Tropomyosin period 3 is essential for enhancement of isometric tension in thin filament-reconstituted bovine myocardium

Abstract

Tropomyosin (Tm) consists of 7 quasiequivalent repeats known as "periods," and its specific function may be associated with these periods. To test the hypothesis that either period 2 or 3 promotes force generation by inducing a positive allosteric effect on actin, we reconstituted the thin filament with mutant Tm in which either period 2 (Delta2Tm) or period 3 (Delta3Tm) was deleted. We then studied: isometric tension, stiffness, 6 kinetic constants, and the pCa-tension relationship. N-terminal acetylation of Tm did not cause any differences. The isometric tension in Delta2Tm remained unchanged, and was reduced to approximately 60% in Delta3Tm. Although the kinetic constants underwent small changes, the occupancy of strongly attached cross-bridges was not much different. The Hill factor (cooperativity) did not differ significantly between Delta2Tm (1.79 +/- 0.19) and the control (1.73 +/- 0.21), or Delta3Tm (1.35 +/- 0.22) and the control. In contrast, pCa(50) decreased slightly in Delta2Tm (5.11 +/- 0.07), and increased significantly in Delta3Tm (5.57 +/- 0.09) compared to the control (5.28 +/- 0.04). These results demonstrate that, when ions are present at physiological concentrations in the muscle fiber system, period 3 (but not period 2) is essential for the positive allosteric effect that enhances the interaction between actin and myosin, and increases isometric force of each cross-bridge.

Figure 1

Tropomyosins used for reconstitution. The structure of E. coli-synthesized Tm used for our study, highlighting the 7 quasirepeat regions. The N-terminal amino acid residue for each region is shown underneath nfTm. Yellow color represents the region(s) deleted in the respective mutants. ∆23Tm was previously studied [13, 14] and is included here for the sake of comparison. nfTm is the full-length protein and was used as a control. The N-terminus of ∆23Tm has the Ala-Ser substitution, whereas the N-termini of ∆2Tm, ∆3Tm, and nfTm do not have this substitution.

Figure 2

Slow pen trace of isometric tension in the standard activating solution (pCa 4.66, 5 mM MgATP, 8 mM Pi, ionic strength 200 mM, 15 mM CP, 320 units/ml CK, pH 7.00) at different temperatures (indicated). Experiments were performed in thin filament-reconstituted cardiac muscle fibers, in which nfTm (A), ∆2Tm (B), and ∆3Tm (C) were added back, together with bovine cardiac Tn. D, activation of native fibers; E, activation of thin filament-extracted fibers after gelsolin treatment for 50–90 minutes; F, activation of actin filament-reconstituted fibers; and G, activation of Tm- and Tn-reconstituted fibers. H, activation at eight different temperatures, as indicated. Between D, E, F, and G, the pen recorder was stopped so that extraction and reconstitution could be performed (at 0°C). Before each activation, the muscle fibers were washed in the standard activating solution (at 0°C), which did not induce tension. Tension was induced by switching the fibers to a bath of the same solution at the higher temperature. The fibers were relaxed in the Rx solution that contained 40 mM BDM at 0°C. Horizontal bars below the pen trace in C indicate that the temperature was 0°C during relaxation. The active tension in F develops as the result of withdrawal of BDM, and the temperature rises to 25°C. The active tension is relaxed as the result of the addition of 40 mM BDM, and the temperature drops to 0°C. Calibrations are 1 min (abscissa) and 10 kPa (ordinate).

Figure 3

pCa-tension curves. (a) pCa-tension plot comparing ∆2Tm-, ∆3Tm-, and nfTm-reconstituted muscle fibers. The tension data were fitted to (1), and the continuous lines are drawn based on best-fit parameters (Table 2). Error bars represent SEM. N = 14 for nfTm, N = 15 for ∆2Tm and ∆3Tm, and N = 9 for acetyl Tm. For acetyl Tm, the fitted parameters were reported in Lu et al. [13]. (b) pCa-stiffness plot, fitted to an equation similar to (1). (c) pCa-tension plot comparing nfTm and acetyl Tm.

Figure 4

pS-tension curves. pS-tension plot (S = [MgATP²⁻]) in (a) and pS-stiffness plot in (b) comparing nfTm, ∆2Tm, and ∆3Tm. The stiffness data were fitted to (2), and continuous lines are drawn based on best-fit parameters. The tension data in (a) are fitted to an equation similar to (2), where only the data to the right of the peak are used for fitting. The experiments were carried out at 8 mM Pi, and in the absence of Ca²⁺. Error bars represent SEM. N = 5 for nfTm and ∆2Tm, and N = 4 for ∆3Tm.

Scheme 1

Six-state cross-bridge model, where A = actin, M = myosin, D = MgADP, S = MgATP, and P = Pi = phosphate.

Figure 5

Frequency plots of complex moduli Y(f). Y(f) values during standard activation at 25°C are plotted as (a) dynamic modulus (= |Y(f)|) versus frequency, (b) phase shift (= argY(f)) versus frequency, and (c) viscous modulus (= Real  Y(f)) versus elastic modulus (= Imag  Y(f)) (Nyquist plot) with the frequency as a variable. Muscle models from three different Tms are compared. The averaged data of 15 (nfTm), 17 (∆2Tm), and 18 (∆3Tm) preparations are shown. Frequencies tested were 0.13, 0.25, 0.35, 0.5, 0.7, 1, 1.4, 2, 3.1, 5, 7, 11, 17, 25, 35, 50, 70, and 100 Hz (clockwise in (c)). The data are fitted to (3). The continuous curves represent represent calculated values from (3) with best-fit parameters. The units of all moduli are T _ac, phase shift is expressed as degree, and frequency in Hz. The resting modulus was not subtracted.

Figure 6

The effect of MgATP on the apparent rate constant 2πc. The data were fitted to (5). The Pi concentration was kept at 8 mM. The continuous lines are based on (5) with best-fit parameters.

Figure 7

The effect of Pi on the apparent rate constant 2πb. To deduce the rate and association constants of steps 4 and 5, the results of Figure 7 were fitted to (6). The MgATP concentration was kept at 5 mM. The continuous lines are based on (6) with best-fit parameters. σ of (7) was calculated based on the K ₁ and K ₂ obtained from the MgATP study (Table 5) and at S = 5 mM.

Figure 8

Cross-bridge distribution among 6 states during full Ca²⁺ activation. See Scheme 1 and its legends for abbreviations of the cross-bridge states. Det is the sum of all detached states (MS and MDP) and weakly attached states (AMS and AMDP). The distribution was calculated based on K ₁, K ₂, K ₄, K ₅ (Table 2), [MgATP] = 5 mM, [Pi] = 8 mM, and (7)–(13) of [43].

Figure 9

Summary of the temperature effect on isometric tension (a) and stiffness (b). The mean and SEM are shown. The number of experiments is nfTm (n = 10), ∆2Tm (n = 10), ∆3Tm (n = 11). Under the experimental conditions used here (standard activation solution and the constant sarcomere length), the larger temperature sensitivity corresponds to an increased hydrophobic interaction between actin and myosin molecules [24].

Figure 10

A photomicrograph of a 13 myocardial preparations generated for biochemical analysis.

Figure 11

SDS-PAGE of reconstituted myocardium with gradient gel. Lane 1: native myocardium. Lane 2: gelsolin-treated myocardium. Lane 3: actin-filament reconstituted myocardium. Lane 4: purified nfTm. Lane 5: myocardium reconstituted with nfTm and Tn after actin-filament reconstitution. Lane 6: purified ∆2Tm. Lane 7: myocardium reconstituted with ∆2Tm and Tn after actin-filament reconstitution. Lane 8: purified ∆3Tm. Lane 9: myocardium reconstituted with ∆3Tm and Tn after actin-filament reconstitution. Lane 10: molecular weight (MW) markers as labeled. Lanes 1, 4 and 5: same gel. Lanes 6 and 7: same gel. Lanes 8, 9 and 10: same gel. α-A c = α-actinin, Ge = gelsolin. Gelsolin is the band just below the α-actinin band in lane 2. ∆2Tm and ∆3Tm run at MW ~30 kD, below the nfTm and native Tm, which run at MW ~34 kD.