The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q

Abstract

Familial hypertrophic cardiomyopathy (FHC) is characterized by severe abnormal cardiac muscle growth. The traditional view of disease progression in FHC is that an increase in the Ca(2+)-sensitivity of cardiac muscle contraction ultimately leads to pathogenic myocardial remodeling, though recent studies suggest this may be an oversimplification. For example, FHC may be developed through altered signaling that prevents downstream regulation of contraction. The mutation L29Q, found in the Ca(2+)-binding regulatory protein in heart muscle, cardiac troponin C (cTnC), has been linked to cardiac hypertrophy. However, reports on the functional effects of this mutation are conflicting, and our goal was to combine in vitro and in situ structural and functional data to elucidate its mechanism of action. We used nuclear magnetic resonance and circular dichroism to solve the structure and characterize the backbone dynamics and stability of the regulatory domain of cTnC with the L29Q mutation. The overall structure and dynamics of cTnC were unperturbed, although a slight rearrangement of site 1, an increase in backbone flexibility, and a small decrease in protein stability were observed. The structure and function of cTnC was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. L29Q reduced the cooperativity of the Ca(2+)-dependent structural change in cTnC in trabeculae under basal conditions and abolished the effect of force-generating myosin cross-bridges on this structural change. These effects could contribute to the pathogenesis of this mutation.

Keywords: Cardiac; Fluorescence spectroscopy; Hypertrophic cardiomyopathy; L29Q; NMR spectroscopy; Troponin C.

Graphical abstract

Fig. 1

Chemical shift perturbations caused by L29Q in cNTnC. (A) An overlay of the ¹H,¹⁵N HSQC spectra of cNTnC (dark blue) and cNTnC(L29Q) (light blue). (B) The chemical shift differences (Δδ) between cNTnC and cNTnC(L29Q) as a function of sequence (and secondary structure). Δδ was calculated using the formula: Δδ = ((Δδ_H)² + (0.2* Δδ_H)²)^1/2. (C and D) Residues that underwent Δδ larger than the mean are shown as spheres (Q29 is shown in red) on the cartoon representation of cNTnC(L29Q). (D) A 180° rotation about the y-axis of figure (C).

Fig. 2

Structure of cNTnC(L29Q). A. The structure of cNTnC(L29Q) is shown in cartoon representation (Ca^2 + ion bound in site II as a black sphere) with the D helix pointed out of the page. B. The structure rotated by 90°. C. A close-up of the AB interhelical interface. Several of the key residues that make of this interface are shown in stick format (residues A23, F27, V44, and L48). D. NOE evidence for the closed conformation of cNTnC(L29Q). A slice from the ¹³C-HSQCNOESY spectrum highlighting the NOEs made by the methyl of A23.

Fig. 3

Comparison of the structure of cNTnC(L29Q) with cNTnC and ScNTnC. A. The structure of cNTnC (PDB: 2CTN, gray) and ScNTnC (PDB: 1R2U, orange) were aligned by their secondary structural elements (residues 5–10,15–27, 35–37, 40–48, 54–64, 71–73, and 74–86) to cNTnC(L29Q) (slate). B. Sequence of site 1 for cNTnC (gray), ScNTnC (orange), and cNTnC(L29Q) (slate). C. The overlay of the average structures of cNTnC(L29Q) with cNTnC (left) and ScNTnC (right) (helices are represented as cylinders). The C_α of residue 29 is shown as a sphere (radius set to 0.5 Å). The structures were aligned to the backbone of residues 15–27 and 41–48 and the RMSD of the C_α in site 1 (residues 28–40) was determined to be 3.20 Å (cNTnC) and 1.77 Å (ScNTnC). All structures are shown in cartoon representation and Ca^2 + ions are depicted as black spheres.

Fig. 4

A comparison of T₁, T₂, NOE, and S² for cNTnC(L29Q) (open symbols) and cNTnC (closed symbols). Relaxation data were collected at a magnetic field strength of 11.7 Tesla (¹H larmor frequency of 500 MHz). Error bars denote SD.

Fig. 5

A comparison of T₁, T₂, NOE, and S² for residues in sites 1 and 2 of cNTnC(L29Q) (open symbols) and cNTnC (closed symbols). Relaxation data were collected at a magnetic field strength of 11.7 Tesla (¹H larmor frequency of 500 MHz). Residues 37, 38, 39, and 40 were fit with the S²-τ_m-R_ex model. The S² for residue 29 is highlighted by a box. Error bars denote SD.

Fig. 6

Ca^2 +-dependence of < P₂ > (open symbols) and force (filled circles) in trabeculae containing A. BR-cTnC_C, B. cTnC(L29Q)_C, C. BR-cTnC_E and D. BR-cTnC(L29Q)_E. Normal trabeculae activation (blue circles) and activation with force inhibition by 25 μM blebbistatin (red squares). Dashed lines are fits of Hill equation to < P₂ > and force, respectively. Error bars denotes SEM for n = 4–6 trabeculae.

Fig. 7

Model of the N-terminus of cTnI interacting with cNTnC and cNTnC(L29Q). The structures of A. cNTnC (gray) and B. cNTnC(L29Q) (slate) were aligned to cNTnC in a model of the thin filament that contained the N-terminus of cTnI (red) bound to cNTnC. The C_α of residue 29 is shown as a sphere. All structures are shown in cartoon representation and Ca^2 + ions are depicted as black spheres.