Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments

Abstract

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.

Figure 1

Cartoon of thin filament regulation. The states refer to the different nomenclatures used with the upper row being based upon structural studies, middle being based upon kinetic and equilibrium binding studies, and the lower being based upon ATPase and/or muscle fiber force studies.

Figure 2

Thin filament states within the half sarcomere. Adapted from the work of Zhang et al. and the current study.

Figure 3

Influence of calcium on labeled Tn exchange pattern in rigor myofibrils. Fluorescently labeled Tn was incubated with myofibrils for different times at pCa 9 or 4 then imaged. The myofibril region of interest (both phase contrast (P) and fluorescent (F)) were cropped and spliced together with the phase contrast being the upper half and the fluorescence image (showing relative amount within a myofibril) being the lower half. Part of the non-overlap region is highlighted within the red rectangle and part of the overlap region is noted by the green rectangle outline. At pCa 9, exchange was in the overlap region that contained rigor cross-bridges and there was a slight increase in the amount in the non-overlap region by 256 min. At pCa 4, exchange was faster in the non-overlap than overlap region at early time-points and the extent of exchange in each region was nearly equal by 256 min.

Figure 4

Influence of Tn subunits on myofibrillar ATPase activity and labeled Tn exchange pattern. Intrinsic Tn (TnCIT) was exchanged out with either TnIT or TnT at pCa 4. ATPase activity was measured at pCa 9 and pCa 4 and expressed relative to TnCIT myofibril pCa 4 activity (a). Labeled Tn was exchanged into these myofibrils as for Figure 2 for 8 min (b). Images are presented as in Figure 2. Exchange of TnIT for TnCIT resulted in complete loss of calcium activation of ATPase activity while exchange of TnT for TnCIT resulted in complete loss of calcium regulation of ATPase activity. There was limited exchange in the non-overlap region for TnIT myofibrils showing that TnC was needed for the calcium dependency of Tn exchange (C state). For TnT myofibrils, non-overlap region exchange (C state) was fast, independent of calcium showing that TnI was needed for B state and TnIC was needed for calcium dependency of switching between the B and C state. Another feature of TnT myofibrils was that exchange was slower in the overlap than non-overlap, suggesting that strong cross-bridges (M state) slowed TnT dissociation.

Figure 5

The rate of Tn dissociation from different states of the thin filament. Troponin T was exchanged for TnCIT as described in Figure 4. Labeled Tn was exchanged into TnCIT and TnT myofibrils as described in Figure 2. Only images from the fluorescence are shown and demonstrate pattern not amount (a). Imaging was done using fixed exposure time (note the noisy signal for the slowest exchanging sample (pCa 9, TnCIT at 1 min). Localization of non-overlap and overlap region is as in Figure 2. The time-dependent change in the pattern is obvious for all myofibrils. Sarcomere intensity increased with time (not shown) while the ratio approached one from either below (pCa 9, TnCIT) or above (pCa 9, TnT; pCa 4, TnCIT and TnT) one. The intensity ratio was fitted to [(1-exp (k_no*t))/(1-exp(k_ol*t))] + C, where k_no is the dissociation rate in the non-overlap region; k_ol is the dissociation rate in the overlap region and C is a constant. Fitted values are given in Table 1.

Figure 6

Intra-sarcomeric relative intensity profiles for TnCIT and TnT myofibrils. The column average (4×44 pixel ROI) was obtained from pCa 9 (red) and 4 (blue), TnCIT (a) and TnT (b) myofibrils at 4 (broken line) and 1024 (continuous line) min from the images in Figure 5(a). For each myofibril, the intensity was normalized to the peak intensity within each sarcomere. Arrowheads mark the intensity peaks for pCa 9 while arrows mark the intensity peaks for pCa 4. For pCa 9, TnCIT myofibrils, there was no change in the position of the peak but there was a change in the intensity in the non-overlap region with increased time. For pCa 4, TnCIT myofibrils, the peak was in the non-overlap region at both 4 and 1024 min and there was an increase in the intensity in the overlap region with increased time. With TnT myofibrils, there was a change in the position of the peak with increased time. At pCa 9, the peak moved from the non-overlap to the A-I band junction while at pCa 4, it moved from the middle of the non-overlap region to nearer the Z-line.

Figure 7

The C-state rates of Tn dissociation are determined by regulatory TnC–TnI interactions. TnIT was exchanged for native Tn, TnCs were added to 0.2 mg/ml TnIT myofibrils at 2 μM, incubated for 30 min, then labeled Tn exchange done at pCa 4 by mixing with an equal volume of 2 μM labeled Tn. Exchange was done for 16 min and samples were processed as for Figure 2. Images were cropped within the footprint of the fluorescence signal and then converted to 8 bit to maximize visualization of subtle differences. Annotations are as for Figure 2. A stick diagram of sarcomere anatomy is shown at the top as a map for the regions within the sarcomere. The nd samples are rabbit skeletal and wild-type chicken TnC, respectively, while 126, 146, 315, 383, and 2075 nM are F78Q TnC^F29W, TnC^F29W, M46Q TnC^F29W, M82Q TnC^F29W, and I62Q TnC^F29W, respectively. Infinity is no TnC added (TnIT myofibril). The rate of Tn dissociation, in the presence of calcium (nominal C state), grades with TnC–TnI regulatory domain affinity. Note the regions of enhanced Tn exchange in the region just proximal to the overlap region for nearly all mutant TnCs.

Figure 8

The C-state rate of Tn dissociation is cooperative with calcium and strong cross-bridges. Labeled Tn exchange was done as for Figure 2, and presented as in Figure 6. The non-overlap region is noted by a red rectangle outline. The rate of Tn dissociation, at sub-maximal calcium (pCa 6.1 to 5.8, the region of cooperative activation of ATPase activity) was not uniform along the length of the non-overlap thin filament at 16 min. A region of enhanced exchange occurred just proximal to the overlap region, and depending on pCa, slowed from the proximal overlap region towards the Z-line. After extensive exchange (1024 min), the intensity was mostly equal in the non-overlap and overlap region except for pCa 9. A subtle sub-region of extra Tn exchange was apparent at pCa 4, just proximal to the Z-line. This was also evident for TnCIT and TnT myofibrils at pCa 4 (Figure 5(a)).

Figure 9

Pathway for Tn dissociation. Protein interactions are noted by lines connecting the letters with A, Tm, T, I, C, being actin, tropomyosin, TnT, TnI and TnC. Steps (1) or (2) can limit rate of Tn dissociation while step (1) is involved in regulation. Step (1) is dissociation of the regulatory domain of TnI from actin-Tm and its binding to the regulatory domain of TnC. Step (2) is TnT dissociation from actin-Tm. Step (1) limits the B-state region the rate of Tn dissociation while step (2) limits C- and M-state region dissociation rate. Calcium and strong cross-bridges influence step (1) while strong cross-bridges also influence step (2).

Figure 10

Cartoon of the signal transduction pathway from the strong cross-bridge to Tn. Adapted from Swartz et al. Actin is represented by circles, Tm the contiguous red line, Tn the blue oblong structure, and the motor domain the dark brown, tadpole-like structure. Strong cross-bridges displace Tm into the M state. Because of Tm’s relative in-elasticity, this displacement is sensed by Tn, to favor TnI (arm-like structure projecting from Tn) dissociation from actin and binding to TnC, which favors calcium binding by TnC.