Titin/connectin-based modulation of the Frank-Starling mechanism of the heart

The basis of the Frank-Starling mechanism of the heart is the increase in active force when muscle is stretched. Various findings have shown that muscle length, i.e., sarcomere length (SL), modulates activation of cardiac myofilaments at a given concentration of Ca²⁺ ([Ca²⁺]). This augmented Ca²⁺ activation with SL, commonly known as “length-dependent activation”, is manifested as the leftward shift of the force-pCa (=−log [Ca²⁺]) relation as well as by the increase in maximal Ca²⁺-activated force. Despite the numerous studies that have been undertaken, the molecular mechanism(s) of length-dependent activation is (are) still not fully understood. The giant sarcomere protein titin/connectin is the largest protein known to date. Titin/connectin is responsible for most passive force in vertebrate striated muscle and also functions as a molecular scaffold during myofibrillogenesis. Recent studies suggest that titin/connectin plays an important role in length-dependent activation by sensing stretch and promoting actomyosin interaction. Here we review and extend this previous work and focus on the mechanism by which titin/connectin might modulate actomyosin interaction.     

Titin-based modulation of active tension and interfilament lattice spacing in skinned rat cardiac muscle

The effect of titin-based passive tension on Ca²⁺ sensitivity of active tension and interfilament lattice spacing was studied in skinned rat ventricular trabeculae by measuring the sarcomere length (SL)-dependent change in Ca²⁺ sensitivity and performing small angle X-ray diffraction studies. To vary passive tension, preparations were treated with trypsin at a low concentration (0.31 g/ml) for a short period (13 min) at 20°C, that resulted in ~40% degradation of the I-band region of titin, with a minimal effect on A-band titin. We found that the effect of trypsin on titin-based passive tension was significantly more pronounced immediately after stretch than at steady state, 30 min after stretch (i.e., trypsin has a greater effect on viscosity than on elasticity of passive cardiac muscle). Ca²⁺ sensitivity was decreased by trypsin treatment at SL 2.25 m, but not at SL 1.9 m, resulting in marked attenuation of the SL-dependent increase in Ca²⁺ sensitivity. The SL-dependent change in Ca²⁺ sensitivity was significantly correlated with titin-based passive tension. Small-angle X-ray diffraction experiments revealed that the lattice spacing expands after trypsin treatment, especially at SL 2.25 m, providing an inverse linear relationship between the lattice spacing and Ca²⁺ sensitivity. These results support the view that titin-based passive tension promotes actomyosin interaction and that the mechanism includes interfilament lattice spacing modulation.     

Interfilament spacing, Ca2+ sensitivity, and Ca2+ binding in skinned bovine cardiac muscle

The length-dependence of myofilament Ca²⁺ sensitivity in cardiac muscle appears to be a function of length-dependent variation in the lateral separation of actin and myosin filaments. The goal of this study was to determine how force, Ca²⁺ sensitivity, and Ca²⁺ binding to troponin C are correlated in skinned bovine ventricular muscle bundles set at sarcomere length 1.9 m and subjected to varying degrees of osmotic compression with Dextran T-500. With 5, 10, and 15% Dextran T-500 the muscle diameter was reduced by 13, 21, and 25%, respectively. Addition of 5% Dextran T-500 caused increases in developed force, Ca²⁺ sensitivity, and in the affinity of Ca²⁺ for the regulatory binding site on troponin C. All of these parameters were reversed back toward control levels with 10% Dextran T-500. With 15% Dextran T-500 all parameters were decreased to below control levels. These data indicate that (1) there is an optimal filament separation at which both Ca²⁺ sensitivity and Ca²⁺ binding are maximized, and (2) Ca²⁺ –troponin C affinity is linked to changes in Ca²⁺ sensitivity rather than to changes in interfilament spacing.     

Spectrofluorometric analysis of length-dependent conformational changes in cardiac troponin C

Length modulation of cardiac muscle is manifested in the Frank–Starling relation of the heart. Recently, it has been shown that length-dependent changes in SH reactivity of cardiac troponin C (cTnC) occurred in association with cross-bridge attachment and Ca²⁺. However, the presence of two SH groups (Cys-35 and Cys-84) in the regulatory region of cTnC complicates efforts to detect conformational changes. In this study skinned porcine cardiac fibers were reacted with 7-diethylamino-3-[4′maleimidylphenyl]-4-methylcoumarin (CPM). Alkaline urea gel electrophoresis, along with protein elution, was used to isolate filament bound cTnC. Analysis of fluorescence measurement showed that there is a Ca²⁺-increased fluorescence for CPM-labeled cTnC in long fibers (sarcomere length = 2.2 ∼ 2.5 μm) but not in short fibers (sarcomere length = 1.6 ∼ 1.8 μm). In addition, the labeled cTnC was measured for the fluorescence decrease over time by adding a non-fluorescence energy acceptor, 4-dimethylaminophenylazophenyl-4′maleimide (DABMI), in the presence and absence of Ca²⁺. Fluorescence quenching by DABMI is not affected by Ca²⁺ in long fibers but it is significantly increased in short fibers. However, the fibers maintained in the relaxed state with 5 mM MgATP and 1 mM Vanadate showed no length effect on the CPM-labeled cTnC in terms of the Ca²⁺-mediated changes in fluorescence spectrum and in fluorescence quenching by DABMI. All together, our results suggest that the relative reactivities of Cys-35 and Cys-84 vary with sarcomere length. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sarcomere length-dependent Ca<Superscript>2+</Superscript> activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca²⁺ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca²⁺ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca²⁺ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca²⁺ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca²⁺ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca²⁺ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

Inhibitory mechanisms for cross-bridge cycling: the nitric oxide-cGMP signal transduction pathway in smooth muscle relaxation

Relaxation follows sequestration of Ca²⁺ mobilized by an excitatory stimulus in striated muscle. Removal of excitatory stimuli also relaxes smooth muscle in vitro after reductions in the myoplasmic [Ca²⁺] and dephosphorylation of the myosin regulatory light chains. However, there are several experimental procedures that produce relaxation in the presence of excitatory stimuli and elevated Ca²⁺-dependent cross-bridge phosphorylation. Of potential widespread physiological importance are treatments that increase myoplasmic [cGMP] owing to the ubiquity of nitric oxide (NO) as a signalling molecule for endothelial-mediated vasodilation and inhibitory nerves in most types of smooth muscle. Several mechanisms are implicated in the NO-cGMP mediated relaxation. Most studies support reductions in myoplasmic Ca²⁺. However, there is evidence that increases in cGMP also lower the Ca²⁺-sensitivity of cross-bridge phosphorylation. This would contribute to a decline in force through actions on the myosin light chain kinase/phosphatase system. In addition, changes in the dependence of force on phosphorylation are observed in tissues partially relaxed by treatments that elevate cGMP. This demonstrates that either the attachment and cycling of phosphorylated cross-bridges is impaired or blocked, or that the formation of dephosphorylated, force-generating cross-bridges (‘latch-bridges’) is reduced. Protein kinase G-catalysed phosphorylation of either a thin filament protein that blocks attachment of cross-bridges or a protein that inhibits myosin light chain phosphatase may explain the NO-induced relaxation with elevated cross-bridge phosphorylation.     

Non-crossbridge calcium-dependent stiffness in slow and fast skeletal fibres from mouse muscle

We showed previously that force development in frog and FDB mouse skeletal muscle fibres is preceded by an increase of fibre stiffness occurring well before crossbridge attachment and force generation. This stiffness increase, referred to as static stiffness, is due to a Ca²⁺-dependent stiffening of a non-crossbridge sarcomere structure which we suggested could be attributed to the titin filaments. To investigate further the role of titin in static stiffness, we measured static stiffness properties at 24 and 35°C in soleus and EDL mouse muscle fibres which are known to express different titin isoforms. We found that static stiffness was present in both soleus and EDL fibres, however, its value was about five times greater in EDL than in soleus fibres. The rate of development of static stiffness on stimulation increased with temperature and was slightly faster in EDL than in soleus in agreement with previously published data on the time course of the intracellular Ca²⁺ transients in these muscles. The present results show that the presence of a non-crossbridge Ca²⁺-dependent stiffening of the muscle fibre is a physiological general characteristic of skeletal muscle. Static stiffness depends on fibre type, being greater and developing faster in fast than in slow fibres. Our observations are consistent with the idea that titin stiffening on contraction improves the sarcomere structure stability. Such an action in fact seems to be more important in EDL fast fibre than in soleus slow fibres.     

Titin Isoform Variance and Length Dependence of Activation in Skinned Bovine Cardiac Muscle

We have explored the role of the giant elastic protein titin in the Frank-Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin-based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform, and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms. Passive tension was also varied in each muscle type by manipulating the pre-history of stretch prior to activation. We found that the SL-dependent increases in Ca²⁺ sensitivity and maximal Ca²⁺-activated tension were markedly more pronounced when titin-based passive tension was high. Small-angle X-ray diffraction experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA and that the lattice spacing is coupled with titin-based passive tension. These results support the notion that titin-based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank-Starling mechanism of the heart by promoting actomyosin interaction in response to stretch.     

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca²⁺ via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca²⁺-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca²⁺ regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca²⁺]. Among the processes studied on myofibrils are: (1) the Ca²⁺-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.     

Calcium-dependent titin–thin filament interactions in muscle: observations and theory

Gaps in our understanding of muscle mechanics demonstrate that the current model is incomplete. Increasingly, it appears that a role for titin in active muscle contraction might help to fill these gaps. While such a role for titin is increasingly accepted, the underlying molecular mechanisms remain unclear. The goals of this paper are to review recent studies demonstrating Ca²⁺-dependent interactions between N2A titin and actin in vitro, to explore theoretical predictions of muscle behavior based on this interaction, and to review experimental data related to the predictions. In a recent study, we demonstrated that Ca²⁺ increases the association constant between N2A titin and F-actin; that Ca²⁺ increases rupture forces between N2A titin and F-actin; and that Ca²⁺ and N2A titin reduce sliding velocity of F-actin and reconstituted thin filaments in motility assays. Preliminary data support a role for Ig83, but other Ig domains in the N2A region may also be involved. Two mechanical consequences are inescapable if N2A titin binds to thin filaments in active muscle sarcomeres: (1) the length of titin’s freely extensible I-band should decrease upon muscle activation; and (2) binding between N2A titin and thin filaments should increase titin stiffness in active muscle. Experimental observations demonstrate that these properties characterize wild type muscles, but not muscles from mdm mice with a small deletion in N2A titin, including part of Ig83. Given the new in vitro evidence for Ca²⁺-dependent binding between N2A titin and actin, it is time for skepticism to give way to further investigation.

     

Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch

The present study examined the contribution of myofilament contractile proteins to regional function in guinea pig myocardium. We investigated the effect of stretch on myofilament contractile proteins, Ca²⁺ sensitivity, and cross-bridge cycling kinetics (K _tr) of force in single skinned cardiomyocytes isolated from the sub-endocardial (ENDO) or sub-epicardial (EPI) layer. As observed in other species, ENDO cells were stiffer, and Ca²⁺ sensitivity of force at long sarcomere length was higher compared with EPI cells. Maximal K _tr was unchanged by stretch, but was higher in EPI cells possibly due to a higher α-MHC content. Submaximal Ca²⁺-activated K _tr increased only in ENDO cells with stretch. Stretch of skinned ENDO muscle strips induced increased phosphorylation in both myosin-binding protein C and myosin light chain 2. We concluded that transmural MHC isoform expression and differential regulatory protein phosphorylation by stretch contributes to regional differences in stretch modulation of activation in guinea pig left ventricle.     

Calcium modulates the influence of length changes on the myofibrillar adenosine triphosphatase activity in rat skinned cardiac trabeculae

The relationship between adenosine triphosphate (ATP) turnover and muscle performance was investigated in skinned cardiac trabeculae of the rat at different [Ca²⁺] and two different sarcomere lengths (1.8 m and 2.2 m) at 20°C. ATP turnover was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of reduced nicotineamide adenine dinucleotide. The trabeculae were studied under isometric conditions and when the length was altered repetitively at a frequency of 23 Hz, with a square wave, by 5% of the initial length. The isometric ATPase activity amounted to 0.48 mM/s. Isometric ATP turnover and force were proportional at different [Ca²⁺]. During length changes at maximal activation (pCa 4.27) and 2.2 m sarcomere length, ATPase activity increased to up to 162% whereas at low [Ca²⁺], ATPase activity decreased with respect to the isometric value at that pCa. At pCa 5.5, ATPase activity was reduced to 33%. These results indicate that during the length changes the apparent cross-bridge detachment rate is increased and the apparent attachment rate is decreased. The findings suggest that the Fenn effect, i. e. the increase in energy turnover above the isometric value during shortening, is present in cardiac trabeculae at high levels of activation, but is absent or reversed at lower levels of activity.     

Characterizations of myosin essential light chain’s N-terminal truncation mutant Δ43 in transgenic mouse papillary muscles by using tension transients in response to sinusoidal length alterations

Cross-bridge kinetics were studied at 20 °C in cardiac muscle strips from transgenic (Tg) mice expressing N-terminal 43 amino acid truncation mutation (Δ43) of myosin essential light chain (ELC), and the results were compared to those from Tg-wild type (WT) mice. Sinusoidal length changes were applied to activated skinned papillary muscle strips to induce tension transients, from which two exponential processes were deduced to characterize the cross-bridge kinetics. Their two rate constants were studied as functions of ATP, phosphate (Pi), ADP, and Ca²⁺ concentrations to characterize elementary steps of the cross-bridge cycle consisting of six states. Our results demonstrate for the first time that the cross-bridge kinetics of Δ43 are accelerated owing to an acceleration of the rate constant k ₂ of the cross-bridge detachment step, and that the number of strongly attached cross-bridges are decreased because of a reduction of the equilibrium constant K ₄ of the force generation step. The isometric tension and stiffness of Δ43 are diminished compared to WT, but the force per cross-bridge is not changed. Stiffness measurement during rigor induction demonstrates a reduction in the stiffness in Δ43, indicating that the N-terminal extension of ELC forms an extra linkage between the myosin cross-bridge and actin. The tension-pCa study demonstrates that there is no Ca²⁺ sensitivity change with Δ43, but the cooperativity is diminished. These results demonstrate the importance of the N-terminal extension of ELC in maintaining the myosin motor function during force generation and optimal cardiac performance.     

Energy turnover for Ca2+ cycling in skeletal muscle

The majority of energy consumed by contracting muscle can be accounted for by two ATP-dependent processes, cross-bridge cycling and Ca²⁺ cycling. The energy for Ca²⁺ cycling is necessary for contraction but is an overhead cost, energy that cannot be converted into mechanical work. Measurement of the energy used for Ca²⁺ cycling also provides a means of determining the total Ca²⁺ released from the sarcoplasmic reticulum into the sarcoplasm during a contraction. To make such a measurement requires a method to selectively inhibit cross-bridge cycling without altering Ca²⁺ cycling. In this review, we provide a critical analysis of the methods used to partition skeletal muscle energy consumption between cross-bridge and non-cross-bridge processes and present a summary of data for a wide range of skeletal muscles. It is striking that the cost of Ca²⁺ cycling is almost the same, 30–40% of the total cost of isometric contraction, for most muscles studied despite differences in muscle contractile properties, experimental conditions, techniques used to measure energy cost and to partition energy use and in absolute rates of energy use. This fraction increases with temperature for amphibian or fish muscle. Fewer data are available for mammalian muscle but most values are similar to those for amphibian muscle. For mammalian muscles there are no obvious effects of animal size, muscle fibre type or temperature.     

Tension generation and relaxation in single myofibrils from human atrial and ventricular myocardium

Fast solution switching techniques in single myofibrils offer the opportunity to dissect and directly examine the sarcomeric mechanisms responsible for force generation and relaxation. The feasibility of this approach is tested here in human cardiac myofibrils isolated from small samples of atrial and ventricular tissue. At sarcomere lengths between 2.0 and 2.3 μm, resting tensions were significantly higher in ventricular than in atrial myofibrils. The rate constant of active tension generation after maximal Ca²⁺ activation (k _ACT) was markedly faster in atrial than in ventricular myofibrils. In both myofibril types k _ACT was the same as the rate of tension redevelopment after mechanical perturbations and decreased significantly by decreasing [Ca²⁺] in the activating solution. Upon sudden Ca²⁺ removal, active tension fully relaxed. Relaxation kinetics were (1) much faster in atrial than in ventricular myofibrils, (2) unaffected by bepridil, a drug that increases the affinity of troponin for Ca²⁺, and (3) strongly accelerated by small increases in inorganic phosphate concentration. The results indicate that myofibril tension activation and relaxation rates reflect apparent cross-bridge kinetics and their Ca²⁺ regulation rather than the rates at which thin filaments are switched on or off by Ca²⁺ binding or removal. Myofibrils from human hearts retain intact mechanisms for contraction regulation and tension generation and represent a viable experimental model to investigate function and dysfunction of human cardiac sarcomeres.     

Mechanism of cross-bridge detachment in isometric force relaxation of skeletal and cardiac myofibrils

Skeletal and cardiac muscle relaxation is governed by the interplay between two macromolecular systems: (i) membrane bound Ca²⁺ transport proteins and (ii) sarcomeric proteins. Photolysis experiments in skinned muscle preparations and fast solution switching studies in single myofibrils offer means for isolating sarcomeric mechanisms of relaxation from those related to myoplasmic Ca²⁺ removal. Single myofibril experiments have recently shown that cross-bridge mechanics and detachment kinetics are the major determinants of the time course of relaxation. Full force decay in myofibrils occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere ‘give’ and dominated by inter-sarcomere dynamics while the former occurs under nearly isometric conditions. Strong evidence has been found that the slow rate of force decay in myofibril relaxation reflects the rate at which cross-bridges leave force-generating states under isometric conditions. Dissection of chemo-mechanical transduction process in myofibrils indicate that both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca²⁺] ([Ca²⁺]_i) due to Ca²⁺ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca²⁺]_i elevation during stimulation depends on: (a) the rate of Ca²⁺ release from the SR, (b) the rate of Ca²⁺ uptake by the SR, and (c) the myoplasmic Ca²⁺ buffering. We have used fluorescent Ca²⁺ indicators to measure [Ca²⁺]_i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca²⁺]_i: β-adrenergic stimulation, which potentiates the SR Ca²⁺ release; application of 2,5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca²⁺ pumps; application of caffeine, which facilitates SR Ca²⁺ release and inhibits SR Ca²⁺ uptake; early fatigue, where the rate of SR Ca²⁺ uptake is reduced; acidosis, which reduces both the myoplasmic Ca²⁺ buffering and the rate of SR Ca²⁺ uptake. Reduced tetanic [Ca²⁺]_i was observed in late fatigue, due to reduced SR Ca²⁺ release, and in alkalosis, due to increased myoplasmic Ca²⁺ buffering. Force is monotonically related to [Ca²⁺]_i, but depends also on the myofibrillar Ca²⁺ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca²⁺]_i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca²⁺ sensitivity and maximum cross-bridge force.     

Cardiac Mechanoenergetics Replicated by Cross-Bridge Model

The aim of this work is to reproduce the experimentally measured linear dependence of cardiac muscle oxygen consumption on stress–strain area using a model, composed of a three-state Huxley-type model for cross-bridge interaction and a phenomenological model of Ca²⁺-induced activation. By selecting particular cross-bridge cycling rate constants and modifying the cross-bridge activation model, we replicated the linear dependence between oxygen consumption and stress–strain area together with other important mechanical properties of cardiac muscle such as developed stress dependence on the sarcomere length and force-velocity relationship. The model predicts that (1) the amount of the passenger cross bridges, i.e., cross bridges that detach without hydrolyzing ATP molecule, is relatively small and (2) ATP consumption rate profile within a beat and the amount of the passenger cross bridges depend on the contraction protocol. © 2000 Biomedical Engineering Society. PAC00: 8719Rr, 8719Ff, 8710+e, 8719Hh     

Mechanical properties of sarcomeres during cardiac myofibrillar relaxation: stretch-induced cross-bridge detachment contributes to early diastolic filling

Sudden Ca²⁺ removal from isometrically contracting cardiac myofibrils induces a biphasic relaxation: first a slow, linear force decline during which sarcomeres remain isometric and then a rapid, exponential decay originating from sequential lengthening, i.e., successive mechanical relaxation, of individual sarcomeres (Stehle et al. 2002; Biophys J 83:2152–2162). Step-stretches were applied to the myofibrils, in order to study the mechanical properties of sarcomeres during this dynamic relaxation process. Stretch applied soon (∼10 ms) after Ca²⁺ removal accelerated the initiation of the rapid, exponential force decay and of the sequential sarcomere lengthening. After the stretch, a short, transient period (∼24 ms) remained, during which time force was enhanced and sarcomeres were homogenously elongated by the stretch. This period was similar to the duration of the switching-off of troponin C in myofibrils, as measured by stopped-flow. In contrast, when the stretch was applied during the rapid, exponential relaxation phase, force quickly decayed after stretch, back to the force level of isometric controls or even lower. Smaller stretches lengthened only those sarcomeres that were located at the wave front of the sequential sarcomere relaxation. The more the stretch-size was increased, the more of the contracting sarcomeres became lengthened by the stretch; those sarcomeres that were relaxed prior to stretch were barely elongated. These results indicate that the stretch accelerates myofibrillar relaxation by forcing the cross-bridges in contracting sarcomeres to detach. Subsequent rapid cross-bridge reattachment occurs during a short period after Ca²⁺ removal until troponin C is switched off. However, this switch off occurs ∼5 times too fast to directly rate-limit the force relaxation under the isometric condition. After troponin C is switched off, stretching induces cross-bridge detachment without subsequent reattachment, and force rapidly decays below the isometric level. This may explain the rapid distention of the ventricular myocardium during early diastolic filling.