Tropomodulin-Tropomyosin Interplay Modulates Interaction Between Cardiac Myosin and Thin Filaments

Abstract

Tropomodulin (Tmod) is an actin-binding protein that interacts with tropomyosin and the actin filament at the pointed end. The influence of Tmod on the thin filament activation in the myocardium is not clear. We studied the interactions of Tmod1 and Tmod4 with the cardiac tropomyosin isoforms Tpm1.1 and Tpm1.2 using size-exclusion chromatography, a pull-down assay, and cross-linking with glutaraldehyde. We found that Tmod1 and Tmod4 form complexes with both Tpm1.1 and Tpm1.2, indicating durable interactions between these proteins. The effects of both Tmods on the actin-myosin interaction were studied using an in vitro motility assay. Tmod did not affect the sliding velocity of bare F-actin. Tmod1 slightly dose-dependently decreased the sliding velocity of F-actin-Tpm1.1 filaments and had no effect on the velocity of F-actin-Tpm1.2 filaments. With ventricular myosin, Tmod1 reduced the calcium sensitivity of the sliding velocity of thin filaments containing Tpm1.1 but did not affect it with filaments containing Tpm1.2. With atrial myosin, Tmod1 decreased the calcium sensitivity of the sliding velocities of thin filaments containing both Tpm1.1 and Tpm1.2. We can conclude that Tmod takes part in the regulation of actin-myosin interactions in the myocardium through interactions with Tpm. The effect of Tmod on the activation of thin filaments depends on the protein isoforms.

Keywords: actin-associated proteins; actin–myosin interaction; calcium regulation; cardiac myosin isoforms; cardiac tropomyosin isoforms; in vitro motility assay; tropomodulin.

Figure 1

The effect of Tmod1 on the sliding velocities of F-actin and F-actin–Tpm filaments on sheep cardiac myosin in the in vitro motility assay. (A) The dependence of the sliding velocity of F-actin on the concentration of Tmod1 over sheep ventricular myosin. (B,C) The dependence of the sliding velocities of F-actin–Tpm filaments containing Tpm1.1 and Tpm1.2 on the Tmod1 concentration on sheep ventricular (B) and atrial (C) myosin. The experimental data for Tmod1 with F-actin–Tpm1.1 filaments and F-actin–Tpm1.2 filaments are approximated by exponential and linear functions, respectively. The experimental values are presented as means ± SDs.

Figure 2

The effect of Tmod1 on the maximum sliding velocities at pCa4 of thin filaments containing F-actin, Tpm, and Tn on sheep cardiac myosin in the in vitro motility assay. (A–D) The dependence of the maximum sliding velocities (pCa4) of thin filaments containing Tpm1.1 (A,C) and Tpm1.2 (B,D) on sheep ventricular (A,B) and atrial (C,D) myosin on the Tmod1 concentration. The experimental data in (A–D) are approximated by exponential functions. The experimental values are presented as means ± SDs.

Figure 3

The effect of Tmod1 on the maximum sliding velocities at pCa4 of thin filaments reconstructed from F-actin, troponin, and Tpm on sheep cardiac myosin in the in vitro motility assay. (A,B) The effect of 500 nM Tmod1 on the calcium dependence of the sliding velocities of thin filaments containing Tpm1.1 (A) and Tpm1.2 (B) over sheep ventricular myosin. (C) The effect of 500 nM Tmod4 on the calcium dependence of the sliding velocity of thin filaments containing Tpm1.1 over sheep ventricular myosin. The experimental values are means ± SDs. The calcium dependence of the filament sliding velocity was approximated by the Hill equation; the values of the parameters are given in Table 1.

Figure 4

The effect of Tmod1 on the interaction of sheep ventricular myosin with native thin filaments. (A) The dependence of the maximum sliding velocity (pCa4) of native thin filaments (NTFs) over sheep ventricular myosin on the Tmod1 concentration in the in vitro motility assay. NTFs and myosin were extracted from the sheep left ventricle. The experimental data are presented as means ± SDs and approximated by a linear function. (B) The effect of 500 nM Tmod1 on the calcium dependence of the sliding velocity of NTFs on sheep ventricular myosin in the in vitro motility assay. The experimental values are means ± SDs. The calcium dependence of the filament sliding velocity was approximated by the Hill equation; the values of the parameters are given in Table 1.

Figure 5

The effect of Tmod1 on the calcium dependence of the sliding velocity of thin filaments on sheep atrial myosin in the in vitro motility assay. (A) The effect of 500 nM Tmod1 on the calcium dependence of the sliding velocity of thin filaments containing Tpm1.1 on sheep atrial myosin. (B) The effect of 500 nM Tmod1 on the calcium dependence of the sliding velocity of thin filaments containing Tpm1.2 on sheep atrial myosin. The experimental values are means ± SDs. The calcium dependence of the filament sliding velocity was approximated by the Hill equation, and the values of the parameters are given in Table 2.

Figure 6

The effect of rat Tmod1 on the calcium dependence of the sliding velocity of thin filaments on rat cardiac myosin in the in vitro motility assay. (A) The effect of 500 nM rat Tmod1 on the calcium dependence of the sliding velocity of thin filaments containing Tpm1.1 on rat ventricular myosin. (B) The effect of 500 nM rat Tmod1 on the calcium dependence of the sliding velocity of thin filaments containing Tpm1.1 on rat atrial myosin. The experimental values are means ± SDs. The calcium dependence of the filament sliding velocity was approximated by the Hill equation, and the values of the parameters are given in Table 3.

Figure 7

The effect of Tmod1 on cross-bridge–cross-bridge (Xb-Xb) cooperativity. (A,B) The effect of 500 nM Tmod1 on the Xb-Xb cooperativity of the interactions of sheep ventricular myosin with F-actin–Tpm filaments containing Tpm1.1 (A) and Tpm1.2 (B). (C) The effect of 500 nM Tmod4 on the Xb-Xb cooperativity of the interaction of sheep ventricular myosin with F-actin–Tpm filaments containing Tpm1.1. (D) The effect of 500 nM Tmod1 on the Xb-Xb cooperativity of the interaction of sheep atrial myosin with F-actin–Tpm filaments containing Tpm1.1. The experimental values are presented as means ± SDs. The experimental values are approximated by the Hill equation.

Figure 8

The effect of 500 nM Tmod1 on the cross-bridge–cross-bridge cooperativity of the interaction of sheep ventricular myosin with thin filaments at a non-saturating calcium concentration (pCa5.2). The experimental values are presented as means ± SDs and approximated by the Hill equation.

Figure 9

The chemical cross-linking of Tpm and Tmod isoforms. (A) Samples subjected to cross-linking before adding glutaraldehyde. (B) Chemical cross-linking by 0.002% glutaraldehyde for 15 min. (C) Chemical cross-linking by 0.008% glutaraldehyde for 15 min. Lanes: 1—Tpm1.1; 2—Tpm1.1 with Tmod1; 3—Tpm1.1 with Tmod4; 4—Tpm1.2; 5—Tpm1.2 with Tmod1; 6—Tpm1.2 with Tmod4; 7—Tmod1; 8—Tmod4. Black arrows mark the positions of Tpm/Tmod complexes. Original images can be found in Supplementary Materials.

Figure 10

Analytical size-exclusion chromatography of Tmod1, Tmod4, Tpm1.1, Tpm 1.2, and their mixtures loaded on a Superose 6 Increase 10/300 GL column. (A) The elution profiles of Tmod1, Tpm1.1, and a mixture of Tmod1 and Tpm1.1 at a concentration ratio of 1:1. (B) The elution profiles of Tmod1, Tpm1.2, and a mixture of Tmod1 and Tpm1.2 at a concentration ratio of 1:1. (C) The elution profiles of Tmod4, Tpm1.1, and a mixture of Tmod4 and Tpm1.1 at concentration ratio of 1:2. (D) The elution profiles of Tmod4, Tpm1.2, and a mixture of Tmod4 and Tpm1.2 at a concentration ratio of 1:2. The digits indicate peak numbers; the peak parameters are presented in Table 5 and Table 6.

Figure 11

The interactions of Tmod4 with Tpm1.1 and Tpm1.2 measured by the pull-down assay. This graph shows the dependence of Tmod4 saturation on Tpm1.1 (black line) and Tpm1.2 (red line) added to the sample in different concentrations. The Tmod4 concentration was 6 µM in all samples, and the Tpm concentrations ranged from 3 to 24 µM. The experimental values are presented as means ± SDs.