Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Abstract

Myosin-binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C includes 11 Ig- and fibronectin-like domains and a cMyBP-C-specific motif. In vitro studies show that in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating thin filament motility. Structural observations of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to actin close to the low Ca(2+) binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by interfering with tropomyosin regulatory movements on actin. To determine directly whether cMyBP-C binding affects tropomyosin position, we have used electron microscopy and in vitro motility assays to study the structural and functional effects of N-terminal fragments binding to thin filaments. 3D reconstructions suggest that under low Ca(2+) conditions, cMyBP-C displaces tropomyosin toward its high Ca(2+) position, and that this movement corresponds to thin filament activation in the motility assay. At high Ca(2+), cMyBP-C had little effect on tropomyosin position and caused slowing of thin filament sliding. Unexpectedly, a shorter N-terminal fragment did not displace tropomyosin or activate the thin filament at low Ca(2+) but slowed thin filament sliding as much as the larger fragments. These results suggest that cMyBP-C may both modulate thin filament activity, by physically displacing tropomyosin from its low Ca(2+) position on actin, and govern contractile speed by an independent molecular mechanism.

Keywords: muscle activation; muscle regulation.

Fig. 1.

Schematic of cMyBP-C and the expressed N-terminal fragments C0C3, C0C2, and C0C1f used in this study. cMyBP-C consists of 8 Ig and 3 Fn domains together with a cMyBP-C-specific M-domain, containing a phosphorylation region (orange) with 4 phosphorylatable serines (P), a ProAla-rich domain, and a cardiac-specific insert (blue) in the C5 domain.

Fig. 2.

Decoration of native thin filaments with C0C2 under low Ca²⁺ conditions. (A, D) Undecorated control. (B, C, E, F) Filaments decorated with C0C2 at A:C0C2 molar ratios of 1:1 (B, E) and 1:3 (C, F). Filaments in D–F have been computationally straightened. [Scale bar (A–C) = 100 nm; (D–F) = 50 nm.)

Fig. 3.

3D reconstructions of native control thin filaments under low and high Ca²⁺ conditions. (A) Low Ca²⁺ filament (gray surface rendering) fitted with ribbon depiction of low-Ca²⁺ A.Tm atomic model (39) (actin monomers, yellow; Tm, red). (B) High-Ca²⁺ reconstruction (yellow surface rendering), fitted with high-Ca²⁺ A.Tm atomic model (actin monomers, yellow; Tm, green). (C) Superposition of A and B demonstrating Tm shift on to inner domain of actin at high Ca²⁺ (note: slight variations in actin contours in A and B cause either gray or yellow to appear on the actin surface in C). (D and E) Transverse sections of low and high Ca²⁺ reconstructions, respectively, showing positioning of Tm (arrows) near the junction of actin SD1 and SD3 in low Ca²⁺, and on SD3 in high Ca²⁺. (F) Superposition of D and E, demonstrating the shift of Tm. Filaments in A–C oriented with pointed end at top; actin subdomains are marked in A and D. (Scale bar = 5 nm.)

Fig. 4.

3D reconstructions of native thin filaments decorated with C0C2 under low Ca²⁺ conditions. (A–C) Reconstructions with the indicated ratios of A:C0C2 fitted with A.Tm atomic models (39), as in Fig. 3, with Tm in blocked (red) or closed (green) position. With low levels of C0C2, there was a small movement of Tm from the blocked position (A and B), whereas with the highest level, Tm shifted to approximately the closed position (C). (E and F) show superposition of B and C, respectively, on the low Ca²⁺ control (D), demonstrating the smaller and larger shifts; black arrows indicate protrusion on SD1 surface, close to Tm, which we attribute to proximal region of C0C2. (G and H) Transverse sections of D and C, respectively, showing the shift of Tm from the blocked position in control (red arrows) to the closed position in C0C2-decorated filament (green arrows). (I) Superposition of G and H. Filaments in A–F oriented with the pointed end up. (Scale bar = 5 nm.)

Fig. 5.

3D reconstructions of native thin filaments decorated with C0C1f under low Ca²⁺ conditions. (A) Low Ca²⁺ control filament (gray surface rendering). (B) C0C1f-decorated filament (blue). (C) Superposition of low Ca²⁺ control (A) and C0C1f-decorated filament (B) showing no Tm shift. (Scale bar = 5 nm.)

Fig. 6.

Effect of N-terminal cMyBP-C fragments on native thin filament sliding in in vitro motility assays. The graph shows “effective activation” (thin filament velocity × fraction of filaments moving) vs. pCa. The black line shows native thin filaments demonstrating a sigmoidal response to Ca²⁺. The red line shows C0C3 activated the thin filaments at low Ca²⁺, increased Ca²⁺ sensitivity, and inhibited maximal velocity. The blue line shows C0C1f had no effect on activation or Ca²⁺ sensitivity but inhibited maximal velocity. See Fig. S8 for individual velocity and fraction-moving data.