649 +/- 90) but not the apparent affinity of the Ca(2+)-ATPase for Ca(2+) (euthyroid, 0.97 +/- 0.02 microM, hyperthyroid, 1.09 +/- 0.04 microM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca(2+) uptake by soleus muscle SR was approximately 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater ( approximately 36%), and the time to peak force and relaxation time were significantly lower ( approximately 30-40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca(2+) pump.

At Physiological Temperatures the ATPase Rates of Shortening Soleus and Psoas Myofibrils Are Similar

We obtained the temperature dependences of the adenosine triphosphatase (ATPase) activities (calcium-activated and relaxed) of myofibrils from a slow muscle, which we compared with those from a fast muscle. We chose rabbit soleus and psoas because their myosin heavy chains are almost pure: isoforms I and IIX, respectively. The Arrhenius plots of the ATPases are linear (4–35°C) with energies of activation for soleus myofibrils 155 kJ mol −1 (activated) and 78 kJ mol −1 (relaxed). With psoas myofibrils, the energies of activation were 71 kJ mol −1 (activated) and 60 kJ mol −1 (relaxed). When extrapolated to 42°C the ATPase rates of the two types of myofibril were identical: 50 s −1 (activated) and 0.23 s −1 (relaxed). Whereas with psoas myofibrils the K m for adenosine triphosphate (activated ATPase) is relatively insensitive to temperature, that for soleus myofibrils increased from 0.3 μM at 4°C to 66.5 μM at 35°C. Our results illustrate the importance of temperature when comparing the mechanochemical coupling in different types of muscle. We discuss the problem of how to reconcile the similarity of the myofibrillar ATPase rates at physiological temperatures with their different mechanical properties.

Effects of pH and temperature on force and stiffness of skeletal muscle fibers during contraction and relaxation in relation to musculoskeletal disorders.

Effects of low pH (6.0, 6.5) were studied at three temperatures (5, 15, 25 degrees C) on the isometric force and stiffness of glycerinated muscle fibers dissected from soleus (type I) and psoas (type IIX) muscles of the Japanese white rabbit. It was observed that the maximum force and stiffness declined as pH decreased, the extent of which was diminished by an increase in temperature in both muscle types. The pH-induced changes in force were greater than those in the stiffness for both muscle types: at 5 degrees C, the psoas showed greater change in stiffness than the soleus. As the pH of the contracting solution decreased, the time to peak stiffness decreased and the time to relaxation increased in both muscle types. At pH 6.0, the latter was significantly longer than the former. The force-stiffness curves indicated that, when pH decreased, force increased as fast as stiffness during contraction and force declined faster than stiffness during relaxation. The results suggested that one of the causes of muscle injury during hard muscular work could be the longer relaxation time in an acidic solution.

Effect of temperature on elementary steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibres.

1. Isometric tension, stiffness and the cross-bridge kinetics in rabbit soleus slow-twitch fibres (STFs) were studied in the temperature range 5-37 degrees C by sinusoidal analysis. 2. The effects of MgATP and phosphate (Pi) on the cross-bridge kinetics were studied, and the temperature dependence of the kinetic constants of elementary steps of the cross-bridge cycle was deduced in the range 20-37 degrees C. 3. The MgATP association constant (K1a) decreased when temperature was increased. The rate constants of the ATP-isomerization step (k1b and k-1b) and the cross-bridge detachment step (k2, and k(-2)) had Q10 values of 3-4, and hence their equilibrium constants (K1b and K2) changed little with temperature. 4. Q10 of the force generation step (k4) was the largest at 6.7; its reversal step (k(-4)) had a Q10 of 2.5, and hence its equilibrium constant (K4) increased significantly with temperature. The Pi association constant (K5) changed little with temperature. 5. The elementary steps of the cross-bridge cycle are more temperature sensitive in soleus STFs than in psoas, which are fast-twitch fibres. This is in accord with a higher temperature sensitivity of the apparent rate constants in STFs.T 6. The temperature dependence of the equilibrium constant of the force generation step (K4) was fitted to the modified Van't Hoff equation to deduce standard enthalpy change (DeltaH degrees; 70 +/- 20 kJ mol(-1)), standard entropy change (DeltaS degrees; 250 +/- 70 J mol(-1) K(-1)), and heat capacity change (DeltaCp; -12 +/- 5 kJ mol(-1) K(-1)). These results indicate that the force generation step is an entropy driven, endothermic reaction that accompanies a burial of large surface area. These observations are consistent with the hypothesis that hydrophobic interaction between residues of actin and myosin and between residues of the myosin head underlies the mechanism of force generation. 7. An increase of isometric tension with temperature is accounted for by the increased number of cross-bridges in tension generating states. Stiffness also increased with temperature, but to a lesser degree.

Relative metabolic utilization of in situ rabbit soleus muscle: thermistor-based measurements and model.

Electrically conditioned skeletal muscle can provide the continuous power source for cardiac assistance devices. Optimization of the available sustained power from in vivo skeletal muscle requires knowledge of its metabolic utilization and constraints. A thermistor-based technique has been developed to measure temperature changes and to provide a relative estimate for metabolic utilization of in situ rabbit soleus muscle. The relative thermistor response, active tension and muscle displacement were measured during cyclic isometric and isotonic contractions across a range of muscle tensions and contraction durations. The thermistor response demonstrated linear relationships versus both contraction duration at a fixed muscle length and active tension at a fixed contraction duration (r(2)=0.90+/-0.14 and 0.70+/-0.21, respectively; means +/- s.d.). A multiple linear regression model was developed to predict normalized thermistor response, DeltaT, across a range of conditions. Significant model variables were identified using a backward stepwise regression procedure. The relationships for the in situ muscles were qualitatively similar to those reported for mammalian in vitro muscle fiber preparations. The model had the form DeltaT=C+at(c)F+bW, where the constant C, and coefficients for the contraction duration t(c) (ms), normalized active tension F and normalized net work W were C=-1.00 (P<0.001), a=5.97 (P<0.001) and b=2.12 (P<0.001).

Measurement of Nucleotide Exchange Rate Constants in Single Rabbit Soleus Myofibrils during Shortening and Lengthening Using a Fluorescent ATP Analog

The kinetics of displacement of a fluorescent nucleotide, 2′(3′)- O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5′-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (Chaen et al., 1997, Biophys. J. 73:2033–2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K m for Cy3-EDA-ATP and the K d for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 μM and 3.8 μM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 μM and 50 μM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 ± 0.007 s −1 ( n = 32) at 10°C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s −1 (isometric) to 0.14 s −1 at 20°C with increasing shortening velocity up to 0.1 myofibril length/s ( V max, the shortening velocity under no load was ∼0.2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.