Instability in the central region of tropomyosin modulates the function of its overlapping ends

Abstract

The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of contiguous Tm dimers, we used transgenic mice (Tm(DM)) that expressed a mutant α-Tm in the heart; S229E and H276N substitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse cardiac troponin T mutants (TnT(1-44Δ) and TnT(45-74Δ)) that have a divergent effect on the overlapping ends of Tm were employed. The S229E-induced instability in the central region of Tm(DM) altered the overlapping ends of Tm(DM), thereby it negated the attenuating effect of H276N on Ca(2+)-activated maximal tension. The rate of cross-bridge detachment (g) decreased in Tm(DM)+TnT(WT) and Tm(H276N)+TnT(WT) fibers but increased in Tm(DM)+TnT(45-74Δ) fibers; however, TnT(45-74Δ) did not alter g, demonstrating that S229E in Tm(DM) had divergent effects on g. The S229E substitution in Tm(DM) ablated the H276N-induced desensitization of myofilament Ca(2+) sensitivity in Tm(DM)+TnT(1-44Δ) fibers. To our knowledge, novel findings from this study show that the structural instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.

Figure 1

Schematic representation of S229E and H276N substitutions in Tm_DM. S229E and H276N substitutions in Tm are represented by black stars and black filled circles, respectively. The central helical region of cTnT (residues 78–193) is denoted by CR. The inter-molecular interaction between the head-to-tail overlapping region of contiguous Tm dimers and the N-terminus of cTnT is denoted by the double-headed arrow (adapted from 47) The orientation of the Tn complex is shown as described previously (48). Because the local instability in Tm does not affect the binding of Tm to Tn (41), and residues 1–77 of cTnT are not known to interact with Tm (47), it is likely that the S229E substitution in Tm affects myofilament function via a long-range conformational effect on the overlapping ends of Tm.

Figure 2

Western blot analysis of reconstituted cardiac muscle fibers. Reconstituted muscle fibers were solubilized in 2% SDS (49). Solubilized fiber samples were electrophoresed on a 10% SDS gel. Protein bands were transferred to a PVDF membrane for Western blot analysis. TnT_WT, TnT_1–44Δ, and TnT_45–74Δ were identified using a monoclonal anti-TnT primary antibody. Panels A and B indicate samples from Tm_WT and Tm_DM fibers, respectively. Lane 1 shows purified recombinant cmyc-tagged TnT_WT and TnT. Lanes 2, 3, and 4 show samples from cmyc-tagged TnT_WT, TnT_1–44Δ, and TnT_45–74Δ reconstituted fibers, respectively.

Figure 3

Effect of Tm_WT and Tm_DM on Ca²⁺-activated maximal tension in TnT_1–44Δ or TnT_45–74Δ reconstituted cardiac muscle fibers. Ca²⁺-activated maximal tension (pCa 4.3) was measured at 20°C. The resting SL was 2.3 μm. Two-way ANOVA revealed a significant Tm-cTnT interaction effect (P < 0.05), indicating that Tm_DM altered the effects of TnT_1–44Δ and TnT_45–74Δ on maximal tension. Post hoc multiple pairwise (Fisher’s LSD) comparisons revealed that tension decreased in TnT_1–44Δ reconstituted fibers, whether the Tm background was Tm_WT or Tm_DM. Maximal tension remained unaffected in Tm_DM+TnT_45–74Δ fibers, but attenuated significantly in Tm_H276N+TnT_45–74Δ fibers (16), suggesting that the S229E-induced instability in the central region of Tm_DM affected the overlapping ends of Tm_DM. Number of determinations was at least six in each group. Values are reported as mean ± SE. ^∗∗∗P < 0.0001.

Figure 4

Effect of Tm_WT and Tm_DM on tension cost in TnT_1–44Δ or TnT_45–74Δ reconstituted cardiac muscle fibers. (A) Effect of Tm_WT on the tension-ATPase relationship in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. (B) Effect of Tm_DM on the tension-ATPase relationship in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. The relationship between steady-state isometric tension and the corresponding ATPase activity was determined at various levels of Ca²⁺ activation (26,27). (C) Tension cost was derived from the slope of the tension-ATPase plot. Two-way ANOVA revealed a significant Tm-TnT interaction effect (P < 0.05), indicating that Tm_DM altered the impact of TnT_1–44Δ and TnT_45–74Δ on tension cost. Post hoc multiple pairwise (Fisher’s LSD) comparisons revealed that tension cost remained unaffected by Tm_WT in either TnT_1–44Δ or TnT_45–74Δ reconstituted fibers but increased significantly by Tm_DM in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. Number of determinations was at least six for each group. Values are reported as mean ± SE. ^∗P < 0.05; ^∗∗∗P < 0.001.

Figure 5

Effect of Tm_WT and Tm_DM on the rate of force redevelopment (k_tr) in TnT_1–44Δ or TnT_45–74Δ reconstituted cardiac muscle fibers. k_tr was measured as described previously (30). The resting SL was 2.3 μm and the temperature was 20°C. (A) Effect of Tm_DM on tension redevelopment following a rapid slack and restretch protocol in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. Data in panel A is shown as normalized tension and traces shown are representative samples from each group. Tension redevelopment was fitted to a single exponential to derive the rate constant k_tr. (B) Representative length trace used in the slack/restretch protocol for k_tr estimation. (C) Effect of Tm_WT and Tm_DM on k_tr in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. Two-way ANOVA revealed no significant interaction effect (P = 0.08) on k_tr. Post hoc multiple pairwise comparisons revealed that Tm_WT did not alter k_tr in either TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. As shown in panels A and C, Tm_DM increased k_tr in TnT_45–74Δ reconstituted fibers but not in TnT_1–44Δ reconstituted fibers. Number of determinations was at least six in each group. Values are reported as mean ± SE. ^∗∗P < 0.01.

Figure 6

Effect of Tm_WT and Tm_DM on the pCa-tension relationships in TnT_1–44Δ or TnT_45–74Δ reconstituted cardiac muscle fibers. Normalized pCa-tension data were fitted to the Hill equation. (A) Effect of Tm_WT on the pCa-tension relationship in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. (B) Effect of Tm_DM on the pCa-tension relationship in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. Arrows in panel B indicate that the S229E substitution in Tm_DM attenuates the desensitizing effect of TnT_1–44Δ on myofilament Ca²⁺ sensitivity (see Tm_WT+TnT_1–44Δ fibers in panel A), thereby ablating the TnT_1–44Δ-induced decrease in myofilament Ca²⁺ sensitivity. Number of determinations was at least six in each group. Values are reported as mean ± SE.

Figure 7

Effect of Tm_DM on myofilament Ca²⁺-sensitivity (pCa₅₀) or cooperativity of tension development (n_H) in TnT_1–44Δ and TnT_45–74Δ reconstituted cardiac muscle fibers. pCa-tension data were fitted to Hill’s equation to derive pCa₅₀ and n_H values. (A) Effect of Tm_WT and Tm_DM on pCa₅₀ in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. Two-way ANOVA revealed a significant Tm-cTnT interaction effect (P < 0.05), indicating that Tm_DM altered the impact of TnT_1–44Δ and TnT_45–74Δ on pCa₅₀. Post hoc multiple pairwise comparisons revealed that pCa₅₀ decreased significantly in Tm_WT+TnT_1–44Δ fibers, but not in Tm_DM+TnT_1–44Δ fibers. TnT_45–74Δ significantly increased pCa₅₀ in the presence of Tm_WT or Tm_DM. (B) Effect of Tm_WT and Tm_DM on n_H in TnT_1–44Δ or TnT_45–74Δ reconstituted fibers. Two-way ANOVA revealed no significant Tm-cTnT interaction effect (P = 0.70) on n_H. Post hoc multiple pairwise comparisons revealed that n_H decreased significantly by TnT_45–74Δ in the presence of Tm_WT or Tm_DM. Number of determinations was at least six in each group. Values are reported as mean ± SE. ^∗∗P < 0.01; ^∗∗∗P < 0.001.