Ghost Muscle Fibers Research Articles

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

The molecular mechanism of muscle dysfunction associated with the R133W mutation in Tpm2.2

Ghost muscle fibres reconstituted with myosin heads labeled with the fluorescent probe 1,5-IAEDANS were used for analysis of muscle fibre dysfunction associated with the R133W mutation in β-tropomyosin (Tpm2.2). By using polarized microscopy, we showed that at high Ca2+ the R133W mutation in both αβ-Tpm heterodimers and ββ-Tpm homodimers decreases the amount of the myosin heads strongly bound to F-actin and the number of switched-on actin monomers, with this effect being stronger for ββ-Tpm. This mutation also inhibits the shifting of the R133W-Tpm strands towards the open position and the efficiency of the cross-bridge work. At low Ca2+, the amount of the strongly bound myosin heads is lower for R133W-Tpms than for WT-Tpms which may contribute to a low myofilament Ca2+-sensitivity of the R133W-Tpms. It is concluded that freezing of the mutant αβ- or ββ-Tpm close to the blocked position inhibits the strong binding of the cross-bridges and the switching on of actin monomers which may be the reason for muscle weakness associated with the R133W mutation in β-tropomyosin. The use of reagents that activate myosin may be appropriate to restore muscle function in patients with the R133W mutation.

The primary cause of muscle disfunction associated with substitutions E240K and R244G in tropomyosin is aberrant behavior of tropomyosin and response of actin and myosin during ATPase cycle

Using the polarized photometry technique we have studied the effects of two amino acid replacements, E240K and R244G, in tropomyosin (Tpm1.1) on the position of Tpm1.1 on troponin-free actin filaments and the spatial arrangement of actin monomers and myosin heads at various mimicked stages of the ATPase cycle in the ghost muscle fibres. E240 and R244 are located in the C-terminal, seventh actin-binding period, in f and b positions of the coiled-coil heptapeptide repeat. Actin, Tpm1.1, and myosin subfragment-1 (S1) were fluorescently labeled: 1.5-IAEDANS was attached to actin and S1, 5-IAF was bound to Tpm1.1. The labeled proteins were incorporated in the ghost muscle fibres and changes in polarized fluorescence during the ATPase cycle have been measured. It was found that during the ATPase cycle both mutant tropomyosins occupied a position close to the inner domain of actin. The relative amount of the myosin heads in the strongly-bound conformations and of the switched on actin monomers increased at mimicking different stages of the ATPase cycle. This might be one of the reasons for muscle dysfunction in congenital fibre type disproportion caused by the substitutions E240K and R244G in tropomyosin.

Deviations in conformational rearrangements of thin filaments and myosin caused by the Ala155Thr substitution in hydrophobic core of tropomyosin

Effects of the Ala155Thr substitution in hydrophobic core of tropomyosin Tpm1.1 on conformational rearrangements of the components of the contractile system (Tpm1.1, actin and myosin heads) were studied by polarized fluorimetry technique at different stages of the actomyosin ATPase cycle. The proteins were labelled by fluorescent probes and incorporated into ghost muscle fibres. The substitution violated the blocked and closed states of thin filaments stimulating abnormal displacement of tropomyosin to the inner domains of actin, switching actin on and increasing the relative number of the myosin heads in strong-binding state. Furthermore, the mutant tropomyosin disrupted the major function of troponin to alter the distribution of the different functional states of thin filaments. At low Ca2+ troponin did not effectively switch thin filament off and the myosin head lost the ability to drive the spatial arrangement of the mutant tropomyosin. The information about tropomyosin flexibility obtained from the fluorescent probes at Cys190 indicates that this tropomyosin is generally more rigid, that obviously prevents tropomyosin to bend and adopt the appropriate conformation required for proper regulation.

Abnormal movement of tropomyosin and response of myosin heads and actin during the ATPase cycle caused by the Arg167His, Arg167Gly and Lys168Glu mutations in TPM1 gene

Amino acid substitutions: Arg167His, Arg167Gly and Lys168Glu, located in a consensus actin-binding site of the striated muscle tropomyosin Tpm1.1 (TM), were used to investigate mechanisms of the thin filament regulation. The azimuthal movement of TM strands on the actin filament and the responses of the myosin heads and actin subunits during the ATPase cycle were studied using fluorescence polarization of muscle fibres. The recombinant wild-type and mutant TMs labelled with 5-IAF, 1,5-IAEDANS-labelled S1and FITC-phalloidin F-actin were incorporated into the ghost muscle fibres to acquire information on the orientation of the probes relative to the fibre axis. The substitutions Arg167Gly and Lys168Glu shifted TM strands into the actin filament centre, whereas Arg167His moved TM towards the periphery of the filament. In the presence of Arg167Gly-TM and Lys168Glu-TM the fraction of actin monomers that were switched on and the number of the myosin heads strongly bound to F-actin were abnormally high even under conditions close to relaxation. In contrast, Arg167His-TM decreased the fraction of switched on actin and reduced the formation of strongly bound myosin heads throughout the ATPase cycle. We concluded that the altered TM-actin contacts destabilized the thin filament and affected the actin-myosin interactions.

Myopathy-causing Q147P TPM2 mutation shifts tropomyosin strands further towards the open position and increases the proportion of strong-binding cross-bridges during the ATPase cycle

The molecular mechanisms of skeletal muscle dysfunction in congenital myopathies remain unclear. The present study examines the effect of a myopathy-causing mutation Q147P in β-tropomyosin on the position of tropomyosin on troponin-free filaments and on the actin–myosin interaction at different stages of the ATP hydrolysis cycle using the technique of polarized fluorimetry. Wild-type and Q147P recombinant tropomyosins, actin, and myosin subfragment-1 were modified by 5-IAF, 1,5-IAEDANS or FITC-phalloidin, and 1,5-IAEDANS, respectively, and incorporated into single ghost muscle fibers, containing predominantly actin filaments which were free of troponin and tropomyosin. Despite its reduced affinity for actin in co-sedimentation assay, the Q147P mutant incorporates into the muscle fiber. However, compared to wild-type tropomyosin, it locates closer to the center of the actin filament. The mutant tropomyosin increases the proportion of the strong-binding myosin heads and disrupts the co-operation of actin and myosin heads during the ATPase cycle. These changes are likely to underlie the contractile abnormalities caused by this mutation.

Aberrant movement of β-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle

We have investigated the effect of the E41K, R91G, and E139del β-tropomyosin (TM) mutations that cause congenital myopathy on the position of TM and orientation of actin monomers and myosin heads at different mimicked stages of the ATPase cycle in troponin-free ghost muscle fibers by polarized fluorimetry. A multi-step shifting of wild-type TM to the filament center accompanied by an increase in the amount of switched on actin monomers and the strongly bound myosin heads was observed during the ATPase cycle. The R91G mutation shifts TM further towards the inner and outer domains of actin at the strong- and weak-binding stages, respectively. The E139del mutation retains TM near the inner domains, while the E41K mutation captures it near the outer domains. The E41K and R91G mutations can induce the strong binding of myosin heads to actin, when TM is located near the outer domains. The E139del mutation inhibits the amount of strongly bound myosin heads throughout the ATPase cycle.

The E117K mutation in β-tropomyosin disturbs concerted conformational changes of actomyosin in muscle fibers

The effect of the skeletal myopathy-causing E117K mutation in human β-tropomyosin on actomyosin structure during the ATPase cycle was studied using fluorescent probes bound to actin subdomain 1 and the myosin head. Multistep changes in flexural rigidity of actin filament and in spatial arrangement of actin subdomain 1 and myosin SH1 helix in troponin-free ghost muscle fibers were revealed. During the ATPase cycle E117K tropomyosin inhibited the rotation of subdomain 1 by 46% and the tilt of the SH1 helix by 49% compared with wild-type. At strong-binding stages the proportion of strong binding sub-states in the actomyosin population is decreased by the mutation. At weak-binding stages abnormally high numbers of switched-on actin monomers were observed, thus indicating a disturbance in concerted conformational changes of actomyosin. These structural alterations are likely to underlie the contractile deficit observed with this mutation.

Gly126Arg substitution causes anomalous behaviour of α-skeletal and β-smooth tropomyosins during the ATPase cycle

To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle β-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of β-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and β-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.

The nemaline myopathy-causing E117K mutation in β-tropomyosin reduces thin filament activation

The effect of the nemaline myopathy-causing E117K mutation in β-tropomyosin (TM) on the structure and function of this regulatory protein was studied. The E117K mutant was found to have indistinguishable actin affinity compared with wild-type (WT) and similar secondary structure as measured by circular dichroism. However the E117K mutation significantly lowered maximum activation of actomyosin ATPase. To explain the molecular mechanism of impaired ATPase activation, WT and E117K TMs were covalently labeled at Cys-36 with 5-iodoacetimido-fluorescein and incorporated into ghost muscle fibers. The changes in the position and flexibility of tropomyosin strands on the thin filaments were observed at simulation of weak and strong binding states of actomyosin at high or low Ca2+ by polarized fluorescence techniques. The E117K mutation was found to shift the tropomyosin strands towards the closed position and restrict the tropomyosin displacement during the transformation of actomyosin from weak to strong binding state thus leading to a reduction in thin filament activation.

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle

Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin–myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin–myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into “ghost muscle fibers”. The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

To understand the molecular mechanism by which the hypertrophic cardiomyopathy-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin alter contractile regulation, we labeled recombinant wild type and mutant α-tropomyosins with 5-iodoacetamide-fluorescein and incorporated them into the ghost muscle fibers. The orientation and mobility of the probe were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and mobility of wild type tropomyosin on the thin filaments during the ATP cycle were observed. Both mutations were found to shift tropomyosin strands further towards the open position and to change the affinity of tropomyosin for actin, with the effect of the Glu180Gly mutation being greater than Asp175Asn, showing an increase in the binding strong cross-bridges to actin during the ATPase cycle. These structural changes to the thin filament are likely to underlie the observed increased Ca 2+-sensitivity caused by these mutations which initiates the disease remodeling.

Molluscan twitchin can control actin–myosin interaction during ATPase cycle

The effect of twitchin, a thick filament protein of molluscan muscles, on actin–myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using fluorescent probes specifically bound to Cys707 of myosin subfragment-1 and Cys374 of actin incorporated into ghost muscle fibers. The multi-step changes in mobility and spatial arrangement of myosin SH1 helix and actin subdomain-1 during the ATPase cycle have been revealed. For the first time, the inhibition of movement of myosin SH1 helix and actin subdomain-1 during the ATPase cycle and the decrease in the myosin head and actin affinity in the presence of unphosphorylated twitchin have been demonstrated. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reversed this effect. These data imply a novel property of twitchin consisting in its ability to regulate in a phosphorylation-dependent manner the actin–myosin interaction during the ATPase cycle by the inhibition of transformation of the weak-binding actomyosin states into the strong-binding ones.

EPR as a Tool to Study the Dynamic of Tropomyosin in the Muscle Fiber - Use of a Bifunctional Spin Label

Tropomyosin (Tm), an alpha-helical coiled-coil protein, is a key regulatory protein in muscle contraction. Little is known about the role of Tm dynamics in muscle regulation and more specifically dynamics in the three states of the thin filament. In this work, the flexibility of four different regions of Tm was determined with Saturation Transfer Electron Paramagnetic Resonance (ST-EPR). The use of bi-functional labels allowed us to immobilize the probe and prevent the motion of the label with respect to protein surface. The rotational correlation time of the bi-functional spin label on Tm was 40 ns compared to 25 ns for a conventional mono-dentate spin label. The spin label was attached to i, i+4 positions of the coiled-coil, obtained by cysteine mutagenesis. The bi-functionally labeled Tm di-mutants were reconstituted into “ghost muscle fibers” from which the myosin filaments and intrinsic regulatory proteins (tropomyosin, troponin) were removed. The filaments were reconsituted with Tn and decorated with myosin S1. We found that there is a gradient of flexibility of Tm along its length in the muscle fiber with the C-terminus mutant A268C/E272C being less mobile (two-fold), as compared to the rest of the mutants. Introduction of troponin decreases the flexibility of the four mutants, specifically the two mid-region mutants H153C/D157C and G188C/E192C by 25% and 30% respectively. The addition of calcium did show a decrease in the flexibility of the C terminus mutant A268C/E272C by 20%. The effect of calcium addition on the two other mutants and the effect of addition of S1 on the four mutants were not significant. Thus, although there is a gradient of flexibility in Tm, its dynamics does not change within different states of thin filament activation.

Effect of hypothyroidism on actin subdomain-1 movement induced by myosin subfragment-1 binding in fast and slow rat skeletal muscles

Orientation and mobility of fluorescent probe N-((iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine)(1.5-IAEDANS)) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of myosin subfragment-1 (S1) in intact rats and in the animals with gradual (during 2-5 weeks) reduction of thyroid hormones synthesis (hypothyreosis development). S1 binding to F-actin of ghost muscle fibers was shown to induce changes in orientation of the dipoles of the fluorescent probe 1.5-IAEDANS and in the relative amount of the randomly oriented fluorophores that indicated changes in actin subdomain-1 orientation and mobility resulting from the formation of its strong binding with S1. This effect is markedly inhibited by hypothyreosis development. The maximal effect of hypothyreosis is observed after 34 days of disease development. It is suggested that the change of thyroid status in the muscle inhibits the ability of F-actin to form strong binding with myosin which is essential for force generation.

The effect of the dilated cardiomyopathy-causing mutation Glu54Lys of α-tropomyosin on actin–myosin interactions during the ATPase cycle

In order to understand how the Glu54Lys mutation of α-tropomyosin affects actomyosin interactions, we labeled SH1 helix of myosin subfragment-1 (S1) and the actin subdomain-1 with fluorescent probes. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. The addition of wild-type α-tropomyosin to actin filaments increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of work of each cross-bridge. The Glu54Lys mutation inhibits this effect. The Glu54Lys mutation also results in the coupling of the weak-binding sub-state of S1 to the strong-binding sub-state of actin thus altering the concerted conformational changes during the ATPase cycle. We suggest that these alterations will result in reduced force production, which is likely to underlie at least in part the contractile deficit observed in human dilated cardiomyopathy.

Dilated cardiomyopathy mutations in α-tropomyosin inhibit its movement during the ATPase cycle

The Glu40Lys and Glu54Lys mutations in α-tropomyosin cause dilated cardiomyopathy (DCM). Functional analysis has demonstrated that both mutations decrease thin filament Ca 2+-sensitivity and that Glu40Lys reduces maximum activation. To understand the molecular mechanism underlying these changes, we labeled wild type α-tropomyosin and both mutants at Cys190 with 5-iodoacetamide-fluorescein and incorporated the labeled proteins into ghost muscle fibers. Using the polarized fluorimetry, the position of the labeled tropomyosins on the thin filament and their affinity for actin were measured and the change in these parameters at different stages of the ATPase cycle determined. Both DCM mutations were found to shift tropomyosin towards the periphery of thin filament and to change the affinity of tropomyosin for actin; during the ATPase cycle the amplitude of tropomyosin movement was reduced and at some stages of the cycle even reversed. The correlation of these structural changes with the observed function effects is discussed.

Dynamics of Bi-Functional Labeled Tropomyosin in Muscle Ghost Fiber Monitored by Saturation Transfer EPR

Tropomyosin (Tm), an alpha-helical coiled-coil protein, is a key regulatory protein in muscle contraction. To date, little is known about the extent of Tm flexibility and the role of Tm dynamics in muscle regulation. In this work, the flexibility of two different regions of Tm was assessed using Saturation Transfer Electron Paramagnetic Resonance (ST EPR). In order to fully immobilize the spin probe on the surface of Tm we used a bi-functional spin label attached to i, i+4 positions of the coiled-coil obtained by cysteine mutagenesis. We have used conventional EPR and ST-EPR to detect wide range of dynamics from the very slow (millisecond) motions to fast sub-nanosecond modes. The labeled Tm mutants were reconstituted into “ghost muscle fibers” from which the myosin filaments and intrinsic regulatory proteins (tropomyosin, troponin) were removed.ST-EPR of the two mid-region mutants Tm H153C/D157C and Tm G188C/E192C as well as the C-terminus mutant Tm A268C/E272 gave a correlation time of 10.5 us ± 4.5 us, 42.5 us ± 27.5 us, and 42.5 us ± 27.5 us respectively (using H”/H and L”/L ratios of V'2 spectra). The difference in correlation time between the different di-mutants is an indication of the differential flexibility of the Tm protein. The study of the N-terminus (L13C/N17C) di-mutant will give us an additional understanding of Tm flexibility. Finally the introduction of Troponin complex (Tn) as well as S1 head of myosin under high and low calcium concentrations will give a complete picture of the dynamics of Tm in muscle regulation.