DCM-related tropomyosin mutants E40K/E54K over-inhibit the actomyosin interaction and lead to a decrease in the number of cycling cross-bridges

Abstract

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu-tion technique. The effects of the Ca²⁺, ATP, phos-phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com-pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T(HC) (high Ca²⁺ ten-sion at pCa 4.66) (E40K: 1.21±0.06 T(a), ±SEM, N = 34; E54K: 1.24±0.07 T(a), N = 28), a significantly lower T(LC) (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T(a), N = 34; E54K: 0.06±0.02 T(a), N = 28), and a significantly lower T(act) (Ca²⁺ activatable tension) (T(act) = T(HC)-T(LC,) E40K: 1.15±0.08 T(a), N = 34; E54K: 1.18±0.06 T(a), N = 28) than WT (T(HC) = 1.53±0.07 T(a), T(LC) = 0.12±0.01 T(a), T(act) = 1.40±0.07 T(a), N = 25). All tensions were normalized to T(a) ( = 13.9±0.8 kPa, N = 57), the ten-sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper-a-tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.

Figure 1. Elementary steps of the cross-bridge cycle.

The uppercase letters K indicate the association or equilibrium constants, and the lowercase letters k the rate constants of the elementary steps. Collectively these are referred to as the “kinetic constants”. A = actin, M = Myosin, D = MgADP, S = MgATP, and P = Pi = Phosphate.

Figure 2. pCa-tension plots.

(A, B) pCa-tension plots comparing mutant Tms and WT-Tm in high-IS solution. N = 25 for WT Tm; N = 34 for E40K; N = 28 for E54K. (C, D) pCa-tension plots comparing mutant Tms and WT-Tm in low-IS solution . N = 8 for WT Tm; N = 10 for E40K; N = 9 for E54K. The means and their SEMs are shown. Curved lines are calculated from Eq. 1, based on best-fit parameters. Tension is normalized to that of actin-filament reconstituted fibers (T_a = 13.9±0.8 kPa, N = 57).

Figure 3. Summary of Tension (A and E), Stiffness (B and F), pCa₅₀ (C and G), and Cooperativity (D and H).

(A–D) Data obtained in high-IS solutions. (E–H) Data obtained in low-IS solutions . (A, E) Low calcium tension (T _LC) represents the tension at pCa 7. High calcium tension (T _HC) represents the tension at pCa 4.66. Active tension (T _act) is the tension developed on Ca²⁺ activation (T _act = T _HC–T _LC). (B, F) Results of the stiffness study. Tension and stiffness were normalized to Ta. (C, G) pCa₅₀ (Ca²⁺ sensitivity). (D, H) Cooperativity. *: p<0.05.

Figure 4. Nyquist plots of the mutants (red) compared to WT (black).

(A–B) Two mutants are compared to WT at pCa 4.66; (C–D) Two mutants are compared to WT at pCa 7.0. Also included in A and B are the baselines taken in the relaxing solution at 0°C (× for the mutant, and + for WT). The data in A–D were obtained in high-IS solutions. (E–F) Two mutants are compared to WT at pCa 4.66 in low-IS solution . Range of frequency used: 0.13–100 Hz. The frequency increases in the clock-wise direction. Note the difference in scale for both axes in panels A–B vs. panels C–D, and panels A–B vs. panels E–F.

Figure 5. Apparent rate constants.

The apparent rate constants for myocardium reconstituted with mutant Tms (red) and WT Tm (black). Symbols represent the mean±SEM. Continuous curves were generated by fitting the data to Eq. 3 (A and B) or Eq. 4 (C and D). (A–B): 2πc (in s⁻¹) is plotted against [MgATP] (mM). (C–D): 2πb (in s⁻¹) is plotted against [Pi] (mM).

Figure 6. Kinetic constants.

The kinetic constants of the cross-bridge cycle (Scheme 1) in reconstituted cardiac fibers are compared among mutant and WT Tms. (A) Rate constants. (B) Equili­brium constants. Note that K ₀ was divided by 20 and K ₅ was multiplied by 5. *: p<0.05.

Figure 7. Cross-bridge distributions.

From the equili­brium constants, the cross-bridge distribution over six states under the standard activating condition (5S8P, Table S2 in [17]) was calculated for two mutant and the WT Tm forms. Att indicates the sum of all strongly attached (force generating) cross-bridges: Att = AMD+AM+AM*S+AM*DP+AM*D.