Cross-bridge Inhibition Research Articles

Work and oxygen consumption of isolated right ventricular papillary muscle in experimental pulmonary hypertension.

Right-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis tested was that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular muscle preparations were subjected to 5Hz sinusoidal length changes at 37°C. Work and suprabasal oxygen consumption ( ) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane and monoamine oxidase and glucose 6-phosphate dehydrogenase activities and phosphatidylglycerol/cardiolipin contents were determined. Mechanical efficiency ranged from 23% to 11% in control (n=6) and from 22% to 1% in MCT-PH (n=15) and correlated with work (r2 =0.68, P<0.0001) but not with (r2 =0.004, P=0.7919). for cross-bridge cycling was proportional to work (r2 =0.56, P=0.0005). Blebbistatin-resistant (r2 =0.32, P=0.0167) and proton permeability of the mitochondrial inner membrane (r2 =0.36, P=0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination r2 =0.79, P=0.0001). Cytosolic cytochrome c correlated inversely with work (r2 =0.28, P=0.0391), but not with efficiency (r2 =0.20, P=0.0867). Glucose 6-phosphate dehydrogenase, monoamine oxidase and phosphatidylglycerol/cardiolipin increased in the right ventricular wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is a result of activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant are discussed. KEY POINTS: Mechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown. Work and oxygen consumption are determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements. Cytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension. Reduced work and mechanical efficiency are related to mitochondrial dysfunction. Upregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is a resiult of the excessive production of reactive oxygen species.

Loss of crossbridge inhibition drives pathological cardiac hypertrophy in patients harboring the TPM1 E192K mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca2+ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin-myosin crossbridge activity at low Ca2+. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca2+ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin-specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.

The Origin of the Force Increase Observed after Active Stretch Beyond Myofilament Overlap in Single Muscle Fibers

At long sarcomere lengths (SLs) beyond myofilament overlap, only passive forces, produced by passive structural elements, are possible. However, Leonard et al. (2010) showed a dramatic increase in force above the passive force when myofibrils were actively stretched beyond actin and myosin filament overlap. It has been suggested that titin's stiffness might increase, or characteristic length decrease, with activation resulting in the increase in force observed when muscles are actively stretched compared to when they are passively stretched. Our purpose was to investigate whether the increase in force after active stretch beyond overlap was caused by a passive component/titin independently of the actin and myosin cross bridges. Therefore muscle fibres were actively stretched to a SL beyond myofilament overlap (5.0 μm) and then deactivated or treated with a cross bridge inhibitor, 2,3-butanedione monoxime (BDM). Fibres were also passively stretched to a SL of 5.0 μm. Results show similar decrease in force, of about 50%, after deactivation and treatment with a BDM solution. However, the forces after deactivation and treatment with BDM are higher than the purely passive stretch force at an average SL of 5.0 μm. The decrease in force after deactivation may be explained assuming that titin is an “activable/deactivable” spring whose stiffness is modulated by activation. However, the decrease in force following BDM exposure cannot be reconciled with a titin based mechanism only. BDM is known to inhibit cross bridge-based force, and thus the decrease in force after BDM exposure is likely associated with cross bridge inhibition. In conclusion, the force increase observed after active stretch beyond myofilament overlap is likely associated with cross-bridge and titin forces.

Force reduction induced by unidirectional transversal muscle loading is independent of local pressure

Transversal unidirectional compression applied to muscles via external loading affects muscle contraction dynamics in the longitudinal direction. A recent study reported decreasing longitudinal muscle forces with increasing transversal load applied with a constant contact area (i.e., leading to a simultaneous increase in local pressure). To shed light on these results, we examine whether the decrease in longitudinal force depends on the load, the local pressure, or both. To this end, we perform isometric experiments on rat M. gastrocnemius medialis without and with transversal loading (i) changing the local pressure from 1.1–3.2Ncm−2 (n=9) at a constant transversal load (1.62N) and (ii) increasing the transversal load (1.15–3.45N) at a constant local pressure of 2.3Ncm−2 (n=7). While we did not note changes in the decrease in longitudinal muscle force in the first experiment, the second experiment resulted in an almost-linear reduction of longitudinal force between 7.5±0.6% and 14.1±1.7%. We conclude that the observed longitudinal force reduction is not induced by local effects such as malfunction of single muscle compartments, but that similar internal stress conditions and myofilament configurations occur when the local pressure changes given a constant load. The decreased longitudinal force may be explained by increased internal pressure and a deformed myofilament lattice that is likely associated with the decomposition of cross-bridge forces on the one hand and the inhibition of cross-bridges on the other hand.

Residual force depression in single sarcomeres is abolished by MgADP-induced activation.

The mechanisms behind the shortening-induced force depression commonly observed in skeletal muscles remain unclear, but have been associated with sarcomere length non-uniformity and/or crossbridge inhibition. The purpose of this study was twofold: (i) to evaluate if force depression is present in isolated single sarcomeres, a preparation that eliminates sarcomere length non-uniformities and (ii) to evaluate if force depression is inhibited when single sarcomeres are activated with MgADP, which biases crossbridges into a strongly-bound state. Single sarcomeres (n = 16) were isolated from rabbit psoas myofibrils using two micro-needles (one compliant, one rigid), piercing the sarcomere externally adjacent to the Z-lines. The sarcomeres were contracted isometrically and subsequently shortened, in both Ca2+- and MgADP-activating solutions. Shortening in Ca2+-activated samples resulted in a 27.44 ± 9.04% force depression when compared to isometric contractions produced at similar final sarcomere lengths (P < 0.001). There was no force depression in MgADP-activated sarcomeres (force depression = −1.79 ± 9.69%, P = 0.435). These results suggest that force depression is a sarcomeric property, and that is associated with an inhibition of myosin-actin interactions.

Shortening-Induced Force Depression in Single Sarcomeres is Abolished by MgADP-Activation

The mechanisms behind the shortening-induced force depression (FD) commonly observed in skeletal muscles remain unclear, but have been associated with sarcomere length non-uniformity and/or cross-bridge inhibition. The purpose of this study was twofold: (i) to evaluate if FD is present in isolated single sarcomeres, a preparation that eliminates sarcomere length non-uniformities and (ii) to evaluate if FD is inhibited when single sarcomeres are activated with MgADP, which biases cross-bridges into a strongly-bound state. Single sarcomeres (n=16) were isolated from rabbit psoas myofibrils using two micro-needles (one compliant, one rigid), piercing the sarcomere externally adjacent to the Z-lines. The sarcomeres were contracted isometrically and subsequently shortened, in both Ca2+- and MgADP-activating solutions. Shortening in Ca2+-activated samples resulted in a 27.44% ± 9.04% force depression when compared to isometric contractions produced at similar final sarcomere lengths (P > 0.001). There was no FD in MgADP-activated sarcomeres (FD = −1.79% ± 9.69%, P = 0.435). These results suggest that FD is a sarcomeric property, and that is associated with an inhibition of myosin-actin interactions.

Measuring Work Loops in Intact Isolated Cardiac Myocytes by Controlling Pre- and Afterload using a New Generation Force Transducer

A recent exciting development in cardiac research is the ability to do mechanical experiments on intact isolated cardiac myocytes. Here we mimic cardiac PV loops by imposing a ‘pre-load’ and ‘after-load’ on the myocyte. For these experiments we developed a new generation force transducer as currently available force transducers were not sufficiently sensitive or had insufficiently stable base line levels to achieve force control. A cantilever with a spring constant of 7 Nm is interrogated using an interferometer via an optical fiber. The resolution of the interferometer/probe system is 2nm, resonance frequency > 2 kHz and force resolution < 10 nN. As the probe is small enough to be fully submerged in water, variations in solution level have no effect on the force measurement, resulting in a stable baseline (drift < 50 nN over a 10 minute period at 21C). Software was written to take in the signal from the force transducer, process it and return a signal to a linear motor that could stretch or shorten the myocyte in order to control force levels. Using this software we were able to achieve a two-sided force clamp (setting ‘pre-load’ and ‘after-load’ ) to measure work loops and re-create the Frank-Starling relation at the single cell level. Experiments show that in isolated cardiac mouse myocytes residual active force at the end of diastole limits the effective work the mycoyte can produce. Small concentrations of BDM, thought to inhibit strong crossbridge formation by stabilizing weak cross bridges, allow the myocyte to relax at end-diastole, shifting the pre-load-sarcomere length relation upwards. The resulting increase in length dependent activation outweighs the effects of crossbridge inhibition, leading to strongly increased mechanical work per contractile cycle.

Enhanced Ca2+ binding of cardiac troponin reduces sarcomere length dependence of contractile activation independently of strong crossbridges

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle and is considered to be the cellular basis of the Frank-Starling law of the heart. SL dependence may involve changes in myofilament lattice spacing and/or myosin crossbridge orientation to increase probability of binding to actin at longer SLs. We used the L48Q cardiac troponin C (cTnC) variant, which has enhanced Ca(2+) binding affinity, to test the hypotheses that the intrinsic properties of cTnC are important in determining 1) thin filament binding site availability and responsiveness to crossbridge activation and 2) SL dependence of force in cardiac muscle. Trabeculae containing L48Q cTnC-cTn lost SL dependence of the Ca(2+) sensitivity of force. This occurred despite maintaining the typical SL-dependent changes in maximal force (F(max)). Osmotic compression of preparations at SL 2.0 μm with 3% dextran increased F(max) but not pCa(50) in L48Q cTnC-cTn exchanged trabeculae, whereas wild-type (WT)-cTnC-cTn exchanged trabeculae exhibited increases in both F(max) and pCa(50). Furthermore, crossbridge inhibition with 2,3-butanedione monoxime at SL 2.3 μm decreased F(max) and pCa(50) in WT cTnC-cTn trabeculae to levels measured at SL 2.0 μm, whereas only F(max) was decreased with L48Q cTnC-cTn. Overall, these results suggest that L48Q cTnC confers reduced crossbridge dependence of thin filament activation in cardiac muscle and that changes in the Ca(2+) sensitivity of force in response to changes in SL are at least partially dependent on properties of thin filament troponin.

New insights into force depression in skeletal muscle

Force depression observed following active shortening is not well understood. Previous research suggested that force depression might be associated with a stress-induced inhibition of cross-bridges in the newly formed overlap zone following shortening. Our aim was to investigate this theory in skinned fibres and determine whether there was an inhibition of the attachment of cross-bridges or a decrease in the force produced per cross-bridge. The stress-induced inhibition of cross-bridge theory gives testable predictions, including: (1) skinned fibres should show proportional force and stiffness depression, (2) force after shortening should not be lower than force before shortening, (3) stiffness following shortening should not be lower than stiffness before shortening and (4) force depression should decrease when the stress during shortening is decreased. In agreement with these predictions, force and stiffness depression were approximately proportional, and force depression decreased with decreasing stress during shortening. However, in contrast to the predictions of the stress-induced inhibition of cross-bridge theory, force after shortening from sarcomere lengths of 2.8 and 3.0 μm to a sarcomere length of 2.4 μm was smaller than force before shortening, and this was not accompanied by a corresponding decrease in stiffness. We conclude that the stress-induced inhibition of cross-bridge theory, as proposed previously, cannot be the only mechanism for force depression, but that there is an additional, stress-induced inhibition of cross-bridges in the old overlap zone. Furthermore, both mechanisms, inhibition of cross-bridge attachment and reduction of force produced per cross-bridge, contribute to force depression. Inhibition and/or reduction of force depend(s) on the amount of stress imposed on actin during the shortening phase.

Sarcomere Length Dependent Contractile Activation is Reduced in Rat Trabeculae Exchanged with cTn Containing the L48Q cTnC Variant Independently of Strong Binding Cross-Bridges

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle. It can also be influenced by maneuvers that alter the distribution of cross-bridges within the cross-bridge cycle. We have demonstrated that cardiac trabeculae have left-shifted and virtually eliminated SL dependence of force-pCa relationship following passive exchange with cTn containing a mutant (L48Q) cTnC (with enhanced TnC-TnI interaction). Here we designed experiments to investigate the importance of strongly bound crossbridges and lattice spacing in modulating the force-pCa relationship of WT and L48Q cTnC-cTn exchanged trabeculae. Using 3% dextran at SL = 2.0 µm to osmotically compress preparations to widths ∼SL = 2.3 µm, we observed increased maximal force but not increased pCa50 in L48Q cTnC-cTn exchanged trabeculae. Conversely, crossbridge inhibition with of 2,3-butanedione monoxime (BDM, 7 mM) at SL 2.3 µm decreased maximal force and Ca2+ sensitivity in native and WT-cTn exchanged trabeculae to levels measured at ∼SL = 2.0 µm. L48QcTnC-cTn exchanged preparations treated with BDM also decreased maximal force to that seen at SL = 2.0 µm, but demonstrated no shift in Ca2+ sensitivity. This result is similar to decrease in maximal force but no shift in Ca2+ sensitivity for L48Q cTnC-cTn exchanged preparations at SL 2.0 vs. 2.3 µm. The combined results further support the idea that L48Q cTnC confers crossbridge independence on thin filament activation. It may also imply that native thin filaments are dependent on strong crossbridge binding for full activation because of relatively weak cTnC-cTnI interaction. Finally, the relative strength of cTnC-cTnI interaction may be an important determinant in length dependent activation of cardiac muscle. Support provided by NIH HL65497 (MR) and T32 HL07828 (FSK).

Influence of enhanced troponin C Ca2+‐binding affinity on cooperative thin filament activation in rabbit skeletal muscle

We studied how enhanced skeletal troponin C (sTnC) Ca2+-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca2+ dissociation, resulting in the following order of rates: wild-type (WT) sTnC-sTn>sTnC(F27W)-sTn>M80Q sTnC-sTn>M80Q sTnCF27W-sTn. Reconstitution of sTnC-extracted fibres increased Ca2+ sensitivity of steady-state force (pCa(50)) by 0.08 for M80Q sTnC, 0.15 for sTnCF27W and 0.32 for M80Q sTnCF27W with minimal loss of slope (nH, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca2+ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnCF27W: xxsTnC. It is interesting that pCa50 increased by approximately 0.1 for M80Q sTnC and approximately 0.3 for M80Q sTnCF27W when near-neighbour RU interactions were reduced; these values are similar in magnitude to those for fibres reconstituted with 100% mutant sTnC. After reconstitution with sTnC: xxsTnC mixtures, nH decreased to a similar value for all mutant sTnCs. Altered sTnC Ca2+-binding properties (M80Q sTnCF27W) did not affect strong crossbridge inhibition by 2,3-butanedione monoxime when near-neighbour thin filament RU interactions were reduced. Together these results suggest increased sTnC Ca2+ affinity strongly influences Ca2+ sensitivity of steady-state force without affecting near-neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca2+ to thin filament activation.

Investigation of thin filament near‐neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle

Ca(2+)-dependent activation of striated muscle involves cooperative interactions of cross-bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild-type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca(2+)-activated force (F(max)) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where F(max) increased in a greater than linear manner with WT sTnC, and suggests that Ca(2+) binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca(2+) sensitivity of force and rate of force redevelopment (k(tr)) was leftward shifted by 0.1-0.2 -log [Ca(2+)] (pCa) units as WT cTn content was increased, but the slope of the force-pCa relation and maximal k(tr) were unaffected by loss of near-neighbour RU interactions. Cross-bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy-ATP) had no greater effect in cardiac muscle with disruption of near-neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca(2+) dissociation was found to be > 2-fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca(2+) binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca(2+) binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca(2+) binding affinity.

EFFECTS OF SEGMENTAL MOTION ON GROUND REACTION FORCE DURING DROP LANDINGS

It is well-recognised that steady-state isometric muscle force is decreased following active shortening (force depression, FD) and increased following active stretch (force enhancement, FE). It has also been demonstrated that passive muscle force is increased following active stretch (passive FE). Several studies have reported that FD increases with shortening amplitude and that FE and passive FE increase with stretch amplitude. Here, we investigate whether these trends continue with further increases in shortening or stretch amplitude. Experiments were performed using in situ cat soleus muscles (n=8 for FD; n=7 for FE and passive FE). FD, FE and passive FE were measured after shortening or stretch contractions that covered as wide a range of amplitudes as practically possible without damaging the muscles. FD increased approximately linearly with shortening amplitude, over the full range of amplitudes investigated. This is consistent with the hypothesis that FD arises from a stress-induced inhibition of crossbridges. FE increased with stretch amplitude only up to a point, and then levelled off. Passive FE, and the transient increase in force at the end of stretch, showed relationships to stretch amplitude that were qualitatively very similar to the relationship for FE, increasing only until the same critical stretch amplitude had been reached. We conclude that FE and passive FE do not increase with stretch amplitude under all circumstances. This finding has important consequences for determining the mechanisms underlying FE and passive FE because any mechanism that is proposed to explain them must be able to predict it.

Residual force depression is not abolished following a quick shortening step

Residual force depression is long lasting, but can be abolished instantaneously when a muscle is deactivated just long enough for force to drop to zero. According to the “cross-bridge inhibition theory” of force depression, this result is predicted as the release of stress on actin during deactivation restores the angular distortion of the actin binding sites, thereby establishing conditions identical to those of a purely isometric contraction. According to this theory, force depression should also be abolished if stress on actin is released through a quick shortening step. For slow (4.5 mm/s) shortening of cat soleus ( n=8), force depression was achieved in all muscles and averaged 5.3% (±1.9%) and 5.8% (±1.3%) for 9 and 18 mm shortening amplitudes. Following quick shortening (200 mm/s) of 18 mm, there was no statistically significant force depression. However, when slow shortening (4.5 mm/s for 9 mm) was followed by quick shortening (200 mm/s for 9 mm) after delays of 0, 1, and 2 s, there was a small but significant force depression in all cases averaging 3.2%, 3.7%, and 4.2%, respectively. We conclude from these results that a small amount of force depression persists following stress release caused by quick shortening, and therefore that the cross-bridge inhibition theory cannot be the sole cause of force depression.

History-dependence of isometric muscle force: Effect of prior stretch or shortening amplitude

It is well-recognised that steady-state isometric muscle force is decreased following active shortening (force depression, FD) and increased following active stretch (force enhancement, FE). It has also been demonstrated that passive muscle force is increased following active stretch (passive FE). Several studies have reported that FD increases with shortening amplitude and that FE and passive FE increase with stretch amplitude. Here, we investigate whether these trends continue with further increases in shortening or stretch amplitude. Experiments were performed using in situ cat soleus muscles ( n=8 for FD; n=7 for FE and passive FE). FD, FE and passive FE were measured after shortening or stretch contractions that covered as wide a range of amplitudes as practically possible without damaging the muscles. FD increased approximately linearly with shortening amplitude, over the full range of amplitudes investigated. This is consistent with the hypothesis that FD arises from a stress-induced inhibition of crossbridges. FE increased with stretch amplitude only up to a point, and then levelled off. Passive FE, and the transient increase in force at the end of stretch, showed relationships to stretch amplitude that were qualitatively very similar to the relationship for FE, increasing only until the same critical stretch amplitude had been reached. We conclude that FE and passive FE do not increase with stretch amplitude under all circumstances. This finding has important consequences for determining the mechanisms underlying FE and passive FE because any mechanism that is proposed to explain them must be able to predict it.

Energetics of small hearts

Little is known about the energetics of the failing heart. In man it is well known that energy metabolism is affected in chronic heart failure (Ingwall & Weiss, 2004). Genetically modified mice are useful in the study of heart disease. A persistent headache has been the lack of basic energetic data. In this issue of The Journal of PhysiologyWiden & Barclay (2006) give us both a method and the first data. For the first time, the basic energetic characteristics of mouse heart muscle activation and contraction have been measured in papillary muscle. Mice can be obtained relatively easily and the mouse genome has been sequenced. The proportion of mouse genes without a human homologue is probably less than 1% (Mouse Genome Sequencing Consortium, 2002). Numerous mouse models for studying heart disease exist already and the number is increasing (Wang et al. 2004). Gene products are processed in cells resulting in many more proteins than genes and a staggering number of possible interactions between the different proteins exist. One important question is then: How does one relate gene function to the final contractile characteristics of cardiomyocytes? Muscles produce mechanical work and heat in different amounts and proportions, depending on the type of muscle and the load (Hill, 1965; Woledge et al. 1985). ATP required for ion pumps and cardiomyocyte shortening is buffered by the Lohmann reaction in the myoplasm. This keeps the ATP concentration constant while the phosphocreatine concentration decreases when the muscle is activated. Net splitting of phosphocreatine releases 34 kJ mol−1 (Woledge et al. 1985). This energy is partly converted into mechanical work during systole when the phosphocreatine concentration decreases and the rest is liberated as heat. A metabolic steady state can only be maintained when creatine is rephosphorylated during one heart cycle. The energy for this is produced by oxidation of fat or carbohydrate, 439 or 473 kJ (mol O2)−1, respectively. Depending on the efficiency of the oxidation (the ∼P/O2 ratio), part of the energy is used to rephosphorylate creatine, and the remainder is liberated as heat. Heat production increases the temperature of the heart muscle preparation, which can be measured using a thermopile. The myothermic technique has a higher time resolution than oxygen consumption or 31P-NMR which are too slow to determine energy fluxes during a single contraction. The interpretation of heat production data requires that the biochemical reaction producing it is known. This is complicated in heart muscle because contraction-related processes, i.e. heat production due to ion pumps and cross-bridge cycling, and oxidative phosphorylation, usually occur simultaneously. Another challenge is that mouse hearts are small (about 0.1–0.2 g) and operate at high metabolic rates (Gibbs & Loiselle, 2001). The risk that the preparation becomes anoxic is considerable. This is a problem because heat production by partly anoxic preparations cannot be interpreted reliably, and because reoxygenation can damage anoxic cardiomyocytes. The maximum rate of oxygen consumption of mouse heart muscle is not known, but can be estimated on the basis of volume density of mitochondria and the rate of oxygen consumption of rat myocardial trabeculae, 0.58 nmol mm−3 s−1 at the maximum heart rate (van der Laarse et al. 2005), 600 beats min−1, which is the resting rate in mice. Mouse cardiomyocytes contain 38% (v/v) mitochondria and rat cardiomyocytes contain 32% (Barth et al. 1992). Assuming the maximum rate of oxygen consumption is proportional to volume density of mitochondria, the maximum rate of oxygen consumption of mouse trabeculae is about 0.7 nmol mm−3 s−1. At this rate of oxygen consumption, the mouse heart becomes anoxic a few seconds after perfusion stops and superfused preparations, e.g. papillary muscles or trabeculae at experimental PO2 of 500 mmHg, must have diameters of less than 160 µm to prevent an anoxic core. Widen & Barclay (2006) solved these problems and determined heat produced by calcium cycling and heat produced by cross-bridge cycling in papillary muscles of Swiss mice. Contraction-related heat was determined during the first three contractions of a series, making use of the relatively slow onset of recovery metabolism at 27°C. Hypoxia was prevented using stimulus periods of 20 s, sufficiently short not to deplete oxygen stored in the muscle, and contraction and activation-related heat were separated by partial inhibition of cross-bridges using butanedione monoxime (Alpert et al. 1989). The results indicate that about one Ca2+ cycles per three cross-bridge cycles, and that the number of cross-bridge cycles per cardiac twitch is half the number of available cross-bridges. How these numbers vary with genotype and intervention remains to be determined.

Does the speed of shortening affect steady-state force depression in cat soleus muscle?

It has been stated repeatedly for the past 50 years that the steady-state force depression following shortening of an activated muscle depends on the speed of shortening. However, these statements were based on results from experiments in which muscles were shortened at different speeds but identical activation levels. Therefore, the force during shortening was changed in accordance with the force–velocity relationship of muscles: that is, increasing speeds of shortening were associated with decreasing forces, and vice versa. Consequently, it is not possible at present to distinguish whether force depression is caused by the changes in speed, as frequently stated, or the associated changes in force, or both. The purpose of this study was to test if force depression depends on the speed of shortening. We hypothesized that force depression was dependent on the force but not the speed of contraction. Our prediction is that the amount of force depression after shortening contractions at different speeds could be similar if the force during contraction was controlled at a similar level. Cat soleus muscles ( n = 7 ) were shortened by 9 or 12 mm at speeds of 3, 9, and 27 mm/s, first with a constant activation during shortening (30 Hz), then with activation levels that were reduced (<30 Hz) for the slow speeds (3 and 9 mm/s) to approximate the shortening forces of the fast speed contractions (27 mm/s). If done properly, force depression could be precisely matched at the three different speeds, indicating that force depression was related to the force during the shortening contraction but not to the speed. However, in order to match force depression, the forces during shortening had to be systematically greater for the slow compared to the fast speeds of shortening, suggesting that force depression also depends on the level of activation, as force depression at constant activation levels can only be matched if the force during shortening, evaluated by the mechanical work, is identical. Therefore, we conclude that force depression depends on the force and activation level during shortening, but does not depend on the speed of shortening as has been assumed for half a century. These results support, but do not prove, the current hypothesis that force depression is caused by a stress-related cross-bridge inhibition in the actin–myosin overlap zone that is newly formed during muscle shortening.

Alterations of myocardial dynamic stiffness implicating abnormal crossbridge function in human mitral regurgitation heart failure.

Mitral regurgitation (MR) causes ventricular dilation, a blunted myocardial force-frequency relation, and increased crossbridge force-time integral (FTI). The mechanism of FTI increase was investigated using sinusoidal length perturbation analysis to compare crossbridge function in skinned left ventricular (LV) epicardial muscle strips from 5 MR and 5 nonfailing (NF) control hearts. Myocardial dynamic stiffness was modeled as 3 parallel viscoelastic processes. Two processes characterize intermediate crossbridge cycle transitions, B (work producing) and C (work absorbing) with Q(10)s of 4 to 5. No significant differences in moduli or kinetic constants of these processes were observed between MR and NF. The third process, A, characterizes a nonenzymatic (Q(10)=0.9) work-absorbing viscoelasticity, whose modulus increases sigmoidally with [Ca(2+)]. Effects of temperature, crossbridge inhibition, or variation in [MgATP] support associating the calcium-dependent portion of A with the structural "backbone" of the myosin crossbridge. Extension of the conventional sinusoidal length perturbation analysis allowed using the A modulus to index the lifetime of the prerigor, AMADP crossbridge. This index was 75% greater in MR than in NF (P=0.02), suggesting a mechanism for the previously observed increase in crossbridge FTI. Notably, the A-process modulus was inversely correlated (r(2)=0.84, P=0.03) with in vivo LV ejection fraction in MR patients. The longer prerigor dwell time in MR may be clinically relevant not only for its potential role as a compensatory mechanism (increased economy of tension maintenance and increased resistance to ventricular dilation) but also for a potentially deleterious effect (reduced elastance and ejection fraction).

The history dependence of force production in mammalian skeletal muscle following stretch-shortening and shortening-stretch cycles

The purpose of this study was to determine the history dependence of force production during and following stretch-shortening and shortening-stretch cycles in mammalian skeletal muscle. Thirty-three different isometric, stretch, shortening, stretch-shortening and shortening-stretch experiments were preformed in cat soleus (n=8) using previously established methods. Stretch-shortening and shortening-stretch cycles are not commutative with respect to the isometric forces following the length changes. Whereas force depression following shortening is virtually unaffected by previous stretching of the muscle, force enhancement following stretch depends in a dose-dependent manner on the amount of muscle shortening preceding the stretch. The history dependence of isometric force following shortening-stretch cycles can conveniently be modelled using an elastic (compressive and tensile) element that engages at the length of muscle activation. Such an “elastic” mechanism has been proposed by Edman and Tsuchiya (1996) (Edman, K.A.P., Tsuchiya, T., 1996. Strain of passive elements during force enhancement by stretch in frog mucle fibres. Journal of Physiology 490. 1, 191–205) based on experimental observations, and has been implemented theoretically in a rheological model of muscle (Forcinito et al., 1997) (Forcinito, M., Epstein, M., Herzog, W., 1997. Theoretical considerations on myofibril stiffness. Biophysics Journal 72, 1278–1286). The history dependence of isometric force following stretch-shortening cycles appears independent of the stretch preceding the shortening, except perhaps, if stretching occurs at very high speeds (i.e. 6–10 times fibre length per second). The results of this study are hard to reconcile with the two major mechanisms associated with history dependence of force production: sarcomere length non-uniformity (Edman et al., 1993) and stress-induced cross-bridge inhibition (Maréchal and Plaghki, 1979) (Maréchal, G., Plaghki, L., 1979. The deficit of the isometric tetanic tension redeveloped after a relase of frog muscle at a constant velocity. Journal of General Physiology 73, 453–467). It appears that studying the history dependence of force production under more functionally relevant conditions than has been done to date may provide new information that contributes to our understanding of possible mechanisms associated with force depression and force enhancement following muscular length changes.