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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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(Received 31 January 2005;
accepted after revision 16 May 2005;
first published online 19 May 2005)
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Introduction |
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Isoform expression of thin filament regulatory proteins is muscle type dependent, and the significance of these isoforms in thin filament regulation is not clear. Mammalian skeletal muscle Tm exists as a double strand containing a mixture of 
and ß Tm isoforms. Two Tm subunits combine to form either a homodimer () or heterodimer (ß); the ßß homodimer is not found in significant quantities in adult mammals (Holtzer et al. 1992). Cardiac Tm in larger mammals also contains a proportion of the ß subunit, while the hearts of smaller mammals such as mice and rats consist entirely of Tm. Chandy et al. (1999) reported the persistence length of cardiac Tm was less than that of skeletal Tm and required more myosin S1 binding to induce similar changes in anisotropy, suggesting isoform-specific differences in Tm mobility on thin filaments that may be related to charge differences between and ß isoforms (Perry, 2001). Among troponin subunits, isoforms vary considerably between cardiac and fast skeletal muscle. Cardiac troponin C (cTnC) contains one low-affinity Ca2+ binding site compared with two for skeletal TnC. Cardiac troponin I (cTnI) has a 32 amino acid extension not present on skeletal TnI and TnI also contains several phosphorylatable residues known to be important in the physiological response of the heart to adrenergic stimulation. There are also several differences in the TnT isoforms present in cardiac and skeletal muscle. A number of reviews have extensively discussed the variations in troponin isoforms (Saggin et al. 1989; Bandman, 1992; Schiaffino & Reggiani, 1996; Solaro & Rarick, 1998; Gomes et al. 2002).
A growing body of evidence suggests that differences between cardiac and skeletal muscle in regulation of force and shortening at the cellular level results, at least in part, from the diverse isoforms for Tn and Tm present in thin filaments. For example, several studies have demonstrated that thin filament activation in cardiac cells is more dependent on strong crossbridge binding than activation in skeletal fibres (Metzger, 1995; Fitzsimons et al. 2001) and that differences in the kinetics of thin filament activation (Regnier et al. 2000, 2004; Adhikari et al. 2004) and the sarcomere length dependence of force development (Regnier et al. 2000, 2004; Adhikari et al. 2004) may be related to Tn and Tm isoforms. Additonally, replacement of native TnC or whole Tn in thin filaments of skinned rabbit psoas muscle fibres with cardiac TnC or whole Tn (respectively) has been shown to influence the mechanics of force and shortening (Piroddi et al. 2003), while over-expression of skeletal Tm in transgenic mouse hearts has shown that Tm isoform can alter the Ca2+ sensitivity of force (Palmiter et al. 1996).
The use of in vitro assays allows one to vary the composition of thin filament proteins with relative ease for the study of thin filament regulation of contractile mechanics. Published studies have utilized a variety of Tn and Tm isoforms to investigate thin filament regulatory mechanisms; however, no systematic analysis of the influence of particular isoforms or isoform concentration dependence on motility has been performed. To date investigations have used a wide range of Tn and Tm concentrations (Fraser & Marston, 1995; Homsher et al. 1996, 2000, 2003; Bing et al. 1997; Gordon et al. 1997; Gorga et al. 2003). Therefore a systematic investigation of the influence of these isoforms on contractile mechanics is warranted. Here we also evaluated the effect of reconstituting thin filaments with varying mixtures of Tn and Tm isoforms on filament sliding and force generation in vitro. We present evidence of differences between cardiac versus skeletal Tn and Tm in regulating these Ca2+-activated thin filament mechanical properties. Our results lead us to conclude that Tn and Tm function best when complexed in thin filaments with their homogeneous counterpart (e.g. skeletal Tn with skeletal Tm) and that the augmentation of force for regulated versus unregulated filaments is greater with skeletal than with cardiac regulatory protein isoforms.
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Methods |
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Fast skeletal myosin was obtained from the psoas muscle of male New Zealand White rabbits (3–3.5 kg). The rabbits were killed with 50 mg kg–1 of sodium pentabarbitol in the marginal ear vein, in accordance with NIH animal care policy and approved by the University of Washington Animal Care Committee. Skeletal myosin was prepared according to the methods of Margossian & Lowey (1982) and stored at –20°C in a high phosphate solution (0.5 mM KCl, 10 mM NaHPO4, 2 mM MgCl2, 1 mM DTT) mixed with 50% glycerol. Aliquots of the glycerinated myosin stock were digested to heavy meromyosin (HMM) with tosyl lysine chloromethyl ketone (TLCK)–chymotrypsin (Sigma); HMM was stored at 4°C and used for no more than a week. Daily aliquots of HMM were also centrifuged with actin to remove denatured enzyme as previously described (Clemmens & Regnier, 2004). Rabbit skeletal f-actin was prepared from acetone powder (Pardee & Spudich, 1982), labelled with rhodamine–phalloidin (Molecular Probes, Eugene, OR, USA) as described by Kron et al. (1991), stored at 4°C and used up to 6 weeks. Skeletal troponin (sTn) was isolated from rabbit psoas muscle and cardiac troponin (cTn) from bovine hearts as described by Potter (1982). Skeletal tropomyosin (sTm) was purified from rabbit back and leg muscle as previously described (Clemmens & Regnier, 2004). Cardiac Tm (cTm) was prepared from bovine hearts according to Smillie (1982). The -Tm isoform of bovine cTm was isolated via a hydroxyapatite column. Solubilized Tn and Tm were aliquoted and stored at –80°C until use.
In vitro motility assays
Experiments were performed according to Clemmens & Regnier (2004) and Gordon et al. (1997) using actin buffer (AB) consisting of (mM): 25 imidazole, 25 KCl, 4 MgCl2, 1 EGTA, 1 DTT at pH 7.4 at 23°C. Filament motility and force data were collected in AB plus calcium (CaB) at a pCa of either 9.0 or 5.0, 2 mM ATP, 50 mM ionic strength, pMg 3, and containing antioxidizing agents (0.018 mg ml–1 catalase, 0.1 mg ml–1 glucose oxidase, 3 mg ml–1 D-glucose, 40 mM DTT) to delay photobleaching of the rhodamine label and inhibit photo-oxidative protein damage. Assay preparation steps were as previously described (Clemmens & Regnier, 2004), except the concentration of Tn and Tm used in the reconstitution step and motility buffer varied with the isoforms used (100 nM for Tn/Tm combinations containing a cardiac isoform; 25 nM for skeletal Tn + skeletal Tm). All experiments were performed at 23°C (range: 22.7–23.3°C), with temperature periodically checked by a thermocouple (model BAT-10R, Physitemp, Clifton, NJ, USA).
Filaments were visualized via fluorescence microscopy and a 100 W HBO Hg lamp (Zeiss). At least six unique areas of the assay chamber were recorded for 30–60-s intervals for each motility assay via a SIT camera and were analysed off-line using Expert Vision software (Motion Analysis Systems, Santa Rosa, CA, USA) as described (Gordon et al. 1997; Clemmens & Regnier, 2004). For mean sliding speeds < 3 µm s–1, data were smoothed using a five-point moving average filter prior to further analysis to yield an apparent sampling rate of 2 fps. In determining the dependence of speed on Tn and Tm concentration (Fig. 1B), normalized speed values include all measured filament paths; no paths are excluded based on the Homsher-Sellers technique (Homsher et al. 1992) so as to not exclude the influence of cardiac versus skeletal isoforms on generating erratic motility. A Homsher-Sellers analysis was performed, however, for reporting the speeds versus pCa (Fig. 2), with a S.D./mean cutoff of 0.5. Non-normalized motility speeds are reported as weighted means ± standard deviations of the daily mean speeds for a given condition. (The method used to calculate the weighted means and standard deviations was as described in Clemmens & Regnier, 2004.) Differences between the weighted means are compared with a standard normal distribution test with confidence intervals at 99%. Weighted mean speed versus pCa data were fitted to a 4-parameter Hill equation using unweighted non-linear regression (SigmaPlot). The reported values are from fits to mean data, rather than mean parameters from individual fits, due to the small number of conditions evaluated on individual days and some variability in control (unregulated) speeds from week to week.
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Microneedle force probe production and calibration, and flow cell construction were as described in Clemmens & Regnier (2004). Microneedles were coated with -actinin, and then positioned in flow cells using a MP-285 micromanipulator (Sutter Instrument Co., Novato, CA, USA). A bolus of 
2 µl rhodamine–phalloidin-labelled filaments (5–30 µm in length) were injected into assay chambers and filaments were ‘caught’ by moving the stage to position the needle near a filament. Once a filament was attached to the needle, it was carefully lowered to a few micrometres above the motility surface using the robotic micromanipulator to permit interactions between the filament and surface-bound myosin motors. Small stage and/or needle movements orientated the filament as perpendicular as possible relative to the needle shaft axis. Experiments were videotaped and stored for offline analysis. An Argus-10 image processor (Hamamatsu, Bridgewater, NJ, USA) was used to measure the length of actin filament in contact with the surface, the steady-state deflection of the needle tip, and the angle of the force vector generated by this interaction. Forces estimated from measured deflections of the needle were corrected for the 2-D angle of pull. Force data are reported as unweighted means of individual steady-state force generation events. Differences between the means are evaluated using Student's t test and are considered significant for P < 0.01.
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Results |
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For all the studies described below we have used skeletal HMM and actin, to assess effects of regulatory protein isoforms on in vitro filament speed and force measurements. To determine the Tn and Tm concentrations that provide the best regulation for our experimental conditions (see Gordon et al. 1997), cardiac and skeletal regulatory protein concentrations were varied in in vitro motility assays. In all cases, the ratio of Tn to Tm for thin filament reconstitution and in the motility buffer was maintained at 1.0. Thin filaments were reconstituted with either all skeletal or all cardiac regulatory proteins for motility assays at either pCa 5 or pCa 9. The relationship between the fraction of filaments moving (fmov, defined as the fraction of filaments moving with a mean speed > 0.5 µm s–1) versus the concentration of Tn and Tm is plotted in Fig. 1A, and the corresponding data for filament sliding speed are shown in Fig. 1B. Speed data were normalized to values obtained with unregulated actin (see Methods) which was 2.7 ± 0.1 µm s–1 for all experiments combined (n = 13 days, motility paths = 6562).
At skeletal Tn and Tm (sTn/sTm) concentrations up to 25 nM thin filament sliding speed at pCa 5 (max Vf) was augmented > 2-fold relative to unregulated filament speed (Fig. 1), similar to the 2-fold increase we recently reported under identical motility conditions (Clemmens & Regnier, 2004). However below 25 nM sTn/sTm filaments were still moving at pCa 9, so Ca2+ regulation was not complete. Full thin filament regulation was achieved at 25 nM sTn/sTm because filament movement at pCa 9 was not different from values obtained in the absence of ATP (rigor). At concentrations above 25 nM sTn/sTm, fmov and max Vf dropped dramatically. This was likely to have been due (at least in part) to an observed bundling of filaments in the assay buffer. At 
100 nM sTn/sTm, very little motility was observed at pCa 5 or pCa 9. These data indicate that Ca2+ regulation of filaments with skeletal Tn and Tm isoforms is best studied using a fairly narrow range of Tn and Tm concentrations, from 10 to < 50 nM.
In contrast, complete regulation of thin filaments and augmentation of max Vf (over unregulated filaments) occurred over a wide range of concentrations with cardiac Tn and Tm (cTn/cTm). As we found with skeletal regulatory proteins (Fig. 1), increasing cTn/cTm concentrations up to 25 nM resulted in a progressive increase in max Vf, while fmov and sliding speed at pCa 9 decreased, such that filaments were completely regulated at 25 nM. However, unlike filaments with sTn/sTm, max Vf and fmov were constant at cTn/cTm concentrations up to 150 nM and filament bundling was not observed at any cTn/cTm concentration. Further, while movement of thin filaments containing cTn/cTm was smooth (see Methods), we consistently observed fewer filaments on the surface at both pCa 9 and pCa 5 compared to assays with filaments containing sTn/sTm. At pCa 9 weak actin–myosin interactions are likely to keep filaments on the surface, supporting the idea that there was less weak crossbridge binding for filaments containing cTn/cTm in the absence of Ca2+. This phenomenon had previously led others and us to add methylcellulose to the motility buffer to prevent filaments from diffusing from the motility surface (Homsher et al. 1992, 1996; Gordon et al. 1997; Chase et al. 2000). Methylcellulose was not used in this study because we wanted similar conditions for speed and force measurements, and the high viscosity of methylcellulose precludes its use for force measurements with microneedles. Under the experimental conditions used in this study (no methylcellulose, 23°C), thin filaments with cTn/cTm remained attached to the surface and moved smoothly at pCa 5 with 100 nM cTn and cTm. Therefore we chose to use 100 nM cTn/cTm and 25 nM sTn/sTm to provide optimal regulation of sliding speed and maximal augmentation of motility speed and force for the experiments described below. Taken together the data in Fig. 1 emphasize that protein concentrations must be considered when designing in vitro motility experiments used to study cardiac versus skeletal thin filament regulation.
Effect of Tn and Tm isoforms on the speed–Ca2+ relationship
To investigate the influence of the cardiac versus skeletal regulatory proteins on the Ca2+ dependence of thin filament sliding speed, we compared the speed of unregulated filaments with filaments reconstituted with cTn/cTm versus sTn/sTm over calcium concentrations from pCa 9 to pCa 5. The data are summarized in Fig. 2A and Table 1. Max Vf (pCa 5) was 14% slower for filaments with cTn/cTm versus filaments with sTn/sTm (Tables 1, P < 0.01). There was no significant shift in the Ca2+ sensitivity of speed (pCa50) of the speed–pCa relationship for filaments with cTn/cTm versus sTn/sTm (Fig. 2A, Table 1), as determined from fitting the data with the Hill equation. In contrast, the apparent slope of the speed–pCa relation for filaments with cTn/cTm was approximately half that for sTn/sTm, although the differences from the fits to the means of the speed data were not significantly different (P > 0.05). On the other hand, below pCa 7.2 the increase of speed for filaments containing cTn/cTm were significantly faster, while at pCa values below 7.2 speed was not significantly different between the isoforms. This suggests that the speed–pCa relation was less steep with cTn/cTm. These data indicate that at optimized concentrations of skeletal and cardiac thin filament proteins, max Vf was greatly enhanced (over actin alone), with the effect being somewhat larger with sTn/sTm. Additionally, both sets of regulatory proteins had a similar ability to Ca2+ regulate thin filament speed, but filaments with cTn/cTm may have exhibited a lower apparent cooperativity (n) of motility speed. This may be related to the fact that cTnC has only a single N-terminal Ca2+ binding (‘trigger’) site while sTnC has two, or to other cardiac versus skeletal differences that affect translation of the Ca2+ activation signal.
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Influence of Tn and Tm isoforms on filament force per unit length
While motility speed gives some indication of the Ca2+ dependence of crossbridge detachment during unloaded sliding, it provides little information about the amount of strong crossbridge binding that can occur with thin filament activation because strong crossbridge binding is thought to be minimal during unloaded shortening. To study this we measured the steady-state force generated per unit filament length interacting with the surface (F/l) in the absence and presence of Tn and Tm. The results for actin alone versus thin filaments with sTn/sTm, cTn/cTm, sTn/cTm, and cTn/sTm are summarized in Fig. 3. The F/l of f-actin (unregulated filaments) was 3.7 ± 0.4 pN µm–1, similar to values we reported recently (Clemmens & Regnier, 2004). The F/l value for filaments with sTn/sTm, reported in Fig. 3, was obtained from experiments recently reported by Clemmens & Regnier (2004) and is provided here for purpose of comparison. As we discuss in that paper, F/l for filaments with sTn/sTm at pCa 5 increased 2.6-fold (9.5 ± 1.3 pN µm–1) compared with unregulated filaments (P < 0.01). This is a much greater augmentation of F/l than the 50% increase reported by Homsher et al. (2000) with purified bovine cTn and cTm using the microneedle assay under similar conditions. To determine if this difference was due to cardiac versus skeletal isoforms of Tn and Tm, we measured the F/l of thin filaments reconstituted with all cardiac regulatory protein isoforms (cTn/cTm) at pCa 5. These filaments produced a 1.78-fold greater F/l than unregulated filaments on skeletal HMM (6.6 ± 0.8 versus 3.7 ± 0.4 pN µm–1), much closer to the 50% increase reported by Homsher et al. (2000). This interesting comparison indicates that augmentation of force by cardiac regulatory proteins is significantly less than augmentation by skeletal regulatory proteins.
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-actinin as an internal load to retard filament sliding in the in vitro motility assay. In their study, thin filaments containing sTn/sTm increased the ‘index of retardation’ (their indirect measure of force) 3-fold, while filaments containing sTn and human cTm had no effect relative to unregulated actin filaments. The F/l for thin filaments with cTn/sTm was also not different from values for actin alone. Thus heterogeneous combinations of regulatory proteins appear to disrupt allosteric protein interactions between thin and thick filaments that result in augmented force production. |
Discussion |
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Combined these results suggest thin filament regulatory proteins do more than control access to myosin binding sites on actin (steric blocking) and are likely to influence actomyosin interactions that underlie muscle force production and shortening, even at saturating levels of Ca2+ (Cassell & Tobacman, 1996; Rosol et al. 2000; Fujita & Kawai, 2002; Lu et al. 2003). Furthermore, there are interesting differences in this regulation for cardiac versus skeletal muscle, which we discuss below.
Thin filament regulation with all cardiac or all skeletal Tn/Tm
This study was the first quantitative assessment of Tn and Tm concentration and isoform interactions in conferring Ca2+-dependent regulation of thin filament motility and force development (Homsher et al. 1992, 1996; Gordon et al. 1997; Chase et al. 2000). The different motility behaviours we observed (both qualitatively and quantitatively) with varying cardiac versus skeletal Tn/Tm concentrations clearly suggest unique interactions of these isoforms with actin and with actomyosin. The tendency of thin filaments with cTn/cTm to dissociate from the surface-bound HMM, particularly at pCa 9, might be explained if the cardiac isoforms reduce the number of weakly bound crossbridges attached to the filament. Conversely, the fact that we did not observe these phenomena for thin filaments reconstituted with sTn/sTm implies that skeletal isoforms did not prevent or inhibit weakly bound crossbridge formation to the extent that cardiac isoforms did at this ionic strength. It was also interesting to note that, at 6.25 nM Tn/Tm (for either cardiac or skeletal proteins), thin filaments were not regulated, as the fraction of moving filaments (fmov) was nearly 1.0 for both pCa 9 and 5. However, filament speeds at pCa 5 were enhanced relative to unregulated filament speed, which suggests that regulatory proteins can influence mechanics even when concentrations are too low to allow complete Ca2+ regulation of actomyosin interactions. This supports the hypothesis that thin filament regulation does not operate solely via steric blocking but that Tn and Tm can directly alter actin interaction with myosin, resulting in increased crossbridge cycling kinetics.
Both cardiac and skeletal muscle Tn/Tm augmented max Vf (Fig. 2). This confirms and extends the finding of Gordon et al. (1998) and Homsher et al. (2000), who reported augmentation of max Vf for filaments containing all skeletal or cardiac regulatory proteins, respectively. The data in Fig. 1 showing augmentation of max Vf with Tn/Tm concentrations too low to inhibit motility in the absence of Ca2+ have important implications for the mechanism(s) by which regulatory proteins modulate actomyosin interactions (Clemmens & Regnier, 2004). If it is assumed that there is a lack of continuity between adjacent Tn/Tm complexes along thin filaments with 6.25 nM Tn/Tm, then the observed augmentation of max Vf implies that of Ca2+ binding to individual regulatory units can extend beyond the seven actins covered by Tm. This is also suggested from both biochemical (Maytum et al. 2001) and muscle fibre studies (Regnier et al. 2002). It also suggests regulatory proteins may induce allosteric structural changes in actin that result in speed augmentation even when concentrations are too low to provide complete Ca2+ regulation.
Figure 2A illustrates that max Vf and Ca2+ sensitivity of speed for thin filaments containing cTn/cTm versus sTn/sTm exhibited similar abilities to augment Ca2+-dependent Vf with some relatively subtle differences. We expected larger differences considering the well-known differences in the Ca2+ binding properties of cTnC and sTnC, as well as structural differences in the corresponding TnI and TnT isoforms (Gordon et al. 2000). However, the influence of regulatory protein isoforms on strong crossbridge binding may be difficult to study with motility assays where there is low strain, and the number of cycling crossbridges is expected to be low (Gordon et al. 1966; Ford et al. 1977; Edman, 1980). Furthermore, we have shown that max Vf is not strongly dependent on the number of cycling crossbridges, especially at high [Ca2+] (Liang et al. 2003). Thus max Vf is likely to underestimate the contribution of myosin to thin filament activation, because relatively low numbers of cycling crossbridges allow maximal sliding speed.
To study regulation of strong crossbridge binding in detail requires measurement of the isometric force produced by thin filament interactions with myosin, where maximal crossbridge binding occurs. As with Vf, both skeletal and cardiac regulatory proteins augmented F/l of filaments compared with actin alone (Fig. 3). However, the augmentation of F/l was much greater with sTn/sTm. Maximal force in muscle cells is linearly dependent on the number of strongly attached crossbridges, so the smaller force generated for cardiac thin filaments can be explained if cTn/cTm limits the number of strong crossbridge that can bind at high [Ca2+] compared with sTn/sTm. An alternative explanation is that allosteric interactions increased the force produced per crossbridge, so that the relatively lower F/l with cardiac regulatory proteins resulted from limited or different allosteric interactions than occurred with skeletal thin filament proteins. We believe that the former is likely to be the case, because in skinned cardiac trabeculae we demonstrated that thin filaments are not completely activated at saturating levels of Ca2+, and it is possible to further increase strong crossbridge binding by replacing ATP with 2 deoxy-ATP as the energy source for contraction (Regnier et al. 2000, 2004). In contrast, dATP does not enhance maximal strong crossbridge binding in either fast or slow skeletal fibres (Regnier & Homsher, 1998; Regnier et al. 1998a,b, 2000). A third possible explanation for lower F/l with cardiac regulatory proteins is phosphorylation. In these studies we did not determine the extent of regulatory protein phosphorylation, but we expect some level of phosphorylation to be present. Phosphorylation of cardiac Tn subunits (cTnI and cTnT) or Tm could modulate the relative ability of cardiac Tn to augment F/l, as suggested by the complex site specific effects of regulatory protein phosphorylation on cardiac contractility (Solaro & Rarick, 1998). Phosphorylation of cardiac regulatory proteins has been shown either to have no effect or to decrease maximum Ca2+-activated force in cellular preparations, depending on the site(s) of phosphorylation. Investigation of the role of phosphorylation on sliding speed and force was beyond the scope of this study, but should be an area for future investigation.
Our observations suggest the lower F/l with cardiac regulatory proteins (Fig. 3) resulted from greater steric blocking by cardiac (versus skeletal) Tn/Tm, even in the presence of saturating Ca2+. Evidence supporting this idea comes from recent electron microscopy and image reconstruction studies (Lehman et al. 2000), which demonstrated that, in the absence of troponin, cardiac () Tm was localized to the inner part of the outer domain of the actin strand. This position corresponds to the ‘blocked’ state (McKillop & Geeves, 1993), while skeletal (–ß mixture) Tm was localized to the ‘closed’ position. Both Tm isoforms were localized to the ‘closed’ state in the presence of Tn and Ca2+ (Lehman et al. 1994; Vibert et al. 1997; Xu et al. 1999). The fact that the cardiac Tm ‘preferred’ the ‘blocked’ position in the absence of Tn and Ca2+ suggests that the energy barrier to Tm movement may be greater for cTm than sTm (Lehman et al. 2000).
To facilitate discussion of regulatory protein isoform effects on Vf and F/l we introduce Scheme 1, a crossbridge model first published in Regnier & Homsher (1998). In this scheme the rate of crossbridge detachment is dependent on steps 7 (ADP release), 1 (ATP binding) and 2 (A–M dissociation). Most evidence suggests step 7 is the rate limiting process during unloaded muscle shortening and actin filament sliding, with transitions 1 and 2 being much faster (Gordon et al. 2000). If so, our data suggest that thin filament allosteric interactions increase step 7. This is supported by the recent work of Homsher et al. (2003), who found the apparent affinity of ADP for actomyosin is reduced in motility assays containing skeletal HMM and thin filaments containing cardiac regulatory proteins (versus actin alone). In contrast, the increases in F/l with addition of regulatory proteins cannot be explained solely by allosteric effects that increase ADP release (step 7), because this would result in reduced, not increased force. In a recent paper we explored the effects of skeletal regulatory proteins on both the rate of a forward, force-generating transition and steps that limit the rate of crossbridge detachment by varying nucleotide conditions and temperature (Clemmens & Regnier, 2005). We concluded that skeletal regulatory proteins increase both the rate(s) into force bearing states (steps 4a and 5) and the rate of crossbridge detachment (steps 7). This interpretation lends insight into our present observations that while sTn/sTm and cTn/cTm similarly enhance Vf (Fig. 2A), F/l is significantly less for actin filaments with cTn/cTm (Fig. 3). In the context of Scheme 1 this implies that cardiac regulatory isoforms allosterically increase step 7 (and consequentially crossbridge detachment) to nearly the same extent as skeletal isoforms, as evidenced by similar max Vf, but they are not as effective at allosterically augmenting the steps in the crossbridge cycle limiting to force generation.
Thin filament regulation with mixtures of cardiac and skeletal Tn and Tm
The heterogeneous mixture sTn/cTm (but not cTn/sTm) lowered Ca2+ sensitivity of speed and slightly reduced max Vf (Fig. 2B) compared with sTn/sTm or cTn/cTm, while Ca2+ regulation of motility was maintained. In comparison F/l (Fig. 3) was more sensitive than Vf to regulatory protein isoform composition (Fig. 2). In fact F/l was not augmented over actin-alone values for either heterogeneous mixture of Tn/Tm. Simply put, it appears that the homogeneous complements of regulatory proteins are more effective at influencing force generation by the HMM motors than the heterogeneous complements (Homsher et al. 2003). As all other experimental conditions were equivalent, including in some instances the same batch of skeletal myosin, effects on Vf and loss of augmented F/l are most likely to be due to less effective communication of the Ca2+-binding signal between the Tn and Tm isoforms.
The greater augmentation of F/l by sTn/sTm, compared to cTn/sTm (Fig. 3) appears to conflict with the recent observations that maximum Ca2+-activated force was the same in skeletal myofibrils in which endogenous sTn was replaced with cTn (Piroddi et al. 2003). However, Piroddi et al. (2003) indicated that Tn exchange was not 100% complete, with myofibrils containing about 7–16% of the endogenous sTn. Our observation that Vf was maximally augmented in filaments when Tn/Tm reconstitution was incomplete (Fig. 1) implies that this remaining endogenous sTn in myofibrils could have allowed some augmentation of maximal force. We have demonstrated that Ca2+ binding to each sTn activates more actin monomers (10–12) than the 7 actins covered by each Tm on thin filaments of muscle fibres (Regnier et al. 2002). Alternatively the different conditions between our in vitro assays and the muscle fibre studies of Piroddi et al. (2003), such as ionic strength, temperature, myosin density, etc., could account for the apparently conflicting results. Interestingly, the decrease in maximum Ca2+-activated force they observed in myofibrils when endogenous skeletal TnC was completely extracted and replaced by cardiac TnC, corresponds to the lower F/l augmentation we observed in filaments with cTn/cTm (Fig. 3). These observations are consistent with our hypothesis that the maximum force-generating capability of regulated thin filaments is influenced by the isoform-specific properties of Tn and Tm.
This study did not address the potential role of myosin (or actin) isoform on Ca2+ regulation of thin filaments. Our experimental protocol was designed to test specifically the role of regulatory protein isoforms on filament mechanics, so we did not covary myosin isoform. Use of skeletal myosin (HMM) for all experiments allowed us to compare our results with a growing body of literature where skeletal myosin has been used as the motor, with various combinations of actin and thin filament regulatory proteins. It would be interesting to investigate whether the differential effects of cardiac versus skeletal regulatory proteins exist in the presence of cardiac myosin.
Significance
Changes in both troponin and tropomyosin isoform expression occur during development and with environmental stressors (Schiaffino & Reggiani, 1996), emphasizing the importance of understanding the role(s) of these isoforms in establishing the characteristics of contraction in striated muscle. Our results demonstrate a clear difference in the ability of homogeneous sets of regulatory proteins to regulate Ca2+-dependent actin filament sliding speed and augment force at saturating Ca2+ concentrations. We also determined that heterogeneous mixtures of cardiac and skeletal Tn and Tm isoforms eliminate the potentiation of force observed with homogeneous mixtures, implying that isoform-specific communication between regulatory proteins is important not only in establishing efficient Ca2+ control of contraction but also in establishing the allosteric interactions that modulate the actomyosin interaction leading to enhanced force and shortening velocity. When combined with published data on the effects of cardiac versus skeletal thin filament regulation of contractile mechanics in demembranated cells (Regnier et al. 1998b, 2000; Regnier et al. 2004), the smaller force measured here for thin filaments containing cardiac (versus skeletal) Tn and Tm isoforms is consistent with the hypothesis that in cardiac muscle thin filament activation is limited even during maximal Ca2+ activation.
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) or rat cardiac Tn and rabbit skeletal Tm (cTn/sTm, 
). Data points are reported as weighted means of the Homsher-Sellers mean assay speeds ± standard deviation (see Methods). In A, continuous (sTn/sTm) and dashed lines (A: cTn/cTm, B: sTn/cTm, cTn/sTm) represent unweighted regression fits of the data to the Hill equation (see Methods). In B, the Hill fit for sTn/sTm (continuous line) is redrawn from A for comparision. Fit parameters are listed in 
Significantly different from A + sTn/sTm. Data point for A + sTn/sTm (grey bar) was previously published in Clemmens & Regnier (2005) and is reproduced here for comparison.