Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization

In striated muscle the pointed ends of polar actin filaments are directed toward the center of the sarcomer. Formed filaments keep a constant length of about 1 μm. As polymerization and depolymerization at free pointed ends are not sufficiently slow to account for the constant length of the filaments, we searched for proteins which occur in sarcomers and can stabilize the pointed ends of actin filaments. We observed that tropornyosintroponin complex reduces the rate of association and dissociation of actin molecules at the pointed ends more than 30-fold. On the average, every 600 s one association or dissociation reaction has been found to occur at the pointed ends near the critical actin monomer concentration.     

Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate

Calcium-dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate-free, low-Ca²⁺ solution (pCa> 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca2²⁺ (pCa 4.5 prior to incubation with vanadate. Transfer back to a high Ca²⁺ solution elicited no additional force (83.2% ± 18.7% of control force). SDS-PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.     

Protein structural dynamics revealed by site-directed spin labeling and multifrequency EPR

Multifrequency electron paramagnetic resonance (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and separation of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resolution to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to separate spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states.     

Approaches to modeling crossbridges and calcium-dependent activation in cardiac muscle

While the primary function of the heart is a pump, ironically, the development of myofilament models that predict developed force have generally lagged behind the modeling of the electrophysiological and Ca²⁺-handling aspects of heart cells. A major impediment is that the basic events in force generating actin–myosin interactions are still not well understood and quantified despite advanced techniques that can probe molecular levels events and identify numerous energetic states. As a result, the modeler must decide how to best abstract the many identified states into useful models with an essential tradeoff in the level of complexity. Namely, complex models map more directly to biophysical states but experimental data does not yet exist to well constrain the rate constants and parameters. In contrast, parameters can be better constrained in simpler, lumped models, but the simplicity may preclude versatility and extensibility to other applications. Other controversies exist as to why the activation of the actin–myosin is so steeply dependent on activator Ca²⁺. More specifically steady-state force–[Ca²⁺] (F–Ca) relationships are similar to Hill functions, presumably as the result of cooperative interactions between neighboring crossbridges and/or regulatory proteins. We postulate that mathematical models must contain explicit representation of nearest-neighbor cooperative interactions to reproduce F–Ca relationships similar to experimental measures, whereas spatially compressing, mean-field approximation used in most models cannot. Finally, a related controversy is why F–Ca relationships show increased Ca²⁺ sensitivity as sarcomere length (SL) increases. We propose a model that suggests that the length-dependent effects can result from an interaction of explicit nearest-neighbor cooperative mechanisms and the number of recruitable crossbridges as a function of SL.     

Phosphorylation of troponin I by protein kinase C: Mechanism of inhibition by calmodulin and troponin C

The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca²⁺, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kianse C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by clamodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Localization of the two tropomyosin-binding sites of troponin T

Troponin T (TnT) binds to tropomyosin (Tm) to anchor the troponin complex in the thin filament, and it thus serves as a vital link in the Ca²⁺ regulation of striated muscle contraction. Pioneer work three decades ago determined that the T1 and T2 chymotryptic fragments of TnT each contains a Tm-binding site. A more precise localization of the two Tm-binding sites of TnT remains to be determined. In the present study, we tested serial deletion constructs of TnT and carried out monoclonal antibody competition experiments to show that the T1 region Tm-binding site involves mainly a 39 amino acids segment in the N-terminal portion of the conserved middle region of TnT. We further employed another set of TnT fragments to locate the T2 region Tm-binding site to a segment of 25 amino acids near the beginning of the T2 fragment. The localization of the two Tm-binding sites of TnT provided new information for the structure-function relationship of TnT and the anchoring of troponin complex on muscle thin filament.     

Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker

Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.     

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by ∼0.1% upon activation relative to the relaxing state and increased by ∼0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca²⁺-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca²⁺-binding and the second induced by actomyosin interaction.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.     

Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments

Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin''s access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion.     

Effects of actin-myosin kinetics on the calcium sensitivity of regulated thin filaments

Activation of thin filaments in striated muscle occurs when tropomyosin exposes myosin binding sites on actin either through calcium-troponin (Ca-Tn) binding or by actin-myosin (A-M) strong binding. However, the extent to which these binding events contributes to thin filament activation remains unclear. Here we propose a simple analytical model in which strong A-M binding and Ca-Tn binding independently activates the rate of A-M weak-to-strong binding. The model predicts how the level of activation varies with pCa as well as A-M attachment, N·k(att), and detachment, k(det), kinetics. To test the model, we use an in vitro motility assay to measure the myosin-based sliding velocities of thin filaments at different pCa, N·k(att), and k(det) values. We observe that the combined effects of varying pCa, N·k(att), and k(det) are accurately fit by the analytical model. The model and supporting data imply that changes in attachment and detachment kinetics predictably affect the calcium sensitivity of striated muscle mechanics, providing a novel A-M kinetic-based interpretation for perturbations (e.g. disease-related mutations) that alter calcium sensitivity.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C

The influences of [Ca(2+)] and Ca(2+) dissociation rate from troponin C (TnC) on the kinetics of contraction (k(Ca)) activated by photolysis of a caged Ca(2+) compound in skinned fast-twitch psoas and slow-twitch soleus fibers from rabbits were investigated at 15 degrees C. Increasing the amount of Ca(2+) released increased the amount of force in psoas and soleus fibers and increased k(Ca) in a curvilinear manner in psoas fibers approximately 5-fold but did not alter k(Ca) in soleus fibers. Reconstituting psoas fibers with mutants of TnC that in solution exhibited increased Ca(2+) affinity and approximately 2- to 5-fold decreased Ca(2+) dissociation rate (M82Q TnC) or decreased Ca(2+) affinity and approximately 2-fold increased Ca(2+) dissociation rate (NHdel TnC) did not affect maximal k(Ca). Thus the influence of [Ca(2+)] on k(Ca) is fiber type dependent and the maximum k(Ca) in psoas fibers is dominated by kinetics of cross-bridge cycling over kinetics of Ca(2+) exchange with TnC.     

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.     

A Periodic Pattern of Evolutionarily Conserved Basic and Acidic Residues Constitutes the Binding Interface of Actin-Tropomyosin

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the “closed state” where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp²⁵-Glu³³⁴-Lys³²⁶-Lys³²⁸) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.     

Functional adaptation between yeast actin and its cognate myosin motors

We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.     

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.