A low-cost 2-D sarcomere model to demonstrate titin-related mechanisms for force production.

Given the recently proposed three-filament theory of muscle contraction, we present a low-cost physical sarcomere model aimed at illustrating the role of titin in the production of active force in skeletal muscle. With inexpensive materials, it is possible to illustrate actin-myosin cross-bridge interactions between the thick and thin filaments and demonstrate the two different mechanisms by which titin is thought to contribute to active and passive muscle force. Specifically, the model illustrates how titin, a molecule with springlike properties, may increase its stiffness by binding free calcium upon muscle activation and reducing its extensible length by attaching itself to actin, resulting in the greater force-generating capacity after an active than a passive elongation that has been observed experimentally. The model is simple to build and manipulate, and demonstration to high school students was shown to result in positive perception and improved understanding of the otherwise complex titin-related mechanisms of force production in skeletal and cardiac muscles.NEW & NOTEWORTHY Our physical sarcomere model illustrates not only the classic view of muscle contraction, the sliding filament and cross-bridge theories, but also the newly discovered role of titin in force regulation, called the three-filament theory. The model allows for easy visualization of the role of titin in muscle contraction and aids in explaining complex muscle properties that are not captured by the traditional cross-bridge theory.

Assessment of sarcomere shortening and calcium transient in primary human and dog ventricular myocytes.

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca2+ dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach. We performed a study to evaluate primary human CM and have them compared to freshly isolated dog cardiomyocytes for their basic function and responses to positive inotropes with well-known mechanisms. Our data showed that simultaneous assessment of sarcomere shortening and Ca2+-transient can be performed with both myocytes using the IonOptix system. Amplitude of sarcomere shortening and Ca2+-transient (CaT) were significantly higher in dog compared to human CM in the basic condition (absence of treatment), while longer duration of sarcomere shortening and CaT were observed in human cells. We observed that human and dog CMs have similar pharmacological responses to five inotropes with different mechanisms, including dobutamine and isoproterenol (ß-adrenergic stimulation), milrinone (PDE3 inhibition), pimobendan and levosimendan (increase of Ca2+sensitization as well as PDE3 inhibition). In conclusion, our study suggests that myocytes obtained from both human donor hearts and dog hearts can be used to simultaneously assess drug-induced effects on sarcomere shortening and CaT using the IonOptix platform.

Estimation of Forces on Actin Filaments in Living Muscle from X-ray Diffraction Patterns and Mechanical Data.

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.

MyoRobot 2.0: An advanced biomechatronics platform for automated, environmentally controlled skeletal muscle single fiber biomechanics assessment employing inbuilt real-time optical imaging.

We present an enhanced version of our previously engineered MyoRobot system for reliable, versatile and automated investigations of skeletal muscle or linear polymer material (bio)mechanics. That previous version already replaced strenuous manual protocols to characterize muscle biomechanics properties and offered automated data analysis. Here, the system was further improved for precise control over experimental temperature and muscle single fiber sarcomere length. Moreover, it also now features the calculation of fiber cross-sectional area via on-the-fly optical diameter measurements using custom-engineered microscope optics. With this optical systems integration, the MyoRobot 2.0 allows to tailor a wealth of recordings for relevant physiological parameters to be sequentially executed in living single myofibers. Research questions include assessing temperature-dependent performance of active or passive biomechanics, or automated control over length-tension or length-velocity relations. The automatically obtained passive stress-strain relationships and elasticity modules are important parameters in (bio)material science. From the plethora of possible applications, we validated the improved MyoRobot 2.0 by assessing temperature-dependent myofibrillar Ca2+ sensitivity, passive axial compliance and Young's modulus. We report a Ca2+ desensitization and a narrowed dynamic range at higher temperatures in murine M. extensor digitorum longus single fibers. In addition, an increased axial mechanical compliance in single muscle fibers with Young's moduli between 40 - 60 kPa was found, compatible with reported physiological ranges. These applications demonstrate the robustness of our MyoRobot 2.0 for facilitated single muscle fiber biomechanics assessment.

Energy cost of isometric force production after active shortening in skinned muscle fibres.

The steady-state isometric force after active shortening of a skeletal muscle is lower than the purely isometric force at the corresponding length. This property of skeletal muscle is known as force depression. The purpose of this study was to investigate whether the energy cost of force production at the steady state after active shortening was reduced compared with the energy cost of force production for a purely isometric contraction performed at the corresponding length (same length, same activation). Experiments were performed in skinned fibres isolated from rabbit psoas muscle. Skinned fibres were actively shortened from an average sarcomere length of 3.0 µm to an average sarcomere length of 2.4 µm. Purely isometric reference contractions were performed at an average sarcomere length of 2.4 µm. Simultaneously with the force measurements, the ATP cost was measured during the last 30 s of isometric contractions using an enzyme-coupled assay. Stiffness was calculated during a quick stretch-release cycle of 0.2% fibre length performed once the steady state had been reached after active shortening and during the purely isometric reference contractions. Force and stiffness following active shortening were decreased by 10.0±1.8% and 11.0±2.2%, respectively, compared with the isometric reference contractions. Similarly, ATPase activity per second (not normalized to the force) showed a decrease of 15.6±3.0% in the force-depressed state compared with the purely isometric reference state. However, ATPase activity per second per unit of force was similar for the isometric contractions following active shortening (28.7±2.4 mmol l-1 mN-1 s mm3) and the corresponding purely isometric reference contraction (30.9±2.8 mmol l-1 mN-1 s mm3). Furthermore, the reduction in absolute ATPase activity per second was significantly correlated with force depression and stiffness depression. These results are in accordance with the idea that force depression following active shortening is primarily caused by a decrease in the proportion of attached cross-bridges. Furthermore, these findings, along with previously reported results showing a decrease in ATP consumption per unit of force after active muscle stretching, suggest that the mechanisms involved in the steady-state force after active muscle shortening and active muscle lengthening are of distinctly different origin.

Fully automated muscle quality assessment by Gabor filtering of second harmonic generation images.

Although structural changes on the sarcomere level of skeletal muscle are known to occur due to various pathologies, rigorous studies of the reduced sarcomere quality remain scarce. This can possibly be explained by the lack of an objective tool for analyzing and comparing sarcomere images across biological conditions. Recent developments in second harmonic generation (SHG) microscopy and increasing insight into the interpretation of sarcomere SHG intensity profiles have made SHG microscopy a valuable tool to study microstructural properties of sarcomeres. Typically, sarcomere integrity is analyzed by fitting a set of manually selected, one-dimensional SHG intensity profiles with a supramolecular SHG model. To circumvent this tedious manual selection step, we developed a fully automated image analysis procedure to map the sarcomere disorder for the entire image at once. The algorithm relies on a single-frequency wavelet-based Gabor approach and includes a newly developed normalization procedure allowing for unambiguous data interpretation. The method was validated by showing the correlation between the sarcomere disorder, quantified by the M-band size obtained from manually selected profiles, and the normalized Gabor value ranging from 0 to 1 for decreasing disorder. Finally, to elucidate the applicability of our newly developed protocol, Gabor analysis was used to study the effect of experimental autoimmune encephalomyelitis on the sarcomere regularity. We believe that the technique developed in this work holds great promise for high-throughput, unbiased, and automated image analysis to study sarcomere integrity by SHG microscopy.

The cross-bridge dynamics is determined by two length-independent kinetics: Implications on muscle economy and Frank-Starling Law.

The cellular mechanisms underlying the Frank-Starling Law of the heart and the skeletal muscle force-length relationship are not clear. This study tested the effects of sarcomere length (SL) on the average force per cross-bridge and on the rate of cross-bridge cycling in intact rat cardiac trabeculae (n=9). SL was measured by laser diffraction and controlled with a fast servomotor to produce varying initial SLs. Tetanic contractions were induced by addition of cyclopiazonic acid, to maintain a constant activation. Stress decline and redevelopment in response to identical ramp shortenings, starting at various initial SLs, was analyzed. Both stress decline and redevelopment responses revealed two distinct kinetics: a fast and a slower phase. The duration of the rapid phases (4.2 ± 0.1 msec) was SL-independent. The second slower phase depicted a linear dependence of the rate of stress change on the instantaneous stress level. Identical slopes (70.5 ± 1.6 [1/s], p=0.33) were obtained during ramp shortening at all initial SLs, indicating that the force per cross-bridge and cross-bridge cycling kinetics are length-independent. A decrease in the slope at longer SLs was obtained during stress redevelopment, due to internal shortening. The first phase is attributed to rapid changes in the average force per cross-bridge. The second phase is ascribed to both cross-bridge cycling between its strong and weak conformations and to changes in the number of strong cross-bridges. Cross-bridge cycling kinetics and muscle economy are length-independent and the Frank-Starling Law cannot be attributed to changes in the force per cross-bridge or in the single cross-bridge cycling rates.

Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding ß-myosin heavy chain (ß-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 µmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.

Mechanical principles of effects of botulinum toxin on muscle length-force characteristics: an assessment by finite element modeling.

Recent experiments involving muscle force measurements over a range of muscle lengths show that effects of botulinum toxin (BTX) are complex e.g., force reduction varies as a function of muscle length. We hypothesized that altered conditions of sarcomeres within active parts of partially paralyzed muscle is responsible for this effect. Using finite element modeling, the aim was to test this hypothesis and to study principles of how partial activation as a consequence of BTX affects muscle mechanics. In order to model the paralyzing effect of BTX, only 50% of the fascicles (most proximal, or middle, or most distal) of the modeled muscle were activated. For all muscle lengths, a vast majority of sarcomeres of these BTX-cases were at higher lengths than identical sarcomeres of the BTX-free muscle. Due to such "longer sarcomere effect", activated muscle parts show an enhanced potential of active force exertion (up to 14.5%). Therefore, a muscle force reduction originating exclusively from the paralyzed muscle fiber populations, is compromised by the changes of active sarcomeres leading to a smaller net force reduction. Moreover, such "compromise to force reduction" varies as a function of muscle length and is a key determinant of muscle length dependence of force reduction caused by BTX. Due to longer sarcomere effect, muscle optimum length tends to shift to a lower muscle length. Muscle fiber-extracellular matrix interactions occurring via their mutual connections along full peripheral fiber lengths (i.e., myofascial force transmission) are central to these effects. Our results may help improving our understanding of mechanisms of how the toxin secondarily affects the muscle mechanically.

A mathematical model of the mouse ventricular myocyte contraction.

Mathematical models of cardiac function at the cellular level include three major components, such as electrical activity, Ca(2+) dynamics, and cellular shortening. We developed a model for mouse ventricular myocyte contraction which is based on our previously published comprehensive models of action potential and Ca(2+) handling mechanisms. The model was verified with extensive experimental data on mouse myocyte contraction at room temperature. In the model, we implemented variable sarcomere length and indirect modulation of the tropomyosin transition rates by Ca(2+) and troponin. The resulting model described well steady-state force-calcium relationships, dependence of the contraction force on the sarcomere length, time course of the contraction force and myocyte shortening, frequency dependence of the contraction force and cellular contraction, and experimentally measured derivatives of the myocyte length variation. We emphasized the importance of the inclusion of variable sarcomere length into a model for ventricular myocyte contraction. Differences in contraction force and cell shortening for epicardial and endocardial ventricular myocytes were investigated. Model applicability for the experimental studies and model limitations were discussed.

In muscle lengthening surgery multiple aponeurotomy does not improve intended acute effects and may counter-indicate: an assessment by finite element modelling.

The goal was to assess the effects of multiple aponeurotomy on mechanics of muscle with extramuscular myofascial connections. Using finite element modelling, effects of combinations of the intervention carried out at a proximal (P), an intermediate (I) and a distal (D) location were studied: (1) Case P, (2) Case P-I, (3) Case P-D and (4) Case P-I-D. Compared to Case P, the effects of multiple interventions on muscle geometry and sarcomere lengths were sizable for the distal population of muscle fibres: e.g. at high muscle length (1) summed gap lengths between the cut ends of aponeurosis increased by 16, 25 and 27% for Cases P-I, P-D and P-I-D, respectively, (2) characteristic substantial sarcomere shortening became more pronounced (mean shortening was 26, 29, 30 and 31% for Cases P, P-I, P-D and P-I-D, respectively) and (3) fibre stresses decreased (mean stress equalled 0.49, 0.39, 0.38 and 0.33 for Cases P, P-I, P-D and P-I-D, respectively). In contrast, no appreciable effects were shown for the proximal population. The overall change in sarcomere length heterogeneity was limited. Consequently, the effects of multiple aponeurotomy on muscle length-force characteristics were marginal: (1) a limited reduction in active muscle force (maximal 'muscle weakening effect' remained between 5 and 11%) and (2) an even less pronounced change in slack to optimum length range of force exertion (maximal 'muscle lengthening effect' distally was 0.2% for Case P-I-D) were shown. The intended effects of the intervention were dominated by the one intervention carried out closer to the tendon suggesting that aponeurotomies done additionally to that may counter-indicated.

Coupling of adjacent tropomyosins enhances cross-bridge-mediated cooperative activation in a markov model of the cardiac thin filament.

We developed a Markov model of cardiac thin filament activation that accounts for interactions among nearest-neighbor regulatory units (RUs) in a spatially explicit manner. Interactions were assumed to arise from structural coupling of adjacent tropomyosins (Tms), such that Tm shifting within each RU was influenced by the Tm status of its neighbors. Simulations using the model demonstrate that this coupling is sufficient to produce observed cooperativity in both steady-state and dynamic force-Ca(2+) relationships. The model was further validated by comparison with reported responses under various conditions including inhibition of myosin binding and the addition of strong-binding, non-force-producing myosin fragments. The model also reproduced the effects of 2.5 mM added P(i) on Ca(2+)-activated force and the rate of force redevelopment measured in skinned rat myocardial preparations. Model analysis suggests that Tm-Tm coupling potentiates the activating effects of strongly-bound cross-bridges and contributes to force-Ca(2+) dynamics of intact cardiac muscle. The model further predicts that activation at low Ca(2+) concentrations is cooperatively inhibited by nearest neighbors, requiring Ca(2+) binding to >25% of RUs to produce appreciable levels of force. Without excluding other putative cooperative mechanisms, these findings suggest that structural coupling of adjacent Tm molecules contributes to several properties of cardiac myofilament activation.

Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro.

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 mum (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 mum, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 mum, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

Nebulin alters cross-bridge cycling kinetics and increases thin filament activation: a novel mechanism for increasing tension and reducing tension cost.

Nebulin is a giant filamentous F-actin-binding protein ( approximately 800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (-log[Ca(2+)]) and force-ATPase relations, as well as the rate of tension re-development (k(tr)) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons(-1) mm(-1) s(-1)) and reductions in k(tr) (4.7 versus 7.3 s(-1)), calcium sensitivity (pCa(50) 5.74 versus 5.90), and cooperativity of activation (n(H) 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.

Assessment by finite element modeling indicates that surgical intramuscular aponeurotomy performed closer to the tendon enhances intended acute effects in extramuscularly connected muscle.

The effects of location of aponeurotomy on the muscular mechanics of extramuscularly connected muscle were assessed. Using finite element modeling, extensor digitorum longus muscle of the rat was studied for the effects of aponeurotomy performed in each of three locations on the proximal aponeurosis: (1) a proximal location (case P), (2) an intermediate location (case I), and (3) a distal location (case D). Proximo-distal force differences were more pronounced for more proximal aponeurotomy. The location also affected proximally and distally assessed muscle length-force characteristics: (1) Muscle optimum length and active slack length shifted differentially to higher lengths, increasing slack to optimum length range (for D to P: distally by 15-44%; proximally by 2-6%). (2) Muscle forces decreased at all lengths (e.g., for D to P distal optimal force=88-68% and proximal optimal force=87-60% of intact values, respectively). Increased length range and force decreases were highest for case P, as were effects on muscle geometry: gap length within the proximal aponeurosis; decreased proximal fiber population pennation angle. Parallel, but not serial, heterogeneity of sarcomere length was highest in case P: (a) For the distal fiber population, sarcomere shortening was highest; (b) for the proximal population, sarcomeres were longer. It is concluded that if aponeurotomy is performed closer to the tendon, intended surgical effects are more pronounced. For bi-articular muscle, mechanics of both proximal and distal joints will be affected, which should be considered in selecting the location of aponeurotomy for optimal results at both joints.

Extramuscular myofascial force transmission alters substantially the acute effects of surgical aponeurotomy: assessment by finite element modeling.

Effects of extramuscular myofascial force transmission on the acute effects of aponeurotomy were studied using finite element modeling and implications of such effects on surgery were discussed. Aponeurotomized EDL muscle of the rat was modeled in two conditions: (1) fully isolated (2) with intact extramuscular connections. The specific goal was to assess the alterations in muscle length-force characteristics in relation to sarcomere length distributions and to investigate how the mechanical mechanism of the intervention is affected if the muscle is not isolated. Major effects of extramuscular myofascial force transmission were shown on muscle length-force characteristics. In contrast to the identical proximal and distal forces of the aponeurotomized isolated muscle, substantial proximo-distal force differences were shown for aponeurotomized muscle with extramuscular connections (for all muscle lengths F (dist) > F (prox) after distal muscle lengthening). Proximal optimal length did not change whereas distal optimal length was lower (by 0.5 mm). The optimal forces of the aponeurotomized muscle with extramuscular connections exerted at both proximal and distal tendons were lower than that of isolated muscle (by 15 and 7%, respectively). The length of the gap separating the two cut ends of the intervened aponeurosis decreases substantially due to extramuscular myofascial force transmission. The amplitude of the difference in gap length was muscle length dependent (maximally 11.6% of the gap length of the extramuscularly connected muscle). Extramuscular myofascial force transmission has substantial effects on distributions of lengths of sarcomeres within the muscle fiber populations distal and proximal to the location of intervention: (a) Within the distal population, the substantial sarcomere shortening at the proximal ends of muscle fibers due to the intervention remained unaffected however, extramuscular myofascial force transmission caused a more pronounced serial distribution towards the distal ends of muscle fibers. (b) In contrast, extramuscular myofascial force transmission limits the serial distribution of sarcomere lengths shown for the aponeurotomized isolated muscle in the proximal population. Fiber stress distributions showed that extramuscular myofascial force transmission causes most sarcomeres within the aponeurotomized muscle to attain lengths favorable for higher force exertion. It is concluded that acute effects of aponeurotomy on muscular mechanics are affected greatly by extramuscular myofascial force transmission. Such effects have important implications for the outcome of surgery performed to improve impeded function since muscle in vivo is not isolated both anatomically and mechanically.

Restoring force development by titin/connectin and assessment of Ig domain unfolding.

Titin/connectin is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is composed of serially-linked immunoglobulin (Ig)-like domains and several unique sequence elements. Here we address the role of titin/connectin in sarcomeres shortened to below the slack length (length attained by an un-activated cell in absence of external forces). Such shortened cells develop so-called restoring forces that re-extend the cells upon relaxation. The experiments that we present are based on a high throughput method with a rapid solution switching system which allows unattached single cardiac myocytes to be activated (resulting in shortening below the slack length) and then to be rapidly relaxed while their maximal re-lengthening velocity is measured at the sarcomere level (dSL/dtmax), with high-resolution imaging techniques. Experiments were carried out on myocytes that express different isoforms of titin/connectin. We measured the relation between dSL/dtmax and the minimal SL during contraction (SLmin) and determined the slope of this relation as a measure of 'restoring stiffness.' We found that the restoring stiffness correlates with the isoform expression profile with myocytes that express high levels of the stiff isoform (N2B) having the highest restoring stiffness. These results support the notion that titin/connectin is a bi-directional spring that develops passive force when stretched above the slack length and restoring force when shortened to below this length. We also discuss in detail the mechanisms that underlie titin/connectin's restoring force development and focus on whether or not unfolding of Ig domains plays a role.

Effects of inter- and extramuscular myofascial force transmission on adjacent synergistic muscles: assessment by experiments and finite-element modeling.

The effects of inter- and extramuscular myofascial force transmission on muscle length force characteristics were studied in rat. Connective tissues at the bellies of the experimental synergistic muscles of the anterior crural compartment were left intact. Extensor digitorium longus (EDL) muscle was lengthened distally whereas tibialis anterior (TA) and extensor hallucis longus (EHL) were kept at constant muscle-tendon complex length. Substantial differences were found in EDL force measured at the proximal and distal tendons (maximally 46% of the proximal force). EDL with intact inter- as well as extramuscular connections had an increased length range between active slack and optimum length compared to EDL with extramuscular connections exclusively: optimum muscle length was shifted by more than 2 mm. Distal EDL lengthening caused the distal force exerted by TA+EHL complex to decrease (approximately 17% of the initial force). This indicates increased intermuscular myofascial force transmission from TA+EHL muscle complex to EDL muscle. Finite-element modeling showed that: (1) Inter- and extramuscular myofascial force transmission leads to a substantial distribution of the lengths of the sarcomeres arranged in series within muscle fibers. Distribution of stress within the muscle fibers showed that the muscle fiber cannot be considered as a unit exerting equal forces at both ends. (2) Increased heterogeneity of mean fiber sarcomere lengths (i.e., a "parallel" distribution of length of sarcomeres among different muscle fibers) is found, particularly at high muscle lengths. This also explains the shift in muscle optimum length to higher lengths. It is concluded that inter- and extramuscular myofascial force transmission has substantial effects on muscle length-force characteristics.

Molecular basis of passive stress relaxation in human soleus fibers: assessment of the role of immunoglobulin-like domain unfolding.

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as "spacers" that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (> approximately 20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.

Image-analysis-based assessment of the effects of the "Ca2+-jump" technique on sarcomere uniformity.

A new image analysis-based technique was used to quantitatively examine the effects of the "Ca2+-jump" activation protocol on the maintenance of fiber quality in skinned rabbit psoas muscle fiber segments. Specifically, contractions in pCa 4.6 were preceded by short-duration "preactivation" soaks in a solution in which EGTA was replaced with the low-Ca2+ buffering capacity analog hexamethylenediamine-N, N, N', N'-tetraacetate, which facilitated rapid Ca2+ equilibration within the fiber segments. Fiber quality was assessed by examining the Fourier spectra of the muscle fiber images before, during, and after activation. Segment lengths were typically below 500 micrometer, thus allowing the majority of the sarcomeres to be visualized in the field of view (x200 and x400 magnification). The preactivation protocol resulted in less deterioration of fiber quality with repetitive activation. In addition, there was also a significant reduction in the time required to reach the 50% level of maximum tension, with no significant change in the maximum tension level.