Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states

Lys-553 of skeletal muscle myosin subfragment 1 (S1) was specifically labeled with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) and fluorescence quenching experiments were carried out to determine the accessibility of this probe at Lys-553 in both the strongly and weakly actin-bound states of the MgATPase cycle. Solvent quenchers of varying charge [nitromethane, (2,2,6, 6-tetramethyl-1-piperinyloxy) (TEMPO), iodide (I(-)), and thallium (Tl(+))] were used to assess both the steric and electrostatic accessibilities of the FHS probe at Lys-553. In the strongly bound rigor (nucleotide-free) and MgADP states, actin offered no protection from solvent quenching of FHS by nitromethane, TEMPO, or thallium, but did decrease the Stern-Volmer constant by almost a factor of two when iodide was used as the quencher. The protection from iodide quenching was almost fully reversed with the addition of 150 mM KCl, suggesting this effect is ionic in nature rather than steric. Conversely, actin offered no protection from iodide quenching at low ionic strength during steady-state ATP hydrolysis, even with a significant fraction of the myosin heads bound to actin. Thus, the lower 50 kD subdomain of myosin containing Lys-553 appears to interact differently with actin in the weakly and strongly bound states.     

Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loop of actin examined by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) experiments were carried out in the absence of nucleotide (rigor) or in the presence of MgADP between fluorescent donor probes (IAEDANS (5((((2-iodoacetyl)amino)ethyl)amino)-naphthalene-1-sulfonic acid) at Cys-374 or DANSYL (5-dimethylamino naphthalene-1-(N-(5-aminopentyl))sulfonamide) at Gln-41 of actin and acceptor molecules (FHS (6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester) at Lys-553 of skeletal muscle myosin subfragment 1. The critical F?rster distance (R(0)) was determined to be 44 and 38 A for the IAEDANS-FHS and DANSYL-FHS donor-acceptor pairs, respectively. The efficiency of energy transfer between the acceptor molecules at Lys-553 of myosin and donor probes at Cys-374 or Gln-41 of actin was calculated to be 0.78 +/- 0.01 or 0.94 +/- 0.01, respectively, corresponding to distances of 35.6 +/- 0.4 A and 24.0 +/- 1.6 A, respectively. MgADP had no significant effect on the distances observed in rigor. Thus, rearrangements in the acto-myosin interface are likely to occur elsewhere than in the lower 50-kDa subdomain of myosin as its affinity for actin is weakened by MgADP binding.     

Alteration of actin-tropomyosin interaction in 2,4-pentanedione-treated rabbit skeletal myofibrils

In previous work, we (El-Saleh, S., Theiret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259, 11014-11021) presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin X Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn X Tm), on acto-heavy meromyosin X Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn X Tm free ("desensitized") myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 nM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD X myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa greater than 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn X Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn X Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that is is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin X Tm interaction following modification of myofibrillar actin most likely at Lys-237.     

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.     

Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges

We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time range from 0.5 to 200 microsecond, indicating that they do not undergo rotational motion in this time range. In the presence of 5 mM MgATP, however, heads undergo complex rotational motion with correlation times of about 5 and 40 microsecond. The motion of heads in relaxed myofibrils is restricted out to 1 ms, as indicated by a nonzero value of the residual anisotropy. The anisotropy decay of eosin-labeled myosin, extracted from labeled myofibrils, also exhibits complex decay on the 200-microsecond time scale when assembled into synthetic thick filaments. The correlation times and amplitudes of heads in filaments (under the same ionic conditions as the myofibril experiments) are unaffected by MgATP and very similar to the values for heads in relaxed myofibrils. The larger residual anisotropy and longer correlation times seen in myofibrils are consistent with a restriction of rotational motion in the confines of the myofibril protein lattice. These are the first time-resolved measurements under physiological conditions of the rotational motions of cross-bridges in the microsecond time range.     

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1.

Bertrand et al. [Bertrand, R., Derancourt, J. & Kassab, R. (1995) Biochemistry 34, 9500-9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the epsilon-amino group of Lys553 reacts with FHS. Addition of 0.4 M KCl reduced the rate of reaction significantly, which indicates ionic strength-dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP.BeF(x), ADP.AlF(4), ADP.V(i) and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M(**)ADP.P(i) state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1.ADP.AlF(4)(-) and S1.ADP.V(i) complexes, which are believed to mimic the M(**)ADP.P(i) state. This indicates that the conformation of the S1-ADP.AlF(4)(-) and S1.ADP.V(i) complexes in the vicinity of Lys553 does not resemble the structure of the M(**)ADP.P(i) state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.     

Effect of nucleotide on interaction of the 567-578 segment of myosin heavy chain with actin

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.     

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.     

Nucleotide turnover rate measured in fully relaxed rabbit skeletal muscle myofibrils

Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 microM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 +/- 0.001 s-1 at 25 degrees C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5 degrees and 42 degrees C, reaching 0.16 +/- 0.04 s-1 and 0.06 +/- 0.005 s-1, respectively, at 39 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.     

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.     

Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Tryptic digestion of rabbit skeletal myofibrils at physiological ionic strength and pH results in cleavage of the myosin heavy chain at one site giving two bands (M_r = 200,000 and 26,000) on sodium dodecyl sulfate/polyacrylamide gels. Following addition of sodium pyrophosphate (to 1 mm) to dissociate the myosin heads from actin, tryptic proteolysis results in production of three bands, 160K², 51K and 26K, with a 74K band appearing as a precursor of the 51K and 26K species. Under these conditions, there is insignificant cleavage of heavy chain to the heavy and light meromyosins. Trypsin-digested myofibrils yield the same amount of rod as native myofibrils when digested with papain. These results indicate that actin blocks tryptic cleavage of the myosin heavy chain at a site 74K from the N terminus. From measurements of the amount of 51K species formed by digestion of rigor fibers at various sarcomere lengths, we estimate that at least 95% of the myosin heads are bound to actin at 100% overlap of thick and thin filaments. Hence all myosin molecules can bind to actin, and consequently both heads of a myosin molecule can interact simultaneously with actin filaments under rigor conditions.     

Direct modeling of X-ray diffraction pattern from contracting skeletal muscle

A direct modeling approach was used to quantitatively interpret the two-dimensional x-ray diffraction patterns obtained from contracting mammalian skeletal muscle. The dependence of the calculated layer line intensities on the number of myosin heads bound to the thin filaments, on the conformation of these heads and on their mode of attachment to actin, was studied systematically. Results of modeling are compared to experimental data collected from permeabilized fibers from rabbit skeletal muscle contracting at 5°C and 30°C and developing low and high isometric tension, respectively. The results of the modeling show that: i), the intensity of the first actin layer line is independent of the tilt of the light chain domains of myosin heads and can be used as a measure of the fraction of myosin heads stereospecifically attached to actin; ii), during isometric contraction at near physiological temperature, the fraction of these heads is ∼40% and the light chain domains of the majority of them are more perpendicular to the filament axis than in rigor; and iii), at low temperature, when isometric tension is low, a majority of the attached myosin heads are bound to actin nonstereospecifically whereas at high temperature and tension they are bound stereospecifically.     

Generation of force by single-headed myosin.

Myosin has two heads, each of which can interact with actin and ATP. We have investigated the possibility that co-operative interactions occur between the heads by measuring the force generated by single-headed myosin in reconstituted actomyosin threads. Myofibrils were digested with papain, actomyosin was extracted from the myofibrils, and one-headed myosin was purified by cycles of sedimentation with actin. The one-headed myosin was approximately 90 to 95% pure as determined by densitometer scans of polyacrylamide gels run in 20 mm-PP₁ (impurities consisted of 1 to 5% of myosin and 1 to 5% myosin rod). The ATPase activity per mole of single-headed myosin was one half that of myosin under conditions where the activity was activated by Ca²⁺, K⁺ or actin. One-headed myosin could also participate in superprecipitation, although with a rate that was at least one order of magnitude slower than that for myosin. Myosin or one-headed myosin was mixed with actin, threads were formed via extrusion into low ionic strength, and the isometric forces and isotonic velocities generated by the threads were measured. The ratio of the isometric tension produced per head by the one-headed myosin to the isometric tension produced per head by myosin was 1·0 ± 0·1. The maximum velocity of thread contraction for the one-headed myosin was also not different from the control myosin. Thus, the absence of one head does not appear to impair the generation of force or motion by the remaining head.     

An electrostatic model with weak actin-myosin attachment resolves problems with the lattice stability of skeletal muscle

The stability of the filament lattice in relaxed striated muscle can be viewed as a balance of electrostatic and van der Waals forces. The simplest electrostatic model, where actin and myosin filaments are treated as charged cylinders, generates reasonable lattice spacings for skinned fibers. However, this model predicts excessive radial stiffness under osmotic pressure and cannot account for the initial pressure (∼1 kPa) required for significant compression. Good agreement with frog compression data is obtained with an extended model, in which S1 heads are weakly attached to actin when the lattice spacing is reduced below a critical value; further compression moves fixed negative charges on the heads closer to the myofilament backbone as they attach at a more acute angle to actin. The model predicts pH data in which the lattice shrinks as pH is lowered and protons bind to filaments. Electrostatic screening implies that the lattice shrinks with increasing ionic strength, but the observed expansion of the frog lattice at ionic strengths above 0.1 M with KCl might be explained if Cl⁻ binds to sites on the motor domain of S1. With myosin-myosin and actin-actin interactions, the predicted lattice spacing decreases slightly with sarcomere length, with a more rapid decrease when actin-myosin filament overlap is very small.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Stepping between Membrane Microdomains

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P₂〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.     

Native connectin from porcine cardiac muscle

Native connectin was isolated from porcine cardiac muscle using the method developed for the preparation of native connectin from chicken breast muscle (Kimura et al. (1984) J. Biochem. 96, 1947-1950). It was not necessary to keep cardiac muscle at 0 degrees C before preparation: the proteolysis of alpha-connectin to beta-connectin proceeded during the preparation of myofibrils. Cardiac connectin showed almost the same properties as those of skeletal muscle connectin: mobility in SDS gel electrophoresis, filamentous structure under an electron microscope, circular dichroism spectra, UV absorption spectra, and amino acid composition. Porcine cardiac connectin cross-reacted with antiserum against chicken breast muscle connectin as revealed by an immunoblot method. Immunoelectron microscopical observations revealed an abundance of connectin antigenic sites around the A-I junction area of cardiac myofibrils. Cardiac connectin also interacted with myosin and actin filaments at low ionic strengths to form aggregates. The extent of interaction was somewhat weaker in the case of cardiac connectin than skeletal muscle connectin, regardless of the origin of myosin and actin (porcine cardiac and rabbit skeletal muscles). In conclusion, cardiac connectin is very similar, but not identical to skeletal muscle connectin.     

The effects of the interaction of myosin essential light chain isoforms with actin in skeletal muscles

In order to compare the ability of different isoforms of myosin essential light chain to interact with actin, the effect of the latter protein on the proteolytic susceptibility of myosin light chains (MLC-1S and MLC-1V - slow specific and same as ventricular isoform) from slow skeletal muscle was examined. Actin protects both slow muscle essential light chain isoforms from papain digestion, similarly as observed for fast skeletal muscle myosin (Nieznanska et al., 1998, Biochim. Biophys. Acta 1383: 71). The effect of actin decreases as ionic strength rises above physiological values for both fast and slow skeletal myosin, confirming the ionic character of the actin-essential light chain interaction. To better understand the role of this interaction, we examined the effect of synthetic peptides spanning the 10-amino-acid N-terminal sequences of myosin light chain 1 from fast skeletal muscle (MLC-1F) (MLCFpep: KKDVKKPAAA), MLC-1S (MLCSpep: KKDVPVKKPA) and MLC-1V (MLCVpep: KPEPKKDDAK) on the myofibrillar ATPase of fast and slow skeletal muscle. In the presence of MLCFpep, we observed an about 19% increase, and in the presence of MLCSpep about 36% increase, in the myofibrillar ATPase activity of fast muscle. On the other hand, in myofibrillar preparations from slow skeletal muscle, MLCSpep as well as MLCVpep caused a lowering of the ATPase activity by about 36%. The above results suggest that MLCSpep induces opposite effects on ATPase activity, depending on the type of myofibrils, but not through its specific N-terminal sequence - which differs from other MLC N-terminal peptides. Our observations lead to the conclusion that the action of different isoforms of long essential light chain is similar in slow and fast skeletal muscle. However the interaction of essential light chains with actin leads to different physiological effects probably depending on the isoforms of other myofibrillar proteins.     

A Mathematical Analysis of Obstructed Diffusion within Skeletal Muscle

Molecules are transported through the myofilament lattice of skeletal muscle fibers during muscle activation. The myofilaments, along with the myosin heads, sarcoplasmic reticulum, t-tubules, and mitochondria, obstruct the diffusion of molecules through the muscle fiber. In this work, we studied the process of obstructed diffusion within the myofilament lattice using Monte Carlo simulation, level-set and homogenization theory. We found that these intracellular obstacles significantly reduce the diffusion of material through skeletal muscle and generate diffusion anisotropy that is consistent with experimentally observed slower diffusion in the radial than the longitudinal direction. Our model also predicts that protein size has a significant effect on the diffusion of material through muscle, which is consistent with experimental measurements. Protein diffusion on the myofilament lattice is also anomalous (i.e., it does not obey Brownian motion) for proteins that are close in size to the myofilament spacing. The obstructed transport of Ca²⁺ and ATP-bound Ca²⁺ through the myofilament lattice also generates smaller Ca²⁺ transients. In addition, we used homogenization theory to discover that the nonhomogeneous distribution in the troponin binding sites has no effect on the macroscopic Ca²⁺ dynamics. The nonuniform sarcoplasmic reticulum Ca²⁺-ATPase pump distribution also introduces small asymmetries in the myoplasmic Ca²⁺ transients.     

Mavacamten has a differential impact on force generation in myofibrils from rabbit psoas and human cardiac muscle

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca²⁺-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca²⁺. Altogether, these effects outline a motor isoform–specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.