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Journal of Physiology (2002), 542.1, pp. 221-229
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.017111

Myosin light chain 2 modulates MgADP-induced contraction in rabbit skeletal and bovine cardiac skinned muscle

Hideaki Fujita *, Daisuke Sasaki *, Kenji Fukuda * and Shin'ichi Ishiwata *†

	ABSTRACT

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Skinned skeletal and cardiac muscle fibres can be activated by MgADP in the presence of MgATP without Ca²⁺; the isometric tension is developed in a sigmoidal manner with the addition of MgADP under relaxing conditions. The critical concentrations of MgADP for this MgADP-induced contraction are about 7.5 and 2.6 mM for skeletal and cardiac muscle fibres, respectively. To investigate whether muscle regulatory proteins, myosin light chain 2 (LC₂) and troponin C (TnC), play a part in the MgADP-induced contraction, these proteins were partly extracted by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), a chelater of divalent cations, and the MgADP-tension relationship was examined in rabbit psoas and bovine cardiac skinned fibres. We found that the sigmoidal MgADP-tension relationship became hyperbolic after a partial extraction of LC₂ (about 30 %) and TnC (about 70 %). Reconstitution with LC₂ restored the sigmoidal MgADP-tension relationship of control fibres almost fully in both skeletal and cardiac fibres, whereas reconstitution with TnC alone had no effect. Furthermore, cardiac fibres reconstituted with skeletal LC₂ exhibited an MgADP-tension relationship intermediate between skeletal and cardiac fibres. The partial extraction of LC₂ and TnC resulted in a reduction of the inhibitory effect of inorganic phosphate (P_i) on the MgADP-activated tension. Reconstitution with LC₂ restored the original P_i-tension relationship, whereas reconstitution with TnC had no effect. In other words, extraction of LC₂ apparently increased the affinity of myosin for MgADP but decreased the affinity for P_i. These results demonstrate that LC₂ modulates MgADP-induced activation of actomyosin interaction.

(Received 16 January 2002; accepted after revision 16 April 2002)
Corresponding author S. Ishiwata: Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan. Email: ishiwata{at}mn.waseda.jp

	INTRODUCTION

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Muscle contraction is initiated by a release of Ca²⁺ from an inner membrane storage site (Ebashi & Endo, 1968), but the molecular mechanism of the Ca²⁺ regulation differs among muscle types. It is generally accepted that, in vertebrate striated muscle cells, the regulatory switch is located on the thin filament (Ebashi & Endo, 1968). Ca²⁺ binds to troponin (strictly speaking, the Ca²⁺-binding subunit of troponin, TnC), which in turn removes the inhibition of tropomyosin and enables actomyosin interaction. In vertebrate smooth muscle cells, the regulatory switch is located on the myosin molecule. Phosphorylation of myosin regulatory light chain by Ca²⁺- and calmodulin-dependent myosin light chain kinase initiates the contraction (cf. Kendrick-Jones & Scholey, 1981; Kamm & Stull, 1985). The role of myosin regulatory light chain (LC₂) in vertebrate striated muscle is still elusive, although recent studies have suggested the possible roles of LC₂ in regulation of muscle contraction. A partial extraction of LC₂ did not affect maximum Ca²⁺-activated tension and stiffness, whereas at submaximum Ca²⁺ activation, the partial extraction increased tension, stiffness and rate of tension development in skinned skeletal muscle fibres (Hofmann et al. 1990; VanBuren et al. 1994; Patel et al. 1996).

Although the thin filament in striated muscle is activated by Ca²⁺, formation of strong-binding cross-bridges is also known to activate the thin filament (Bremel & Weber, 1972; Greene & Eisenberg, 1980; McKillop & Geeves, 1993; Swartz et al. 1996) and enhance actomyosin interaction. Rigor cross-bridges formed by the reduction of MgATP concentration can 'turn on' the thin filament in a cooperative manner, allowing neighbouring MgATP-bound cross-bridges to interact with actin and produce tension (Kawai & Brandt, 1976). Lowering the MgATP concentration increases maximum Ca²⁺-activated tension and increases Ca²⁺ sensitivity in crayfish muscle fibres (Brandt et al. 1972), skeletal muscle fibres (Godt, 1974) and cardiac muscle fibres (Fabiato & Fabiato, 1975; Best et al. 1977). Similarly, exogenously added MgADP activates the thin filament in the absence of Ca²⁺, and increases maximum Ca²⁺-activated tension and Ca²⁺ sensitivity both in skeletal (Hoar et al. 1987; Shimizu et al. 1992) and cardiac muscle fibres (Fukuda et al. 1996, 1998).

In the present study, we have investigated the effect of partial extraction of LC₂ on isometric tension induced by MgADP in the absence of Ca²⁺ (MgADP-activated tension) to further clarify the regulatory roles of LC₂. After the partial extraction of LC₂, the sigmoidal relationship between the MgADP concentration and tension (MgADP-tension relationship) became hyperbolic with a concomitant increase in the apparent affinity of MgADP for cross-bridges. This effect was reversed by reconstitution with LC₂. In addition, the inhibitory effect of P_i on the MgADP-activated tension was reduced by the partial extraction of LC₂ and recovered by reconstitution with LC₂. These results indicate that, in striated muscles, LC₂ modulates the MgADP-induced activation mechanism by cross-bridges.

	METHODS

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Solutions

The solutions used were as follows (mM): rigor solution, 170 KCl, 1.0 MgCl₂, 1.0 EGTA and 10 3-(N-morpholino) propanesulphonic acid (Mops) (pH adjusted to 7.0 with HCl); relaxing solution, 117 KCl, 5.0 MgCl₂, 4.0 ATP, 1.0 EGTA, 10 Mops (pH adjusted to 7.0 with HCl), 20 2,3-butanedione 2-monoxime (BDM); ADP assay solutions, 34-120 KCl (ionic strength 150 mM), 4.3-20 MgCl₂ (2.0 mM free Mg²⁺), 2.2 ATP (2.0 mM MgATP), 0-22.8 ADP (0-15 mM MgADP), 2.0 EGTA, 10 Mops (pH adjusted to 7.0 with HCl) and 50 µM P¹,P⁵-di(adenosine-5')pentaphosphate (AP₅A); P_i assay solutions, 18-58 KCl (ionic strength 150 mM), 14.2 MgCl₂ (2.0 mM free Mg²⁺), 2.2 ATP (2.0 mM MgATP), 16.4 ADP (10 mM MgADP), 0-10 P_i, 2.0 EGTA, 10 Mops (pH adjusted to 7.0 with HCl) and 50 µM AP₅A; Ca²⁺-activating solutions, 117 KCl, 4.3 MgCl₂, 2.2 ATP, 2.0 EGTA, 20 Mops (pH adjusted to 7.0 with HCl) and 1.9 CaCl₂ (pCa 4.7) or 0.77 CaCl₂ (pCa 6.5). ATP (disodium salt), ADP (potassium salt) and AP₅A were purchased from Boehringer Ingelheim (Ingelheim, Germany); EGTA and Mops were from Dojindo Laboratories (Kumamoto, Japan). All chemicals were of reagent grade. The concentrations of free Mg²⁺, free Ca²⁺, MgATP, MgADP, P_i and other ionic species were calculated by a computer program using published stability constant values (Horiuti, 1986).

Muscle fibres and proteins

Skeletal muscle fibres were prepared from rabbit psoas muscle according to the Guidelines for the Care and Use of Laboratory Animals in the Human Science Department of Waseda University. Cardiac muscle fibres were prepared from a straight portion of bovine left ventricular papillary muscle obtained from a local abattoir (Fukuda et al. 1996). Muscle bundles (for skeletal muscle, approximately 7 cm in length and 3 mm in diameter; for cardiac muscle, approximately 5 cm in length and 5 mm in diameter) were excised and both ends were tied to a glass rod and the muscle was incubated in glycerol solution (rigor) composed of 50 % (v/v) glycerol, 0.5 mM NaHCO₃, 5.0 mM EGTA (pH adjusted to 7.0 with HCl) and 2.0 mM leupeptin at 0 °C, overnight. Fibres were then stored in fresh glycerol solution at -20 °C. Glycerinated fibres were used between 2 and 8 weeks after preparation. TnC was prepared from rabbit white skeletal muscle (sTnC) and from bovine cardiac muscle (cTnC) according to the method of Ebashi (1974) and column purified using DEAE Sephadex A-25 (Pharmacia, Stockholm, Sweden). LC₂ was prepared from rabbit white skeletal muscle (sLC₂) and from bovine cardiac muscle (cLC₂) according to the method of Weeds & Lowey (1971) and free 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) was removed using DEAE Sephadex G-25 (Pharmacia).

Tension measurement

For tension measurement (cf. Fujita & Ishiwata, 1999), a glycerinated thin bundle (~1 mm in length, 100 µm in diameter) was removed with a pair of forceps under a stereomicroscope just before the experiment. To prepare a thin bundle, dissection was carried out in glycerol solution at about -10 °C (Fukuda et al. 1996). Both ends of the muscle fibres were fixed to thin tungsten wires with enamel and one of the wires was attached to a tension transducer (AE-801, SensoNor a.s., Holten, Norway). The muscle was then immersed in the rigor solution containing 1 % (v/v) Triton X-100 for 20 min to remove the residual internal membrane system. Triton X-100 was washed out with rigor solution before experiments. Sarcomere length was set at 2.6-2.7 µm for skeletal and 2.0-2.1 µm for cardiac fibres. For tension measurement, fibres were immersed in relaxing solution and then active tension was measured by immersing the fibres in an activating solution. Measurements were recorded with a pen recorder (VP-6533A, National, Osaka, Japan). The tension measurement was carried out in a chamber made from a silicon-coated aluminum block (10 cm 10 cm 1 cm) with several small holes (5 mm in diameter) filled with approximately 0.4 ml each of the experimental solutions (Horiuti, 1986).

SDS-PAGE

Fibres were collected and dissolved in lysis solution (7.5 % SDS, 10 % (v/v) glycerol, 1.0 mM dithiothreitol (DTT) and 10 mM Tris-HCl (pH 6.8) and heated for 3 min at 90 °C. SDS-PAGE was carried out according to the method of Laemmli (1970) with 5-20 % gradient running gel. Proteins were fluorescently stained with SYPRO Ruby Protein gel stain (Molecular Probes Inc., Eugene, OR, USA), which is more sensitive than Coomassie Brilliant Blue R. The gel pattern was analysed by using a densitometre (model AE-6920-V, ATTO Inc., Tokyo, Japan) after the linearity of staining against the amount of proteins was confirmed.

Extraction and reconstitution of LC₂ and TnC

To extract LC₂ and TnC, fibres were immersed in a solution composed of 5.0 mM trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), 40 mM Tris-HCl (pH 8.4), 0.6 mM NaN₃ and 0.1 mM DTT for 90 min for skeletal and for 120 min for cardiac fibres at 25 °C. CDTA was washed out for 10 min in the solution composed of 90 mM KCl, 10 mM Mops (pH 7.0), 1.0 mM NaN₃ and 0.1 mM DTT. To reconstitute LC₂ and/or TnC, fibres were immersed in relaxing solution containing 1.2 mg ml^-1 LC₂ and/or 1.5 mg ml^-1 TnC for 60 min at 25 °C. Figure 1 shows SDS-PAGE analysis of fibres before and after the CDTA treatment and after the reconstitution with LC₂ and TnC. The LC₂ content was determined from the LC₂/(LC₁ + LC₃) ratio for skeletal fibres and the LC₂ : LC₁ ratio for cardiac fibres. The TnC content was determined from the TnC : actin ratio. After the CDTA treatment, approximately 30 % of LC₂ and 70 % of TnC were removed from the fibres, based on the densitometric trace of the gel stained with the fluorescent dye. Similarly, the densitometric trace showed that almost 100 % of LC₂ and TnC were recovered after the reconstitution. Our LC₂ preparation contained a small amount of TnC. Because of this, some TnC may have been reconstituted when LC₂ was added. Contamination was not detected in our TnC preparation.

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Figure 1. SDS-PAGE of muscle fibres before and after CDTA treatment and after reconstitution with LC2 and TnC Lanes 1-4, skeletal fibres; lanes 5-8, cardiac fibres; lanes 1 and 5, control; lanes 2 and 6, after CDTA treatment; lanes 3 and 7, after reconstitution with LC2; lanes 4 and 8, after reconstitution with TnC and LC2. LC2 and TnC used for lanes 3 and 4 were prepared from skeletal muscle and LC2 and TnC used for lanes 7 and 8 were prepared from cardiac muscle. Abbreviations: A, actin; LC1, LC2 and LC3, myosin light chains 1, 2 and 3, respectively; TnC, troponin C.

	RESULTS

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Effect of LC₂ and TnC on MgADP-induced contraction in skeletal fibres

Figure 2 shows a pen trace of MgADP-activated isometric tension developed by skeletal muscle fibres in the absence of Ca²⁺. With increasing MgADP concentration, fibres generated increased tension in a sigmoidal manner, which is in good agreement with previous results in skeletal (Shimizu et al. 1992) and cardiac muscle fibres (Fukuda et al. 1996).

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Figure 2. Recordings of isometric tension at various MgADP concentrations in control skeletal muscle fibres Arrowheads indicate the exchange of solutions. The MgADP concentrations of activating solutions are indicated below the arrowheads. The fibre was relaxed after each activation by immersion in relaxing solution. Spikes are artefacts due to solution exchange. Tension was measured at 25 °C. Vertical and horizontal bars, 5 10-5 N and 2 min, respectively.

Figure 3 shows pen traces of isometric tension at various MgADP concentrations (indicated) or in pCa solutions (pCa 6.5 or 4.7) after treatment with CDTA (Fig. 3A), after reconstitution with sTnC (Fig. 3B) and then after reconstitution with sLC₂ (Fig. 3C). These records were made sequentially from the same muscle fibre. To avoid the deterioration of fibres due to rigor tension development upon the exchange of relaxing solution with rigor solution of low ionic strength for CDTA treatment (see Methods), control experiments were performed using different fibres. The MgADP-activated tension at 15 mM did not differ significantly after reconstitution with sLC₂. Active tension at pCa 6.5 and 4.7 in the absence of MgADP was also measured to compare the results with earlier studies (Hofmann et al. 1990). The maximum Ca²⁺-activated tension at pCa 4.7 did not change, whereas tension at pCa 6.5, which was developed after the removal of LC₂, disappeared again upon reconstitution with sLC₂.

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Figure 3. Recordings of isometric tension at various MgADP concentrations in skeletal muscle fibres Recordings were made after treatment with CDTA (A), after reconstitution with sTnC (B) and after reconstitution with sLC2 (C). All tension records were taken from the same fibre sequentially. Arrows and arrowheads indicate the exchange of solutions. The MgADP concentrations of activating solutions are indicated below the arrowheads. The pCa values of Ca2+-activating solution are indicated below the arrows. The fibre was relaxed after each activation by immersion in relaxing solution. Spikes are artefacts due to solution exchange. Tension was measured at 25 °C. Vertical and horizontal bars, 5 10-5 N and 2 min, respectively.

Figure 4A summarises the MgADP-tension relationship of skeletal muscle fibres before and after treatment with CDTA and after LC₂ reconstitution. Control fibres generated active tension in a sigmoidal manner on increasing the MgADP concentration (Fig. 4A, ). In contrast, tension developed in a hyperbolic manner (Fig. 4A, ) in the CDTA-treated fibres. After reconstitution with sLC₂, the MgADP-tension relationship became sigmoidal, which was not different from that of control fibres (Fig. 4A, ).

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Figure 4. Effect of MgADP on the isometric tension in skeletal muscle fibres Fibres were treated with CDTA for 90 min before reconstitution except for control fibres. Control (), after CDTA treatment for 90 min (), reconstituted with sLC2 (), reconstituted with sTnC (), reconstituted with sTnC and then with sLC2 (), and reconstituted with sLC2 and then with sTnC (). Lines in A are fitted for control (continuous line) and CDTA-treated fibres (dashed line) by the Hill equation (cf. Fukuda & Ishiwata, 1999) using the values in Table 1. To fit the data, we assumed that tension at 15 mM MgADP was 90 % of the saturated tension value. The same lines as in A were drawn in B for comparison. Reconstitution with LC2 or TnC was achieved by immersing the CDTA-treated fibres in solution containing 1.2 mg ml-1 LC2 or 1.5 mg ml-1 TnC for 60 min at 25 °C. Tension was measured in ADP assay solution at 25 °C and normalized to that obtained at 15 mM MgADP (the relative value was assumed to be 0.9) for each fibre tested. Data points and vertical bars show mean and S.D. calculated from 5 experiments using different preparations.

Since CDTA treatment partly extracts TnC (Fig. 1) as well as LC₂, the effect of TnC reconstitution on the MgADP-activated tension was also examined. The CDTA-treated fibres reconstituted with sTnC showed a hyperbolic MgADP-tension relationship (Fig. 4B, ), which was not different from that of CDTA-treated fibres. Only when sLC₂ was added to the sTnC-reconstituted fibres could the MgADP-tension relationship of control fibres be restored (Fig. 4B, ). The order of reconstitution did not affect the result (Fig. 4B, ), indicating that TnC has little effect on the modulation of MgADP-induced tension.

Effect of LC₂ and TnC on MgADP-induced contraction in cardiac fibres

The effect of LC₂ extraction and reconstitution on the MgADP-tension relationship was also examined in cardiac fibres. With the addition of MgADP, cardiac fibres developed isometric tension in a sigmoidal manner similar to skeletal fibres, but the critical MgADP concentration (MgADP₅₀) was much lower (Fig. 5A, ) and the Hill coefficient (n_H) was smaller (Table 1), consistent with a previous report (Fukuda et al. 1998). With extraction of LC₂ and TnC, the MgADP-tension relationship became hyperbolic (Fig. 5A, ), and reconstitution with cLC₂ restored the sigmoidal MgADP-tension relationship (Fig. 5A, ; cf. Table 1). These results are qualitatively the same as those of skeletal fibres. Interestingly, when the CDTA-treated cardiac fibres were reconstituted with sLC₂, the MgADP-tension relationship also became sigmoidal, but MgADP₅₀ became larger than that of cardiac control fibres (Fig. 5A, ) and assumed a value intermediate between skeletal and cardiac control fibres (Table 1).

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Figure 5. Effect of MgADP on isometric tension in cardiac muscle fibres Fibres were treated with CDTA for 120 min before reconstitution except for control fibres. Control (), after CDTA treatment for 120 min(), reconstituted with cLC2 (), reconstituted with sLC2 (), reconstituted with cTnC (), reconstituted with cTnC and then with cLC2 (), reconstituted with cLC2 and then with cTnC(), reconstituted with cTnC and then with sLC2 (), and reconstituted with sLC2 and then with cTnC (). Lines in A are fitted for control (continuous line), CDTA-treated (dashed line), and sLC2-reconstituted fibres (dotted line) by the Hill equation using the values in Table 1. Other methods are the same as in Fig. 4, except that tension was normalised to that obtained at 10 mM MgADP, of which the relative value was assumed to be 0.9 for each fibre tested. For fibres reconstituted with sLC2, tension was nomalised to that obtained at 15 mM MgADP (the relative value was assumed to be 0.9). Data points and vertical bars show mean and S.D. calculated from 3-5 experiments using different preparations.

The effect of TnC on the MgADP-tension relationship was also examined in cardiac fibres (Fig. 5B). Reconstitution with cTnC in the CDTA-treated cardiac fibres did not change the hyperbolic MgADP-tension relationship (Fig. 5B, ). When cLC₂ was added to the cTnC-reconstituted fibres, the MgADP-tension relationship became sigmoidal (Fig. 5B, ). The MgADP-tension relationship of cLC₂-reconstituted fibres remained unchanged on addition of cTnC (Fig. 5B, ). Similar results were obtained for fibres reconstituted with sLC₂ either before (Fig. 5B, ) or after the cTnC reconstitution (Fig. 5B, ). These fibres exhibited an MgADP₅₀ value intermediate between those of control skeletal and cardiac fibres. These results indicate that TnC does not modulate the MgADP-tension relationship either in cardiac or skeletal fibres.

Effect of LC₂ and TnC on inhibitory role of P_i in MgADP-induced contraction

In the following experiments, we examined the effect of P_i on MgADP-activated tension before and after partial extraction of LC₂ and TnC. Figure 6A summarises the effect of P_i on control (), CDTA-treated () and sLC₂-reconstituted () skeletal fibres in the presence of 10 mM MgADP. In control fibres, the MgADP-activated tension decreased markedly with the addition of P_i and reached a plateau at approximately 20 % at 7 mM P_i. In contrast, in the CDTA-treated fibres, the extent of tension reduction was less and reached a plateau at approximately 40 % at 7 mM P_i. On reconstitution with sLC₂, this P_i-tension relationship regained its original shape. The effect of sTnC reconstitution on the P_i-tension relationship is summarised in Fig. 6B. Reconstitution with sTnC had no effect on the P_i-tension relationship in CDTA-treated () and sLC₂-reconstituted fibres (). Only when sLC₂ was added to the TnC-reconstituted fibres () did the P_i-tension relationship resume the shape of that of control fibres.

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Figure 6. Effect of Pi on (10 mM) MgADP-induced isometric tension in skeletal muscle fibres Fibres were treated with CDTA for 90 min before reconstitution except for control fibres. Control (), after CDTA treatment for 90 min (), reconstituted with sLC2 (), reconstituted with sTnC (), reconstituted with sTnC and then with sLC2 (), and reconstituted with sLC2 and then with sTnC (). Lines in A are fitted by eye. The same lines were drawn in B for comparison. Reconstitution with LC2 or TnC was achieved by immersing the CDTA-treated fibres in solution containing 1.2 mg ml-1 LC2 or 1.5 mg ml-1 TnC for 60 min at 25 °C, respectively. Tension was measured in Pi assay solutions at 25 °C and normalized to that obtained at 0 mM Pi for each fibre tested. Data points and vertical bars show mean and S.D. calculated from 5 experiments using different preparations.

Figure 7A summarises the effect of P_i in cardiac fibres. In control fibres, tension inhibition by P_i was much smaller than that in skeletal fibres, reaching a plateau at approximately 65 % at 10 mM P_i (), consistent with previous results (Fukuda et al. 1998; Fukuda & Ishiwata, 1999). On treatment with CDTA, the extent of reduction in tension became even less, reaching a plateau at approximately 75 % at 10 mM P_i (). The original P_i- tension relationship recovered on reconstitution with cLC₂ (). sLC₂-reconstituted fibres also showed a P_i-tension relationship resembling that of control cardiac fibres (). As summarised in Fig. 7B, the reconstitution of cTnC did not change the P_i-tension relationship significantly in CDTA-treated fibres () and also in cLC₂- and sLC₂-reconstituted fibres ( and , respectively). Addition of either cLC₂ or sLC₂ to the cTnC-reconstituted fibres ( and , respectively) produced P_i-tension relationships similar to that of control cardiac fibres. In contrast to the higher effectiveness of sLC₂ in the MgADP-tension relationship (Fig. 5), the effectiveness of sLC₂ in the inhibitory role of P_i in the MgADP-activated tension was indistinguishable from that of cLC₂ (Fig. 7).

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Figure 7. Effect of Pi on (10 mM) MgADP-induced isometric tension in cardiac muscle fibres Fibres were treated with CDTA for 120 min before reconstitution except for control fibres. Control (), after CDTA-treatment for 120 min (), reconstituted with cLC2 (), reconstituted with sLC2 (), reconstituted with cTnC (), reconstituted with cTnC and then with cLC2 (), reconstituted with cLC2 and then with cTnC (), reconstituted with cTnC and then with sLC2 (), and reconstituted with sLC2 and then with cTnC (). Lines in A are fitted by eye. The same lines were drawn in B for comparison. Other methods are the same as in Fig. 6. Data points and vertical bars show mean and S.D. calculated from 3-5 experiments using different preparations.

	DISCUSSION

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Extraction of LC₂ and TnC by treatment with CDTA

As demonstrated from the analysis of SDS-PAGE (Fig. 1), treatment with CDTA removed approximately 30 % of LC₂ and 70 % of TnC from skeletal and cardiac muscle fibres. These proteins were fully reconstituted into the fibres by immersing the CDTA-treated fibres in relaxing solution containing purified LC₂ and TnC. After reconstitution with TnC, active tension at 15 mM MgADP (without Ca²⁺) was ~85 % of the maximum Ca²⁺-activated tension (P₀), which was in good agreement with previous results that the fibres fully activated by MgADP (without Ca²⁺) produced ~90 % of P₀ (Shimizu et al. 1992). This shows that TnC was fully reconstituted into the fibres.

To further confirm the removal and reconstitution of LC₂, tension at submaximal Ca²⁺ activation (pCa 6.5) was compared to that at maximal Ca²⁺ activation (pCa 4.7). It has been reported that Ca²⁺ sensitivity was increased by the removal of LC₂ in skeletal muscle fibres (Hofmann et al. 1990). In skeletal muscle fibres, active tension does not develop at pCa 6.5 (data not shown) at pH 7.0. When LC₂ was partly removed (TnC-reconstituted fibres), active tension at pCa 6.5 was 14 % P₀. This result is consistent with a previous report that LC₂-extracted fibres increased Ca²⁺ sensitivity and generated 16 % of P₀ at pCa 6.5 (Hofmann et al. 1990). Active tension at pCa 6.5 after the reconstitution with LC₂ was only < 1 % of P₀, confirming the reconstitution of LC₂ into the fibre.

The active tension at 15 mM MgADP (without Ca²⁺) was reduced by approximately 5 % after reconstitution with TnC and by 20 % after reconstitution with LC₂ (Fig. 3). Because active tension tends to decrease after large tension development, we conclude that the removal and reconstitution of TnC and LC₂ did not affect the MgADP-activated tension. This is in agreement with previous results that removal of LC₂ did not affect the maximal Ca²⁺-activated tension (Hofmann et al. 1990), and thus did not affect the force-generating ability of cross-bridges.

In this study, we used CDTA as a chelater of divalent cations, which resulted in a dissociation of LC₂ from myosin. This procedure extracted only 30 % of LC₂. If the removal of LC₂ occurs at random, 30 % reduction in LC₂ means that 42 % (= 2 0.3 0.7) of myosin lost one LC₂, 9 % (= 0.3 0.3) of myosin lost 2 LC₂ molecules, but 49 % (= 0.7 0.7) of myosin was still intact. However, a previous study suggested that the second LC₂ is more difficult to extract than the first LC₂ in a procedure using EDTA (Kendrick-Jones et al. 1976). Thus, if such a cooperative removal of LC₂ also occurs with our CDTA treatment, approximately 60 % of myosin could have lost one of the two LC₂ molecules.

The extent of LC₂ removal could be enhanced by treatment with DTNB and EDTA, a procedure that was reported to be capable of complete LC₂ removal (Szczesna et al. 1996). We did not use this procedure because oxidized SH-groups produced by the DTNB treatment may remain oxidized even after DTT treatment. Even though this is the case, complete removal of LC₂ using DTNB and EDTA would be worth investigating to enhance our present results.

Effect of LC₂ on MgADP activation

Removal of LC₂ shifted the critical MgADP concentration (MgADP₅₀) towards a lower value, by 3.0 and 1.0 mM in skeletal (Fig. 4) and cardiac fibres (Fig. 5), respectively. This change in MgADP₅₀ can be interpreted to mean that myosin molecules without LC₂ have higher binding affinity to the thin filament in muscle fibres (Hofmann et al. 1990). This result is consistent with that reported in solution (Wagner, 1984). Because LC₂ is located at the head-rod junction of the myosin molecule, which is distal to MgADP or actin-binding site (Rayment et al. 1993), the increase in affinity for the thin filament is attributable to allosteric regulation of the ADP- and actin-binding sites of the myosin head through structural change in the head-rod junction. The difference between skeletal and cardiac muscle fibres in the MgADP-tension relationship is attributable to the difference in the LC₂ isoforms, since addition of skeletal LC₂ to the LC₂-extracted cardiac fibres shifted the MgADP₅₀ towards that of skeletal fibres (Fig. 5 and Table 1). It is notable that extraction of only 30 % of LC₂ was sufficient to transform the qualitative change in the MgADP-tension relationship from sigmoidal to hyperbolic. Such a large effect on MgADP activation by a relatively small change in the protein composition suggests that the allosteric effect of LC₂ is highly cooperative.

Effect of LC₂ on deactivating effect of P_i in MgADP-activated fibres

It is known that MgADP-activated fibres are deactivated by P_i in skeletal (Shimizu et al. 1992) and cardiac fibres (Fukuda et al. 1996), but the extent of deactivation is larger in skeletal than in cardiac fibres. With partial extraction of LC₂, inhibition of tension by P_i decreased significantly both in skeletal and cardiac fibres (Fig. 6 and Fig. 7). The CDTA-treated cardiac fibres reconstituted with sLC₂ showed the P_i-tension relationship of control cardiac fibres rather than skeletal fibres (Fig. 7). This observation is in contrast to the other observation that sLC₂ altered the MgADP-binding affinity of cardiac fibres towards the skeletal type (Fig. 5). The reason why sLC₂ did not modify the P_i effects in cardiac fibres may be attributable to the fact that the proportion of LC₂ exchanged was not large enough to overcome the high affinity for MgADP of cardiac myosin containing a cLC₂ isoform. In the presence of 10 mM MgADP, the effect of MgADP may be saturated, such that the apparent affinity of P_i was maintained.

An alternative and interesting explanation for the above contrasting observation is to assume the following hypotheses, in which the cooperativity of two heads of the myosin molecule is considered (cf. Tokiwa & Morales, 1971; Chaen et al. 1986). (1) Both heads should bind MgADP to turn on the thin filament, which is in the inhibitory state in the absence of Ca²⁺ (Fig. 8, top) and, conversely, (2) both MgADP heads should bind exogenous P_i to lose the ability to turn on the thin filament (Fig. 8, bottom). The high MgADP₅₀ value of the MgADP-tension relationship observed in the sLC₂-reconstituted cardiac fibres (Fig. 5) is consistent with hypothesis (1), because the myosin molecule containing sLC₂ cannot turn on the thin filament until the sLC₂-bound head, having a lower affinity to MgADP, binds MgADP. The contrasting result that the substitution of sLC₂ in cardiac fibres was not sufficient to produce inhibition of the MgADP-activated tension by P_i (Fig. 7), can be explained by hypothesis (2), such that tension does not decrease until the cLC₂-bound head binds P_i because this head has a lower affinity for P_i. Hypothesis (2) also explains the sigmoidal relationship between the P_i concentration and the MgADP-activated tension shown in Fig. 7. Note that the sigmoidal feature was not evident in skeletal fibres (Fig. 6), probably because the MgADP concentration (10 mM) was not high enough to saturate the myosin heads with MgADP. In fact, the sigmoidal feature has been observed even in skeletal fibres when the concentration of MgADP was high, e.g. 15 mM (see Fig. 7 of Shimizu et al. 1992).

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Figure 8. A schematic illustration showing the activation mechanism by MgADP-bound cross-bridges (top) and deactivation mechanism by Pi (bottom) Actin protomers shown by open circles are in the off-state, whereas those shown by shaded circles are in the on-state. Thin filament is assumed to be cooperatively activated over a certain distance when cross-bridges of which both heads bind MgADP are formed (a), so that neighbouring MgATP-bound cross-bridges can interact with actin and produce force (b). Both cross-bridges with only one head binding MgADP (c) and those without MgADP but with MgATP (d) cannot activate the thin filament, so that MgATP-bound cross-bridges cannot produce force in this region. The activated region of the thin filament is still activated when one head binds Pi when the other head binds MgADP (e) and deactivated only when both heads bind Pi ( f ). Rigor heads are practically absent. Abbreviations: D, MgADP; T, MgATP; and P, Pi.

There is a possibility that two-headed myosin is not necessary for the ADP activation to occur. For example, it is known that exogenously added N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) is sufficient to activate muscle fibres (Swartz & Moss, 1992). Thus, the binding of two independent ADP-bound myosin heads to the regulatory unit may be enough to activate the thin filament, although the cooperativity may be different.

Physiological significance

In summary, the ability of LC₂ to modulate MgADP-induced contraction as presented here, together with abilities to decrease the Ca²⁺ sensitivity of tension development (Hofmann et al. 1990) and to increase the rate of tension development at submaximal Ca²⁺ activation (Metzger & Moss, 1992; Patel et al. 1996), enables us to conclude that LC₂ plays substantial roles in the modulation of muscle contraction.

In skeletal muscle in normal physiological conditions, the MgADP concentration may not reach such high levels that ADP activation becomes significant because of the ATP-regenerating system. However, in myocardium, the accumulation of submillimolar concentrations of ADP that occurs in ischaemia may cause abnormal tension development, because cardiac muscle has a higher affinity for MgADP (compare Fig. 4 and Fig. 5). In this respect, the regulatory role of LC₂ in lowering the affinity for ADP may be important, especially in myocardium.

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Acknowledgements

We thank Professor M. Kawai of the University of Iowa for his critical reading of the manuscript. This research was partly supported by Grants-in-Aid for Scientific Research, for Scientific Research on Priority Areas and for the High-tech Research Center Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan. H. Fujita is the recipient of a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists.

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