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* Department of Bioengineering, University of Washington, Seattle, Washington 98195; and Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Correspondence: Address reprint requests to Michael Regnier, Dept. of Bioengineering, Box 357962, School of Medicine, University of Washington, Seattle, WA 98195-7962. Tel.: 206-616-4325; Fax: 206-685-3300; E-mail: mregnier{at}u.washington.edu.
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INTRODUCTION |
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) and the level of force developed is determined by the availability of myosin binding sites on thin filaments, which is ultimately controlled by the distribution of tropomyosin (Tm) between positions on the surface of thin filaments that either block or uncover binding sites on actin for myosin cross-bridge attachment. Transduction of the Ca2+ signal is through the troponin complex, resulting in increased mobility of Tm over the surface of actin and exposure of strong binding sites for myosin. This shifts the thin filament from a "blocked" to a "closed" state (McKillop and Geeves, 1993), thereby allowing further myosin cross-bridge formation. Strong cross-bridge binding is then thought to stabilize Tm in an "open" state and the thin filament is considered "activated." In effect, the level of contractile activation appears to result from a balance of the effects of Ca2+ binding to TnC and cross-bridges on Tm state. The growing amount of evidence suggests that cross-bridge displacement of Tm into an activating position may play a more prominent role in the regulation of cardiac contraction (Fuchs and Smith, 2001).
Like force, the rate of force development is Ca2+ dependent, but the mechanisms that determine the thin filament activation dependence of force kinetics are less well established. In demembranated skeletal muscle fibers the relationship between steady-state force and the rate that force redevelops (ktr) after a release-restretch cycle is Ca2+ sensitive, being slow (1–2 s–1 at 10–15°C) at low levels of force and increasing 10–15-fold as force rises to maximum at high levels of Ca2+ activation (Brenner, 1986, 1988). Based on these and other findings it was suggested that Ca2+ controls the rate-limiting process in force development rather than simply controlling the level of cross-bridge recruitment (Brenner, 1986, 1988). However, we and others have shown that in skeletal muscle when the Ca2+-activated force is high the kinetics of actin-myosin interaction are likely limiting to the rate of force development, whereas at submaximal force the rate-limiting process may be the kinetics of thin filament activation (Chase et al., 1994; Regnier et al., 1996, 1998; Fitzsimmons et al., 2001a,b). This limiting process is greatest at low levels of activation, as most of the increase in ktr comes at Ca2+ concentrations that produce >50% of maximal Ca2+-activated steady-state force.
Similar to reports for fast skeletal muscle, ktr is Ca2+ dependent in demembranated cardiac muscle. However, most studies have reported smaller magnitude (one- to fivefold) change in ktr (compared with skeletal muscle) as the level of Ca2+ activation is varied (Wolff et al., 1995; Palmer and Kentish, 1998; Patel et al., 2001), though one recent report suggest a >8-fold change (Fitzsimons et al., 2001). These studies mostly show that at similar temperatures ktr is similar or faster in cardiac versus skeletal muscle at low Ca2+ concentrations, and slower at high levels of Ca2+ activation. This suggests the lower Ca2+ dependence of ktr results from different actin-myosin cycling kinetics (the main determinant of maximal ktr in skeletal muscle) and/or the relative kinetics of thin filament processes versus actin-myosin cycling kinetics is different than in skeletal muscle (Hancock et al., 1997). Coupled with evidence that thin filament activation level is more dependent on strong cross-bridge binding in cardiac (versus skeletal) muscle, it also suggests that Ca2+ binding to Tn may result in less complete activation of cardiac thin filaments (Regnier et al., 2000b).
We have been studying the relative contribution of Ca2+ binding to TnC and cross-bridge binding on both the level of thin filament activation and the rate that force develops in cardiac and skeletal muscle. A particularly successful approach has been to alter cross-bridge binding and cycling rates by using nucleotide analogs of ATP (NTPs) as the substrate for contraction. We have shown that one of these analogs, 2 deoxy-ATP (dATP), has an affinity for skeletal myosin similar to that of ATP and increases both solution NTPase and unloaded shortening velocity of rabbit skeletal and rat cardiac muscle (Regnier and Homsher, 1998; Regnier et al., 1998, 2000b). Interestingly, dATP enhances both maximal Ca2+-activated steady-state force (Fmax) and ktr in cardiac trabeculae from both untreated rats, containing mostly 
-MHC (fast cardiac myosin), and PTU-treated rats, containing ß-MHC (slow cardiac myosin) (Regnier et al., 2000b). This contrasts with our findings in skeletal muscle where dATP has no effect on Fmax, but increases maximal ktr in rabbit psoas fibers (fast skeletal myosin) and rat soleus fibers (slow skeletal myosin) (Regnier et al., 1998). These results indicate that cross-bridge cycling rate per se cannot explain these cardiac versus skeletal muscle differences, and suggests that the properties of cardiac thin filament regulatory proteins may limit cross-bridge cycling. Therefore we have hypothesized that (compared to ATP) dATP augments the level of thin filament activation and/or strong cross-bridge binding at saturating Ca2+ concentration in demembranated cardiac muscle, resulting in an increase in Fmax and maximal ktr over that attained with ATP as the contractile substrate. This hypothesis is supported by our finding that dATP also enhances both force and ktr at subsaturating levels of Ca2+ activation in skeletal muscle (Regnier et al., 1998).
In this study we hypothesized that the relatively low activation dependence of ktr in cardiac muscle (compared with skeletal muscle) reflects a relative limitation of Ca2+ binding to TnC to activate thin filaments at all levels of Ca2+. Consequently, this would give cross-bridge binding a more prominent role in the activation process. To test this hypothesis we compared steady-state force and stiffness, and ktr in rat cardiac trabeculae as Ca2+ concentration was varied with either 5 mM ATP or 5 mM dATP as the contractile substrate. In addition, we tested the hypothesis that thin filament kinetics can limit the rate of force development during maximal Ca2+ activation. To do this we compared the rate force develops after a step increase in Ca2+ concentration with photorelease of Ca2+ from NP-EGTA (kCa) (Ellis-Davies and Kaplan, 1994) to maximal ktr, where Ca2+ binding to thin filaments should be in (near) equilibrium during force development. We found that dATP enhanced steady-state force and stiffness at all levels of Ca2+ activation. We also found that at similar submaximal levels of steady-state force there was no increase in ktr with dATP, but dATP enhanced ktr at higher Ca2+ concentrations, resulting in an extension (not elevation) of the curvilinear force-ktr relationship. Similar results were obtained for rat trabeculae with either -MHC or ß-MHC isoforms, indicating this effect was not due to the absolute rate of cross-bridge cycling. Significantly, we found that ktr is faster than kCa with maximal Ca2+ activation, and that both are increased a similar amount by dATP. This finding suggests the kinetics and/or level of Ca2+-mediated thin filament activation may limit the rate that force develops in rat cardiac muscle, even at saturating levels of Ca2+. In a subsequent article, (Adhikari et al., 2004), we show that this property of cardiac thin filaments may at least partially explain the greater sarcomere length-dependence of force found in cardiac (versus skeletal) muscle. Preliminary reports of this work have been published previously (Regnier et al., 2000a; Martin et al., 2002).
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Solutions
Experimental solutions contained (in mM): 15 phosphocreatine, 15 EGTA, at least 40 3-(N-morpholino)propanesulfonic acid (MOPS), 1 free Mg2+, 135 Na+ + K+, 1 dithiothreitol (DTT), 250 units ml–1 creatine kinase (CK, Sigma, St. Louis, MO), and either 5 ATP or 5 mM 2 deoxy-ATP (Sigma, St. Louis, MO) at pH 7.0 and 15 ± 1°C. Ionic strength was 0.2 M. Affinities of dATP and ATP for Mg2+ were assumed to be the same (Regnier and Homsher, 1998). For activation solutions, the Ca2+ level (expressed as pCa = –log [Ca2+]) was set by adjusting Ca(propionate)2.
Individual chemically demembranated ("skinned") trabeculae were wrapped in aluminum foil T-clips for attachment to a force transducer (either Model 400A, 2.2 kHz-resonant frequency (Cambridge Technology, Watertown, MA) or Model AE801, 
5 kHz resonant frequency (SensoNor, Horton, Norway)) and a servo-motor (model 300, Cambridge Technology, Watertown, MA) tuned for a 300 µs-step response. For most experiments trabecular sarcomere length (Ls) was measured with helium-neon laser diffraction (Chase et al., 1993) and set to 2.25 µm at pCa 9.2. At this Ls trabeculae lengths ranged from 1.2–2.3 mm and diameters averaged 140 ± 16 µm, with no significant difference between trabeculae from normal versus PTU-treated rats. Stiffness was determined by small amplitude (0.05% Ls) sinusoidal length oscillations (1 kHz). Steady-state isometric force is reported as kN/M2 in Results, assuming circular geometry and the rate of isometric tension redevelopment (ktr) was determined from the halftime of force recovery after a rapid release-restretch transient, as previously described (Regnier et al., 1998). For comparisons, force and ktr are reported normalized to measurements obtained during maximal Ca2+ activation with ATP as the contractile substrate.
Comparisons of maximal ktr with the rate of force development (kCa) after photorelease of Ca2+ from NP-EGTA were made during the same activation done in a separate set of experiments under identical conditions, except temperature was 17°C instead of 15°C. kCa was measured as described in Martin et al. (2004). Briefly, after initial Ca2+ activations in either ATP or dATP, during which control measurements of ktr were performed at maximal activation (ktr control), the preparation was transferred through a series of NTP containing solutions to a final photolysis solution containing 5 mM NTP, 2mM NP-EGTA, and 
1.8 mM added calcium. The output from a frequency doubled ruby laser producing 150 mJ in a 50 ns pulse at a wavelength of 347 nm (Laser Applications, Winter Park, FL) was focused onto the trabeculae. Incident energy was measured by deflecting 10% of the beam energy onto a Scientech Energy Meter (365, Boulder, CO); 2 s after peak force was attained, ktr was measured (ktr photolysis) and the tissue relaxed. For these experiments, all elicited transients were collected for subsequent analysis but only those resulting in maximal activations within 5% of maximal Ca2+-activated force at pCa 4.0 (Fmax) have been included in the data.
Data analysis
Force-pCa and ktr-pCa relationships are fit with the Hill equation:
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From each Hill fit, the pCa at half-maximal force or ktr (pCa50) and the slope (nH) are determined, and reported pCa50 and nH values represent the means of the values from the individual fits, ± SE. The time course of force development from ktr and kCa protocols was determined by exponential fits or from t1/2 values, as previously described in (Chase et al., 1994), both yielding similar results. Summarized data are reported as mean ± SE and means were compared using Student's t-test, with significance at the 95% confidence level.
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). dATP increased the Ca2+-sensitivity of force (pCa50) of trabeculae from both untreated and PTU-treated rats, but had no significant effect on nH. For trabeculae from untreated rats, dATP increased the Ca2+-sensitivity of force (P < 0.05) by 0.13 pCa units from 5.41 ± 0.01 (ATP) to 5.54 ± 0.03 (dATP), whereas corresponding nH values were 5.4 ± 0.6 (ATP) and 4.6 ± 1.2 (dATP). For trabeculae from PTU-treated rats, pCa50 with ATP was similar to untreated rats, whereas the increase of Ca2+ sensitivity of force with dATP was greater than found for untreated rats, increasing 0.26 pCa units from 5.43 ± 0.01 (ATP) to 5.69 ± 0.03 (dATP). nH was unaffected by dATP in PTU treated trabeculae (3.2 ± 0.2 (ATP) vs. 3.5 ± 0.6 (dATP)). The Hill fit parameters (pCa50 and nH) for the force-pCa relations for trabeculae from untreated and PTU-treated rats included in Table 1.
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2 s later a ktr measurement was made (third arrow) and the trabecula was relaxed. In several preparations the ktr measurement was repeated in control activation solution (not shown) to confirm that photolysis produced maximal Ca2+-activation conditions. The kCa 
ktr procedure was then repeated for activations with dATP as the contractile substrate (right side of Fig. 6, upper panel). The order of NTP testing was varied between preparations. Comparisons of ktr and kCa were made only for maximal Ca2+ activation and then only if the photolysis of NP-EGTA produced Ca2+ activation within 5% of the force level in control activations. For this example trabecula it is obvious this is the case and that Fmax was increased by dATP in both control and NP-EGTA activations.
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DISCUSSION |
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). These studies provide information about how altering cross-bridge cycling influences cross-bridge binding and thin filament activation at submaximal levels of Ca2+ in cardiac muscle. The results also allow us to compare results in cardiac muscle with previous work in fast and slow skeletal muscle, where we have extensively studied the effects of cross-bridge binding and cycling rates versus thin filament dynamics on the Ca2+ activation dependence of steady-state force and force development kinetics (Regnier et al., 1996, 1998, 1999, 2000a,b). Those studies provided strong evidence that thin filament dynamics are limiting to the level and rate of force development in skeletal muscle during submaximal Ca2+ activation.
The existing structural sequences and conservation information for myosin heavy chains offer no explanation about how dATP might increase Fmax in cardiac muscle, but not in skeletal muscle (Regnier et al., 2000b). Indeed the increase in Fmax with dATP may be peculiar to mammalian cardiac muscle in general, as we have also found similar results in chemically demembranated trabeculae from mouse and dog heart (unpublished observations). In previous studies we suggested that dATP might increase the number of force-generating cross-bridges during submaximal Ca2+ activation in skeletal muscle (Regnier et al., 1998) and during maximal Ca2+ activation in cardiac muscle (Regnier et al., 2000b) by increasing one or more transition rates associated with cross-bridge attachment and force generation while increasing the rate of cross-bridge detachment to a lesser extent. In this article, we now propose this enhanced cross-bridge attachment leads to increased activation, which is apparent at submaximal Ca2+ in both muscle types and in maximal force in cardiac muscle.
Steady-state force
Proportional increases in steady-state force and stiffness were found at all levels of Ca2+ activation with dATP. This apparent increase of thin filament activation could result from recruitment of strongly bound force-generating cross-bridges, from an increase in force per cross-bridge, or both. Since dATP increases cross-bridge cycling rate, it suggests the cross-bridge detachment rate (gapp) is increased, which further implies the rate of strong cross-bridge attachment (fapp) must increase to a greater extent to provide a higher level of steady-state force and stiffness (Regnier et al., 2000b). This effect of dATP appears to be partially or completely independent of the overall cross-bridge cycling rate, as there were increases in both maximal force and Ca2+ sensitivity of force for trabeculae from untreated versus PTU-treated rats (Fig. 1; Table 1). This suggests the effects of dATP are largely independent of myosin kinetics (Figs. 3 and 4), implying that enhanced strong cross-bridge binding is the important contribution of dATP to increased activation. Whatever the detailed mechanism of force enhancement by dATP, the net result is an apparent elevation of thin filament activation at all levels of Ca2+ in cardiac muscle with either - or ß-MHC. Interestingly, dATP increased the Ca2+ sensitivity of force (pCa50) for trabeculae with ß-MHC twice as much as for trabeculae with -MHC (0.26 vs. 0.13 pCa units, respectively; Fig. 1 and Table 1). This suggests enhanced cross-bridge binding and/or cycling rate may improve activation in cardiac muscle with slow myosin. Our steady-state force-pCa results (Fig. 1) contrast with those reported by Metzger et al. (1999) in their study of myocytes from untreated versus PTU-treated rats. Although we report no difference in the Ca2+ sensitivity of force (pCa50) for trabeculae from untreated versus PTU-treated rats, they observed a decrease in pCa50 by 0.17 units with no change in the slope (Metzger et al., 1999). It is unclear why our results differ, but the focus and novel discovery of our study is that increased cross-bridge binding (with dATP) increases pCa50 of force for both untreated and PTU-treated trabeculae without changing the apparent cooperativity (nH) of the force-pCa relationship (Fig. 1).
In cardiac muscle changes in cross-bridge binding have been shown to increase Ca2+ binding to troponin C (Hofmann and Fuchs, 1987; Wang and Fuchs, 1994; Martyn and Gordon, 2001). Our experiments do not allow us to determine if enhanced Ca2+ sensitivity of force with dATP is correlated with increased Ca2+ binding to troponin C or via Ca2+-independent increases in strong cross-bridge binding (or both). The increased force at saturating [Ca2+], however, is most likely explained by increased cross-bridge binding alone because of proportional increases in trabeculae stiffness. To determine if force enhancement by dATP directly results in a shifting of thin filament toward the "on" state will require a more direct measurement of the state of tropomyosin on thin filaments.
Rate of force development (ktr, kCa)
In this study the Ca2+ sensitivity of ktr was increased with dATP (Fig. 4; Table 1), similar to steady-state force. When ktr was plotted versus the steady-state force produced at varying [Ca2+], it was apparent that ktr was similar at force and stiffness levels common to both substrates. However, with dATP both ktr and force rose substantially above maximal levels for ATP for trabeculae from both untreated (Fig. 5 a) and PTU-treated (Fig. 5 b) rats, indicating the effect was independent of cardiac myosin isoforms (Regnier et al., 2000b). Changes in the force-ktr relationship at high levels of Ca2+ with dATP could result from greater activation of thin filaments by cross-bridges, as suggested by the increased stiffness (Fig. 2). Evidence for elevation of the rate force develops during submaximal Ca2+ activation by enhanced cross-bridge binding was provided by Fitzsimons et al. (2001), who found that NEM S-1 binding to thin filaments at submaximal forces raised ktr to levels measured during maximal Ca2+ activations. Since NEM S-1 cross-bridges are strong binding and slowly dissociating, they suggested that thin filaments with bound NEM S-1 were at a much higher activation level than in the absence of NEM S-1 at the same level of submaximal Ca2+, thus allowing a faster rate of force development. An additional effect of dATP may be directly attributed to a faster rate of cross-bridge cycling with dATP since maximal ktr is the sum of fapp + gapp, both of which could be increased by dATP (Regnier et al., 2000b). However, the similar effect of dATP on ktr-force relations (Fig. 5; Table 1) for trabeculae from untreated (mostly -MHC) and PTU-treated (ß-MHC) rats indicates that cross-bridge cycling rate cannot be the only factor limiting the level and rate of force development in cardiac muscle. If ktr at submaximal levels of activation is limited by the intrinsic acto-myosin NTPase rate of cross-bridges, we should have observed an elevation of ktr at all levels of force. In contrast, Fig. 5 shows similar values of ktr for ATP and dATP at the same level of force and stiffness, imply that a noncross-bridge cycling mechanism is limiting the rate that force can develop. An alternative interpretation of our results would be that the amount of force generated per cross-bridge is enhanced with dATP (compared with ATP). However, our previous observation that maximal Ca2+-activated force is not elevated by dATP in either fast or slow soleus skeletal muscle fibers argues against this. Slow skeletal fibers contain the same myosin isoform (ß-MHC) present in trabeculae from PTU-treated rats, indicating dATP-induced elevation of force does not result from an increased level of force per cross-bridge. This suggests that the elevation of force and ktr by dATP result from cross-bridge dependent enhancement of thin filament activation.
In cardiac muscle the relatively low Ca2+-activation dependence of ktr (a two- to fivefold change compared with a 10–15-fold change for skeletal muscle) could result from an intrinsically lower acto-myosin ATPase activity at maximal activation or a relative limitation of the level of thin filament activation, even at saturating [Ca2+]. The last possibility is supported by the observations that 1), cardiac TnC (cTnC) has a single regulatory Ca2+-binding site (Johnson et al., 1980) compared with the two regulatory Ca2+-binding site for fast skeletal muscle; 2), cTnC undergoes less and slower conformational change upon Ca2+-binding than skeletal TnC (Sia et al., 1997; Hazard et al., 1998); 3), the Ca2+ induced conformational change associated with TnI interaction is slow for cardiac TnC (perhaps as low as 4–6 s–1) (Dong et al., 1996, 1997); and 4), when cardiac TnC is fully substituted for endogenous TnC in skeletal fibers maximum force is significantly lower (Chase et al., 1994; Morris et al., 2001). An additional consideration is that the signal transmission of Ca2+ binding to TnC to tropomyosin could be slow relative to cross-bridge cycling rates, especially at low Ca2+ concentrations. If the level or rate of thin filament activation is limited in cardiac muscle, interventions that elevate thin filament activation might be expected to enhance both maximum force and ktr, as we observe with dATP. We have previously demonstrated that thin filament activation dynamics are rate limiting to force development at low levels of Ca2+ in skeletal fibers (Chase et al., 1994; Regnier et al., 1998). We now suggest this mechanism may also apply to cardiac muscle.
This hypothesis is further supported by comparisons of ktr and kCa during the same activation of trabeculae at pCa 4.5. For both ATP and dATP kCa was consistently only about half as fast as ktr (Table 2, Figs. 6 and 7), indicating the dynamics of thin filament activation by Ca2+ limit the rate of force development. However, because force develops from different levels during ktr and kCa measurements, differences in time courses of force rise could be influenced by the distribution of compliance between cross-bridges and noncross-bridge components (Huxley et al., 1994; Luo et al., 1994; Higuchi et al., 1995; Martyn et al., 2002). Comparison of the force traces in Fig. 7 indicates that the ktr force redevelops from a nonzero value, whereas it rises from relaxed levels after the Ca2+ transient. As a result, in ktr measurements the compliance of noncross-bridge structures would be high relative to cross-bridge compliance at the beginning of force development, resulting in an apparent slowing of force kinetics (Luo et al., 1994; Martyn et al., 2002) and thus would underestimate the difference between ktr and kCa. In comparison, during kCa measurements noncross-bridge compliance would be lowest at the beginning of force rise, thus the initial rate of force development would be faster, slowing as cross-bridge compliance decreases with increasing force. However, there was no indication of an initial rapid rise of force that approached ktr during kCa measurements. Thus differences in distribution of compliance between cross-bridge and noncross-bridge components do not explain the different time course of ktr and kCa.
If cross-bridge cycling kinetics were limiting the rate of force development, we would expect kCa and ktr to be similar. The thin filament activation state should be in near equilibrium during tension redevelopment with the ktr protocol. Changes in myoplasmic [Ca2+] during tension transients (Vandenboom et al., 1998; Wang and Kerrick, 2002) or Ca2+ binding when force is inhibited in cardiac muscle (Wang and Fuchs, 1994; Martyn and Gordon, 2001) indicate that some Ca2+ dissociates from thin filaments during the release-restretch transients of ktr measurements. However, this amount is likely to be a small fraction of Ca2+ bound to troponin C at pCa 4.5 (Martyn and Gordon, 2001). Furthermore, any thin filament deactivation during the release phase of the release-restretch transient would lead to a slower value of maximal ktr, and thus underestimate differences between ktr and kCa. In contrast, force development during the kCa measurement begins with near zero levels of Ca2+ bound to thin filaments. Thus our observation that kCa is always slower than ktr supports a hypothesis that thin filament activation by Ca2+ is rate limiting for force development even at saturating [Ca2+]. Previously Palmer and Kentish (1998) reported no difference between kact (reported here as kCa) and ktr in rat and guinea pig trabeculae at 22°C. However these measurements were not obtained during the same activation and may have been from separate trabeculae. Additionally, their kact values in rat trabeculae varied almost fourfold with some activations producing more force than the control activations, and maximum ktr varied by more than twofold. This suggests some of their measurements may have been made in submaximally activating conditions. The argument that thin filament regulatory protein dynamics limit the rate that force develops in cardiac muscle is further supported by the observation that both rates were elevated 60% by dATP, whereas kCa remained slower than ktr (Table 1). Thus, even though cross-bridge cycling kinetics can be increased by dATP, the kinetics of thin filament activation by Ca2+ may limit the rate force develops in rat cardiac muscle. If so, mechanisms that enhance cross-bridge binding at a given submaximal level of intracellular Ca2+, such as phosphorylation of myosin-binding protein C could be important in increasing the power output of the heart during sympathetic stimulation. This mechanism would not be available to skeletal muscle, where Ca2+ binding to TnC appears to be much more successful in fully activating thin filaments and where contraction usually results from action potential trains that allow muscles to contract at the upper end of the force-pCa relationship.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Institutes of Health (HL61683) to M. Regnier and (HL67071) to D. A. Martyn, and a grant from the American Heart Association (0140040N) to M. Regnier. M. Regnier is an Established Investigator of the American Heart Association.
Submitted on December 22, 2003; accepted for publication June 3, 2004.
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REFERENCES |
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Brenner, B. 1986. The cross-bridge cycle in muscle. Mechanical, biochemical, and structural studies on single skinned rabbit psoas fibers to characterize cross-bridge kinetics in muscle for correlation with the actomyosin-ATPase in solution. Bas. Res. Cardiol. 81:1–15.[CrossRef][Medline]
Brenner, B. 1988. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc. Natl. Acad. Sci. USA. 85:3265–3269.
Chase, P. B., D. A. Martyn, and J. D. Hannon. 1994. Isometric force redevelopment of skinned muscle fibers from rabbit with and without Ca2+. Biophys. J. 67:1994–2001.
Chase, P. B., D. A. Martyn, M. J. Kushmerick, and A. M. Gordon. 1993. Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit. J. Physiol. (Lond.). 460:231–246.
Dong, W., S. S. Rosenfeld, C. K. Wang, A. M. Gordon, and H. C. Cheung. 1996. Kinetic studies of calcium binding to the regulatory site of troponin C from cardiac muscle. J. Biol. Chem. 271:688–694.
Dong, W., C. K. Wang, A. M. Gordon, S. S. Rosenfeld, and H. C. Cheung. 1997. A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle. J. Biol. Chem. 272:19229–19235.
Ellis-Davies, G. C., and J. H. Kaplan. 1994. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc. Natl. Acad. Sci. USA. 91:187–191.
Fitzsimmons, D. P., J. R. Patel, K. S. Campbell, and R. L. Moss. 2001a. Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers. J. Gen. Physiol. 117:133–148.
Fitzsimons, D. P., J. R. Patel, and R. L. Moss. 2001b. Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament. J. Physiol. 530:263–272.
Fuchs, F., and S. H. Smith. 2001. Calcium, cross-bridges, and the Frank-Starling relationship. News Physiol. Sci. 16:5–10.
Gordon, A. M., E. Homsher, and M. Regnier. 2000. Regulation of contraction in striated muscle. Physiol. Rev. 80:853–924.
Hancock, W. O., L. L. Huntsman, and A. M. Gordon. 1997. Models of calcium activation account for differences between skeletal and cardiac force redevelopment kinetics. J. Muscle Res. Cell Motil. 18:671–681.[CrossRef][Medline]
Hazard, A. L., S. C. Kohout, N. L. Stricker, J. A. Putkey, and J. J. Falke. 1998. The kinetic cycle of cardiac troponin C: calcium binding and dissociation at site II trigger slow conformational rearrangements. Prot. Sci. 7:2451–2459.[Abstract]
Higuchi, H., T. Yanagida, and Y. E. Goldman. 1995. Compliance of thin filaments in skinned fibers of rabbit skeletal muscle. Biophys. J. 69:1000–1010.
Hofmann, P. A., and F. Fuchs. 1987. Effect of length and cross-bridge attachment on Ca2+ binding to cardiac troponin C. Am. J. Physiol. 253:C90–C96.[Medline]
Huxley, H. E., A. Stewart, H. Sosa, and T. Irving. 1994. X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle. Biophys. J. 67:2411–2421.
Johnson, J. D., J. H. Collins, S. P. Robertson, and J. D. Potter. 1980. A fluorescent probe study of Ca2+ binding to the Ca2+-specific sites of cardiac troponin and troponin C. J. Biol. Chem. 255:9635–9640.
Luo, Y., R. Cooke, and E. Pate. 1994. Effect of series elasticity on delay in development of tension relative to stiffness during muscle activation. Am. J. Physiol. 267:C1598–C1606.[Medline]
Martin, H., R. J. Barsotti, D.-G. C. Ellis, and M. Regnier. 2002. Activation of rat cardiac muscle studied with caged calcium and deoxy-ATP (dATP). Biophys. J. 82(1):364a.
Martin, H., M. G. Bell, G. C. Ellis-Davies, and R. J. Barsotti. 2004. Activation of skinned cardiac muscle by laser photolysis of nitrophenyl-EGTA. Biophys. J. In press.
Martyn, D. A., P. B. Chase, M. Regnier, and A. M. Gordon. 2002. A simple model with myofilament compliance predicts activation-dependent cross-bridge kinetics in skinned skeletal fibers. Biophys. J. 83:3425–3434.
Martyn, D. A., and A. M. Gordon. 2001. Influence of length on force and activation-dependent changes in troponin C structure in skinned cardiac and fast skeletal muscle. Biophys. J. 80:2798–2808.
McKillop, D. F., and M. A. Geeves. 1993. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys. J. 65:693–701.
Metzger, J. M., P. A. Wahr, D. E. Michele, F. Albayya, and M. V. Westfall. 1999. Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes. Circ. Res. 84:1310–1317.
Morris, C. A., L. S. Tobacman, and E. Homsher. 2001. Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin-C mutant. J. Biol. Chem. 276:20245–20251.
Palmer, S., and J. C. Kentish. 1998. Roles of Ca2+ and cross-bridge kinetics in determining the maximum rates of Ca2+ activation and relaxation in rat and guinea pig skinned trabeculae. Circ. Res. 83:179–186.
Patel, J. R., D. P. Fitzsimons, S. H. Buck, M. Muthuchamy, D. F. Wieczorek, and R. L. Moss. 2001. PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin. Am. J. Physiol. Heart Circ. Physiol. 280:H2732–H2739.
Regnier, M., and E. Homsher. 1998. The effect of ATP analogs on posthydrolytic and force development steps in skinned skeletal muscle fibers. Biophys. J. 74:3059–3071.
Regnier, M., D. A. Martyn, and P. B. Chase. 1996. Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle. Biophys. J. 71:2786–2794.
Regnier, M., D. A. Martyn, and P. B. Chase. 1998. Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle. Biophys. J. 74:2005–2015.
Regnier, M., D. A. Martyn, and P. B. Chase. 2000a. The calcium sensitivity of steady-state force and force development kinetics in cardiac muscle is enhanced by 2-deoxy-ATP. Biophys. J. 78(1):141A.
Regnier, M., A. J. Rivera, P. B. Chase, L. B. Smillie, and M. M. Sorenson. 1999. Regulation of skeletal muscle tension redevelopment by troponin C constructs with different Ca2+ affinities. Biophys. J. 76:2664–2672.
Regnier, M., A. J. Rivera, Y. Chen, and P. B. Chase. 2000b. 2-deoxy-ATP enhances contractility of rat cardiac muscle. Circ. Res. 86:1211–1217.
Sia, S. K., M. X. Li, L. Spyracopoulos, S. M. Gagne, W. Liu, J. A. Putkey, and B. D. Sykes. 1997. Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain. J. Biol. Chem. 272:18216–18221.
Vandenboom, R., D. R. Claflin, and F. J. Julian. 1998. Effects of rapid shortening on the rate of force regeneration and myoplasmic [Ca2+] in intact frog skeletal muscle fibres. J. Physiol. 511.1:171–180.
Wang, Y., and W. G. Kerrick. 2002. The off rate of Ca2+ from troponin C is regulated by force-generating cross bridges in skeletal muscle. J. Appl. Physiol. 92:2409–2418.
Wang, Y. P., and F. Fuchs. 1994. Length, force, and Ca2+-troponin C affinity in cardiac and slow skeletal muscle. Am. J. Physiol. 266:C1077–C1082.[Medline]
Wolff, M. R., K. S. McDonald, and R. L. Moss. 1995. Rate of tension development in cardiac muscle varies with level of activator calcium. Circ. Res. 76:154–160.
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) enhances the Ca2+-sensitivity of steady-state force in skinned trabeculae. Force was normalized to maximal force at pCa 4.5 with ATP (•) for trabeculae from normal (a) and PTU-treated (b) rats for comparisons. Data are reported as the mean ± SE for each pCa. Data were fit to 
) and dATP (
) data yielded identical slopes of 0.96 with r2 values >0.97. The values for dATP >1.0 demonstrate that trabeculae stiffness is elevated proportionally with force over values for ATP, suggesting an increase in the number of strongly bound, force-producing cross-bridges at high levels of Ca2+ activation.
). Measurements of ktr were made at pCa 4.5 in control solution (ktr control) and during maximal activation induced by photolysis of NP-EGTA (ktr photolysis). kCa was determined from the time course of the transient after NP-EGTA photolysis. (Lower panel) Rapid time base recordings from upper panel. (a) ktr photolysis for ATP determined immediately after activation by photolysis of NP-EGTA. (b) kCa induced by photolysis of NP-EGTA with ATP. (c) ktr control for ATP. Rates are determined from the t1/2 of the respective transients. (d) Force records from a and b superimposed to show relative rates.