Unloaded Shortening Research Articles

Myocyte contractility can be maintained by storing cells with the myosin ATPase inhibitor 2,3 butanedione monoxime.

Isolated intact myocytes can be used to investigate contractile mechanisms and to screen new therapeutic compounds. These experiments typically require euthanizing an animal and isolating fresh cells each day or analyzing cultured myocytes, which quickly lose their rod-shaped morphology. Recent data suggest that the viability of canine myocytes can be prolonged using low temperature and N-benzyl-p-toluene sulfonamide (an inhibitor of skeletal myosin ATPase). We performed similar studies in rat myocytes in order to test whether the cardiac myosin ATPase inhibitors 2,3-Butanedione monoxime (BDM) and blebbistatin help to maintain cell-level function over multiple days. Myocytes were isolated from rats and separated into batches that were stored at 4°C in a HEPES-buffered solution that contained 0.5 mmol L−1 Ca2+ and (1) no myosin ATPase inhibitors; (2) 10 mmol L−1 BDM; or (3) 3 μmol L−1 blebbistatin. Functional viability of myocytes was assessed up to 3 days after the isolation by measuring calcium transients and unloaded shortening profiles induced by electrical stimuli in inhibitor-free Tyrode's solution. Cells stored without myosin ATPase inhibitors had altered morphology (fewer rod-shaped cells, shorter diastolic sarcomere lengths, and membrane blebbing) and were not viable for contractile assays after 24 h. Cells stored in BDM maintained morphology and contractile function for 48 h. Storage in blebbistatin maintained cell morphology for 72 h but inhibited contractility. These data show that storing cells with myosin ATPase inhibitors can extend the viability of myocytes that will be used for functional assays. This may help to refine and reduce the use of animals in experiments.

Cross-bridge Kinetics Impairment In Elite Octogenarian Masters Athletes

The association between adult normal aging and impairments of the neuromuscular system as well as the application of exercise training to attenuate these problems have already been reported by several studies. However, it is still not clear whether intrinsic cross-bridge kinetics, especially of the ‘slow’ resilient fibre type, are affected in aging muscle, and if this kinetics would be better preserved in high functioning elderly athletes. PURPOSE: to compare cross-bridge kinetics in healthy non-athlete octogenarians to age-matched world class masters athletes, and young controls. METHODS: Skinned muscle fibres obtained from vastus lateralis biopsies of young (∼23y), old non-athlete (NA) adults (∼80y), and age-matched world class masters athletes (MA; ∼80y) were first isometrically activated to assess force per cross-sectional (Po), then submitted to slack test, and shortening-re-stretch protocol, by which unloaded shortening velocity (Vo), and the rate constant of force redevelopment (ktr) were respectively obtained. A post-hoc analysis was performed, and only the mechanical properties of ‘slow type’ fibres based on unloaded shortening velocity measurements were used (the level of significance was set at P<0.05). RESULTS: MA and NA produced ∼55% and 45% lower Po than young. Vo for both MA and NA old groups were ∼62% and 42% slower, respectively, compared with young. Both MA and NA adults had ∼2 times slower values for ktr compared with young. CONCLUSION: The lower ktr in both old groups relative to young suggests that impaired cross-bridge kinetics was, at least in part, responsible for impaired single fibre contractile properties, i.e Vo, Po, with aging. Apparently, masters athletes’ higher performance cannot be attributed to their intrinsic cross-bridge kinetics.

Omecamtiv Mecarbil modulates the kinetic and motile properties of porcine β-cardiac myosin.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

Redefining the Latch-State in Human Airway Smooth Muscle

Rationale: The latch-state describes the ability of tonic smooth muscle to maintain force at reduced ATP consumption and crossbridge cycling rate. In animal models this is evidenced by reduced unloaded shortening velocity (Vmax), presumably indicative of crossbridge cycling rate, and reduced myosin light chain phosphorylation (pMLC). In this study we aim to assess this phenomenon in human airway smooth muscle (hASM).Methods: hASM tissues were dissected from trachea procured from the International Institute for the Advancement of Medicine. Force-velocity curves and pMLC measurements were performed at several time points in multiple prolonged methacholine 10-6M contractions. Rat ASM strips were tested with the same protocol. A mathematical model was developed to explain the findings.Results: Despite a 40% drop in pMLC between the 1st to the 20th min after the peak contraction, hASM Vmax did not show any change. Conversely, rat ASM Vmax decreased by 50%. A mathematical model of the cross-bridge cycle, combining strain-dependent ADP affinity of myosin with actin regulated binding of dephosphorylated myosin heads, was capable of reproducing our findings.Conclusion: As our results contradict the classical latch-bridge theory, we suggest an alternative model. We propose that Vmax, as calculated from standard force-velocity curves, is not indicative of the crossbridge cycling rate during latch. Instead, Vmax is reduced due to drag caused by dephosphorylated myosin heads binding to actin, with their binding force regulated by actin regulatory proteins and their activation state. The increase in dephosphorylated myosin late in contraction decreases the measured Vmax, unless its binding force is down-regulated by regulatory proteins. Our modelling further shows that force maintenance can be explained by phosphorylated myosin heads remaining attached to actin in a locked configuration, unable to release ADP because of strain dependence of the ADP affinity of myosin.

Abstract 40: Genetic Deletion Of Mir-208a Induces Pathological Remodeling And Heart Failure

Introduction: miR-208a, a small non-coding RNA expressed only in the heart, has a profound influence on cardiac gene expression. It has been previously demonstrated that genetic deletion of miR-208a does not affect viability or induce gross morphological heart defects, suggesting it is not required for normal cardiac growth and function (van Rooij et al., 2007; Callis et al., 2009). However, recent data from our lab indicate that miR-208a knock-out (KO) mice (developed in the laboratory of Da-Zhi Wang and subsequently maintained by inbreeding) display gross morphological cardiac abnormalities and dramatically reduced survival. Therefore, this investigation sought out to determine the mechanism(s) responsible for the maladaptive growth. Methods: Wild-type (WT) and miR-208a KO littermate mice (3-5 mon old) were used for this investigation. Cardiac function was assessed via echocardiography, cardiac fibrosis by collagen fiber staining, and gene expression by real-time PCR and Western blotting. We also measured basal Ca2+ transients and unloaded cell shortening in isolated mouse ventricular myocytes. Results: We observed KO mice have significantly lower survival rates, developed marked cardiac hypertrophy, fibrosis and have significantly reduced cardiac function. Compared to WT controls, cardiomyocytes from KO mice exhibited a significantly lower percent of cell shortening, slower rates of relaxation and contraction, lower amplitude, and slower kinetics (tau) of Ca2+ transients. Additionally, there was a reduced phosphorylation of phospholamban (Ser16) in miR-208 KO mice indicating a lower Ca2+ affinity of the SR Ca2+-ATPase. Conclusions: These data provide evidence that the observed reduction in cardiac function in miR-208a KO mice is likely due, in part, to alterations in cellular Ca2+ fluxes. To our knowledge, this is the first study to demonstrate genetic deletion of miR-208a induces a maladaptive phenotype at baseline. Our findings indicate the importance of considering variables such as genetic/environmental constraints when perturbing the expression of the miR-208a in the heart.

Actin sliding velocity on pure myosin isoforms from hindlimb unloaded mice.

Notwithstanding the widely accepted idea that following disuse skeletal muscles become faster, an increase in shortening velocity was previously observed mostly in fibres containing type 1 myosin, whereas a decrease was generally found in fibres containing type 2B myosin. In this study, unloaded shortening velocity of pure type 1 and 2B fibres from hindlimb unloaded mice was determined and a decrease in type 2B fibres was found. To clarify whether the decrease in shortening velocity could depend on alterations of myosin motor function, an in vitro motility assay approach was applied to study pure type 1 and pure type 2B myosin from hindlimb unloaded mice. The latter approach, assessing actin sliding velocity on isolated myosin in the absence of other myofibrillar proteins, enabled to directly investigate myosin motor function. Actin sliding velocity was significantly lower on type 2B myosin following unloading (2.70 ± 0.32 μm s(-1)) than in control conditions (4.11 ± 0.35 μm s(-1)), whereas actin sliding velocity of type 1 myosin was not different following unloading (0.89 ± 0.04 μm s(-1)) compared with control conditions (0.84 ± 0.17 μm s(-1)). Myosin light chain (MLC) isoform composition of type 2B myosin from hindlimb unloaded and control mice was not different. No oxidation of either type 1 or 2B myosin was observed. Higher phosphorylation of regulatory MLC in type 2B myosin after unloading was found. Results suggest that the observed lower shortening velocity of type 2B fibres following unloading could be related to slowing of acto-myosin kinetics in the presence of MLC phosphorylation.

CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression

Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T-helper 2 (Th2) inflammatory response. CD4(+) T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4(+) T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen-stimulated CD4(+) T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co-incubation with antigen-stimulated CD4(+) T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4(+) T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co-incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper-responsiveness and asthma.

Dynamic coupling of regulated binding sites and cycling myosin heads in striated muscle

In an activated muscle, binding sites on the thin filament and myosin heads switch frequently between different states. Because the status of the binding sites influences the status of the heads, and vice versa, the binding sites and myosin heads are dynamically coupled. The functional consequences of this coupling were investigated using MyoSim, a new computer model of muscle. MyoSim extends existing models based on Huxley-type distribution techniques by incorporating Ca(2+) activation and cooperative effects. It can also simulate arbitrary cross-bridge schemes set by the researcher. Initial calculations investigated the effects of altering the relative speeds of binding-site and cross-bridge kinetics, and of manipulating cooperative processes. Subsequent tests fitted simulated force records to experimental data recorded using permeabilized myocardial preparations. These calculations suggest that the rate of force development at maximum activation is limited by myosin cycling kinetics, whereas the rate at lower levels of activation is limited by how quickly binding sites become available. Additional tests investigated the behavior of transiently activated cells by driving simulations with experimentally recorded Ca(2+) signals. The unloaded shortening profile of a twitching myocyte could be reproduced using a model with two myosin states, cooperative activation, and strain-dependent kinetics. Collectively, these results demonstrate that dynamic coupling of binding sites and myosin heads is important for contractile function.

In Vitro Smooth Muscle Hypercontractility Induced by Cd4+ T Cells is Transient

Smooth muscle (SM) hypercontractility is a characteristic of several pathologies, including asthma. Inflammatory cells and/or the cytokines they release may alter contractile protein expression, thereby also altering the SM mechanics. We characterized the maximal unloaded shortening velocity (Vmax), stress (force/cross sectional area), and contractile protein expression of Brown Norway rat airway SM co-cultured with activated CD4+ T cells for 24h and 48h. After 24h incubation, Vmax (mean±SEM) was significantly increased (0.15±0.01 l/s vs. 0.29±0.02 l/s; control vs. T cells, p = 0.0012). However, this alteration in Vmax was transient and disappeared after 48h of co-incubation (0.21±0.01 vs. 0.25±0.01 l/s, p = 0.099). No significant differences in stress were observed at 24h or 48h. Western blot analysis showed a significant increase in the levels of MLCK (0.20±0.03 vs. 0.41± 0.09; control vs. T cells; p<0.05, p=0.018) and the (+)insert SM myosin isoform (0.55±0.04 vs. 0.81±0.05; p = 0.0023) after 24h incubation with the CD4+ T cells. These alterations were also transient; all values returned to baseline after 48h exposure to the CD4+ T cells. Thus, the increase in Vmax was correlated with the increase in MLCK and (+)insert isoform expression. Conversely, there were no differences in levels of total SM myosin heavy chain or α-smooth muscle actin, suggesting there was no increase in muscle mass. These results show that indeed inflammatory cells and/or their mediators can alter SM contractile protein expression and SM function, but that these effects may be transient. This transient behavior may be important to consider when studying SM function in disease.Supported by: CIHR, NIH-RO1HL103405.

Effects of Chronic Myocardial Infarction on Cardiac Muscle Performance and Structure In-Vivo and In-Vitro

After chronic myocardial infarction (CMI) the myocardium undergoes remodeling which is described initially as ‘hypertrophic’ followed by ‘de-compensation’. We have studied this phenomenon at the sarcomeric level to understand how muscle mechanics, post-translational modifications, tissue morphology and sarcomeric structures are affected in the remaining well-perfused tissue.We use a rat model of left anterior descending (LAD) ligation to create phenotypic chronic myocardial infarction (CMI). A specialized temperature jump (T-jump) approach assess changes to myocardial muscle function during physiological shortening in saturating (32μM) and physiological (1μM) calcium concentrations. During ‘compensation’ we observe higher power (45%) compared to control. During ‘de-compensation’ we observe a 50% reduction in peak power and 40% reduction in maximal unloaded shortening velocity (Vmax). We exploit second harmonic generation (SHG) by two-photon excitation microscopy (TPE) to assess collagen abundance and infiltration. Global type I collagen abundance and infiltration increases 2-fold post-AMI but does not change during disease progression. Phos-Tag SDS-PAGE is used to assess Troponin I (TnI), Myosin Binding Protein C (MyBP-C) and Regulatory Light Chain (RLC) phosphorylation. Electron microscopy is used to assess sarcomeric organization and mitochondrial health and abundance. Sarcomeric disorder becomes evident with disease progression as well as a global reduction in functional mitochondria. This study demonstrates that sarcomeric protein modification is an important modifier of cardiac muscle function during the evolution of disease, which mirrors the route of progression from ‘compensated’ to ‘decompensated’ phenotypes that is part of the wider picture of heart failure that encompasses changes to tissue structure and organelle morphology.

Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.

Longitudinal decline of lower extremity muscle power in healthy and mobility-limited older adults: influence of muscle mass, strength, composition, neuromuscular activation and single fiber contractile properties

This longitudinal study examined the major physiological mechanisms that determine the age-related loss of lower extremity muscle power in two distinct groups of older humans. We hypothesized that after ~3 years of follow-up, mobility-limited older adults (mean age: 77.2 ± 4, n = 22, 12 females) would have significantly greater reductions in leg extensor muscle power compared to healthy older adults (74.1 ± 4, n = 26, 12 females). Mid-thigh muscle size and composition were assessed using computed tomography. Neuromuscular activation was quantified using surface electromyography and vastus lateralis single muscle fibers were studied to evaluate intrinsic muscle contractile properties. At follow-up, the overall magnitude of muscle power loss was similar between groups: mobility-limited: -8.5 % vs. healthy older: -8.8 %, P > 0.8. Mobility-limited elders had significant reductions in muscle size (-3.8 %, P < 0.01) and strength (-5.9 %, P < 0.02), however, these parameters were preserved in healthy older (P ≥ 0.7). Neuromuscular activation declined significantly within healthy older, but not in mobility-limited participants. Within both groups, the cross-sectional areas of type I and IIA muscle fibers were preserved while substantial increases in single fiber peak force (>30 %), peak power (>200 %) and unloaded shortening velocity (>50 %) were elicited at follow-up. Different physiological mechanisms contribute to the loss of lower extremity muscle power in healthy older and mobility-limited older adults. Neuromuscular changes may be the critical early determinant of muscle power deficits with aging. In response to major whole muscle decrements, major compensatory mechanisms occur within the contractile properties of surviving single muscle fibers in an attempt to restore overall muscle power and function with advancing age.

Abstract 345: R403Q Mutation Increases the Rate of Force Redevelopment in 2 Month Mice

Familial hypertrophic cardiomyopathy is a primary disease of the sarcomere. The R403Q mutation resides at the actin-interaction site on myosin and leads to progressive hypertrophic cardiomyopathy which progresses towards heart failure. Along with deteriorating cardiac function, these hearts experience an overall change in metabolic landscape, suggesting altered energetic function in hearts that express the R403Q mutation. We tested the hypothesis that the R403Q mutation intrinsically increases the energetic cost of contraction. To do this, we determined myofilament function in demembranated cardiac trabeculae from male wild-type (WT) and R403Q mice at 2 months of age, prior to overt signs of cardiac pathology. Firstly, steady-state Ca2+ sensitivity of force generation was not significantly different between male R403Q (n=4) and WT counterparts (n=2) consistent with previous findings. Secondly, the rate of force redevelopment (ktr) in skinned cardiac tissue was measured following unloaded isotonic shortening and a rapid re-stretch to 15% of the original muscle length at a sarcomere length of 2.0μm. R403Q mice display an increased rate of force redevelopment (49.89 s-1 ± 8.13, n = 4) compared to WT counterparts (24.52 ± 4.29, n = 6) at maximal activation indicating an increase in the apparent rates of crossbridges entering and leaving force-generating states (p &lt; 0.05). In conclusion, the R403Q mutation does not impact steady-state Ca2+ sensitivity of force but increases total crossbridge cycling rate suggesting a higher energy cost of force generation. Future studies are aimed at determining the energetic cost of contraction in R403Q hearts and how this increased energetic cost leads to hypertrophic cardiomyopathy.

Detection of an aging-related increase in advanced glycation end products in fast- and slow-twitch skeletal muscles in the rat

Glycation, a non-enzymatic addition of reducing sugars to ε-amino groups of proteins, is a post-translational modification that results in the formation of irreversible advanced glycation end products (AGEs). Ageing related decline in myofibrillar protein function is effected by a number of structural and functional modifications including glycation. Functional properties of skeletal muscles, such as maximum velocity of unloaded shortening, are known to be profoundly affected by ageing at the motor unit, cellular and tissue levels. However, the contribution of protein modifications to a decline in muscle function is not well understood. In this study we measured AGEs of intracellular and sarcolemmal proteins, using an anti-AGE antibody in soleus (SOL) and extensor digiotorum longus (EDL) muscles of male and female rats of five different age groups. Using a fluorescent secondary antibody to visualize AGEs in the confocal microscope, we found that myosin is glycated in both fiber types in all age groups; an ageing related increase in AGEs was observed in both intracellular and sarcolemmal regions in all age groups, with the exception of sarcolemma of SOL (unchanged) and EDL (reduced) in female rats; the greatest concentration of AGEs was found intracellularly in the SOL of the oldest age group (27-30) of females. While an ageing related decline in motor properties can be partially attributed to the observed increase in myofibrillar protein glycation, our results also indicate that intracellular and the less well studied sarcolemmal protein modification likely contribute to an aging-related decline in muscle function. Further studies are required to establish a link between the observed ageing related increase in glycation and muscle function at the motor unit, cellular and tissue levels.

Enhancement of Force Generated by Individual Myosin Heads in Skinned Rabbit Psoas Muscle Fibers at Low Ionic Strength

Although evidence has been presented that, at low ionic strength, myosin heads in relaxed skeletal muscle fibers form linkages with actin filaments, the effect of low ionic strength on contraction characteristics of Ca2+-activated muscle fibers has not yet been studied in detail. To give information about the mechanism of muscle contraction, we have examined the effect of low ionic strength on the mechanical properties and the contraction characteristics of skinned rabbit psoas muscle fibers in both relaxed and maximally Ca2+-activated states. By progressively decreasing KCl concentration from 125 mM to 0 mM (corresponding to a decrease in ionic strength μ from 170 mM to 50 mM), relaxed fibers showed changes in mechanical response to sinusoidal length changes and ramp stretches, which are consistent with the idea of actin-myosin linkage formation at low ionic strength. In maximally Ca2+-activated fibers, on the other hand, the maximum isometric force increased about twofold by reducing KCl concentration from 125 to 0 mM. Unexpectedly, determination of the force-velocity curves indicated that, the maximum unloaded shortening velocity Vmax, remained unchanged at low ionic strength. This finding indicates that the actin-myosin linkages, which has been detected in relaxed fibers at low ionic strength, are broken quickly on Ca2+ activation, so that the linkages in relaxed fibers no longer provide any internal resistance against fiber shortening. The force-velocity curves, obtained at various levels of steady Ca2+-activated isometric force, were found to be identical if they are normalized with respect to the maximum isometric force. The MgATPase activity of muscle fibers during isometric force generation was found not to change appreciably at low ionic strength despite the two-fold increase in Ca2+-activated isometric force. These results can be explained in terms of enhancement of force generated by individual myosin heads, but not by any changes in kinetic properties of cyclic actin-myosin interaction.

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Hormone replacement therapy improves contractile function and myonuclear organization of single muscle fibres from postmenopausal monozygotic female twin pairs

Ageing is associated with a decline in muscle mass and strength leading to increased physical dependency in old age. Postmenopausal women experience a greater decline than men of similar age in parallel with the decrease in female sex steroid hormone production. We recruited six monozygous female twin pairs (55-59 years old) where only one twin pair was on hormone replacement therapy (HRT use = 7.8 ± 4.3 years) to investigate the association of HRT with the cytoplasmic volume supported by individual myonuclei (myonuclear domain (MND) size,) together with specific force at the single fibre level. HRT use was associated with a significantly smaller (∼27%; P < 0.05) mean MND size in muscle fibres expressing the type I but not the IIa myosin heavy chain (MyHC) isoform. In comparison to non-users, higher specific force was recorded in HRT users both in muscle fibres expressing type I (∼27%; P < 0.05) and type IIa (∼23%; P < 0.05) MyHC isoforms. These differences were fibre-type dependent, i.e. the higher specific force in fast-twitch muscle fibres was primarily caused by higher force per cross-bridge while slow-twitch fibres relied on both a higher number and force per cross-bridge. HRT use had no effect on fibre cross-sectional area (CSA), velocity of unloaded shortening (V0) and relative proportion of MyHC isoforms. In conclusion, HRT appears to have significant positive effects on both regulation of muscle contraction and myonuclei organization in postmenopausal women.

Contact between Cd4+ T Cells and Smooth Muscle Tissue Enhances Contractile Function

Smooth muscle (SM) hyper-contractility is central to the pathophysiology of several diseases, including asthma. Inflammatory cells and/or their products may alter the expression of contractile proteins, thereby enhancing SM mechanics. The goal of the current study was to characterize the maximum unloaded shortening velocity (Vmax), stress (force/cross sectional area), and contractile protein expression of Brown Norway (BN) rat airway SM in the presence of activated CD4+ T cells. T cells from OVA-sensitized BN rats were co-cultured with tracheal rings for 24h. The SM strips were then mounted in a mechanical testing apparatus, equilibrated and supramaximally stimulated with methacholine. A significant increase in Vmax (mean±S.E.) (0.15±0.01 l/s for control, 0.29±0.02 l/s for sensitized, p=0.0012) was observed whereas no significant changes in stress (84.5±13.9 kPa for control, 107.8±4.8 kPa for sensitized) were seen. Western blot analysis showed that levels of the (+)insert myosin heavy chain isoform and myosin light chain kinase (MLCK) were significantly increased. To determine whether contact between T cells and SM cells is required for this enhanced contractility, CD4+T cells were placed in the upper chamber of a transwell, separated by a membrane from the muscle strips placed below. The membrane had 0.4 μm pores that allowed the cytokines released from the CD4+T cells to reach the SM but prevented direct contact between cells. We found a significant increase in Vmax when the SM were in contact with the T cells (0.32±0.02 l/s) compared to the co-culture without contact (0.25±0.01 l/s) or compared to the SM alone (0.24±0.01 l/s; p=0.01). Thus, contact between SM and CD4+T cells is necessary for modulation of SM contractile properties and this may be mediated, at least in part, by increases in (+)insert myosin heavy chain and MLCK expression. Supported by: CIHR, NIH-RO1HL103405.

Evidence that Actin-Myosy Cycling in Muscle may not Pass through Rigor Configuration

In 1989, Sutoh, Tokunaga and Wakabayashi (J Mol Biol 206:357) prepared a monoclonal antibody (IgG) directed to junctional peptide between 50- and 20-kDa segments of myosin heavy chain, and showed that the antibody attaches around the tip of myosin heads facing actin filaments in muscle. using the same antibody, we successfuly position-mark the tip of myosin heads in synthetic myosin filaments kept in hydrated state in the gas environmental chamber, and succeeded in recording ATP-induced myosin head movement electron microscopically (Sugi et al. PNAS 105: 17396, 2008). We have examined the effect of this antibody on both ATP-dependent in vitro actin-myosin sliding and Ca-activated contraction of permealized rabbit psoas muscle fibers. Unexpectedly, the antibody (concentration, up to 2mg/ml) had no appreciable effect on the velocity profile of actin-myosin sliding and the contraction characteristics of muscle fibers, including magnitude of the maximum Ca-activated isometric force, the maximum unloaded shortening velocity, ans MgATPase activity during contraction. Since the junctional peptide is located between the two main actin- binding sites of myosin heads during formation of rigor linkages, attachment of massive antibodies to myosin heads completely covers their actin- binding sites. The possibility that the antibody does not diffuse into muscle fibers is excluded by the fact that other kinds of antibody diffuse into muscle fibers to inhibit Ca-activated force generation (e.g., Sugi et al. PNAS 89: 6134, 1992). Our work, therefore, suggests strongly that attachment-detachment cycle between actin and myosin in muscle may not pass through rigor configuration. It seems possible that so-called strong actin-myosin binding might be due to some electrostatic mechanism.

Factors Beyond Detachment Kinetics that Influence Unloaded Shortening Velocities of Muscle

Recent studies demonstrate the influence of actin-myosin attachment kinetics on in vitro actin filament sliding velocities (V), but it is unclear what factors determine whether velocities are detachment (d/τon) or attachment limited (Yengo et al. 2012. J Muscle Res Cell Motil.). We have developed a muscle model with molecular detail that suggests three key mechanisms by which attachment kinetics can influence unloaded shortening velocities. First, the saturation of myosin binding sites on actin by weakly bound myosin heads can result in attachment limited velocities that are slower than the detachment limit. Essentially, at high myosin densities the actin-activated ATPase activity (attachment limited) in the motility assay saturates before mechanical saturation (detachment limited). Second if actin-bound myosin heads that resist actin movement have a relatively low stiffness, unloaded shortening velocities can exceed the detachment limit. The non-linear compliance of positively and negatively strained myosin heads was demonstrated experimentally (Kaya and Higuchi. 2010. Science. 329: 686-9). Finally, our model shows that mechanically accelerated ADP release kinetics can result in velocities that exceed the detachment limit. These simulations accurately describe the observation of hypermotile velocities (Hooft et al. 2007. Biochemistry. 46:3513-20., Jackson and Baker. 2009. PCCP. 11:4808-14.). In summary, unloaded shortening velocities can be influenced by many factors other than detachment kinetics. Thus a mutation or perturbation that results in a change in V need not result from a change in only d or τon.