Kinetics of cardiac thin-filament activation probed by fluorescence polarization of rhodamine-labeled troponin C in skinned guinea pig trabeculae

Abstract

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.

FIGURE 1

Detection of rhodamine-labeled cTnC exchanged into chemically skinned trabeculae of the guinea pig. Dilution series from trabecular homogenates along with standards made from labeled and unlabeled cTnC-Cys-84 were spotted onto a nitrocellulose Fast Slide as described in Methods. The figure illustrates four pads of an eight-pad slide. Each sample was spotted in triplicate from left to right and in increasing concentration from bottom row to top row. The right three columns of panel A (std in the figure) are rhodamine-labeled cTnC standards. The right three columns of panel B (std) are unlabeled cTnC standards. The top row of each pad and the first three columns of panel B were spotted with buffer (b) to estimate background fluorescence. The slide was scanned for rhodamine fluorescence (shown here), and the fluorescence of the labeled Tn subunit in the homogenate was measured directly against known concentrations of labeled protein standard. The pads were then incubated with an antibody to cTnC followed by a Cy-5-conjugated secondary antibody and scanned for Cy-5 fluorescence (not shown), providing a measure of the total (labeled plus unlabeled) cTnC in the trabecular homogenates. The extent of passive exchange of labeled cTnC-Cys-84 into guinea pig skinned trabeculae was 0.67 ± 0.01 (mean ± SE, n = 14). Panels C and D duplicate the sample dilution series of panels A and B, demonstrating the repeatability of the spotting and scanning method within the same slide.

FIGURE 2

Confocal optical section through the core of a trabecula in which the endogenous cTnC was exchanged for rhodamine-labeled cTnC-Cys-84 as described in Methods. The trabecula was in relaxing conditions during the confocal imaging. Z-lines are indicated by arrows.

FIGURE 3

Confocal image showing cTnC and actin in a trabecula in which the endogenous cTnC was exchanged for cTnC-Cys-84-rhodamine then labeled for actin with FITC-conjugated phalloidin. The red channel (left panel) shows the TnC distribution, and the green channel (right panel) shows phalloidin-actin. The cTnC-Cys-84-rhodamine appears to be highly colocalized with actin.

FIGURE 4

Dependence of isometric tension (circles) and cTnC-Cys-84 probe peak angle (triangles) on free [Ca²⁺] in the absence (solid symbols, solid lines) and presence of 1 mM Vi (open symbols, dashed lines) expressed as a relative change from relaxed to maximally Ca²⁺-activated. Relative angle in rigor is also shown (solid squares), with that at pCa 4.5 horizontally offset for clarity. Relative values were calculated for each trabecula before pooling the data from all trabeculae. Data at pCa 9 and 4.5 correspond to those in Table 1. The maximal isometric tension was 31 ± 2.1 (mean ± SE, n = 13). The solid curve through the tension data was generated by fitting a Hill equation to the data; the pCa₅₀ was 5.8 and the Hill coefficient was 2.4 in this series. Angular dispersion (not shown) did not significantly change from relaxed conditions across the full pCa range in the absence or presence of Vi or in rigor. The curves through the angle data were generated by fitting to the model illustrated in Fig. 8 as described in Discussion. Each point is mean ± SE of measurements in 7–13 trabeculae.

FIGURE 5

Rapid activation of skinned trabeculae by photolysis of caged-Ca²⁺. Shown are time courses of the polarization ratios, Q_‖ and Q_⊥, analyzed as peak axial angle and Gaussian dispersion along with isometric tension resulting from a step in [Ca²⁺] from approximately pCa 5.9–5.3. The photolysis occurred at time = 0, indicated by the arrow. Data points were also taken at 2.5 s during the asymptotes of slow components in tension and polarization (not shown). Pre- and postphotolysis pCa were estimated from comparison to the steady-state pCa-tension relation (13). The halftime of the initial rapid change in probe conformation was ∼4 ms at 21°C.

FIGURE 6

Rapid activation of skinned trabecula at two different pre- and postflash [Ca²⁺]. Time courses of the relative change in peak probe angle (upper panel) and relative tension (lower panel) are shown. Preflash [Ca²⁺] was established by the Ca²⁺-loading level of NP-EGTA, and the magnitude of the [Ca²⁺] step varied with laser energy. Postflash pCa are indicated on the plots, estimated from comparison to the steady-state pCa-tension relation. In the trial corresponding to final pCa ≅ 5.2, initial pCa was likewise estimated at ∼6.1. In the trial corresponding to final pCa ≅ 6.2, initial tension was near zero, thus initial pCa was estimated at 7.5 from the known Ca²⁺ binding affinity of NP-EGTA under these conditions.

FIGURE 7

[Ca²⁺] dependence of components of the angle change time course for TMRIA at cTnC-Cys-84. The rate of the rapid component was plotted against relative final tension, which provides an estimate of the sensitivity of Ca²⁺-cTnC binding. The rate of the slow component of the angle change was plotted against the slow component of the tension rise to demonstrate their correlation.

FIGURE 8

Model to assist in the interpretation of the observed pCa-tension relation of TMRIA at cTnC-Cys-84. The probe's fluorescent transition dipole is disposed at some angle with respect to a torsional axis of the protein (cTnC) to which it is attached. In response to increasing Ca²⁺ binding or cross-bridge formation, cTnC undergoes a rotation about its local axis, swinging the dipole first away from (less axial, labeled “partial activation”), and then at higher activation levels, closer to the long axis of the thin filament. FP reports these changes as an increase and then a decrease in the dipole's angle with respect to the filament axis.