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Am J Physiol Heart Circ Physiol 293: H2580-H2587, 2007. First published August 10, 2007; doi:10.1152/ajpheart.00039.2007
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Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities

Catalina Norman, Jack A. Rall, Svetlana B. Tikunova, and Jonathan P. Davis

Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio

Submitted 10 January 2007 ; accepted in final form 7 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated whether changing thin filament Ca2+ sensitivity alters the rate of contraction, either during normal cross-bridge cycling or when cross-bridge cycling is increased by inorganic phosphate (Pi). We increased or decreased Ca2+ sensitivity of force production by incorporating into rat skinned cardiac trabeculae the troponin C (TnC) mutants V44QTnCF27W and F20QTnCF27W. The rate of isometric contraction was assessed as the rate of force redevelopment (ktr) after a rapid release and restretch to the original length of the muscle. Both in the absence of added Pi and in the presence of 2.5 mM added Pi 1) Ca2+ sensitivity of ktr was increased by V44QTnCF27W and decreased by F20QTnCF27W compared with control TnCF27W; 2) ktr at submaximal Ca2+ activation was significantly faster for V44QTnCF27W and slower for F20QTnCF27W compared with control TnCF27W; 3) at maximum Ca2+ activation, ktr values were similar for control TnCF27W, V44QTnCF27W, and F20QTnCF27W; and 4) ktr exhibited a linear dependence on force that was indistinguishable for all TnCs. In the presence of 2.5 mM Pi, ktr was faster at all pCa values compared with the values for no added Pi for TnCF27W, V44QTnCF27W, and F20QTnCF27W. This study suggests that TnC Ca2+ binding properties modulate the rate of cardiac muscle contraction at submaximal levels of Ca2+ activation. This result has physiological relevance considering that, on a beat-to-beat basis, the heart contracts at submaximal Ca2+ activation.

force; thin filament

CARDIAC MUSCLE CONTRACTION is initiated by Ca2+ binding to troponin C (TnC), which triggers conformational changes on the thin filament, exposing myosin-binding sites on actin. After the myosin heads (cross bridges) attach to actin, the thin filaments slide along the thick filaments and the muscle contracts (18). The kinetics of cross-bridge cycling can be studied with several approaches. One approach has been developed by Brenner (3, 4). According to this protocol, a rapid shortening-restretch maneuver mechanically detaches the cross bridges, and the rate at which the cross bridges reattach and generate force is a measure of the rate of contraction, known as the rate of force (tension) redevelopment (ktr).

In both cardiac and skeletal muscle, it is well established that ktr becomes faster with increasing levels of activation by Ca2+ (5, 6, 13, 28, 46). Many studies have investigated the role Ca2+ plays in the activation dependence of ktr (for review, see Ref. 13). It has been proposed that the effects of Ca2+ on ktr occur either as a direct effect of Ca2+ on the cross bridges or indirectly by activation of the thin filament, which subsequently allows cross bridges to cycle from non-force-generating states to force-generating states.

Studies in skeletal muscle investigated the hypothesis that Ca2+ has a direct effect on the cross bridge cycle. Caged inorganic phosphate (Pi) experiments suggest that Ca2+ does not regulate the kinetics of Pi release but rather the distribution of cross bridges between non-force-generating and force-generating states (25, 45). Similarly, in vitro motility assays have shown that Ca2+, through binding to TnC, controls the number of cross bridges interacting with actin rather than directly controlling the rate of ATP hydrolysis or the filament sliding speed (14, 17). Moreover, Ca2+ does not control ktr through binding to the regulatory light chains (24). Overall, these studies suggest that Ca2+ does not influence the rate of contraction through a direct effect on cross-bridge cycling.

Alternatively, it was suggested that in skeletal muscle Ca2+ influences the rate of contraction by modulating the level of thin filament activation. For instance, calmidazolium sensitizes muscle to Ca2+ by increasing the Ca2+ binding affinity of TnC without any direct effect on cross bridges (36). In the presence of calmidazolium ktr was increased at submaximal Ca2+ activation, but it was unchanged at maximal activation. Thus modulating the thin filament Ca2+ activation by changing TnC Ca2+ binding properties can ultimately influence the rate of contraction.

In cardiac muscle, interesting results have been observed by measuring the rate of contraction on the same preparation with two protocols, ktr and photolysis of caged Ca2+ (kCa). One study did not find any difference between ktr and kCa and concluded that the activation rate is determined solely by the kinetics of cross-bridge cycling (28). Recent studies in isolated cardiac myofibrils also showed that there was no difference between ktr and the rate of contraction induced by rapid Ca2+ switching results confirmed in mice, guinea pig, and human myofibrils from either atrial or ventricular regions (31, 38). Another study reported that the rate of contraction measured by kCa was slower than ktr (35). That study suggested that the slower rate of contraction during the kCa protocol results from the dynamics of Ca2+ binding and activating the thin filament, especially at low Ca2+ concentrations, in addition to the kinetics of cross-bridge cycling.

Our novel approach was to directly change the level of thin filament Ca2+ activation by incorporating into rat skinned cardiac trabeculae TnC mutants with increased or decreased Ca2+ binding affinities. We investigated the influence of changing the thin filament Ca2+ activation on the rate of force generation, using the ktr protocol. Our study suggests that ktr dependence on Ca2+ is modulated by both thin filament Ca2+ binding properties and the kinetics of cross-bridge cycling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rat skinned cardiac trabeculae and experimental apparatus. All protocols were approved by the Institutional Animal Care and Use Committee. Male LBN-F1 rats (175–200 g) were anesthetized with intraperitoneal injection of pentobarbital sodium (Nembutal, 50 mg/kg). The thoracic cavity was opened, and heparin (0.1 ml of 10,000 U/ml bottle) was injected intracardially. The heart was rapidly excised and placed in relaxing solution (see Standard solutions) at room temperature. Unbranched trabeculae were harvested from the right ventricle and placed overnight at 4°C in relaxing solution containing 1% Triton X-100. The skinned trabeculae were used within 48 h. The skinned trabeculae were mounted between the arms of a high-speed length controller (model 322C, Aurora Scientific) and an isometric force transducer (model 403A, Aurora Scientific) in the experimental chamber containing relaxing solution by means of aluminum T clips, as previously described (34). The resting sarcomere length was set at ~2.2 µm as determined by the first-order diffraction pattern from a HeNe laser directed through the trabeculae. A reticule on the eyepiece of the dissecting microscope was used to measure the width and depth of the trabecula. Cross-sectional area was calculated from the depth and width measurements by assuming an elliptical circumference. Force per cross-sectional area (F/CSA) was calculated as an average of two maximal activations at the beginning of the experiment. Each trabecula was activated at the beginning of the experiments in a pCa 4.0 solution, and a rapid slack (1 ms, 20% of the total length) was applied when the isometric force reached a plateau. This resulted in a rapid drop in force below the resting force baseline. The trabecula was then returned to pCa 9.0, held at the slack length for 3 s, and restretched back to the original length in a 5-s ramp. The same procedure was used, before the maximal activation, in a pCa 9.0 solution to obtain the resting force. The active force generated by the trabeculae in various pCa solutions was calculated as the total force minus the resting force. The mean F/CSA of a total of 52 trabeculae used for this study was 54.6 ± 3.3 mN/mm2. The output of the force transducer was recorded with real-time data collection LabView software (version 6.1) with in-house programming. All experiments were performed at 15°C.

TnC extraction and reconstitution protocol. TnC was extracted by soaking the trabeculae for 30 min in an extraction solution containing (in mM) 10 HEPES, 5 EDTA, and 0.5 trifluoperazine dihydrochloride (TFP) at pH 7.0. The trabeculae were then transferred to a pCa 9.0 solution and washed three times for 5 min to remove residual TFP. The residual force in pCa 4.0 solution was 3.7 ± 0.6% of the maximal force for all TnCs used in this study (n = 38). TnCs were reconstituted into the trabeculae by soaking the extracted trabeculae for 30 min in a pCa 9.0 solution containing 16.7 µM TnC (control or mutant). The trabeculae were then activated in a pCa 4.0 solution, and the force generated was expressed as a percentage of the maximum preextraction force. This ratio represented the percent recovery of postextraction force. The TnCF27W mutant was used previously to follow the fluorescence changes of isolated TnC in solution (41), and it was the control for all the studies performed here.

ktr Protocol. The trabecula was rapidly slackened (1 ms, 20% of total length), held at the slack length for 20 ms, and then rapidly restretched (1 ms) back to the original length. The movement of the length controller arm was initiated by an in-house programming algorithm using LabView software. Force redeveloped to levels similar to those before the slack-restretch (100.2 ± 0.4%, n = 118). This protocol was repeated in pCa solutions ranging from pCa 6.2 to pCa 4.0. To average out the rundown of the preparation throughout the experiment, maximum pCa activations were repeated in the middle and at the end of the experiments. The average force rundown in unextracted trabeculae and trabeculae reconstituted with TnCF27W, V44QTnCF27W, and F20QTnCF27W was 9 ± 3%, 8 ± 3%, 5 ± 3%, and 6 ± 2%, respectively. The redeveloped force traces were imported into Fig. P 2.7 analysis software for further analysis. The rate of force redevelopment, estimated from the time to half-activation, was very similar to the rate fitted with a monoexponential relationship (r2 = 0.93). Therefore, all the traces were fit with a monoexponential relationship. The fitted rate represented the rate of force redevelopment, ktr. The ktr at levels of force <60 µN was difficult to measure accurately; therefore, this represented the cutoff for calculating the ktr values. This protocol allowed us to calculate, for each pCa solution, the force produced before slack-restretch, which was used to generate the force vs. pCa relationships. Also, ktr values at each Ca2+ activation were used to characterize the ktr vs. pCa relationships and ktr vs. force relationships. The ktr protocol was applied in six different trabeculae reconstituted with either TnC mutant or control in experiments done in solutions with no added Pi. For the experiments using added-Pi solutions, the ktr protocol was done in the same fiber at the same pCa solutions without added Pi and then repeated with added Pi (matched experiments). This sequence of activation was repeated in unextracted trabeculae and in trabeculae reconstituted with TnC mutants in the presence of 2.5 mM Pi.

Isometric force vs. pCa relationship and ktr vs. pCa relationship. The relationships between force and pCa and ktr and pCa were fitted with a logistic sigmoid relationship mathematically equivalent to the Hill equation, as previously described (41, 42).

Standard solutions. The solutions for skinned trabecula experiments were prepared as previously described (23, 34). Large batches of pCa 9.0 and 4.0 solutions were made from stocks, divided into aliquots, and kept at –80°C. From these stock solutions, varied amounts of pCa 9.0 and 4.0 solutions were thawed and mixed to make solutions with intermediate Ca2+ concentrations (intermediate pCa), which were used within 1 wk. All added-Pi solutions were made fresh daily at the beginning of the experiments from a 0.5 M Pi stock (from potassium phosphate, monobasic, anhydrous; Sigma).

Protein mutagenesis and purification. The pET3a plasmid encoding human cardiac TnC was a generous gift from Dr. Lawrence B. Smillie (University of Alberta, Edmonton, AB, Canada). TnCF27W and its mutants were constructed from the TnC plasmid as previously described (23, 41). The pET3a plasmid encoding Cys-less human cardiac troponin I (TnIC79S,C96S) was also a generous gift from Dr. Lawrence B. Smillie. The TnIS149C,C79S,C96S mutant was constructed from the TnIC79S,C96S plasmid by primer-based site-directed mutagenesis with a Stratagene (La Jolla, CA) QuikChange site-directed mutagenesis kit. The mutation was confirmed by DNA sequence analysis. This construct is designated TnIS149C. The plasmid encoding TnIS149C was transformed into Escherichia coli Rosetta (DE3)pLysS cells (Novagen, San Diego, CA). Expression of TnIS149C was induced by adding 0.4 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG) when the bacterial cell density reached an optical density at 600 nm of 0.8–1.0. The purification of TnIS149C was carried out by standard laboratory techniques (15, 22).

Designing a fluorescent TnI. To follow Ca2+ binding to TnC-TnI complexes, Ser149 in TnIC79S,C96S was mutated to Cys and labeled with the fluorescent probe IAANS, making TnI Formula. In TnI, Ser149 is located in the beginning of the switch region (residues 149–158), which interacts with the hydrophobic patch on the N-domain of TnC (39). The switch region plays a key role in transmission of the Ca2+ signal from the N-domain of TnC onto other components of the thin filament system (39). When TnIFormula was complexed with TnC, its fluorescence was sensitive to the Ca2+-dependent interactions of TnI with the regulatory domain of TnC.

Labeling of TnIS149C. TnIS149C was reacted with three-to fivefold molar excess of IAANS for 3–5 h at 4°C with constant shaking in labeling buffer (50 mM Tris, 90 mM KCl, 1 mM EGTA, 6 M urea, pH 7.5). The labeling reaction was stopped by addition of 2 mM DTT, and the labeled protein was exhaustively dialyzed against labeling buffer at 4°C to remove unreacted label.

Reconstitution of the TnC-TnI complexes. The TnC-TnI complexes were prepared following a protocol described by Tobacman and Lee (43).

Determination of Ca2+ dependence of conformational changes in IAANS-labeled TnC-TnI complexes. All steady-state fluorescence measurements were performed with a Perkin-Elmer LS55 spectrofluorimeter at 15°C. IAANS fluorescence was excited at 330 nm and monitored at 450 nm as microliter amounts of CaCl2 were added to 2 ml of each IAANS-labeled TnC-TnI complex (0.17 µM) in (mM) 200 MOPS (to prevent pH changes on addition of Ca2+), 150 KCl, 2 EGTA, 1 DTT, and 3 MgCl2, with 0.04% Tween 20, pH 7.0 at 15°C. Free Ca2+ concentration was calculated with the computer program EGCA02 developed by Robertson and Potter (37). The Ca2+ sensitivities of conformational changes were reported as a pCa50, representing a mean ± SE of three or four separate titrations. The data were fit with a logistic sigmoid function as stated above (42).

Statistical analysis. We determined the statistical significance by applying an unpaired two-sample t-test or a paired t-test (for Pi studies) with the statistical analysis software Minitab (State College, PA). Linear regression analysis for slopes and intercepts of the ktr vs. relative force relationships was performed with GraphPad Prism 4.0. Statistical significance was established at a P value ≤0.05. All data are shown as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of TnC mutations on Ca2+ binding to IAANS-labeled TnC-TnI complexes. Ca2+-induced changes in IAANS fluorescence that occur when Ca2+ binds to the regulatory domains of wild-type (WT) TnC-TnIFormula, TnCF27W-TnIFormula, F20QTnCF27W-TnIFormula, and V44QTnCF27W-TnIFormula complexes are shown in Fig. 1. WT TnC-TnIFormula and TnCF27W-TnIFormula exhibited an increase in IAANS fluorescence with pCa50 of 5.73 ± 0.02 (Hill coefficient of 1.79 ± 0.06) and pCa50 of 5.74 ± 0.01 (Hill coefficient of 1.62 ± 0.02), respectively. For F20QTnCF27W-TnIFormula and V44QTnCF27W-TnIFormula, Ca2+ induced a decrease in IAANS fluorescence that occurred with pCa50 values of 5.33 ± 0.02 (Hill coefficient of 1.10 ± 0.02) and 5.98 ± 0.02 (Hill coefficient of 0.99 ± 0.04), respectively. Thus F20QTnCF27W and V44QTnCF27W produced TnC-TnI complexes with ~2.6-fold lower and ~1.7-fold higher Ca2+ binding sensitivities, respectively, compared with the TnCF27W-TnI complex. Interestingly, F20QTnCF27W and V44QTnCF27W induced decreases in IAANS fluorescence of TnIFormula, as opposed to increases observed with WT TnC and TnCF27W. These data suggest that the F20Q and V44Q mutations located in the hydrophobic pocket of the regulatory domain of TnC affected the local environment of the fluorescent probe.


Figure 1
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  		Fig. 1. Effect of troponin C (TnC) mutations on Ca2+ binding to TnCF27W- TnIFormula complexes. The Ca2+-dependent changes in IAANS fluorescence are shown for wild-type (WT) TnC (open squares), TnCF27W- TnIFormula(open hexagons), and V44QTnCF27W-TnIFormula(open triangles) as a function of pCa. IAANS fluorescence was excited at 330 nm and monitored at 450 nm: 100% IAANS fluorescence corresponds to the highest fluorescence value, whereas 0% fluorescence corresponds to the lowest fluorescence value for each individual TnC-TnI complex. Each data point represents the mean ± SE of 3 or 4 titrations fit with a logistic sigmoid function.

 
Effect of TnC mutants on Ca2+ sensitivity of force development. Rat skinned cardiac trabeculae were reconstituted with TnC mutants V44QTnCF27W or F20QTnCF27W. For the control TnCF27W, V44QTnCF27W, and F20QTnCF27W in the experiments with no added Pi, the percent recovery of force was 86% ± 7, 68% ± 5, and 66% ± 9, respectively (n = 6). Compared with endogenous TnC (TnCendog), the TnCF27W mutant increased the Ca2+ sensitivity of force development ~1.3-fold (Fig. 2A). In agreement with the Ca2+ binding affinities of the TnC-TnI complex, V44QTnCF27W and F20QTnCF27W increased ~1.6-fold and decreased ~1.5-fold, respectively, the Ca2+ sensitivity of force development (pCa50 = 6.00 ± 0.02 and 5.61 ± 0.03) compared with control TnCF27W (pCa50 = 5.80 ± 0.03; Fig. 2B). Thus it was possible to sensitize or desensitize cardiac muscle to Ca2+ on incorporating TnC mutants with different Ca2+ binding affinities into skinned cardiac trabeculae.


Figure 2
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  		Fig. 2. Isometric force vs. pCa relationship of rat skinned cardiac trabeculae containing endogenous or mutant TnCs. A: rat endogenous TnC (TnCendog, open squares) and rat cardiac trabeculae reconstituted with TnCF27W mutant (filled squares). B: rat cardiac trabeculae reconstituted with V44QTnCF27W (open triangles), with F20QTnCF27W (open hexagons), and TnCF27W (filled squares). Each data point represents the mean ± SE for an average of 6 trabeculae for each TnC. Force values are normalized against force at pCa 4.0 (F/F4.0).

 
Effect of Ca2+ concentration on ktr. For all the TnCs, ktr became progressively faster as the level of Ca2+ activation increased from pCa 6.2 to pCa 4.0 (Fig. 3). For this range of Ca2+ concentrations, the ktr values ranged from 3.0 ± 0.4 to 9.3 ± 0.8 s–1 for TnCendog, from 1.8 ± 0.1 to 11.0 ± 0.9 s–1 for TnCF27W, from 3.0 ± 0.2 to 10.5 ± 0.9 s–1 for V44QTnCF27W, and from 3.7 ± 0.5 to 9.3 ± 0.9–1 for F20QTnCF27W (n ≥ 6). Representative traces from three trabeculae reconstituted with TnCF27W, V44QTnCF27W, or F20QTnCF27W are shown in Fig. 3, A–C.


Figure 3
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  		Fig. 3. Rates of force redevelopment (ktr) in trabeculae reconstituted with TnC mutants. A–C: representative traces of rat skinned cardiac trabeculae reconstituted with control TnCF27W, V44QTnCF27W, and F20QTnCF27W at pCa 6.0, 5.8, 5.6, or 4.0. The fitted curve is represented as a smooth curve overlaying the trace. D: ktr vs. pCa relationships for control TnCF27W (filled squares), V44QTnCF27W (open triangles), and F20QTnCF27W (open hexagons). Each data point represents the mean ± SE for an average of 5 or 6 trabeculae for each TnC. The data were fit with a sigmoidal relationship, equivalent to the Hill equation. The y-intercept of the ktr vs. relative force relationship for each TnC was used as the minimal value for the ktr vs. pCa relationships (see Fig. 4B).

 
Figure 3D shows that the TnC mutants exhibited ktr vs. pCa relationships with a pCa50 of 5.77 ± 0.01 for TnCF27W, 6.00 ± 0.03 for V44QTnCF27W, and 5.61 ± 0.01 for F20QTnCF27W. Thus V44QTnCF27W increased the Ca2+ dependence of ktr ~1.6-fold, and F20QTnCF27W decreased the Ca2+ dependence of ktr ~1.4-fold. For control TnCF27W, V44QTnCF27W, and F20QTnCF27W, maximum ktr values were not significantly different. Therefore, by sensitizing or desensitizing cardiac muscle to Ca2+, ktr at submaximal levels of Ca2+ could be increased or decreased, respectively, whereas at saturating Ca2+ ktr was similar.

Relationship between ktr and relative force. Figure 4A shows that when TnC-reconstituted trabeculae generated similar amounts of relative force at different Ca2+ concentrations, ktr was similar. Figure 4B shows the relationship between average ktr values and average relative force at each Ca2+ concentration for TnCendog, TnCF27W, V44QTnCF27W, and F20QTnCF27W. The ktr vs. force relationship was fitted with a linear relationship, with no better fit provided by a curvilinear relationship. The slopes of the ktr vs. relative force relationships and the y-intercepts were not significantly different among all TnCs (see Fig. 4).


Figure 4
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  		Fig. 4. The dependence of ktr on relative force for trabeculae reconstituted with TnC mutants. A: control TnCF27W (blue), V44QTnCF27W (red), and F20QTnCF27W (green) have similar rates of contraction at matched levels of relative force, at maximal, intermediate, or low levels of Ca2+ concentration. B: average ktr values at each intermediate pCa are plotted vs. average levels of relative force generated at the corresponding pCa. TnCendog (black, open squares), control TnCF27W (blue, filled squares), V44QTnCF27W (red, filled triangles), and F20QTnCF27W (green, filled circles) are shown on the same plot. Each data point represents the mean ± SE for an average of 6 trabeculae for each TnC. The ktr dependence on relative force was fitted with a linear relationship (not shown for clarity). The y-intercept (I) and slope (S) values for all TnCs are TnCendog: I = 0.94 ± 1.33, S = 9.28 ± 1.70; TnCF27W: I = 0.56 ± 1.08, S = 10.5 ± 1.30; V44QTnCF27W: I = 1.20 ± 0.66, S = 10.07 ± 0.87; F20QTnCF27W: I = 2.03 ± 0.84, S = 8.08 ± 1.07.

 
Effect of Pi on force generated in unextracted trabeculae. It has been shown that in the presence of added Pi force is depressed in cardiac muscle preparations (2, 16, 20). Consistent with the literature, Fig. 5A demonstrates the effect of two Pi concentrations on the relative force in unextracted trabeculae. At 2.5 mM and 5.0 mM Pi, the force generated at maximal activation was 80 ± 5% and 65 ± 5%, respectively, of the force generated at maximal activation in the absence of added Pi. For the unextracted trabeculae, the Ca2+ sensitivity of force production in the presence of 2.5 mM Pi was similar to that in the absence of added Pi and significantly decreased in the presence of 5.0 mM Pi (Table 1).


Figure 5
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  		Fig. 5. Effects of inorganic phosphate (Pi) on force production for unextracted trabeculae and trabeculae reconstituted with TnC mutants. A: force vs. pCa relationship for unextracted trabeculae with no added Pi (open squares), 2.5 mM Pi (filled circles), and 5 mM Pi (filled diamonds). Each data point represents the mean ± SE for an average of 6–8 unextracted trabeculae. The force values for added Pi are normalized against the maximum force for no added Pi for each trabecula. B: force vs. pCa relationship for TnCF27W (filled squares), V44QTnCF27W (open triangles), and F20QTnCF27W (open hexagons) in the presence of 2.5 mM Pi. The force values for added Pi are normalized against the maximum force for no added Pi for each trabecula. Each data point represents the mean ± SE (n ≥ 6).

 

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  		Table 1. Effect of added Pi on Ca2+ sensitivity of force production for unextracted trabeculae and trabeculae reconstituted with TnCF27W mutants

 
Effect of Pi on force generated in trabeculae reconstituted with TnCF27W mutants. For trabeculae reconstituted with TnCF27W mutants, only the effects of 2.5 mM Pi on ktr were studied, since 5.0 mM Pi decreased the amount of force at submaximal Ca2+ activations to levels at which ktr values would have been difficult to measure accurately. In the presence of 2.5 mM added Pi, the maximum force generation in trabeculae reconstituted with control TnCF27W, F20Q TnCF27W, and V44QTnCF27W decreased to 76 ± 3%, 65 ± 4% and 76 ± 3%, respectively, from maximum force in the absence of added Pi (Fig. 5B). For trabeculae reconstituted with TnCF27W and F20QTnCF27W, Ca2+ sensitivity of force production in the presence of 2.5 mM added Pi and in the absence of Pi were similar (Table 1). For the V44QTnCF27W-reconstituted trabeculae, the Ca2+ sensitivity of force production was significantly decreased ~1.4-fold in the presence of 2.5 mM Pi (Table 1). However, V44QTnCF27W and F20QTnCf27W still sensitized and desensitized, respectively, the muscle to Ca2+ compared with TnCF27W (see Fig. 5B and Table 1). Thus, in accordance with the literature (2, 20), adding Pi to the activation solution decreased force production and desensitized cardiac muscle to Ca2+ in unextracted trabeculae in the presence of 5.0 mM added Pi and in V44QTnCF27W-reconstituted trabeculae in the presence of 2.5 mM added Pi.

Effect of 2.5 mM Pi on ktr for trabeculae reconstituted with control TnCF27W, V44QTnCF27W, and F20Q TnCF27W. Figure 6, A–C, shows that for trabeculae reconstituted with control TnCF27W, V44QTnCF27W, and F20Q TnCF27W, ktr was increased at all relative forces by the addition of 2.5 mM Pi. In the presence of 2.5 mM Pi, the ktr vs. relative force relationships were similar for trabeculae reconstituted with the TnCF27W mutants. Linear regression analysis showed that the slopes in the presence and in the absence of added Pi were not significantly different among all TnCs used in the study, but the y-intercepts in the absence of added Pi were significantly lower than the y-intercepts in the presence of added Pi. The y-intercepts in the presence or absence of added Pi were not statistically different for all trabeculae reconstituted with TnC mutants and control. At low Ca2+ activation, insufficient data were collected to obtain a reliable fit of the ktr vs. pCa relationship because of the low force production with 2.5 mM Pi. However, in the presence of 2.5 mM added Pi, Ca2+ sensitivities of force production for the TnCs were different (see Fig. 5B), but the ktr vs. relative force relationships were the same (see Fig. 6D). These results suggest that, in the presence of faster cross-bridge cycling, TnC Ca2+ binding properties can alter ktr at the same Ca2+ concentration.


Figure 6
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  		Fig. 6. Effects of 2.5 mM Pi on ktr for trabeculae reconstituted with TnC mutants. A–C: ktr vs. relative force relationship in the absence of added Pi (open symbols) and in the presence of 2.5 mM Pi (filled symbols). The ktr dependence on relative force was fitted with a linear relationship. A: TnCF27W. B: V44QTnCF27W. C: F20QTnCF27W. Each data point represents the mean ± SE (n ≥ 6). The y-intercept (I) and slope (S) values for the Pi-matched experiments are shown in each panel. D: ktr vs. relative force relationships in the absence of added Pi (open symbols) and in the presence of 2.5 mM Pi (filled symbols) for TnCF27W (squares), V44QTnCF27W (triangles), and F20QTnCF27W(hexagons).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main objective of this study was to investigate the effect of directly increasing or decreasing Ca2+ binding affinity of TnC on the rate of force generation. Previously (9, 41), we generated a number of skeletal and cardiac TnC mutants with a wide range of Ca2+ binding affinities, by individually substituting hydrophobic residues with polar Gln. The data indicated that the effect of the mutation on the Ca2+ dependence of skeletal muscle force generation could be better predicted from the Ca2+ affinity of TnC in the presence of TnI than from that of isolated TnC (9). Consistent with the results observed in skeletal muscle, the V44QTnCF27W and F20QTnCF27W mutations increased and decreased, respectively, the Ca2+ sensitivity of the TnCF27W-TnI Formula complex and cardiac muscle force generation. Thus V44QTnCF27W and F20QTnCF27W were used to investigate the influence of the thin filament Ca2+ binding properties on ktr in skinned cardiac trabeculae. The main observation of this study is that ktr is modulated by the Ca2+ binding properties of TnC during normal and accelerated cross-bridge cycling at submaximal Ca2+ activation but not at maximal Ca2+ activation.

ktr in cardiac vs. skeletal muscle. Huxley (18) developed a two-state cross bridge model that was later adopted by Brenner (4) to interpret ktr. According to this model, the rate of force redevelopment (ktr) is described as the sum of forward and backward rates: ktr = fapp + gapp, where fapp is the sum of the forward transition of cross bridges from detached or weakly attached, non-force-generating states to attached, force-generating states and gapp represents the backward transition and return to non-force-generating states. It was proposed that Ca2+ modulates ktr by regulating fapp (4). The Ca2+-dependent increase in ktr was observed for both skeletal and cardiac muscle. In skeletal muscle ktr increases ~10-fold from low to high levels of Ca2+ concentration with a curvilinear ktr vs. relative force relationship (13), whereas in cardiac muscle ktr increases ~2- to 5-fold (1, 13, 28, 46) with either a linear (35, 46) or a curvilinear (1, 28, 30) ktr vs. force relationship. In our study, the ktr values for the unextracted trabeculae increased approximately threefold with increasing levels of Ca2+ concentration and the ktr vs. relative force relationship was linear.

Ca2+ binding properties of TnC modulate ktr at submaximal levels of Ca2+ concentration. Figure 3D shows that, at submaximal levels of Ca2+ concentration, ktr was increased or decreased, respectively, by sensitizing or desensitizing the myofilaments to Ca2+. Nevertheless, at matched levels of relative force production (reached at different Ca2+ concentrations), the ktr vs. relative force relationship was the same for trabeculae reconstituted with the TnC mutants (Fig. 4). This result is in agreement with similar observations reported by other studies (10, 31, 44). In slow skeletal muscle fibers bepridil sensitized the muscle to Ca2+, but the ktr vs. relative force relationship in the absence and in the presence of bepridil was the same (44). The same observation was confirmed in recent studies using bepridil in skeletal myofibrils (10) and in human cardiac myofibrils (31). In fast skeletal myofibrils reconstituted with cardiac troponin (cTn) or a Ca2+-sensitizing Tn chimera (slow skeletal TnI-cTn), ktr vs. relative force relationships for Tnendog, cTn, and Tn chimera were similar (10). Thus ktr dependence on relative force is the same regardless of the amount of Ca2+ required to achieve a particular level of force.

It would appear that the ktr dependence on relative force at submaximal levels of Ca2+ concentration is mainly correlated with the number of attached cross bridges in force-generating states. However, at any given Ca2+ concentration, the level of force is determined by the level of Ca2+ activation of the thin filament, which mainly modulates the probability of cross bridges to enter force-generating states and to bind to the available actin sites (33). Thus we conclude that, at submaximal levels of Ca2+ concentration, ktr correlates with the level of activation of the thin filament, which can be modulated by TnC Ca2+ binding properties.

Ca2+ binding properties of TnC do not modulate ktr at high Ca2+ concentration. At high levels of Ca2+ concentration, the maximum ktr was similar for unextracted trabeculae and trabeculae reconstituted with control TnCF27W, V44QTnCF27W, or F20Q TnCF27W (Fig. 3D). However, the maximal force recoveries for V44QTnCF27W and F20Q TnCF27W (~68% and ~66%, respectively) were different than the force recovery for control TnCF27W (~86%). Our observation that at maximal Ca2+ ktr remains elevated, independent of the level of relative force production, is supported by several studies in both skeletal and cardiac muscle (12, 26). The minimum number of actin monomers that can be activated by Ca2+ and facilitate cross-bridge attachment represents a functional unit (FU) (11, 12). Gillis and collaborators (12) showed that decreasing the number of FU, and thus the relative level of force production, did not affect the maximal ktr in cardiac muscle. The same observation was made in skeletal muscle (26, 27). Thus maximal ktr can be dissociated from the relative level of force production in both cardiac and skeletal muscle.

Summary of ktr dependence on Ca2+ at all levels of Ca2+ activation. By reducing or enhancing Ca2+ binding properties of TnC within a FU, we showed that a Ca2+-dependent process is able to modulate ktr at submaximal levels of Ca2+ activation. This result suggests that ktr is correlated with the relative activation state of the FU such that, at low Ca2+, the Ca2+ binding properties within a FU would influence the availability of myosin-binding sites on actin and the overall probability of cross bridges to attach and generate force. At saturating Ca2+, independent of the number of FUs (12) or the Ca2+ binding properties of the FU (our study), the probability of cross bridges to bind is increased to such an extent that the overall ktr becomes maximal. However, even at high Ca2+ concentration, the maximum ktr in skeletal muscle can be modulated by the isoform of TnC (26, 27, 32).

Effects of Pi on cardiac muscle contractile performance and ktr. Up to now, the majority of Pi studies have been performed in skeletal muscle (3, 7, 8, 21, 29, 40). It has been shown that increasing Pi is associated with a reversal of the power stroke, which shifts the population of cross bridges from force-generating to non-force-generating states (16). This results in less force production, Ca2+ desensitization of force, and faster ktr (for review, see Ref. 13). Similarly, our experiments in rat skinned cardiac trabeculae at 15°C showed that increasing the Pi concentration progressively decreased the force production in unextracted trabeculae, desensitized the muscle to Ca2+ in the presence of 5.0 mM Pi, and increased ktr. These results are consistent with previous studies in cardiac muscle, despite differences in temperature, species, solution composition, and cardiac preparation (2, 16, 20).

Effect of 2.5 mM Pi on ktr in trabeculae reconstituted with TnC mutants and control. In the presence of 2.5 mM added Pi (Fig. 6) the maximum ktr increased approximately twofold and the y-intercept of the ktr vs. relative force relationship (which approximates gapp) increased approximately fourfold for unextracted trabeculae and trabeculae reconstituted with TnC mutants. In the presence of 2.5 mM Pi, V44QTnCF27W and F20QTnCF27W, which sensitized or desensitized the muscle to Ca2+, did not change the ktr vs. relative force relationship. This result implies that, even in the presence of faster cross-bridge cycling, at submaximal levels of Ca2+, ktr can still be modulated by the level of the thin filament Ca2+ activation. Again, maximum ktr was similarly increased in trabeculae reconstituted with TnCF27W mutants in the presence of 2.5 mM Pi, suggesting that Ca2+ binding properties of these TnCs do not affect ktr at high Ca2+.

Conclusion and physiological implications. We showed that the rate of submaximal force generation in cardiac muscle could be modulated via changes in the level of thin filament activation. This observation has important physiological and pathological implications, since the heart primarily contracts submaximally. On a beat-to-beat basis, the heart adjusts its contractions according to the levels of stress, physical activity, emotional stimuli, changes in body temperature, etc. A recent review on Ca2+-sensitizing agents in the heart suggests that the thin filament proteins can be potential targets for inotropic drugs (19). Accordingly, as our data suggest, directly sensitizing the thin filament to Ca2+ might prove beneficial in conditions in which the heart has lost this adaptability and is failing, by increasing the force production and accelerating the rate of rise of force.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants AR-020792 (J. A. Rall) and HL-087462 (S. B. Tikunova) and American Heart Association Grants 0415071B (C. Norman) and 0735079N (J. P. Davis).


   ACKNOWLEDGMENTS
 
We thank Dr. Lawrence Smillie for the generous gift of human TnC and TnI plasmids. We also thank Dr. Peter Reiser for critical reading of the manuscript and Laszlo Sarkozy for technical assistance with LabView programming.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. P. Davis, Ohio State Univ., Dept. of Physiology and Cell Biology, 1645 Neil Ave., 400 Hamilton Hall, Columbus, OH 43210 (e-mail: davis.812{at}osu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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J. P. Davis and S. B. Tikunova
Ca2+ exchange with troponin C and cardiac muscle dynamics
Cardiovasc Res, March 1, 2008; 77(4): 619 - 626.
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