Ultrastructural basis of airway smooth muscle contraction

The sliding filament theory of contraction that was developed for striated muscle is generally believed to be also applicable to smooth muscle. However, the well-organized myofilament lattice (i.e., the sarcomeric structure) found in striated muscle has never been clearly delineated in smooth muscle. There is evidence that the myofilament lattice in some smooth muscles, such as airway smooth muscle, is malleable; it can be reshaped to fit a large range of cell dimensions while the maximal overlap between the contractile filaments is maintained. In this review, some early models of the structurally static contractile apparatus of smooth muscle are described. The focus of the review, however, is on the recent findings supporting a model of structurally dynamic contractile apparatus and cytoskeleton for airway smooth muscle. A list of unanswered questions regarding smooth muscle ultrastructure is also proposed in this review, in the hope that it will provide some guidance for future research.     

Models of cardiac electromechanics based on individual hearts imaging data Image-based electromechanical models of the heart

Current multi-scale computational models of ventricular electromechanics describe the full process of cardiac contraction on both the micro- and macro- scales including: the depolarization of cardiac cells, the release of calcium from intracellular stores, tension generation by cardiac myofilaments, and mechanical contraction of the whole heart. Such models are used to reveal basic mechanisms of cardiac contraction as well as the mechanisms of cardiac dysfunction in disease conditions. In this paper, we present a methodology to construct finite element electromechanical models of ventricular contraction with anatomically accurate ventricular geometry based on magnetic resonance and diffusion tensor magnetic resonance imaging of the heart. The electromechanical model couples detailed representations of the cardiac cell membrane, cardiac myofilament dynamics, electrical impulse propagation, ventricular contraction, and circulation to simulate the electrical and mechanical activity of the ventricles. The utility of the model is demonstrated in an example simulation of contraction during sinus rhythm using a model of the normal canine ventricles.     

Geometrical factors influencing muscle force development. I. The effect of filament spacing upon axial forces.

The influence of geometry on the force and stiffness measured during muscle contraction at different sarcomere lengths is examined by using three specific models of muscle cross-bridge geometry which are based upon the double-hinge model of H. E. Huxley (Science [Wash. D.C.]. 1969, 164:1356-1366) extended to three dimensions. The force generated during muscle contraction depends upon the orientation of the individual cross-bridge force vectors and the distribution of the cross-bridges between various states. For the simplest models, in which filament separation has no effect upon cross-bridge distribution, it is shown that changes in force vectors accompanying changes in myofilament separation between sarcomere lengths 2.0 and 3.65 microgram in an intact frog skeletal muscle fiber have only a small effect upon axial force. The simplest models, therefore, produce a total axial force proportional to the overlap between the actin and myosin filaments and independent of filament separation. However, the analysis shows that it is possible to find assumptions that produce a cross-bridge model in which the axial force is not independent of filament spacing. It is also shown that for some modes of attachment of subfragment-1 (S1) to actin the azimuthal location of the actin site is important in determining the axial force. A mode of S1 attachment to actin similar to that deduced by Moore et al. (J. Mol. Biol., 1970, 50:279-294), however, exhibits rather constant cross-bridge behavior over a wide range of actin site location.     

Effect of bronchial smooth muscle contraction on lung compliance.

Lung compliance is generally considered to represent a blend of surface and tissue forces, and changes in compliance in vivo are commonly used to indicate changes in surface forces. There are, however, theoretical arguments that would allow contraction of airway smooth muscle to affect substantially the elasticity of the lung. In the present study we evaluated the role of conducting airway contraction on lung compliance in vivo by infusing methacholine (MCh) at a constant rate into the bronchial circulation. With a steady-state MCh infusion of 2.4 micrograms/min into the bronchial perfusate (perfusate concentration = 0.7 microM), there was an approximate doubling of lung resistance and a 50% fall in dynamic compliance. There were also significant decreases in chord compliance measured from the quasi-static pressure-volume curves and in total lung capacity and residual volume. When the same infusion rate was administered into the pulmonary artery, no changes in lung mechanics were observed. These results indicate that the conducting airways may have a major role in regulating lung elasticity. This linkage between airway contraction and lung compliance may account for the common observation that pharmacological challenges given to the lung usually result in similar changes in lung compliance and airway conductance. Our results also suggest the possibility that the lung tissue resistance, which dominates the measurement of lung resistance in many species, might in fact reflect the physical properties of conducting airways.     

Interfilament spacing is preserved during sarcomere length isometric contractions in rat cardiac trabeculae

It is generally assumed that the myofilament lattice in intact (i.e., nonskinned) striated muscle obeys constant volume. However, whether such is the case during the myocardial contraction is unknown. Accordingly, we measured interfilament spacing by x-ray diffraction in ultra-thin isolated rat right ventricular trabeculae during a short 10 ms shuttered exposure either just before electrical stimulation (diastole), or at the peak of the contraction (systole); sarcomere length (SL) was held constant throughout the contraction using an iterative feedback control system. SL was thus varied in a series of SL-clamped contractions; the relationship between SL and interfilament spacing was not different between diastole and systole within 1%; this was true also over a wide range of inotropic states induced by varied [Ca(2+)](o). We conclude that the cardiac myofilament lattice maintains constant volume, and thus constant interfilament spacing, during contraction.     

Nonlinear Actomyosin Elasticity in Muscle?

Cyclic interactions between myosin II motor domains and actin filaments that are powered by turnover of ATP underlie muscle contraction and have key roles in motility of nonmuscle cells. The elastic characteristics of actin-myosin cross-bridges are central in the force-generating process, and disturbances in these properties may lead to disease. Although the prevailing paradigm is that the cross-bridge elasticity is linear (Hookean), recent single-molecule studies suggest otherwise. Despite convincing evidence for substantial nonlinearity of the cross-bridge elasticity in the single-molecule work, this finding has had limited influence on muscle physiology and physiology of other ordered cellular actin-myosin ensembles. Here, we use a biophysical modeling approach to close the gap between single molecules and physiology. The model is used for analysis of available experimental results in the light of possible nonlinearity of the cross-bridge elasticity. We consider results obtained both under rigor conditions (in the absence of ATP) and during active muscle contraction. Our results suggest that a wide range of experimental findings from mechanical experiments on muscle cells are consistent with nonlinear actin-myosin elasticity similar to that previously found in single molecules. Indeed, the introduction of nonlinear cross-bridge elasticity into the model improves the reproduction of key experimental results and eliminates the need for force dependence of the ATP-induced detachment rate, consistent with observations in other single-molecule studies. The findings have significant implications for the understanding of key features of actin-myosin-based production of force and motion in living cells, particularly in muscle, and for the interpretation of experimental results that rely on stiffness measurements on cells or myofibrils.     

A simple model with myofilament compliance predicts activation-dependent crossbridge kinetics in skinned skeletal fibers

The contribution of thick and thin filaments to skeletal muscle fiber compliance has been shown to be significant. If similar to the compliance of cycling cross-bridges, myofilament compliance could explain the difference in time course of stiffness and force during the rise of tension in a tetanus as well as the difference in Ca(2+) sensitivity of force and stiffness and more rapid phase 2 tension recovery (r) at low Ca(2+) activation. To characterize the contribution of myofilament compliance to sarcomere compliance and isometric force kinetics, the Ca(2+)-activation dependence of sarcomere compliance in single glycerinated rabbit psoas fibers, in the presence of ATP (5.0 mM), was measured using rapid length steps. At steady sarcomere length, the dependence of sarcomere compliance on the level of Ca(2+)-activated force was similar in form to that observed for fibers in rigor where force was varied by changing length. Additionally, the ratio of stiffness/force was elevated at lower force (low [Ca(2+)]) and r was faster, compared with maximum activation. A simple series mechanical model of myofilament and cross-bridge compliance in which only strong cross-bridge binding was activation dependent was used to describe the data. The model fit the data and predicted that the observed activation dependence of r can be explained if myofilament compliance contributes 60-70% of the total fiber compliance, with no requirement that actomyosin kinetics be [Ca(2+)] dependent or that cooperative interactions contribute to strong cross-bridge binding.     

THE RELATIONSHIP BETWEEN MYOFILAMENT PACKING DENSITY AND SARCOMERE LENGTH IN FROG STRIATED MUSCLE

In cross-sections of single fibers from the frog semitendinosus muscle the number of thick myofilaments per unit area (packing density) is a direct function of the sarcomere length. Our data, derived from electron microscopic studies, fit well with other data derived from in vivo, low-angle X-ray diffraction studies of whole semitendinosus muscles. The data are consistent with the assumption that the sarcomere of a fibril maintains a constant volume during changes in sarcomere length. The myofilament lattice, therefore, expands as the sarcomere shortens. Since the distance between adjacent myofilaments is an inverse square root function of sarcomere length, the interaction of the thick and the thin myofilaments during sarcomere shortening may occur over distances which increase 70 A or more. The "expanding-sarcomere, sliding-filament" model of sarcomere shortening is discussed in terms of the current concepts of muscle architecture and contraction.     

Residual force enhancement exceeds the isometric force at optimal sarcomere length for optimized stretch conditions.

Residual force enhancement (FE) following stretch of an activated muscle is a well accepted property of skeletal muscle contraction. However, the mechanism underlying FE remains unknown. A crucial assumption on which some proposed mechanisms are based is the idea that forces in the enhanced state cannot exceed the steady-state isometric force at a sarcomere length associated with optimal myofilament overlap. Although there are a number of studies in which forces in the enhanced state were compared with the corresponding isometric forces on the plateau of the force-length relationship, these studies either did not show enhanced forces above the plateau or, if they did, they lacked measurements of sarcomere lengths confirming the plateau region. Here, we revisited this question by optimizing stretch conditions and measuring the average sarcomere lengths in isolated fibers, and we found that FE exceeded the maximal isometric reference force obtained at the plateau of the force-length relationship consistently (mean+/-SD: 4.8+/-2.1%) and by up to 10%. When subtracting the passive component of FE from the total FE, the enhanced forces remained greater than the isometric plateau force (mean+/-SD: 4.3+/-2.0%). Calcium-induced increases in passive forces, known to be present in single fibers and myofibrils, are too small to account for the FE observed here. We conclude that FE cannot be explained exclusively with a stretch-induced development of sarcomere length nonuniformities, that FE in single fibers may be associated with the recruitment of additional contractile force, and that isometric steady-state forces in the enhanced state are not uniquely determined by sarcomere lengths.     

An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.     

Modelling the mechanical properties of cardiac muscle

A model of passive and active cardiac muscle mechanics is presented, suitable for use in continuum mechanics models of the whole heart. The model is based on an extensive review of experimental data from a variety of preparations (intact trabeculae, skinned fibres and myofibrils) and species (mainly rat and ferret) at temperatures from 20 to 27°C. Experimental tests include isometric tension development, isotonic loading, quick-release/restretch, length step and sinusoidal perturbations. We show that all of these experiments can be interpreted with a four state variable model which includes (i) the passive elasticity of myocardial tissue, (ii) the rapid binding of Ca²⁺ to troponin C and its slower tension-dependent release, (iii) the kinetics of tropomyosin movement and availability of crossbridge binding sites and the length dependence of this process and (iv) the kinetics of crossbridge tension development under perturbations of myofilament length.     

The community effect and ectoderm-mesoderm interaction inXenopus muscle differentiation

Community effects are believed to play an important role in the patterning of many tissues during development. They involve an interaction between neighbouring equivalent cells that is necessary for them to proceed to their fully differentiated state. However, the mechanisms underlying these effects remain unclear. In this paper, diffusion-based mathematical models are constructed and analysed in order to study possible mechanisms for the community effect inXenopus muscle differentiation. These models differ from each other in the assumptions that are made about the nature of an inhibitory effect that ectodermal tissue has been observed to have on muscle differentiation. It is possible to construct consistent models based on all the forms of inhibition considered. However, each model requires the diffusible factors on which it is based to have different properties. The current data from tissues reaggregate experiments are insufficient to determine the mechanisms underlying the community effect; the work presented here suggests that quantitative analysis of a further series of reaggregate experiments will make it possible to distinguish between the proposed mechanisms.     

Redox regulation of diaphragm proteolysis during mechanical ventilation

Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox-sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control, 2) 6 h of MV, 3) 6 h of MV with infusion of the antioxidant Trolox, 4) 18 h of MV, and 5) 18 h of MV with Trolox. Trolox did not attenuate MV-induced increases in diaphragmatic levels of ubiquitin-protein conjugation, polyubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome alpha-subunit 7, 14-kDa E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities in the diaphragm after 18 h of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (mug/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.     

The myosin motor: muscle contraction and in vitro movement

The molecular mechanism of in vitro movement is assumed, by most investigators, to be identical to that of muscle contraction. We discuss this view, which raises various problems. We believe there are mechanisms for muscle contraction (in this case considerable forces are developed, with small displacements) and other mechanisms for in vitro movement (giving large displacements, without necessarily generating substantial forces). Hybrid models may explain muscle contraction. The traditional swinging-crossbridge model may explain in vitro movement. For muscle contraction, movement may result partly from the swinging-crossbridge mechanism and partly from other factors. Comparisons of different fibres at different moments of the Mg-ATPase cycle suggest that both the value of the isometric force in muscle and in vitro and that of the Mg-ATPase activity used in vitro need to be reconsidered. The recently reported dependence of the isometric active tension of smooth skinned fibres on temperature appears to be weaker than predicted by the swinging-crossbridge theory alone. This recent observation is compatible with the existence of other forces (electrostatic repulsions) decreasing with temperature as has been known for some years. From recent experimental data, we think the biochemistry of myosin and actomyosin should be reassessed, to try to find new details of the mechanisms of muscle contraction and in vitro motility.     

Intramolecular interactions in the N-domain of cardiac troponin C are important determinants of calcium sensitivity of force development

Myocardial contraction is initiated when Ca2+ binds to site II of cardiac troponin C. This 12-residue EF-hand loop (NH2-DEDGSGTVDFDE-COOH) contains six residues (bold) that coordinate Ca2+ binding and six residues that do not appear to influence Ca2+ binding directly. We have introduced six single-cysteine substitutions (italics) within site II of cTnC to investigate whether these residues are essential for Ca2+ binding affinity in isolation and Ca2+ sensitivity of force development in single muscle fibers. Ca2+ binding properties of mutant proteins were examined in solution and after substitution into rat skinned soleus fibers. Except for the serine mutation, cysteine substitution had no effect on Ca2+ binding on cTnC in solution. However, as part of the myofilament, the threonine mutation reduced Ca2+ sensitivity while the phenylalanine mutation increased Ca2+ sensitivity. Analysis of the available crystal and NMR structures reveals specific structural mechanisms for these effects.     

Comparison of the compliance of resting and contracting muscle]

Frog sartorius muscles are stretched at rest and during maximal tetanic contractions. Parallel compliance decreases when the length increases. The relationship between compliance and length is linear in double-logarithmic scale. The compliance of the active muscle (tetanic contraction) is not related to the length. The series-compliance is calculated from the parallel compliance and the active one. It increases with the length of muscle. These results are discussed on the basis of the sliding-filaments theory.     

In vivo modular control analysis of energy metabolism in contracting skeletal muscle

We used (31)P MRS (magnetic resonance spectroscopy) measurements of energetic intermediates [ATP, P(i) and PCr (phosphocreatine)] in combination with the analytical tools of metabolic control analysis to study in vivo energy metabolism in the contracting skeletal muscle of anaesthetized rats over a broad range of workload. According to our recent MoCA (modular control analysis) used to describe regulatory mechanisms in beating heart, we defined the energetic system of muscle contraction as two modules (PCr-Producer and PCr-Consumer) connected by the energetic intermediates. Hypoxia and electrical stimulation were used in this in vivo study as reasonably selective modulations of Producer and Consumer respectively. As quantified by elasticity coefficients, the sensitivities of each module to PCr determine the control of steady-state contractile activity and metabolite concentrations. The magnitude of the elasticity of the producer was high (4.3+/-0.6) at low workloads and decreased 5-fold (to 0.9+/-0.2) at high workloads. By contrast, the elasticity of the consumer remained low (0.5-1.2) over the range of metabolic rates studied. The control exerted by each module over contraction was calculated from these elasticities. The control of contraction was found on the consumer at low workloads and then swung to the producer, due to the workload-dependent decrease in the elasticity of producer. The workload-dependent elasticity and control pattern of energy production in muscle is a major difference from heart. Since module rate and elasticity depend on the concentrations of substrates and products, the absence of homoeostasis of the energetic intermediates in muscle, by contrast with heart, is probably the origin of the workload-dependent elasticity of the producer module.     

Energetic consequences of mechanical loads

In this brief review, we have focussed largely on the well-established, but essentially phenomenological, linear relationship between the energy expenditure of the heart (commonly assessed as the oxygen consumed per beat, oxygen consumption (VO2)) and the pressure-volume-area (PVA, the sum of pressure-volume work and a specified 'potential energy' term). We raise concerns regarding the propriety of ignoring work done during 'passive' ventricular enlargement during diastole as well as the work done against series elasticity during systole. We question the common assumption that the rate of basal metabolism is independent of ventricular volume, given the equally well-established Feng- or stretch-effect. Admittedly, each of these issues is more of conceptual than of quantitative import. We point out that the linearity of the enthalpy-PVA relation is now so well established that observed deviations from linearity are often ignored. Given that a one-dimensional equivalent of the linear VO2-PVA relation exists in papillary muscles, it seems clear that the phenomenon arises at the cellular level, rather than being a property of the intact heart. This leads us to discussion of the classes of crossbridge models that can be applied to the study of cardiac energetics. An admittedly superficial examination of the historical role played by Hooke's Law in theories of muscle contraction foreshadows deeper consideration of the thermodynamic constraints that must, in our opinion, guide the development of any mathematical model. We conclude that a satisfying understanding of the origin of the enthalpy-PVA relation awaits the development of such a model.     

HDAC4 and PCAF bind to cardiac sarcomeres and play a role in regulating myofilament contractile activity

Reversible acetylation of lysine residues within a protein is considered a biologically relevant modification that rivals phosphorylation ( Kouzarides, T. (2000) EMBO J. 19, 1176-1179 ). The enzymes responsible for such protein modification are called histone acetyltransferases (HATs) and deacetylases (HDACs). A role of protein phosphorylation in regulating muscle contraction is well established ( Solaro, R. J., Moir, A. J., and Perry, S. V. (1976) Nature 262, 615-617 ). Here we show that reversible protein acetylation carried out by HATs and HDACs also plays a role in regulating the myofilament contractile activity. We found that a Class II HDAC, HDAC4, and an HAT, PCAF, associate with cardiac myofilaments. Primary cultures of cardiomyocytes as well as mouse heart sections examined by immunohistochemical and electron microscopic analyses revealed that both HDAC4 and PCAF associate with the Z-disc and I- and A-bands of cardiac sarcomeres. Increased acetylation of sarcomeric proteins by HDAC inhibition (using class I and II HDAC inhibitors or anti-HDAC4 antibody) enhanced the myofilament calcium sensitivity. We identified the Z-disc-associated protein, MLP, a sensor of cardiac mechanical stretch, as an acetylated target of PCAF and HDAC4. We also show that trichostatin-A, a class I and II HDAC inhibitor, increases myofilament calcium sensitivity of wild-type, but not of MLP knock-out mice, thus demonstrating a role of MLP in acetylation-dependent increased contractile activity of myofilaments. These studies provide the first evidence that HATs and HDACs play a role in regulation of muscle contraction.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.