The length of cooperative units on the thin filament in rabbit psoas muscle fibres

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1 The length of cooperative units on the thin filament in rabbit psoas muscle fibres To elicit a biological function, multiple molecules of the same or similar kind are often assembled together, and they work in concert. This is called molecular cooperativity, where the transition from an inactive state to an active state is more abruptly achieved than in its absence. A classical example is O 2 binding to haemoglobin, where 2nd, 3rd and 4th bindings become progressively easier than the first binding (Monod et al. 1963). Another example of cooperativity can be seen in the activation of striated muscles. Here, cooperativity is known to exist on the thin filament (Bremel & Weber, 1972; Tobacman & Sawyer, 1990) and between the thin filament and crossbridges (Güth & Potter, 1987; Gordon et al. 1988; Pan et al. 1989; Brandt et al. 1990; Metzger, 1995). With regard to thin filament cooperativity, the basic cooperative unit consists of seven actin monomers, one tropomyosin dimer and one troponin (Tn) complex that contains TnT, TnI and TnC molecules (Ebashi & Endo, 1968). Twenty-five cooperative units make up one strand of the thin filament in vertebrate striated muscles such as in rabbit psoas (Ebashi & Endo, 1968). Two such strands make up one thin filament. Two opposing hypotheses have been proposed with regard to the length of thin filament cooperativity. One hypothesis Wei Ding, Hideaki Fujita and Masataka Kawai * Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, IA 52242, USA (Manuscript received 4 July 2002; accepted 11 September 2002) The length of cooperative units along the thin filament of rabbit psoas single muscle fibres was determined by reducing filament length by treatment with the thin filament severing protein, gelsolin, in the presence of Ca 2+ and 2,3-butanedione 2-monoxime (BDM). The average time for 50 % reduction in isometric tension was 6.7 min at 22 C. The pca tension relationship was measured at 22 C, ph 7.00 and ionic strength 200 mm, and the data were fitted to the Hill equation to determine the half-saturation point (K) and the cooperativity (n). Our results demonstrate that the cooperativity does not change much when the remaining isometric tension was in the range %. The cooperativity quickly diminished when the remaining tension was reduced to less than 20 %. Our results further demonstrate that the change in the pk value was minimal and averaged (less Ca 2+ sensitive) as the thin filament length was reduced. We infer from the first observation that the thin filament cooperativity extends up to 0.2 mm, which includes the maximum of about four basic cooperative units consisting of seven actin molecules, one tropomyosin dimer and one troponin complex. We infer from the second observation that the Ca 2+ sensitivity is slightly reduced by removal of the cooperative interaction between neighbouring cooperative units. Experimental Physiology (2002) 87.6, states that thin filament cooperativity extends over the entire length of the filament (Brandt et al. 1987) and the other that thin filament cooperativity is limited to 10 % of the filament length (Geeves & Lehrer, 1994). It is therefore of interest to determine the length of thin filament cooperativity with experiments using muscle fibres. To achieve this goal, we reduced thin filament length using the enzyme gelsolin. The cooperativity is measured by the Hill factor, which represents the steepness of the pca tension relationship. Gelsolin (sometimes called brevin) is an 86 kda plasma protein that severs the thin filament in the presence of Ca 2+ at a random location (Yin et al. 1979; McLaughlin et al. 1993). By treating rabbit psoas muscle fibres with gelsolin for different periods of time, we can obtain fibres with differential thin filament lengths. The remaining maximum tension represents the length of the thin filament that overlaps with cross-bridges, assuming the sliding filament theory as proposed by Gordon et al. (1966). Using gelsolin, we reduced thin filament length, and observed how the cooperativity and Ca 2+ sensitivity changed by looking at pca tension curves. It was interesting that the cooperativity remained the same if the isometric tension was between 20 and 100 %, but decreased significantly when the isometric Publication of The Physiological Society * Corresponding author: masataka-kawai@uiowa.edu 2448

2 692 W. Ding, H. Fujita and M. Kawai Exp. Physiol tension was reduced to less than 20 %. A preliminary account of the present results was reported earlier (Ding & Kawai, 1999). METHODS Solutions The thin filament extracting solution contained (mm): KCl 117, MgCl , ATP 2.2, Mops (3-(N-morpholino) propanesulfonic acid) 20, EGTA 2, CaCl 2 1.9, (pca 5.05; ionic strength, 143mM), BDM (2,3-butanedione 2-monoxime) 20, leupeptin 2 and 0.8 mg ml _1 gelsolin (Fujita et al. 1996, 2002). For the source of EGTA, an ultra pure grade EGTA manufactured by Amaresco (Solon, OH, USA) was used, hence no correction to the pca values was applied (cf. Miller & Smith, 1984). BDM was used to prevent tension development in the extraction solution, which contains Ca 2+. The activating solution contained (mm): Na 2 MgATP 5.83, Na 2 K 2 ATP 1.36, disodium creatine phosphate (Na 2 CP) 15, K 2 EGTA 6, sodium propionate (NaProp) 11, potassium propionate (Kprop) 92, Mops 10 and 160 unit ml _1 creatine kinase (CK); ionic strength was adjusted to 200 mm. The total [Ca] was adjusted to obtain a desired pca value (pca = _log[ca 2+ ]) by keeping the total [EGTA] at 6 mm. Our activating solutions contained a series of pca values: 6.6, 6.4, 6.2, 6.0, 5.9, 5.8, 5.7, 5.6, 5.4, 5.2, 5.0, and The activating solution with the pca value of 4.66 is called the control activating solution. The relaxing solution contained (mm): EGTA 6, Na 2 MgATP 2, Na 2 K 2 ATP 5, KH 2 PO 4 4, K 2 HPO 4 4, NaProp 62, KProp 48 and Mops 10. Although the relaxing solution did not contain CP/CK, the fibres were washed with the solution that contained 15 mm CP and 160 units ml _1 CK before the Ca 2+ concentration was increased. The ph of all solutions was adjusted to Gelsolin preparation Gelsolin was purified from bovine serum according to the method of Kurokawa et al. (1990). We obtained mg of gelsolin from 1 l of bovine blood. Gelsolin was analysed by SDS- PAGE, which demonstrated that the gelsolin band was about 96 % of the total stainable proteins. Muscle preparations Single skinned fibres from male rabbit (4 5 kg) psoas muscle were used for the studies reported here. Rabbits were killed by intravenous injection of sodium pentobarbital at 150 mg kg _1 in the ear vein. The use of rabbits conformed with the current Guide for the Care and Use of Laboratory Animals (NIH publication DHSS/USPHS), and was approved by the Institutional Animal Care and Use Committee. Muscle bundles (50 mm in length and 3mm in diameter) were tied to wooden sticks at in situ length, and chemically skinned in a solution that contained (mm): EGTA 5, MgATP 2, free ATP 5, NaProp 132, imidazole 6, BDM 20 and leupeptin 2 (ph 7.00) at 2 C for 24 h. The Na + -based skinning solution was used to avoid K + -induced depolarization that causes a contracture on solution change. The fibre bundles were then stored at _20 C in a solution that contained (mm): EGTA 5, MgATP 2, free ATP 5, KProp 132, imidazole 6, BDM 20, leupeptin 2 (ph 7.00) and 50 % (v/v) glycerol. Single muscle fibres that were about 100 mm in diameter and 2 3mm in length were dissected from the stock bundles, and glued between two insect pins with nail polish. One pin was attached to a gauge element (AE-801; SensoNor, Horten, Norway) for tension measurement, and the other pin was attached to a micromanipulator for the length adjustment. The volume of the muscle chamber was about 200 ml. The sarcomere length was adjusted to about 2.2 mm by optical diffraction. After treatment with gelsolin for a given period of time, the tension was measured by gradually increasing the Ca 2+ concentration, and pca tension Figure 1 A, 4 15 % gradient SDS-PAGE stained with silver. Lanes 1 3, single rabbit psoas control fibre; lanes 4 6, single fibres after treatment with gelsolin (0.3mg ml _1 ) for 80 min at 0 C. MHC, myosin heavy chain; Ge, gelsolin; A, actin; Tm-a, a-tropomyosin; Tm-b, b-tropomyosin; LC1, light chain 1, LC2, light chain 2; LC3, light chain 3. The proteins were identified according to their molecular weights. B, the integrated optical density (Intensity) of lane 2 (control fibre) and lane 6 (gelsolin-treated fibre) in A was measured and plotted.

3 Exp. Physiol Length of cooperative units on the thin filament 693 curves were constructed. All experiments including gelsolin treatment were performed at 22 C, unless otherwise stated. Biochemical analysis of muscle preparations The muscle fibres used for mechanical experiments were dissolved in the loading buffer that consisted of 7.5 % sodium dodecyl sulfate (SDS), 10 % glycerol, 1 mm dithiothreitol (DTT) and 10 mm Tris-HCI, (ph 6.8), and boiled for 3min. SDS PAGE was carried out according to the method of Laemmli (1970) with 5 % polyacrylamide as the stacking gel and BioRad 4 15 % gradient SDS-PAGE, and stained with silver (Giulian et al. 1983). The stained gel was scanned and the intensity of each protein band was analysed with NIH image 1.62b (image analysis software package). RESULTS Biochemical analysis of gelsolin-treated fibres Figure 1A demonstrates the results from 4 15 % gradient gel of single muscle fibres with silver stain. Lanes 1 3 are control single fibres (no gelsolin treatment). Lanes 4 6 are single fibres after gelsolin treatment (0.3mg ml _1 ) for 80 min at 0 C. Gelsolin still severs the thin filament at this low temperature, but the severing action takes longer. In Fig. 1, the proteins are identified according to their molecular weight. Because of a difference in the amount of protein loaded to each lane, as shown by different staining in the myosin heavy chain (MHC) band, a good comparison can be made between lanes 2 and 6 of Fig. 1. The integrated optical density (intensity) was measured in each of these two lanes and plotted in Fig. 1B. It is clear from this graph that the intensity of MHC and two light chains (LC1 and LC2) are about the same in lanes 2 and 6 (before and after gelsolin treatment). In contrast, the intensity of thin filament proteins actin, TnT, Tm-a and Tm-b are reduced significantly in lane 6 compared to lane 2. The amount of a-actinin is not very different in the two lanes, although it is slightly reduced after gelsolin treatment. The presence of gelsolin is apparent in lanes 4 6 (Fig. 1A). Because light Figure 2 The time course of gelsolin treatment. Single psoas fibres about 100 mm in diameter and 2 3mm in length were treated with 0.8 mg ml _1 gelsolin. The normalized remaining tension at a saturating Ca 2+ concentration (pca 4.66) is plotted against the duration of the treatment. The continuous line represents a single exponential fit with a half-decay time of 6.7 min. chain 3 (LC3) is close to the front of the gel, its density is not reliable, hence not included in Fig. 1B. Analysis of SDS-PAGE indicates that gelsolin removes only thin filament proteins without extracting other proteins. This result is comparable to earlier studies which used gelsolin to remove the thin filament from rabbit psoas fibres (Funatsu et al. 1994) and from bovine myocardium (Fujita et al. 1996, 2002). Figure 3 Thin filament extraction procedure by gelsolin and tension time course in response to solutions containing various pca levels. A single skinned rabbit psoas fibre, 2 mm in length and 100 mm in diameter, was mounted. An upward arrow (,) indicates a change to the pca 4.66 solution, and a downward arrow (.) indicates a change to the relaxing solution. A vertical line indicates a change to an individual pca solution. * indicates the start of the pca series that includes (left to right) pca 6.6, 6.4, 6.2, 6.0, 5.9, 5.8, 5.7, 5.6, 5.4, 5.2, 5.0 and The activation at the far left-hand side is the control activation. G 10, gelsolin treatment for 10 min; G 20, gelsolin treatment for an additional 20 min. Pen recorder was stopped during the gelsolin treatment.

4 694 W. Ding, H. Fujita and M. Kawai Exp. Physiol Time course of gelsolin treatment Isometric tension of single psoas fibres was initially measured in the control activating solution (pca 4.66). This was followed by gelsolin treatment (0.8 mg ml _1 ) for a specified duration up to 35 min, and the remaining tension was measured in the control activating solution. The data were normalized to the initial tension and plotted in Fig. 2. The data are fitted to a single exponential time course (continuous line). From this fit we found that the halfdecay time was 6.7 min. The scatter of the data in Fig. 2 is Figure 4 A, measured tension from Fig. 3 is plotted against pca after normalization at pca B, data in A were fitted to eqn (1) and their theoretical projection is shown. A single psoas fibre was treated with gelsolin for 0, 10 and 30 min, and tension was measured at each pca. Relative maximum tension is indicated in A as % (middle, right). primarily based on the activity of gelsolin, which is different in each preparation. pca tension measurements Figure 3 demonstrates the tension time course in response to various pca levels and gelsolin treatments. A single fibre was mounted and the initial tension was tested with the control activating solution (far left-hand side). Ca 2+ concentration was then gradually increased starting at pca 6.6 (*) and ending at pca 4.66 (,). The fibre started to show a threshold tension at pca , and isometric tension became saturated by pca 5.2. After treatment with gelsolin (0.8 mg ml _1 ) for 10 min, pca tension relationships were measured again. The fibre was treated with gelsolin for a further 20 min, and the pca tension measurements were repeated. Measured tension from Fig. 3 is plotted against pca in Fig. 4A. The relative remaining tension at pca 4.66 was 100 %, 52 % and 13%, after treating fibres with gelsolin for a total of 0, 10 and 30 min, respectively. Tension was normalized to the maximum tension at pca 4.66 before plotting. The data were fitted to the Hill equation (eqn (1); Brandt et al. 1980, 1982) to determine the cooperativity (n) and the Ca 2+ dissociation constant (K): 1 Tension =. (1) n K 1+ [Ca 2+ ] For the control activation, we found n to be 5.53± 0.18 and pk (=_log 10 K) to be 5.83± 0.03(mean ± S.E.M., N = 11, where N is number of experiments). Figure 4B is a plot of eqn (1) with best fit parameters. Eleven further experiments similar to the one shown in Fig. 3 were performed and n and K values were obtained. Of these, six experiments were from one time treatment with gelsolin. The results are plotted in Fig. 5 against the remaining tension. In Fig. 5 the same symbols indicate the data from the same preparation. The Hill factor n, which represents the cooperativity, is plotted in Fig. 5A. At a remaining tension higher than 20 %, the Hill factor did not change much. However, the Hill factor dropped significantly when the remaining tension was less than 20 %. This observation indicates that the cooperativity diminished rapidly if the thin filament length was reduced so that the thick-to-thin filament overlap region was reduced below 20 % of the control condition. In contrast, the change in pk was limited to _0.22, and it averaged _0.075 ± (N = 10) when the remaining tension was between 20 and 51 %, and _0.075 ± (N = 13) when the remaining tension was less than or equal to 20 %. This observation indicates that Ca 2+ sensitivity decreases slightly when the thin filament length is reduced, but there is no associated change in the Ca 2+ sensitivity when the cooperativity diminishes dramatically for reductions in thin filament length below 20 %.

5 Exp. Physiol Length of cooperative units on the thin filament 695 DISCUSSION The purpose of our study was to determine the extent of cooperativity in the thin filament in single fibres of rabbit psoas muscle. To achieve this goal, we used a plasma protein, gelsolin, to reduce the thin filament length. By following the steepness of the pca tension curve, we aimed to find a point where the steepness suddenly drops as the thin filament length is reduced. Figure 6 is a schematic diagram of a sarcomere structure describing our thin filament extraction system. The Z-line, the thin filament, the thick filament and cross-bridges are shown. The dimensions of these elements are based on values summarized by Higuchi et al. (1995). As the thin filament length is reduced by gelsolin treatment, the maximum isometric tension is expected to become smaller. Because isometric tension is proportional to the amount of overlap between thick and thin filaments (Gordon et al. 1966) where the cross-bridges are present, 100 %, 50 %, 30 % and 10 % isometric tension is expected in Fig. 6A, B, C and D, respectively. Biochemical analysis of gelsolinextracted fibres demonstrates that only the thin filament was extracted from the fibres (Fig. 1) as in previous reports (Funatsu et al. 1994; Fujita et al. 1996, 2002). Cooperativity in the contractile apparatus can happen within the thin filaments (Bremel & Weber, 1972; Tobacman & Sawyer, 1990), within the cross-bridges or between the thin filament and the cross-bridges (Güth & Potter, 1987; Gordon et al. 1988; Pan et al. 1989; Brandt et al. 1990; Metzger, 1995). In our investigation, we focused on the thin filament cooperativity. The differential extraction of the thin filament with gelsolin made the skeletal muscle fibre a good model for studying the thin filament cooperativity. In this system, the cooperative effects within Figure 5 A, the Hill factor n, which represents the cooperativity, is plotted against remaining tension after normalization. At higher remaining tension, the n value does not change much, but drops significantly when the remaining tension becomes less than 20 %. The continuous line was drawn by eye. B, a change in the Ca 2+ dissociation constant (K) is plotted in log units against remaining tension. This figure demonstrates that the change in the Ca 2+ dissociation constant is limited to the range 0 to _0.22 (average, _0.075) in DpK units, indicating that there is a small decrease in Ca 2+ sensitivity with the reduction of thin filament length. The same symbols indicate the same fibre preparation. Figure 6 Schematic diagram of a sarcomere describing the experimental system. The sarcomere length is set to 2.2 mm in this diagram. The Z-line, the thin filament, the thick filament and cross-bridges are shown. As the thin filament length is reduced by gelsolin treatment, the maximum isometric tension becomes smaller (A, 100 %; B, 50 %; C, 30 %; D, 10 %). Remaining tension corresponds to the overlap region of thick and thin filaments where the cross-bridges are present.

6 696 W. Ding, H. Fujita and M. Kawai Exp. Physiol the thin filament were gradually destroyed during the extraction, while the thick filament including cross-bridges remained intact. If the thin filament cooperativity exists over the entire length, we should see a gradual and continuous decline of the Hill factor. If the cooperativity is limited to a certain length, then we should expect a break point which corresponds to an outer limit of the cooperativity. Above the break point, the Hill factor should remain the same, and below the break point, the Hill factor should decline. We found that the cooperativity did not change much if the remaining tension was between 20 and 100 %, but the cooperativity dropped significantly when the remaining tension was less than 20 % (Fig. 5A). This result indicates that the cooperativity extends up to about 20 % of the overlap length between the thin filament and crossbridges. Because in one strand of thin filament there are about 20 basic cooperative units (~ (1.63_ 0.16)/2/0.036) that interact with cross-bridges to generate force at a sarcomere length of 2.2 mm, we calculate that the thin filament cooperativity extends to about four basic cooperative units (= 20 w 0.2). This calculation is based on the fact that the thin filament length is 1.12 mm, the thick filament length is 1.63 mm, and the bare zone is 0.16 mm (Higuchi et al. 1995). The basic cooperative unit consists of seven actin monomers, one tropomyosin dimer and one troponin complex that includes TnC, TnI and TnT. This result is similar to the prediction of Geeves & Lehrer (1994), and at variance with the results of Brandt et al. (1987). Geeves & Lehrer (1994) predicted that the cooperativity would extend to perhaps about 10 % of the thin filament length, that is about three cooperative units (~ 0.1 w 1.12/0.036), whereas Brandt et al. (1987) predicted that the cooperativity would extend to the entire length of the thin filament. The decrease in the Ca dissociation constant (pk) is ± log units (N = 23) (Fig. 5B) when the remaining tension is less than 51 %, indicating that there is a slight decrease in Ca 2+ sensitivity (by 16 %) with reduction in thin filament length. However, there is no clear break point in pk as in the case of cooperativity. The diagram of Fig. 6 is based on an ideal experiment and known lengths of structural elements (Higuchi et al. 1995). However, this may not be the case in reality after the gelsolin treatment. This is because gelsolin is not a pointed end depolymerizer, but rather it cuts the thin filament at a random position (McLaughlin et al. 1993). Therefore, it would be more appropriate to state that the remaining isometric tension after gelsolin treatment reflects the average length of the thin filament that overlaps with myosin cross-bridges. The actual filament lengths must be distributed around the average length. The distribution of the thin filament length would cause less than perfect results. The fact that the data shown in Fig. 5A are inclined to the left between 20 and 100 % tension implies that the thin filament length was indeed distributed around the average length. However, even with this imperfection in the experimental model, we can safely conclude that the cooperativity extends to about 20 % of the thin filament length, because of the presence of the clear break point in the Hill factor at about 20 % remaining tension (Fig. 5A). In conclusion, we studied thin filament cooperativity experimentally, and found that it extends up to 20 % (equivalent to four cooperative units) of the filament length. BRANDT, P. W., COX, R. N. & KAWAI, M. (1980). Can the binding of Ca 2+ to two regulatory sites on troponin-c determine the steep pca/tension relationship of skeletal muscle? 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