Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation

Figure 1. Effect of the E173A mutation in γ- tropomyosin (Tpm) on sensitivity of the thin filaments to activating Ca2+ concentrations. Ca2+-dependence was determined for fully regulated reconstituted thin filaments. The acto-S1 ATPase was measured in the presence of wild-type (WT) Tpm (squares), and E173A-Tpm (stars) at 25 °C. Error bars indicate ±SEM. pCa values were calculated from data averaged from 3 experiments. Conditions are given in Section 3.

Figure 2. Verification of the incorporation of recombinant Tpm into the ghost muscle fibres. The SDS-PAGE shows the results of electrophoretic separation of muscle fibre isolated from m. psoas of rabbit (lanes 1 and 4), fibres after extraction of myosin and regulatory proteins of thin filaments (ghost fibres, lane 2), ghost fibres reconstituted with the recombinant wild-type (lane 6) and mutant (lane 8) tropomyosin incorporated in thin filaments, and the recombinant tropomyosin preparations per se (lanes 5 and 7) as well as molecular weight markers (Sigma-Aldrich, St. Louis, MO, USA, lane 3). Designations: MyBP-C—myosin-binding protein C; LC1 and LC3—myosin light chains; Tpm1.1, Tpm2.2 and Tpm3.12—tropomyosin isoforms; troponin (TN)-T, TN-I and TN-C—troponin subunits. Scanning densitometry (Image Lab 6.0) revealed that ghost fibres consist of 71% F-actin. The quantitative assessment of the Tpm binding to actin filaments in ghost fibres showed the content of the mutant Tpm bound to actin is not less than that of the WT-Tpm. Thus, the mutant Tpm can incorporate within the thin filaments in vivo. The electrophoretic mobility of the mutant Tpm differs significantly from WT-Tpm, that was…

Figure 3. The effect of troponin and Ca2+ on the values of ФЕ (a,b) of the polarized fluorescence for 5-iodoacetamidofluorescein (5-IAF) linked with WT-Tpm and E173A-Tpm was revealed in glycerol-skinned fibres under conditions simulating the sequential steps of the actomyosin ATPase cycle. The relative amount of the WT and mutant Tpms, as well as the amount of S1 in all experiments were monitored by measuring the fluorescence intensity of proteins in the actin-WT-Tpm-S1 and actin-E173A-Tpm-S1 complexes. When simulating rigor and in the presence of Mg-adenosine 5′-diphosphate (MgADP), the molar ratio of S1 to actin was 1:(2.4 ± 0.6) and the molar ratio of tropomyosin to actin was 1:(7.0 ± 0.5). In the presence of Mg-adenosine 5′-triphosphate (MgATP), a reduction in the molar ratio of S1 to actin was seen, which was similar for the WT and mutant Tpms (Section 3). It is supposed that the same fraction of actin monomers bound S1 in each case. The values of ФЕ were corrected in order to take into account the changes in conformation of actin monomers. The first and second entries from the left in each panel present the data obtained in the absence of S1. The data represent the mean values for…

Figure 4. A schematic view of the changes in tropomyosin localization and spatial rearrangements of actin caused by the E173A substitution in Tpm, as follows from the data of polarized fluorimetry. The changes in the value for ΦE (the angle of orientation of the emission dipoles of the probe bound to Tpm or actin monomers) are interpreted as the shift of Tpm relative to the inner and outer domains of actin and the rotation of actin monomers in actin filament. The changes in the value for bending stiffness ε are shown as the alterations in the persistence length of the Tpm strands and actin filament. Ghost fibres were composed of actin and Tpm (A,B) or of actin, Tpm and TN (C,D). (A) The position of the mutant Tpm and actin conformation in the region of the dye binding do not differ from that of the wild-type (WT)-Tpm in ghost fibres containing only Tpm. (B) However, the mutation decreases the persistence length of Tpm and increases that of actin. (C) The effect of the mutation emerges in the presence of troponin—at low Ca2+ Tpm shifts towards the inner domains of actin and actin monomers switch on. (D) The persistence length is lower for the mutant Tpm than for the WT-Tpm. The increase in…

Figure 5. The effect of troponin and Ca2+ on the values of ΦE (a) and ɛ (b) of the polarized fluorescence for FITC-phalloidin-actin, in the presence of WT-Tpm or E173A-Tpm under conditions simulating the sequential steps of the myosin ATPase cycle. The first and second entries from the left in each panel present the data obtained in the absence of S1. The relative amount of the WT and mutant Tpms, as well as the amount of S1 in all experiments was similar in the Actin-WT-Tpm-S1 and Actin-E173A-Tpm-S1 complexes (see Section 3), suggesting that the same fraction of actin monomers bound S1 in each case. Calculations of ΦE and ɛ values, preparation of the fibres, their composition and the conditions of the experiments are described in Section 3. The data represent the mean values for 8–10 fibres for each experimental condition. Both in the absence and in the presence of nucleotides the ΦE and ɛ values for WT-Tpm and E173A-Tpm are significantly altered by troponin and Ca2+ (p < 0.05). Statistically insignificant differences in the values of ΦE, ɛ and N between WT and mutant Tpms are indicated by the symbol “ns”. Error bars indicate ±SEM. The values of N were close to 0.1.

Figure 6. A schematic view of the changes in tropomyosin localization and spatial rearrangements of actin and S1, as follows from the data of polarized fluorimetry. The changes in the value for ΦE (the angle of orientation of the emission dipoles of the probe bound to Tpm, actin monomers or the myosin heads) at simulation of different conformational states of Actin-Tpm-TN-S1 complex are interpreted as the shift of Tpm relative to the inner and outer domains of actin, the rotation of actin monomers in actin filament and the azimuthal tilt of the myosin motor domain (a,c,d,g). The left and right panels show the transition of actin-myosin from the state in the absence of nucleotide to the state in the presence of ATP at high and low Ca2+ concentrations, respectively. The changes in the value of bending stiffness ε are shown as the alterations in the persistence length of Tpm strands and actin filament (b,f). The effect of the E173A mutation in Tpm is discussed in the text. Briefly, the mutant Tpms shift towards the inner domains of actin, which cause the abnormal switching on of actin monomers and the appearance of the myosin heads strongly bound with actin under relaxing conditions…

Figure 7. The effect of troponin and Ca2+ on the values of ΦE (a), ɛ (b) and N (c) of the polarized fluorescence of N-(iodoacetaminoethyl)-1-naphthyl-amine-5-sulfonic acid (1,5-IAEDANS) bound to S1 (AEDANS-S1), in the presence of WT-Tpm or E173A-Tpm was revealed in glycerol-skinned fibres under conditions simulating the sequential steps of the actomyosin ATPase cycle. The data represent the mean values for 5–7 fibres for each experimental condition. The ΦE, ɛ and N values in the absence of troponin and in the presence of Ca2+ and nucleotides are significantly altered by the E173A mutation in Tpm (p < 0.05). Statistically insignificant differences in the values of ΦE, ɛ and N between the WT and mutant Tpms are indicated by the symbol “ns”. Error bars indicate ±SEM.

Figure 8. The effect of troponin and Ca2+ on the values of ΦE (a), ɛ (b) and N (c) of the polarized fluorescence of 1,5-IAEDANS bound to S1 (AEDANS-S1), in the presence of the E173A-Tpm or E173A-Tpm with 50 μM of W7 revealed in glycerol-skinned fibres under conditions simulating the sequential steps of the actomyosin ATPase cycle. The data represent the mean values for 5–7 fibres for each experimental condition. The ΦE, ɛ and N values in the presence of troponin are significantly altered by W7 (p < 0.05). Statistically insignificant differences in the values of ΦE, ɛ and N between the WT and mutant Tpms are indicated by the symbol “ns”. Error bars indicate ±SEM.