Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle.

Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This "interacting-heads motif" (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3-4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3-4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.

Mn2+ -Phos-Tag Polyacrylamide for the Quantification of Protein Phosphorylation Levels.

This paper provides a guideline for optimizing and utilizing Mn2+ Phos-tag gel technology to separate phosphorylated proteins from their unphosphorylated counterparts. It provides key insights into methods for careful sample preparation and experimental directions for determining the appropriate Phos-tag gel compositions and electrophoresis and western blotting conditions. This protocol has been used to successfully resolve proteins extracted from cardiac and skeletal muscles. The guidelines can be extended for optimizing protocols to resolve proteins from other cells or tissue sources. With this, phosphoproteomics and the elucidation of underlying mechanisms of disease progression can be accelerated. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

Regulatory Light Chains in Cardiac Development and Disease.

The role of regulatory light chains (RLCs) in cardiac muscle function has been elucidated progressively over the past decade. The RLCs are among the earliest expressed markers during cardiogenesis and persist through adulthood. Failing hearts have shown reduced RLC phosphorylation levels and that restoring baseline levels of RLC phosphorylation is necessary for generating optimal force of muscle contraction. The signalling mechanisms triggering changes in RLC phosphorylation levels during disease progression remain elusive. Uncovering this information may provide insights for better management of heart failure patients. Given the cardiac chamber-specific expression of RLC isoforms, ventricular RLCs have facilitated the identification of mature ventricular cardiomyocytes, opening up possibilities of regenerative medicine. This review consolidates the standing of RLCs in cardiac development and disease and highlights knowledge gaps and potential therapeutic advancements in targeting RLCs.

Functional and Molecular Characterisation of Heart Failure Progression in Mice and the Role of Myosin Regulatory Light Chains in the Recovery of Cardiac Muscle Function.

Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes (i.e., isometric force, calcium sensitivity and sarcomeric protein phosphorylation) was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca2+ sensitivity measurement, while the LV myocardium was used to assay sarcomeric proteins' (RLC, troponin I (TnI) and myosin binding protein-C (MyBP-C)) phosphorylation levels and enzyme (myosin light chain kinase (MLCK), zipper interacting protein kinase (ZIPK) and myosin phosphatase target subunit 2 (MYPT2)) expression levels. Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.

How cognitive engagement fluctuates during a team-based learning session and how it predicts academic achievement.

The objective of the paper is to report findings of two studies that attempted to find answers to the following questions: (1) What are the levels of cognitive engagement in TBL? (2) Are there differences between students who were more exposed to TBL than students who were less exposed to TBL? (3) To which extent does cognitive engagement fluctuate as a function of the different activities involved in TBL? And (4) How do cognitive engagement scores collected over time correlate with each other and with academic achievement? The studies were conducted with Year-1 and -2 medical students enrolled in a TBL curriculum (N = 175, 62 female). In both studies, six measurements of cognitive engagement were taken during the distinct TBL activities (preparation phase, individual/team readiness assurance test, burning questions, and application exercises). Data were analysed by means of one-way repeated-measures ANOVAs and path modelling. The results of the repeated-measures ANOVA revealed that cognitive engagement systematically fluctuated as a function of the distinct TBL activities. In addition, Year-1 students reported significantly higher levels of cognitive engagement compared to Year-2 students. Finally, cognitive engagement was a significant predictor of performance (ß = .35). The studies presented in this paper are a first attempt to relate the different activities undertaken in TBL with the extent to which they arouse cognitive engagement with the task at hand. Implications of these findings for TBL are discussed.

The Closed State of the Thin Filament Is Not Occupied in Fully Activated Skeletal Muscle.

Muscle contraction is powered by actin-myosin interaction controlled by Ca2+ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 µm length of a thin filament. In the absence of Ca2+, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca2+ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15-0.275 nm-1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.

Tropomyosin movement is described by a quantitative high-resolution model of X-ray diffraction of contracting muscle.

Contraction of skeletal and cardiac muscle is controlled by Ca2+ ions via regulatory proteins, troponin (Tn) and tropomyosin (Tpm) associated with the thin actin filaments in sarcomeres. In the absence of Ca2+, Tn-C binds actin and shifts the Tpm strand to a position where it blocks myosin binding to actin, keeping muscle relaxed. According to the three-state model (McKillop and Geeves Biophys J 65:693-701, 1993), upon Ca2+ binding to Tn, Tpm rotates about the filament axis to a 'closed state' where some myosin heads can bind actin. Upon strong binding of myosin heads to actin, Tpm rotates further to an 'open' position where neighboring actin monomers also become available for myosin binding. Azimuthal Tpm movement in contracting muscle is detected by low-angle X-ray diffraction. Here we used high-resolution models of actin-Tpm filaments based on recent cryo-EM data for calculating changes in the intensities of X-ray diffraction reflections of muscle upon transitions between different states of the regulatory system. Calculated intensities of actin layer lines provide a much-improved fit to the experimental data obtained from rabbit muscle fibers in relaxed and rigor states than previous lower-resolution models. We show that the intensity of the second actin layer line at reciprocal radii from 0.15 to 0.3 nm-1 quantitatively reports the transition between different states of the regulatory system independently of the number of myosin heads bound to actin.

Revisiting Frank-Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr ) in a length-dependent fashion.

KEY POINTS: Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (ktr ) at submaximal [Ca(2+) ]. Increasing RLC phosphorylation ∼50% from the in vivo level in maximally [Ca(2+) ]-activated cardiac trabecula accelerates ktr . Decreasing RLC phosphorylation to ∼70% of the in vivo control level slows ktr and reduces force generation. ktr is dependent on sarcomere length in the physiological range 1.85-1.94 µm and RLC phosphorylation modulates this response. We demonstrate that Frank-Starling is evident at maximal [Ca(2+) ] activation and therefore does not necessarily require length-dependent change in [Ca(2+) ]-sensitivity of thin filament activation. The stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank-Starling law in the heart. These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. ABSTRACT: Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin-associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (ktr ) in maximally activating [Ca(2+) ]. Activation was achieved by rapidly increasing the temperature (temperature-jump of 0.5-20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85-1.94 µm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC-exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates ktr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows ktr by ∼50%, relative to the ktr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of ktr following step shortening or ramp shortening. Using a two-state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross-bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca(2+) ] and are not explained by changes in the Ca(2+) -sensitivity of acto-myosin interactions. The length-dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank-Starling law of the heart.

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.

Instrumentation to study myofibril mechanics from static to artificial simulations of cardiac cycle.

Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable myofibrils can be obtained from frozen tissue.•We are describing a versatile technique for measuring the contractility and its Ca(2+) regulation in single myofibrils. The control of myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently.•This single myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca(2+) dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.

Phosphorylation of the regulatory light chain of myosin in striated muscle: methodological perspectives.

Phosphorylation of the regulatory light chain (RLC) of myosin modulates cellular functions such as muscle contraction, mitosis, and cytokinesis. Phosphorylation defects are implicated in a number of diseases. Here we focus on striated muscle where changes in RLC phosphorylation relate to diseases such as hypertrophic cardiomyopathy and muscular dystrophy, or age-related changes. RLC phosphorylation in smooth muscle and non-muscle cells are covered briefly where relevant. There is much scientific interest in controlling the phosphorylation levels of RLC in vivo and in vitro in order to understand its physiological function in striated muscles. A summary of available and emerging in vivo and in vitro methods is presented. The physiological role of RLC phosphorylation and novel pathways are discussed to highlight the differences between muscle types and to gain insights into disease processes.

The dilated cardiomyopathy-causing mutation ACTC E361G in cardiac muscle myofibrils specifically abolishes modulation of Ca(2+) regulation by phosphorylation of troponin I.

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca(2+) sensitivity and increases the rate of Ca(2+) release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca(2+)-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca(2+) regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca(2+) sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca(2+) sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca(2+) sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca(2+) sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca(2+) sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.

Why muscle is an efficient shock absorber.

Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.

Mechanical and energetic properties of papillary muscle from ACTC E99K transgenic mouse models of hypertrophic cardiomyopathy.

We compared the contractile performance of papillary muscle from a mouse model of hypertrophic cardiomyopathy [α-cardiac actin (ACTC) E99K mutation] with nontransgenic (non-TG) littermates. In isometric twitches, ACTC E99K papillary muscle produced three to four times greater force than non-TG muscle under the same conditions independent of stimulation frequency and temperature, whereas maximum isometric force in myofibrils from these muscles was not significantly different. ACTC E99K muscle relaxed slower than non-TG muscle in both papillary muscle (1.4×) and myofibrils (1.7×), whereas the rate of force development after stimulation was the same as non-TG muscle for both electrical stimulation in intact muscle and after a Ca²âº jump in myofibrils. The EC50 for Ca²âº activation of force in myofibrils was 0.39 ± 0.33 µmol/l in ACTC E99K myofibrils and 0.80 ± 0.11 µmol/l in non-TG myofibrils. There were no significant differences in the amplitude and time course of the Ca²âº transient in myocytes from ACTC E99K and non-TG mice. We conclude that hypercontractility is caused by higher myofibrillar Ca²âº sensitivity in ACTC E99K muscles. Measurement of the energy (work + heat) released in actively cycling heart muscle showed that for both genotypes, the amount of energy turnover increased with work done but with decreasing efficiency as energy turnover increased. Thus, ACTC E99K mouse heart muscle produced on average 3.3-fold more work than non-TG muscle, and the cost in terms of energy turnover was disproportionately higher than in non-TG muscles. Efficiency for ACTC E99K muscle was in the range of 11-16% and for non-TG muscle was 15-18%.

Myosin regulatory light chain (RLC) phosphorylation change as a modulator of cardiac muscle contraction in disease.

Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.

Stretch of contracting cardiac muscle abruptly decreases the rate of phosphate release at high and low calcium.

The contractile performance of the heart is linked to the energy that is available to it. Yet, the heart needs to respond quickly to changing demands. During diastole, the heart fills with blood and the heart chambers expand. Upon activation, contraction of cardiac muscle expels blood into the circulation. Early in systole, parts of the left ventricle are being stretched by incoming blood, before contraction causes shrinking of the ventricle. We explore here the effect of stretch of contracting permeabilized cardiac trabeculae of the rat on the rate of inorganic phosphate (P(i)) release resulting from ATP hydrolysis, using a fluorescent sensor for P(i) with millisecond time resolution. Stretch immediately reduces the rate of P(i) release, an effect observed both at full calcium activation (32 µmol/liter of Ca(2+)), and at a physiological activation level of 1 µmol/liter of Ca(2+). The results suggest that stretch redistributes the actomyosin cross-bridges toward their P(i)-containing state. The redistribution means that a greater fraction of cross-bridges will be poised to rapidly produce a force-generating transition and movement, compared with cross-bridges that have not been subjected to stretch. At the same time stretch modifies the P(i) balance in the cytoplasm, which may act as a cytoplasmic signal for energy turnover.

Mutations of ventricular essential myosin light chain disturb myosin binding and sarcomeric sorting.

AIMS: We tested the hypothesis that mutations in the human ventricular essential myosin light chain (hVLC-1) that are associated with hypertrophic cardiomyopathy (HCM) affect protein structure, binding to the IQ1 motif of cardiac myosin heavy chain (MYH) and sarcomeric sorting in neonatal cardiomyocytes. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties and protein-protein interactions of a recombinant head-rod fragment of rat cardiac ß-MYH (amino acids 664-915) with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ2(ala4)) and normal or five mutated (M149V, E143K, A57G, E56G, R154H) hVLC-1 forms. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced normal and E56G-mutated (hVLC-1(E56G)) hVLC-1 constructs in neonatal rat cardiomyocytes. Fluorescence lifetime imaging microscopy was applied to map the microenvironment of normal and E56G-mutated hVLC-1 in permeabilized muscle fibres. Affinity of M149V, E143K, A57G, and R154H mutated hVLC-1/rß-MYH(664-915)IQ2(ala4) complexes was significantly lower compared with the normal hVLC-1/rß-MYH(664-915)IQ2(ala4) complex interaction. In particular, the E56G mutation induced an ∼30-fold lower MYH affinity. Sorting specificity of E56G-mutated hVLC-1 was negligible compared with normal hVLC-1. Fluorescence lifetime of fibres replaced with hVLC-1(E56G) increased significantly compared with hVLC-1-replaced fibres. CONCLUSION: Disturbed myosin binding of mutated hVLC-1 may provide a pathomechanism for the development of HCM.

Millisecond-scale biochemical response to change in strain.

Muscle fiber contraction involves the cyclical interaction of myosin cross-bridges with actin filaments, linked to hydrolysis of ATP that provides the required energy. We show here the relationship between cross-bridge states, force generation, and Pi release during ramp stretches of active mammalian skeletal muscle fibers at 20°C. The results show that force and Pi release respond quickly to the application of stretch: force rises rapidly, whereas the rate of Pi release decreases abruptly and remains low for the duration of the stretch. These measurements show that biochemical change on the millisecond timescale accompanies the mechanical and structural responses in active muscle fibers. A cross-bridge model is used to simulate the effect of stretch on the distribution of actomyosin cross-bridges, force, and Pi release, with explicit inclusion of ATP, ADP, and Pi in the biochemical states and length-dependence of transitions. In the simulation, stretch causes rapid detachment and reattachment of cross-bridges without release of Pi or ATP hydrolysis.

Semi-automated analysis of organelle movement and membrane content: understanding rab-motor complex transport function.

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.

The fraction of myosin motors that participate in isometric contraction of rabbit muscle fibers at near-physiological temperature.

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca(2+) at 4°C were heated to 31-34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41-43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ~6.3 pN.