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J Physiol (2003), 550.1, pp. 205-215
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.040899

Thin filament activation and unloaded shortening velocity of rabbit skinned muscle fibres

Carl A. Morris, Larry S. Tobacman* and Earl Homsher

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

The unloaded shortening velocity of skinned rabbit psoas muscle fibres is sensitive to [Ca²⁺]. To determine whether Ca²⁺ affects the unloaded shortening velocity via regulation of crossbridge kinetics or crossbridge number, the shortening velocity was measured following changes in either [Ca²⁺] or the number of active thin filament regulatory units. The native troponin C (TnC) was extracted and replaced with either cardiac TnC (cTnC) or a mixture of cTnC and an inactive mutant cardiac TnC (CBMII TnC). The unloaded shortening velocity of the cTnC-replaced fibres was determined at various values of [Ca²⁺] and compared with different cTnC:CBMII TnC ratios at a saturating [Ca²⁺]. If Ca²⁺ regulates the unloaded shortening velocity via kinetic modulation, differences in the velocity-tension relationship between the cTnC fibres and the cTnC:CBMII TnC fibres would be apparent. Alternatively, Ca²⁺ control of the number of active crossbridges would yield similar velocity-tension relationships when comparing the cTnC and cTnC:CBMII TnC fibres. The results show a decline in the unloaded shortening velocity that is determined by the relative tension, defined as the level of thin filament activation, rather than the [Ca²⁺]. Furthermore, at lower levels of relative tension, the reduction in unloaded shortening is not the result of changes in any cooperative effects of myosin on Ca²⁺ binding to the thin filament. Rather, it may be related to a decrease in crossbridge-induced activation of the thin filament at the level of the individual regulatory unit. In summary, the results suggest that Ca²⁺ regulates the unloaded shortening velocity in skinned fibres by reducing the number of crossbridges able to productively bind to the thin filament without affecting any inherent property of the myosin.

(Resubmitted 4 February 2003; accepted after revision 4 April 2003; first published online 2 May 2003)
Corresponding author C. A. Morris: Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania, 19104, USA. Email: camorris{at}mail.med.upenn.edu

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Huxley's (1957) two-state model of muscle contraction predicts that the unloaded shortening velocity (V_u) in maximally activated muscle is dependent on the rate of crossbridge detachment and is determined by the balance of positively and negatively strained crossbridges. It has been hypothesized that isometric force is directly proportional to the number of attached crossbridges, while V_u is independent of crossbridge number. Gordon et al. (1966) tested this by varying the sarcomere length of intact muscle fibres and observed only a small decline (~15 %) in the shortening velocity of fibres shortening isotonically under low load (4 % maximal isometric tension, P₀). These results were confirmed using the slack test to measure V_u (Edman, 1979), suggesting that changes in this parameter under different conditions stem from changes in the crossbridge dissociation rate or power-stroke size rather than alterations in the number of attached and cycling crossbridges. The speed of shortening during an unloaded contraction in a maximally activated muscle fibre is then set by the rate-limiting step in the crossbridge cycle; either ADP release or an isomerization of a step immediately preceding it (Cooke & Pate, 1985; Siemankowski et al. 1985; Metzger, 1996).

It was originally thought that Ca²⁺ binding to the thin filament regulatory proteins acts as a simple switch, providing crossbridges access to the actin binding sites (Ebashi & Endo, 1968). Once the troponin (Tn) complex is activated and the actin sites are accessible, [Ca²⁺] should not affect the kinetics of the crossbridge cycle, and thus V_u should be unaffected by reductions in the [Ca²⁺]. In living fibres, Podolsky & Teichholz (1970) reported that V_u was unaffected by reduced [Ca²⁺], while Julian (1971) observed a reduction in the shortening velocity as [Ca²⁺] was lowered. In skinned muscle fibres, V_u decreases as [Ca²⁺] is reduced (Moss, 1986; Martyn et al. 1994; Metzger, 1996). During submaximal activations (reduced [Ca²⁺]), skinned fibres exhibit a biphasic shortening during the slack test (Moss, 1986), but living fibres do not seem to exhibit this behaviour (Edman, 1979). The biphasic shortening consists of two distinct linear phases, characterized by an initial fast phase of shortening (V_u1) and a second slower phase (V_u2) with an apparent break point at ~8-12 % length change (Moss, 1986; Metzger & Moss, 1988). The reason for the biphasic shortening behaviour during submaximal activation is not understood. Several mechanisms have been proposed to account for this observation: (1) the existence of a constant passive internal load (Brenner, 1980; Gulati & Babu, 1985); (2) the presence of long-lived, negatively strained, strongly bound crossbridges (Moss, 1986; Martyn et al. 1994); (3) weakly bound crossbridges imposing a negative strain on shortening (Stehle & Brenner, 2000); (4) shortening-induced deactivation of the thin filament (Iwamoto, 1998; Josephson & Edman, 1998). V_u in skinned muscle fibres is influenced by addition of inorganic phosphate and ADP (Metzger, 1996), nucleotides (Regnier et al. 1998), partial extraction of either TnC (Moss, 1986) or C-protein (Hoffman et al. 1991).

Iwamoto (1998) examined the shortening velocity in skinned fibres using a protocol in which the muscle was activated and allowed to develop force isometrically and then was repeatedly shortened by ~20 % at speeds near V_u. Following the initial shortening period, the fibre was immediately and rapidly restretched to its original length and again allowed to shorten. The shortening velocity in submaximally activated fibres exhibited biphasic behaviour (both fast and slow phases) only during the initial shortening of the multiple shortening protocol, while subsequent releases produced only slow-phase shortening. Iwamoto suggested that crossbridge cooperativity (i.e. attached crossbridges promoting activation of the thin filament) is reduced by shortening and deactivates the thin filament that only becomes evident during submaximal activations. In support of this, solution studies using isolated actin, S-1, and regulatory proteins or isolated myofibrils, demonstrated that S-1 binding to thin filaments occurs in a cooperative manner in the absence of Ca²⁺, with the cooperativity greatly reduced at saturating levels of Ca²⁺ (Bremel & Weber, 1972; Greene & Eisenberg, 1980; Swartz et al. 1996).

Although it is clear that lowering [Ca²⁺] causes a reduction in the V_u of skinned muscle fibres, the mechanism is not well understood. It is possible that Ca²⁺ activates the fibre by increasing the population of actively cycling crossbridges without altering any kinetic properties. Alternatively, activation by Ca²⁺ may directly modulate the kinetics of the crossbridge cycle. To investigate the potential regulatory mechanisms involved, we extracted the endogenous skeletal TnC (sTnC) and reconstituted the thin filament with mixtures of cardiac TnC (cTnC) and an inactivated cTnC mutant, CBMII TnC (Putkey et al. 1989; Huynh et al. 1996). We recently showed that incorporation of CBMII TnC produced a direct reduction in skinned fibre isometric tension (Morris et al. 2001). Thus, by varying the relative amounts of cTnC and CBMII TnC incorporated into the thin filament, we altered only the number of crossbridges that can interact with the thin filament at saturating [Ca²⁺]. This method allows a direct comparison of the effects of reduced levels of fibre activation due to lowered [Ca²⁺] and reduction in the number of actively cycling crossbridges on V_u.

If Ca²⁺ does not regulate attached crossbridge kinetics, then the behaviour of V_u at reduced [Ca²⁺] and at reduced levels of thin filament activation with saturating [Ca²⁺] will be the same (i.e. plots of V_u vs. relative force varied by the two methods will superimpose. If Ca²⁺ controls crossbridge kinetics, the plots should exhibit significant differences including alterations in the biphasic shortening behaviour observed at low [Ca²⁺]. The results presented below show a decline in V_u that is dependent only on the extent of activation, not on [Ca²⁺]. This suggests that shortening velocity is dependent on the number of crossbridges attaching productively to the thin filament, with little or no Ca²⁺-dependent modulation of crossbridge cycle kinetics.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Solutions

All fibre solutions were maintained at 200 mM ionic strength and contained 100 mM Bes (pH 7.1 at 15 °C), 5 mM MgATP, 1 mM Mg²⁺ (added as magnesium acetate), 20 mM potassium acetate, 15 mM creatine phosphate, 200 U ml^-1 creatine phosphokinase, and 1 mM DTT. Relaxing and activating solutions also contained 20 mM EGTA. Ca²⁺ was added as Ca²⁺-K⁺-EGTA, and the pCa was varied by adjusting the relative amounts of K⁺-EGTA and Ca²⁺-K⁺-EGTA, while maintaining total EGTA at 20 mM. The preactivating solution contained 2 mM K⁺-EGTA and 18 mM 1,6-diaminohexane-N,N,N',N'-tetraacetic acid. The composition of the solutions was calculated using the QuickBasic program, SOLUTION (Millar & Homsher, 1990).

Fibre preparation and mechanical apparatus

Skinned fibres were prepared from female New Zealand White rabbit psoas muscle as described previously (Goldman et al. 1984; Millar & Homsher, 1990). The rabbits were killed by overdose with pentobarbitone (120 mg kg^-1 I.V.) according to protocols approved by the Chancellor's Animal Research Committee at UCLA. Single skinned fibres were isolated in silicon oil with the aid of a dissection microscope, and T-clips were loosely attached. The fibre ends were fixed by flowing a stream of 1 % gluteraldehyde in 50 % glycerol over each end for ~20 s to reduce end-end compliance (Chase & Kushmerick, 1988). For mechanical measurements, fibre segments (1.5-3.3 mm) were attached via the T-clips at one end to a force transducer (SensoNor AE801 strain gauge), while the other end was attached to a length driver (Ling 100A shaker motor). After mounting the fibre, the sarcomere length was set to 2.6 µm using a He:Ne laser diffraction pattern, the fibre width was measured at three or four different sites along the length of the fibre, and the fibre length was measured, all under relaxing conditions (Millar & Homsher, 1990). The sarcomere length was not measured during active contractions.

V_u was measured using the 'slack test' method (Edman, 1979). The fibre was shortened by variable lengths (from a constant fibre length) after reaching steady-state tension (L). The time required for redevelopment of force after each release was measured (t). Only data from fibres whose maximal isometric tension fell by < 10 % during the course of the experiment were included in the study. The fibre apparatus was modified to enable multiple length changes during a single activation, based on the method described by Brenner (1983). During measurements of V_u, the fibre was shortened and tension allowed to redevelop at the shorter length. Rapidly stretching a fibre while it is fully activated frequently damages the fibre irreversibly. To circumvent this problem, an extra release was included immediately before the fibre was returned to its initial length, reducing force to zero (as most crossbridges detach), thereby limiting the possibility of fibre damage during the restretch. This simple modification allowed measurement of an entire V_u plot (five to nine separate length changes) during a single activation.

V_u was determined as described by Moss (1986). Plots of L vs. t for each fibre, under each condition, were fitted to either one or two straight lines, with V_u calculated in muscle lengths (ML) s^-1 from the slope of the lines. Generally, changes in V_u between the slopes of L < 10 % and L > 10 % indicated that the data were better fitted with two lines (Moss, 1986).

Data acquisition

All tension signals were recorded and analysed using KFIT (a QuickBASIC program written by N. Millar) running on a Gateway 66 MHz PC (Millar & Homsher, 1990; Regnier et al. 1995; Morris et al. 2001) and SigmaPlot 4.0. Fibre data are reported as means ± S.E.M.

TnC extraction and reconstitution

Endogenous sTnC was extracted using a combination of previously described methods (Metzger et al. 1989; Yates et al. 1993). The sarcomere length was increased to > 3.0 µm in relaxing solution and the fibres were incubated in a solution containing 5 mM EDTA, 20 mM Tris-HCl (pH 7.2), and 0.5 mM trifluoperazine dihydrochloride at 15-17 °C for 6-8 min. If the Ca²⁺-activated isometric tension did not fall to less than 10 % P₀, a second incubation was performed. Reconstitution with sTnC, bovine ventricular cTnC or a mixture of CBMII TnC and cTnC (CBMII TnC-cTnC) was achieved by incubating the fibre in relaxing solution containing 0.5 mg ml^-1 TnC for 1 min followed by 30 s in relaxing solution alone. This cycle was repeated four times prior to initial force measurement at pCa 4.5. In most cases, four to six 1 min cycles in the TnC-containing solution were sufficient to maximize the recovery of Ca²⁺-dependent isometric force.

The sTnC was kindly provided by Marion Greaser (University of Wisconsin at Madison) and was obtained by methods described in Greaser & Gergely (1973). The cTnC (bovine ventricular cTnC and CBMII cTnC) was produced and purified as described previously (Huynh et al. 1996). The TnC content of the muscle fibres was determined using SDS-PAGE and densitometry following published methods (Giulian et al. 1983; Moss et al. 1985; Moss, 1986) and as shown for CBMII TnC (Morris et al. 2001). The TnC content was measured both in the fibres used for mechanical measurements and in separate fibres. The TnC content was normalized to the myosin light chain (LC) 1 (LC₁) content. The LC₂/(LC₁ + LC₃) ratio (mean ± S.E.M.; n = 3) was similar for control (0.66 ± 0.03), extracted (0.63 ± 0.05), cTnC-reconstituted (0.67 ± 0.06) and CBMII TnC-cTnC-reconstituted fibres (e.g. 0.65 ± 0.06 for 25 % CBMII TnC-cTnC; 0.63 ± 0.05 for 50 % CBMII TnC-cTnC). These results suggest that the extraction procedure was specific for TnC, with no observable loss of myosin light chains, and are consistent with previous studies (Moss et al. 1986a; Metzger & Moss, 1988; Metzger et al. 1989; Martyn et al. 1994; Regnier et al. 2002).

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Extraction of the native sTnC in single muscle fibres reduced the maximal isometric tension from 142.5 ± 5.6 kN m^-2 (mean ± S.E.M.; n = 21) to 10.0 ± 1.0 kN m^-2 (n = 18), a decrease to 0.07 P₀. Reconstitution with purified sTnC returned maximal force to 0.89 P₀ (126.9 ± 11.3 kN m^-2; n = 3), while replacement with 100 % cTnC returned force to 0.72 P₀ (102.1 ± 6.8 kN m^-2; n = 7) of the tension obtained prior to extraction (Table 1), results similar to those obtained previously (Morris et al. 2001).

P₀ in fibres reconstituted with CBMII TnC-cTnC was plotted as a function of the relative amount of cTnC (cTnC/(cTnC + CBMII TnC)). The decline in tension was fit with a straight line yielding a slope of 95.6 ± 4.6 kN m^-2 per unit cTnC/(cTnC + CBMII TnC), with a y intercept of 4.1 ± 2.9 kN m^-2. These results suggest that varying the ratio of cTnC to CBMII TnC reduces the isometric tension in proportion to the amount of CBMII TnC added, as shown previously (Morris et al. 2001).

Steady-state measurements were performed at varying [Ca²⁺] prior to extraction of the native sTnC and following reconstitution with cTnC to determine the force-pCa relationship. The sTnC and cTnC force-pCa data were fitted to the Hill equation (P = P₀/(1 + 10^{nH(pCa50 - pCa)}; where pCa₅₀ is [Ca²⁺] that results in 50% of the maximum force) to determine if there were any changes in Ca²⁺ sensitivity. The fibres exhibited normal Ca²⁺ sensitivity with the expected shift in pCa₅₀ from 6.74 ± 0.02 to 6.63 ± 0.03 and a reduction in the Hill coefficient from 2.43 ± 0.14 to 1.87 ± 0.10 observed following extraction of the native TnC and replacement with cTnC. These results are in agreement with previous work (Moss et al. 1986b; Metzger, 1996).

V_u of extracted and reconstituted fibres

The V_u of skinned skeletal muscle fibres was determined using the slack test (Edman, 1979). A length change was applied to an isometrically contracting muscle fibre and the time required for the fibre to take up the slack and redevelop tension was determined. The applied length changes resulted in the tension dropping to zero with the time required to remove the slack (t) varying with the size of the length change (L). Figure 1 shows a plot of L vs. t, at pCa 4.5, for a fibre containing endogenous sTnC and the same fibre following extraction of the native sTnC and reconstitution with purified sTnC. Extraction of the endogenous sTnC and replacement with purified sTnC did not affect V_u. Table 1 shows the V_u data for native, sTnC- and cTnC-reconstituted fibres. There was no difference observed between the V_u at pCa 4.5 before extraction and following replacement with sTnC (P < 0.6) or cTnC (P < 0.8). In addition, no difference between the sTnC- and cTnC-reconstituted shortening velocities was observed (P < 0.5). These results are similar to those found previously (Moss, 1986) and suggest that the extraction and replacement procedure did not significantly affect the V_u of the muscle fibre.

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Figure 1. Plot of slack test before extraction of endogenous sTnC and after replacement with purified sTnC The inset shows original tension records for the three length changes performed in the native fibres and following replacement with purified sTnC, corresponding to L values of 330, 390 and 450 µm. 1* and 2* show the similarity for L 330 µm before and after the extraction and replacement of TnC. No change in Vu is observed (2.3 ML s-1 before extraction vs. 2.4 ML s-1 after replacement). Isometric force is reduced by ~15 % following replacement with purified sTnC.

Effect of Ca²⁺ on V_u

To examine the effect of [Ca²⁺] on unloaded shortening, V_u was measured at maximal and submaximal [Ca²⁺]. Figure 2 shows that a plot of L vs. t is well fitted by a straight line at pCa 4.5. At reduced levels of activation (pCa 6.6 and pCa 6.8 in Fig. 2) the plot of length change vs. slack time becomes biphasic and is better fitted by two straight lines. The observation of a non-linear relationship between t and L at submaximal activations (increased pCa) is consistent with results obtained previously (Gulati & Podolsky, 1981; Brenner, 1986; Moss, 1986; Metzger & Moss, 1988; Martyn et al. 1994; Metzger, 1996; Iwamoto, 1998).

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Figure 2. Plots of slack-test measurements in a single skinned fibre at three different values of [Ca2+] The inset shows examples of original records of both length (upper) and tension (lower) for the slack test at pCa 4.5. The plot indicates a linear Vu at pCa 4.5, but it is apparent at pCa 6.6 and 6.8 that the data is better fitted by two linear phases. The Vu at pCa 4.5 was 2.6 ML s-1. Vu1 and Vu2 were 1.9 and 0.65 ML s-1 at pCa 6.6 and 1.7, and 0.32 ML s-1 at pCa 6.8. Maximal tension was 130 kN m-2 and fibre length was 3.1 mm at 2.6 µm sarcomere-1.

Figure 3A shows the V_u in native sTnC- and cTnC-replaced fibres as a function of pCa. Reducing the [Ca²⁺] produced similar and characteristic changes in the V_u in both sTnC- and cTnC-containing fibres. At submaximal activations, the initial phase of shortening (V_u1) for both sTnC and cTnC remained relatively constant until the pCa exceeded 6.7, while the second phase (V_u2) declined at pCa 6.0 and greater. The sTnC and cTnC V_u1 data were fitted with the Hill equation: P = P₀/(1 + 10^{nH(pCa50 - pCa)}), with a Hill coefficient of 3.95 ± 1.0 for the sTnC fibres and 2.16 ± 0.53 for the cTnC fibres. The pCa₅₀ values were similar for the sTnC and cTnC fibres, 6.93 ± 0.05 and 6.86 ± 0.04, respectively (Table 1). As expected, the pCa₅₀ values for V_u were higher when compared to those for steady-state tension, indicating that less Ca²⁺ is required to maintain the initial phase of the shortening velocity than maximal tension. Figure 3B shows a plot of V_u as a function of the relative tension and indicates that V_u1 changes only gradually until the force is reduced to ~40 % for sTnC and ~45 % for cTnC (P < 0.05), supporting the hypothesis that V_u1 (or V_max) is relatively independent of crossbridge number, as reported previously (Huxley, 1957; Moss, 1986). The conversion from pCa to relative force was determined using force data at different [Ca²⁺] and normalizing to the force obtained at pCa 4.5 with the same TnC isoform (Morris et al. 2001). Comparison of the V_u data of the sTnC and cTnC fibres indicate only minor differences in either the fast or slow phases of shortening between the TnC isoforms.

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Figure 3. Unloaded shortening velocity A, Vu in skinned fibres containing either sTnC (filled symbols) or cTnC (open symbols) as a function of pCa. The circles indicate the initial phase of shortening (Vu1) and the squares represent the slower shortening phase (Vu2). The data are shown as means ± S.E.M., with n > 5 for each point. The individual Vu1 data for the sTnC and cTnC were fitted with the Hill equation, yielding a Hill coefficient of 3.95 ± 1.0 and 2.16 ± 0.72, and a pCa50 of 6.93 ± 0.04 and 6.86 ± 0.04, respectively (dashed line, sTnC; continuous line, cTnC). B, Vu vs. relative tension from fibres containing sTnC (filled symbols) or cTnC (open symbols). The tension was varied by altering the pCa and is shown relative to the maximal tension obtained at pCa 4.5. The circles represent Vu1 and the squares, Vu2; the lines represent fits to the data, dashed lines, sTnC; continuous lines, cTnC. The Vu1 data were fitted with the equation y = KmVmax/Km + Vmax only to show the similarities in the data. The Vu2 data were fitted with a straight line and the two data sets again show good agreement. Each point represents the mean ± S.E.M. with n > 5.

Effect of CBMII TnC on V_u

To learn whether the reduction in V_u is caused by either a reduction in the number of cycling crossbridges or by an effect of Ca²⁺ per se, we measured the V_u following incorporation of CBMII TnC into the thin filament at a saturating [Ca²⁺]. The isometric force and V_u were measured at various [Ca²⁺] prior to the extraction and replacement protocol. Following replacement with the mixture of CBMII TnC and cTnC, the force correlated directly with the relative amount of cTnC. The V_u was then determined using the slack test at saturating [Ca²⁺]. Plots of L vs. t at pCa 4.5 for two fibres before and after replacement with either 25 or 50 % CBMII TnC are shown in Fig. 4. In both plots, the biphasic shortening is apparent after extraction and replacement with the inactive TnC. In Fig. 4A, the initial V_u was 2.4 ML s^-1, while following extraction and replacement with 25 % CBMII TnC, V_u1 and V_u2 were 2.4 and 1.3 ML s^-1, respectively. Following extraction and replacement with 50 % CBMII TnC (Fig. 4B), the V_u was reduced from 3.1 to 2.3 ML s^-1 and V_u2 was 0.4 ML s^-1.

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Figure 4. Plots of L vs. t for skinned fibres before extraction of sTnC and after replacement with 25 % (A) and 50 % CBMII TnC (B) The inset in A shows original tension records before and after replacement with 25 % CBMII TnC. The two points labelled 1* represent a length change of 240 µm before and after replacement, indicating no change in the time required to remove the slack at this shorter length change. The points labelled 2* and 3* correspond to a similar length change (420 µm) before and after replacement, showing a reduction in shortening velocity following replacement with 25 % CBMII TnC. In A, the Vu before extraction was 2.4 ML s-1, and after replacement Vu1 and Vu2 were 2.4 and 1.3 ML s-1, respectively. In B, Vu was 3.1 ML s-1 and Vu1 and Vu2 were 2.3 and 0.4 ML s-1 after replacement with 50 % CBMII TnC. Note the different time scales in A and B. The fibres in A and B had maximal tensions of 133 and 126 kN m-2, with lengths of 2.8 and 2.9 mm, respectively, at a sarcomere length of 2.6 µm.

Comparison of V_u as a function of relative tension (Fig. 5) shows that V_u1 and V_u2 were reduced similarly as relative tension declined, regardless of the method used to lower the level of activation, either by varying [Ca²⁺] or CBMII TnC incorporation. Replacement with CBMII TnC reduced the force produced by the fibre in direct proportion to the amount of CBMII TnC added (Morris et al. 2001). However, V_u1 remained near maximal until the relative tension had been reduced to ~0.5 P₀, below which it only fell to 30 % of maximal at the lowest level of activation measured (~0.2 P₀). V_u2 in both cTnC and CBMII TnC fibres displayed no such separation from the relative tension, as it fell linearly with the level of thin filament activation. Importantly, the two methods used to reduce the level of activation, CBMII TnC incorporation and varying [Ca²⁺] yielded superimposable changes in both V_u1 and V_u2.

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Figure 5. Vu as a function of relative tension in fibres containing 100 % cTnC (filled symbols) or ratios of CBMII:cTnC (open symbols) The tension was varied in the fibres containing 100 % cTnC by altering the pCa, the tensions being shown relative to that measured at pCa 4.5 after replacement. The CBMII TnC-containing fibres are shown relative to 72 % of the tension measured prior to extraction (sTnC) at pCa 4.5. All data are represented as means ± S.E.M. with n > 5 for each point. The lines (continuous lines, cTnC; dotted lines, CBMII TnC) show fits to the data as described in Fig. 3 and indicate that both phases of unloaded shortening decline similarly for both TnC isoforms. The cTnC data (open symbols; dotted lines) are the same as those shown in Fig. 3.

These results suggest that the unloaded shortening is dependent on fractional thin filament activation, not Ca²⁺ per se. The observed behaviour of both V_u1 and V_u2 with relative tension (Fig. 5) is the same regardless of the mechanism deactivating the thin filament. The data therefore suggest that neither the free [Ca²⁺] nor the dynamics of Ca²⁺ binding directly affects the kinetic behaviour of the attached and detaching crossbridges.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

We altered the level of thin filament activation by clamping fractional Ca²⁺ binding at subsaturating levels and determined the functional effects of reduction in the number of crossbridges on V_u in skinned muscle fibres, and compared the results observed at various values of [Ca²⁺]. The data imply that it is the level of thin filament activation (i.e. the number of attached and cycling crossbridges), not the [Ca²⁺], controls V_u.

Extraction and replacement with CBMII TnC-cTnC

Following extraction of the native TnC and reconstitution with either purified sTnC or cTnC, the maximal force was reduced. It is unclear why isometric tension does not return to pre-extraction levels. Other studies have reconstituted the isometric tension with cTnC to levels ranging from 0.6 P₀ (Martyn et al. 1994) to 1.0 P₀ (Metzger, 1996). However, regardless of the mechanism, it does not appear to affect V_u. Maximal V_u in the presence of saturating Ca²⁺ is unaffected by cTnC incorporation and is the main focus of the current work.

It is likely that incomplete incorporation of the exogenous TnC is partially responsible for the reduction in P₀. However, incorporation of TnC will be randomly distributed along the length of the thin filament and not isolated to the overlap or non-overlap region. To reduce the potential for selective extraction, the fibres were lengthened to > 3.0 µm sarcomere^-1 during the extraction procedure to maximize removal of native TnC, as extraction occurs more readily in the non-overlap region (Yates et al. 1993; Swartz et al. 1997). Reconstitution was performed under relaxing conditions where no differences between TnC incorporation in overlap and non-overlap regions were observed in myofibrils (Swartz et al. 1997). Martyn et al. (1994) suggested that incorporation of TnC was distributed evenly across the width of the fibre, but distribution of exogenous TnC along the length of the thin filament is not well characterized, and under relaxing conditions it is thought to be random (Swartz et al. 1997).

A brief survey of published work indicates that replacement with cTnC into skeletal muscle fibres generally reduces the maximal isometric tension to ~75 % (Kerrick et al. 1985; Metzger et al. 1989; Harrison & Bers, 1990; Moss et al. 1991; Martyn et al. 1994; Metzger, 1996). This suggests reconstitution with cTnC into the skeletal muscle fibre may alter interactions within the Tn complex, tropomyosin and actin. However, it should be noted that full recovery of isometric tension following replacement with cTnC has been obtained for all reconstituted fibres (Metzger, 1996) and in a subset of reconstituted fibres (Moss et al. 1991), suggesting that it is possible to obtain maximal force following reconstitution with the cTnC isoform in skinned skeletal muscle fibres.

Regnier et al. (2002) recently published steady-state force data using a mutant sTnC that could not bind Ca²⁺, similar to CBMII TnC. They determined steady-state force as a function of the active TnC fraction and the results indicated a greater than linear response in tension to the increase in the fraction of active TnC units, whereas the use of cTnC produces a linear relationship between isometric force and the fraction of active TnC. The only major difference between the two studies was the use of cTnC or sTnC. This suggests that cTnC is not able to communicate as effectively with the sTnI-sTnT complex to fully activate the thin filament. Furthermore, Pogessi et al. (C. Poggesi, personal communication) observed that substitution of the entire cardiac Tn complex into an extracted skeletal myofibril does not alter maximal isometric force or the rate of force redevelopment. However, reconstitution with a Tn complex containing cTnC and sTnI-sTnT produces changes in the maximal isometric force and the rate of force redevelopment.

During this work, great care was taken to completely extract the native sTnC from the muscle fibre. By reducing the isometric force to < 0.1 P₀, reconstitution with the cTnC would enable replacement to occur randomly along the length of the entire thin filament and is likely to result in a homogeneous regulatory system. Though every effort was made to fully reconstitute the thin filament with a full complement of cTnC or CBMII TnC, it is possible that incomplete incorporation of TnC occurred. Alternatively, the loss of P₀ and the observed shift in Ca²⁺ sensitivity may be a result of undetected sarcomere length changes, as we did not monitor the active sarcomere length. As discussed above, we suggest that the addition of cTnC to a thin filament containing sTnI-sTnT does not fully activate the regulatory complex and therefore limits the force recovered following reconstitution with the cTnC isoforms, although it is unclear what limits isometric tension and it is evident that further investigation is necessary. Extraction of TnC has been shown to alter the Ca²⁺ sensitivity of muscle fibres (Moss et al. 1985, 1991), and this could be partially responsible for the shift in pCa₅₀ observed in the present study. However, our results are similar to pCa₅₀ changes obtained by Metzger (1996) in cTnC-reconstituted fibres exhibiting 0.99 P₀ of the pre-extraction tension and are also consistent with our previous results (Morris et al. 2001) and those of others (Moss et al. 1986). Therefore, the change in Ca²⁺ sensitivity is probably due to the addition of the cTnC, as described previously (Moss et al. 1986; Metzger, 1996); however, incomplete reconstitution cannot be ruled out.

Our results indicate that reconstitution with various ratios of CBMII TnC and cTnC provides a method by which to reduce the isometric tension independently of the [Ca²⁺] while greatly increasing the likelihood of all the thin filament regulatory units containing the full complement of TnC, TnI and TnT.

The effects of fibre activation on V_u

Two studies have suggested that shortening velocity correlates well with the crossbridge cycling rate. Barany (1967) measured the actomyosin ATPase activity of myosin from a variety of muscles and found that V_u is roughly proportional to the ATPase rate. Siemankowski et al. (1985) observed a direct correlation between the ADP dissociation rate and V_u in different muscle types ranging in speeds over two orders of magnitude, suggesting that ADP release limits V_u. These studies imply that during maximal activations, V_u is dependent on the crossbridge cycling rate, which is a property of myosin. If the shortening velocity is due to cycling crossbridge kinetics, which is defined by the myosin isoform, why does a reduction in the [Ca²⁺] or level of thin filament activation slow muscle shortening? The slack test, as described by Edman (1979), monitors the time required to take up the slack generated by the imposed change in fibre length. Generally, the fibre is shortened at one end from a specific sarcomere length by a varying amount. The time taken to begin redeveloping force should be similar and is dependent solely on the amount of slack induced by the length change. However, during submaximal activations there is an appreciable decline in V_u over extended length changes. The slowing of V_u can only be due to crossbridges taking longer to remove the slack imposed by the length change, either directly through reductions in the myosin cycling rate or via thin filament regulatory mechanisms.

Martyn et al. (1994) examined this question using a modified cTnC that remained in an active conformation independent of [Ca²⁺]. Reductions in both phases of V_u were observed that were dependent on the level of thin filament activation. Their results also suggest that Ca²⁺ binding to proteins other than TnC, such as the myosin light chains, does not greatly affect V_u. Reductions in the level of activation by varying [Ca²⁺], partial extraction of TnC (Moss, 1986; Metzger, 1996), and partial replacement with activated TnC (Martyn et al. 1994) or CBMII TnC do not limit the number of crossbridges in the same way as varying the degree of filament overlap (Gordon et al. 1966; Edman, 1979). In the present study, thin filament activation is modified directly and randomly along the entire filament length, thereby limiting crossbridge attachment throughout the thin filament, while altering the degree of overlap maintains complete thin filament activation where the crossbridges can attach, providing normal thin filament access. The [Ca²⁺] affects the proportion of thin filament units that are switched on and accessible to cycling crossbridges at any given time. The partial extraction of TnC produces a distribution of TnI molecules that will prevent the necessary Tn-tropomyosin movement necessary for active crossbridge cycling. Similarly, incorporation of CBMII TnC produces regulatory units that inhibit crossbridge binding along the length of the thin filament, limiting any potential long-range cooperative effects of strong crossbridge binding. Thus, during submaximal unloaded shortening, the inhibition of crossbridge cycling imposed by the thin filament regulatory system must reduce the overall shortening velocity in a manner not observed during maximal contractions (i.e. Gordon et al. 1966; Barany, 1967; Edman, 1979; Siemankowski et al. 1985).

Comparison with studies in intact fibres

Studies of V_u in intact fibres reveal little or no effect of the number of attached crossbridges on the shortening velocity (Gordon et al. 1966; Edman, 1979; Vandenboom et al. 1998). However, in skinned fibres, reduction in the [Ca²⁺] slows the shortening velocity primarily by reducing the speed of shortening at larger length changes, yielding a biphasic shortening velocity at submaximal activations. This separation of V_u into the two distinct phases observed here and in previous studies (Moss, 1986; Martyn et al. 1994; Metzger, 1995; Iwamoto, 1998) is not evident in the work using intact fibres (Gordon et al. 1966; Edman, 1979; Vandenboom et al. 1998). The discrepancy between the intact fibre studies and skinned fibre studies raises a potential problem in that an effect is seen in an in vitro model (skinned fibres) but not in an in vivo system (intact fibres).

Gordon et al. (1966) and Edman (1979) varied the degree of thick and thin filament overlap altering only the number of crossbridges that can interact with the thin filament, which does not change the number of attached crossbridges per unit length of thin filament. The level of fibre activation was reduced based on the length-tension curve and was therefore independent of any effects due to the level of thin filament activation. During fibre activation, all of the thin filament regulatory units, including those beyond the reach of the myosin heads, were fully active, and the actin sites within the overlap region were completely accessible to the cycling crossbridges. Similarly, Vandenboom et al. (1998) also observed a linear shortening velocity in fully active fibres, so no reduction in V_u was expected.

A reduction in the level of activation to ~25 % by the use of a pharmacological agent dantrolene produced no change in V_u (Edman, 1979). The lack of an effect of dantrolene on V_u by inhibiting Ca²⁺ release into the sarcoplasm seems to imply that V_u is unaffected by the level of activation. However, it is unclear whether the effect of dantrolene is homogenous within the filament lattice or whether it produces a Ca²⁺ gradient across the fibre width that produces this effect. Also, during measurements of V_u the muscle fibre was released by less than 10 % of its length, corresponding to only the initial, Ca²⁺-independent shortening phase (V_u1) in skinned fibres. The apparent discrepancies between intact and skinned muscle fibres are not necessarily due to differences in the experimental system, but may stem from the different parameters and methods used in the experiments.

Potential mechanisms for reducing V_u

The presence of long-lived negatively strained crossbridges could reduce V_u (Moss, 1986). It has been suggested that a small population of AM.ADP crossbridges may remain attached for an extended time, thereby producing a negative strain and reducing the shortening velocity (Cooke & Pate, 1985; Siemankowski et al. 1985; Metzger, 1996). Metzger (1996) observed biphasic shortening at pCa 4.5 with added MgADP, probably because it competes with ATP for binding to the rigor crossbridges (AM*) and prolongs the existence of an AM.ADP state during shortening. In agreement with these results, a slowing of the sliding speed of actin filaments has been observed in the presence of MgADP in the in vitro motility assay (Homsher et al. 1992). It has been suggested that under some circumstances the concentration of MgADP reaches ~0.5 mM at the centre of the fibre, decreasing to ~0.01 mM at the surface of the fibre (Lu et al. 1993). So it is possible that a build-up of MgADP within the filament lattice could affect the V_u in skinned fibres.

Iwamoto's experiments in skinned muscle fibres using a multiple shortening procedure, whereby the fibre was shortened at various loads, restretched and then allowed to shorten again, allow investigation of the shortening velocity over a much greater length change range than is possible using the slack test (Iwamoto, 1998). At reduced [Ca²⁺], the biphasic shortening velocity is apparent only during the first shortening; in the later shortenings, the first phase (V_u1) is not observed and the shortening velocity is reduced. The existence of long-lived, negatively strained crossbridges causing the slow second phase shortening velocity is unlikely, as the latter shortenings are monitored following forceful crossbridge detachment after restretch to the original fibre length. When a time period of isometric contraction was inserted between the first and second shortenings, V_u1 increased, with the degree of recovery dependent on the level of tension redevelopment at low [Ca²⁺]. Thus Iwamoto suggested that strong crossbridge binding cooperatively activates the muscle fibre and shortening deactivates the thin filament. Bremel & Weber (1972) suggested that strong crossbridge binding cooperatively activates the thin filament. Also, it has been shown that cooperativity is most readily apparent at low [Ca²⁺] and is greatly diminished at saturating [Ca²⁺] (Greene & Eisenberg, 1980; Lehrer & Morris, 1982; Swartz et al. 1996; Butters et al. 1997). The model of Huxley (1957) identifies the shortening velocity as a function of positively and negatively strained crossbridges, the greater the ratio of positively to negatively strained crossbridges, the greater the speed of shortening. Maximal V_u is obtained when the rate of formation of positively strained crossbridges is just balanced by the rate of removal of the retarding, negatively strained crossbridges (Huxley, 1957). During shortening, the reduction in the number of crossbridges entering the cycle will shift the balance between these two processes, favouring the negatively strained crossbridges and therefore slowing the shortening velocity. Josephson & Edman (1998) describe the effect as being due to the proportion of positively strained crossbridges always lagging behind the negatively strained crossbridges. The absolute rate of crossbridge attachment is not the determining factor; rather, the acceleration or slowing in the rate of attachment controls the relative ratio of pulling and dragging crossbridges. During shortening, the rate of ADP release is accelerated, in turn increasing the rate of crossbridge detachment. This shifts the rate-limiting step to the Ca²⁺-dependent weak-to-strong transition that precedes force generation. At saturating [Ca²⁺], the thin filament is likely to remain active, allowing sufficient crossbridges to bind, even though shortening reduces the crossbridge number. However, at submaximal [Ca²⁺], the initial rate of crossbridge attachment is reduced and as shortening proceeds, the rate of attachment is further decreased, limiting the number of crossbridges entering the cycle and slowing the shortening velocity. If the deactivation is continual, a progressive decline in the number of attaching crossbridges will maintain the proportion of negatively strained crossbridges always greater than the positively strained crossbridges. Alternatively, the number of crossbridges binding to the thin filament may be reduced to a level that is not capable of maintaining the shortening velocity (Uyeda et al. 1990; Homsher et al. 1996, 2000).

The V_u at maximal activation is correlated directly with the rate of ADP release, but what happens when the [Ca²⁺] is reduced? Our results show that reductions in crossbridge access to the thin filament produce the same slowing of the shortening velocity, independent of whether [Ca²⁺] or the number of active regulatory units is reduced (Fig. 5). In isometrically contracting muscle fibres, it has been estimated that 20-40 % of the crossbridges are attached to the thin filament during maximal activation (Huxley, 1957; Cooke, 1990) and there is a further 70 % reduction in the number of strongly bound crossbridges as the muscle shortens during V_u (Ford et al. 1985). Further reductions in crossbridge number at submaximal [Ca²⁺] or a lower proportion of active regulatory units do not greatly affect the initial phase of the shortening velocity until isometric force is reduced by ~50 %. Therefore, during shortening the number of attached crossbridges can be reduced to 3-6 % of the total crossbridge population before an effect is observed, a result consistent with the hypotheses that shortening is independent of crossbridge number. The results obtained using the motility assay also suggest that the sliding speed (or shortening velocity) is independent of crossbridge number above a given value (Homsher et al. 2000).

Shortening-induced deactivation of the muscle fibre could be responsible for the second phase of shortening observed in skinned fibres at submaximal levels of activation (Iwamoto, 1998; Josephson & Edman, 1998; Swartz & Moss, 2001). Rapid shortening reduces the number of positively strained, force-generating crossbridges to such an extent that they no longer balance the force exerted by the negatively strained crossbridges, and thus the muscle shortens more slowly (Josephson & Edman, 1998). Reducing the [Ca²⁺] or adding CBMII TnC decreases the number of thin filament units that will bind crossbridges strongly, while the shortening itself produces a further reduction in the number of crossbridges reattaching to the thin filament. A decreased rate of crossbridge reattachment during steady-state shortening slows the speed in a manner similar to that proposed by Huxley (1957). A progressive reduction in the number of crossbridges entering the cycle as the muscle shortens will produce a balance of forces that favours the negatively strained crossbridges, and therefore results in a slowing of the muscle shortening velocity until a new equilibrium is reached.

What, then, is the mechanism that underlies the biphasic shortening behaviour? According to the shortening-induced deactivation hypothesis, the initial attachment occurs under 'isometric' conditions (i.e. the fibre length is reduced but the crossbridges must attach for shortening to begin). Once the crossbridges bind to the thin filament they produce force and begin to remove the imposed slack, thereby shortening the fibre. As the fibre begins to shorten, the process of thin filament deactivation begins and the rate of crossbridge detachment accelerates, while the crossbridge attachment rate falls. The 75-90 nm (half-sarcomere)^-1 break-point probably represents the distance through which the initial attached crossbridges can influence the level of thin filament activation. Through the addition of exogenous strongly bound S1 (NEM-S1), Swartz & Moss (2001) were able to extend the distance of crossbridge influence and eliminate the second phase of shortening usually observed during activations at submaximal [Ca²⁺]. Finally, the present experiments advance understanding of the mechanism underlying this deactivation. One possibility is that decreased crossbridge attachment reduces fractional Ca²⁺ binding, since crossbridges promote Ca²⁺ binding to the thin filament (Bremel & Weber, 1972; Butters et al. 1997). This possibility can now be excluded. In Fig. 5, the same pattern is observed whether partial activation occurs by a decrease in the free [Ca²⁺] or instead by keeping the [Ca²⁺] high and incorporating CBMII TnC-cTnC mixtures. Therefore, thin filament deactivation must not greatly depend on the dissociation of Ca²⁺ from the thin filament. This leaves the alternative possibility that the state of the thin filament is changing. As more crossbridges are detached, less of the thin filament will remain in the fully active, or 'M' state, and (where Ca²⁺ is bound) more will exist in the 'C' state, in which tropomyosin has a less activating position on actin (Vibert et al. 1997; Lehman et al. 2000; Craig & Lehman, 2001). We suggest that the biphasic shortening behaviour involves this structural transition in the thin filament.

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Acknowledgements

This work was supported by National Institutes of Health grants AR-39968 (E.H.) and HL-38834 and HL-67734 (L.T.), and partially by a NIH Chemistry/Biology Interface Training Grant GM08496 (C.M.).

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