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EDITORIAL FOCUS

NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Calcium regulation of troponin and its role in the dynamics of contraction and relaxation

Institute of Vegetative Physiology, University of Cologne, Köln, Germany, and Center of Molecular Medicine Cologne, University Cologne, Köln, Germany

CA²⁺ SENSITIVITY OF STRIATED muscle contraction is modulated by a number of factors, the mechanism by which force is altered by Ca²⁺ appears to be complex. If we focus on the level of the regulatory, heterotrimeric troponin complex (Tn = TnC·TnI·TnT), not only an altered Ca²⁺ affinity of troponin C (TnC) but also developmental changes in troponin I (TnI) isoforms manifest themselves in altered Ca²⁺ sensitivity. For example, neonatal hearts, which express the slow skeletal TnI (ssTnI), exhibit an increased Ca²⁺ sensitivity compared with adult hearts containing the cardiac TnI (cTnI) isoform (19, 21, 28).

In this issue of AJP-Regulatory, Integrative and Comparative Physiology, the article "Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics" by de Tombe and coworkers (6) investigated whether specific interventions on TnC and TnI leading to Ca²⁺ sensitization of contraction affect force kinetics in myofibrils. To alter the regulatory function of Tn, they used two straightforward approaches: 1) treatment of the myofibrils with bepridil, a Ca²⁺ sensitizer known to specifically bind to TnC; and 2) replacement of the endogeneous, fast skeletal Tn (fsTn) in the rabbit psoas myofibril by either cTnC·cTnI·cTnT or cTnC·ssTnI·cTnT. Bepridil and both types of Tn replacements increased the Ca²⁺ sensitivity (pCa₅₀) of myofibrillar force generation, whereby ssTnI increased it more than cTnI, in agreement with studies on skinned fibers (21, 36). Under all conditions, the authors find that Ca²⁺ activation induces a monoexponential rise of force with a rate constant k_act. Yet, importantly, de Tombe et al. (6) found that none of these interventions altered the maximum Ca²⁺-activated force nor produced a different k_act-force relationship, i.e., for a given active force, the observed kinetics of contraction were similar. Even more intriguingly, neither Tn exchange nor bepridil caused any significant alterations in the kinetics of force decay following rapid Ca²⁺ removal; no slowing down of mechanical relaxation was observed as one might have expected from the elevated Ca²⁺ sensitivity induced by these interventions. We learn from these results that the nature of Tn defines the Ca²⁺ sensitivity of contraction, while it does not influence the kinetics of myofibrillar contraction and relaxation, provided that the complex is able to completely turn off and fully turn on.

To better understand the implications of these results, in terms of the kinetic mechanism of Ca²⁺-induced contraction, one has to consider why the rate constant of force development is Ca²⁺ dependent at all. This feature was first approached by Brenner (2) by investigating the Ca²⁺ dependence of the rate constant of force redevelopment (k_tr) to demonstrate that Ca²⁺ regulation of force generation results from the gradual increase in the apparent turnover rate constant (f_app) by which cross- bridges enter force-generating states. This was inconsistent with the hypothesis that Ca²⁺-dependent thin-filament activation controls cross-bridge cycling in an all-or-nothing manner, i.e., by a simple mechanism in which an increase in Ca²⁺ activation recruits new, so far noncycling, cross bridges. There is a foresighted remark from Brenner (2) in his discussion of how rate modulation of cross-bridge turnover kinetics can be explained: "Regulation of muscle contraction through turnover kinetics is a natural consequence if it is assumed that the turned-on and turned-off forms of the regulated actin units are in a dynamic equilibrium with fast rate constants compared with cross-bridge turnover." Later on, Brenner and Chalovich (3) confirmed this hypothesis of highly dynamic thin-filament behavior by measuring the switch on/off kinetics of N-{[2-(lodoacetoxy)ethyl]-N-methyl}amino-7-nitrobenz-2-oxa-1,3-diazole-labeled fast skeletal TnI incorporated to rabbit psoas fibers. Kinetics of thin-filament inactivation were mechanically induced by a rapid change from isometric contraction to unloaded active shortening. This protocol rapidly shifts cross bridges from force-generating to non-force-generating states, thereby releasing their effect of contribution to thin-filament activation. Though the kinetics of fsTnI-switching were not directly induced by Ca²⁺, the rate constants for the switch on (k_on) and switch off (k_off) were obtained by performing the mechanical perturbation under different steady Ca²⁺ concentrations. Such obtained values of k_on and k_off were at least 10-fold higher than the ones for the force redevelopment. According to the model of Brenner and Chalovich (3), rate modulation of force redevelopment kinetics by Ca²⁺ activation results from the continuous switch on and switch off of the thin filament. The rate constants of the cross-bridge ATPase cycle itself are Ca²⁺ independent. However, the probability (i.e., the apparent rate constant) by which cross bridges enter force-generating states increases with elevated Ca²⁺ activation because it is a function of the forward step that rate-limits transition to the force states multiplied by the occupancy of the state preceding this step. Only the latter is Ca²⁺ dependent by Tn and tropomyosin (Tm) regulating the acto-myosin interaction. As force will be proportional to the probability by which cross-bridges enter force-generating states, it is a natural consequence that any fully functional Tn will always lead to the same k_tr-force relationship as long as 1) it does not directly intervene in the cross-bridge ATPase cycle, and 2) it switches between off and on states with kinetics faster than the kinetics of the step in the cross-bridge cycle, which rate limits the transition to force-generating states.

In their study, de Tombe and colleagues (6) demonstrate that these facets are true by showing that the k_act-force relationship remains the same after Tn replacements leading to enhanced Ca²⁺ sensitivity. Since myofibrils are thin enough to prevent diffusion limits (17), they are a powerful tool to study the time course of contraction and relaxation under defined control of calcium concentration ([Ca²⁺]) by using rapid switches (<10 ms) between the Ca²⁺-activating and relaxing solutions (5). Previous studies provided evidence that k_act is rate limited by cross-bridge turnover kinetics. Similar to, as indicated in de Tombe's paper (see Tables 1 and 2 in Ref. 6), studies on skeletal myofibrils (33), cardiac trabeculae (23), and cardiac myofibrils (29, 30, 26) revealed, that under all conditions investigated, k_act is similar to the rate constant of force redevelopment (k_tr) following a mechanical slack-restretch maneuver during steady Ca²⁺ activation. This implies that the Ca²⁺-induced switch on of the regulatory system occurs before the rise in force and does not rate limit Ca²⁺-induced force development. Furthermore, it had been shown that exchange of a functional troponin complex in skeletal (25) and cardiac myofibrils (11) does not alter force kinetics of contraction and relaxation upon maximum Ca²⁺ activation and upon Ca²⁺ removal, respectively. Incorporation of human cTn complex into murine cardiac myofibrils does not slow down myofibrillar activation-relaxation kinetics (11), despite the fact that human cardiac myofibrils have about 10 times slower force kinetics than murine cardiac myofibrils (30). This suggests that troponin isoforms existing in species with inherently slower myofibrillar contraction-relaxation dynamics can switch on and off sufficiently rapidly to not rate limit force kinetics, even in a myofibrillar environment with much faster cross-bridge kinetics. The new important finding in the study of de Tombe et al. (6) is that this maintains true under very critical conditions to test the validity of the model hypothesis of dynamic thin-filament behavior, under low Ca²⁺ activation and after exchanges of Tn's, which lead to significant increases in Ca²⁺ sensitivity. At low [Ca²⁺], the Ca²⁺-regulated on rate of cTn becomes negligible, and the overall dynamics of the thin filament (k_on + k_off) among off and on states become slowest, i.e., just determined by k_off (7). Troponin containing ssTnI has been shown to have a three times slower rate of Ca²⁺ dissociation from TnC (k_off) compared with troponin containing cTnI (8). This is in agreement with the enhanced Ca²⁺ sensitivity of myofibrils containing ssTnI, compared to myofibrils containing cTnI found by de Tombe et al. (6). That they find no change in the k_act-force relationship at low forces when ssTnI is incorporated into the myofibril therefore strongly supports their conclusion that Tn dynamics do not rate limit force kinetics.

Studies on cTnC mutant forms show that by decreasing the Ca²⁺ dissociation rate without changing the Ca²⁺ association rate, TnC exhibits an increased Ca²⁺ affinity (4, 10, 24). From these kinds of studies, Luo et al. (18) provided direct evidences that decreasing the off-rate by site-directed mutagenesis can slow down the relaxation kinetics in skinned fibers, while increasing the off-rate of TnC does not affect relaxation kinetics, probably, because any accelerating effect on relaxation would be damped by the rate limit of cross-bridge detachment kinetics. The work of de Tombe et al. (6) includes an alternative approach to test whether the off rate of cTnC could rate limit relaxation kinetics. They used bepridil to enhance the thin-filament responsiveness to Ca²⁺. Bepridil contributes to the full opening of the "regulatory" NH₂ lobe as the "switch" region of cTnI (cTnI_147–163) does when cTnC is Ca²⁺ saturated. Two bepridil molecules located in the hydrophobic cavity between the domains I and II pull the NH₂ and COOH lobes of cTnC together to result in a compact architecture, and a third bepridil molecule stabilizes the open cTnC NH₂ lobe conformation (15). Both, binding of Ca²⁺ and bepridil or Ca²⁺ and cTnI_147–163 to the cTnC NH₂ lobe act in a synergetic manner to open completely the hydrophobic pocket and to initiate a strong cTnC-cTnI interaction. Bepridil shields the NH₂ lobe hydrophobic cavity exposure from the solvent, thereby slowing down the rate of Ca²⁺ release without excluding cTnI_147–163-binding to cTnC (16). This also applies to skeletal TnC, where the presence of two Ca²⁺ on the domains I and II is sufficient to fully open the NH₂ lobe (14), leading to net enhancement of the Ca²⁺ affinity for sTnC, as confirmed by de Tombe et al. (6) by the increase in pCa₅₀. It was therefore a useful and specific pharmacological tool for their study to show that reducing TnC switch-off kinetics does not necessarily slow down relaxation kinetics.

In myofibrils, the force decay following Ca²⁺ removal is defined by two clear-cut phases. Previous studies demonstrated that during the initial, slow phase of force relaxation, all sarcomeres remain isometric, whereas the fast phase results from rapid sequential lengthening of the sarcomeres (27, 29). Comparison of the kinetics of the slow force decay (k_rel,slow) with force development kinetics suggests that k_rel,slow represents the turnover rate by which cross bridges leave force-generating states (27, 29, 33). The isometric, slow-relaxation phase lasts until the moment when the first, mechanically weakest, sarcomere in myofibril elongates. This perturbs the strain of cross-bridges in all sarcomeres, thereby catalyzing their detachment from actin and accelerating the force decay (29). This complex process of muscle relaxation is independent of the level of thin-filament activation prior to Ca²⁺ removal, but rather depends on the final [Ca²⁺] after rapid solution switch (27, 33). As de Tombe's results indicate, the different TnI isoforms do not alter the observed rate by which cross bridges leave force-generating states under isometric conditions (k_rel,slow) and the strain-induced accelerated cross-bridge detachment rates by sarcomere dynamics (k_rel,fast).

While incorporation of recombinant wild-type or purified Tn complexes to myofibrils had not revealed any significant changes in k_act, k_tr, k_rel,slow, and k_rel,fast (6, 11, 25, 27), slight modifications of Tn by single-site directed mutagenesis can produce dramatic changes. Krüger et al. (12) showed by cTn exchange in cardiac myofibrils that the FHC-related mutation R145G in human cTnI (hcTnI^R145G) lowers the range of Ca²⁺-dependent changes in k_act, which was increased at low and decreased at high [Ca²⁺] compared with myofibrils with wild-type hcTnI. These effects of the mutation were associated with an incomplete inhibition of active force at low [Ca²⁺] and a reduced maximum Ca²⁺-activated force. R145 is located in the inhibitory peptide of cTnI, which, in the absence of Ca²⁺, interacts with actin, whereas in the presence of Ca²⁺, it interacts with cTnC (13, 34). Impairment of cTnI's essential functions to fully inhibit and to fully activate the acto-myosin interaction thereby restricts the rate modulation of k_act at low and high [Ca²⁺], respectively. Reduced inhibitory function of hcTnI by the R145G mutation and likewise by the truncation of hcTnI to hcTnI1–192, the predominant degradation product in the stunned myocardium, inevitably slows down relaxation kinetics manifesting in a delayed onset and decreased rate constant of the fast relaxation phase (12, 22). That these two relaxation parameters are very sensitively affected by only slight, persistent activation is in agreement with the similar effects in native myofibrils following a slight incomplete Ca²⁺ removal (33).

The study of de Tombe et al. (6) nicely shows that the dynamic contractile properties appear to be determined by cross-bridge kinetics regardless of the nature of TnI, as long as TnI preserves its full regulatory capacity. On the other hand, a cardiomyopathy-associated mutation or truncation of TnI can alter the dynamics of contraction and/or relaxation, whereby the important findings of de Tombe et al. corroborate the previous interpretation that these alterations do not result from Tn affecting contraction or relaxation in a direct, rate-limiting manner (12).

Nevertheless, the global mechanism that can describe coupling of cross-bridge kinetics to thin-filament activation and inactivation dynamics has still to be elaborated. One may think that the mechanism is clear from the well-known steric-blocking model (1, 35); Ca²⁺ binds to TnC, TnI switches from actin to TnC, and TnT and Tm undergo conformational changes enabling cross bridges to interact in a force-generating manner with actin. However, it remains unclear in which order these events should be reversed during relaxation. The rate constant of the initial, slow myofibrillar force decay (k_rel,slow) following Ca²⁺ removal suggests that no force-generating cross-bridges become newly formed after Ca²⁺ removal (26, 27, 29, 30, 32, 33). This is corroborated by the findings of de Tombe et al (6) that k_rel,slow and t_rel,slow are insensitive to interventions that decrease the off rate of Tn. Hence, force kinetics suggest that the inactivation of the thin filament is completed well before the onset of the rapid, major force decay. However, this causes a new problem: if, according to the steric-blocking model, Tm controls the acto-myosin interface of force-generating cross bridges, how can Tm move back to a position where it prevents new binding of cross bridges as long as force-generating cross bridges are still there? Rapid blocking could only occur if Tn-Tm would be able to rapidly switch off with its own intrinsic dynamics, i.e., dependent on Ca²⁺ binding to cTnC but independent of the number of force-generating cross bridges. Indeed, myofibrillar force kinetics seem to indicate this, as k_rel,slow is neither altered by the level of force nor by the degree of Ca²⁺ activation preceding the Ca²⁺ removal (27, 30, 31, 33).

The most direct way to explain force development (k_act) and relaxation kinetics (k_rel,slow) is that Tn-Tm regulates the transition of cross bridges to force-generating states. The most simple model to describe this is that Ca²⁺·Tn-Tm controls cross-bridge attachment to a preforce-generating state without needing to directly control the binding of force-generating cross bridges. In this way, Tn-Tm could freely switch off, i.e., unhindered by force-generating cross bridges. Such a consideration differs from models derived from numerous structural and kinetic studies using reconstituted thin filaments and S1, and measurements on skinned fibers using NEM-S1, low [ATP] and high [ADP], which together clearly show that strongly-bound cross bridges have a strong impact on thin-filament activation (9, 20). Therefore, a positive feedback mechanism to activate the thin filament had been also proposed for force-generating cross bridges (9). The latter had been extremely useful to explain the high cooperativity of the force-pCa relation during Ca²⁺ activation. However, the same mechanism is counterproductive for enabling relaxation.

In summary, we might believe we understand the mechanism of thin-filament activation, but we are far from understanding the one of inactivation. Perhaps, in contrast to strongly-bound cross bridges, cycling force-generating cross bridges can hardly activate the thin filament. Likewise, the capability of force-generating cross bridges to keep the thin filament activated might be inversely related to the strain of cross-bridges. Perhaps, thin-filament models have to be modified in the way that the on-state of Tn-Tm more strongly controls actin binding of cross-bridges to the preforce-generating state than the actin-binding of force-generating states. To elaborate, a global, consistent model describing how thin-filament regulation controls cross-bridge cycling under both activation and relaxation, open questions that need to be answered: what takes place first, the regulatory switch off and thereafter cross-bridge detachment or vice versa? To what extent do force-generating cross bridges and Tn subunits rate limit the switch off? Therefore, kinetics of thin-filament inactivation need to be investigated directly during muscle relaxation. Studies like de Tombe et al's (6) show that we are on the right course.

GRANTS

This report was funded by the German Research Council (SFB612-A2) and the Center of Molecular Medicine Cologne (A2).

FOOTNOTES

Address for reprint requests and other correspondence: R. Stehle, Institute of Vegetative Physiology, Univ. of Cologne, 50931 Köln, Germany (e-mail: robert.stehle{at}uni-koeln.de)

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This Article Full Text (PDF) All Versions of this Article: 292/3/R1125    most recent 00841.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Stehle, R. Articles by Pfitzer, G. Search for Related Content PubMed PubMed Citation Articles by Stehle, R. Articles by Pfitzer, G.