The structural basis of alpha-tropomyosin linked (Asp230Asn) familial dilated cardiomyopathy

Abstract

Recently, linkage analysis of two large unrelated multigenerational families identified a novel dilated cardiomyopathy (DCM)-linked mutation in the gene coding for alpha-tropomyosin (TPM1) resulting in the substitution of an aspartic acid for an asparagine (at residue 230). To determine how a single amino acid mutation in α-tropomyosin (Tm) can lead to a highly penetrant DCM we generated a novel transgenic mouse model carrying the D230N mutation. The resultant mouse model strongly phenocopied the early onset of cardiomyopathic remodeling observed in patients as significant systolic dysfunction was observed by 2months of age. To determine the precise cellular mechanism(s) leading to the observed cardiac pathology we examined the effect of the mutation on Ca²⁺ handling in isolated myocytes and myofilament activation in vitro. D230N-Tm filaments exhibited a reduced Ca²⁺ sensitivity of sliding velocity. This decrease in sensitivity was coupled to increase in the peak amplitude of Ca²⁺ transients. While significant, and consistent with other DCMs, these measurements are comprised of complex inputs and did not provide sufficient experimental resolution. We then assessed the primary structural effects of D230N-Tm. Measurements of the thermal unfolding of D230N-Tm vs WT-Tm revealed an increase in stability primarily affecting the C-terminus of the Tm coiled-coil. We conclude that the D230N-Tm mutation induces a decrease in flexibility of the C-terminus via propagation through the helical structure of the protein, thus decreasing the flexibility of the Tm overlap and impairing its ability to regulate contraction. Understanding this unique structural mechanism could provide novel targets for eventual therapeutic interventions in patients with Tm-linked cardiomyopathies.

Keywords: Cardiomyopathy; Dilated; Mice; Mutation; Transgenic; Tropomyosin.

Figure 1

Atomistic Model of the Cardiac Thin Filament. (A) Average structure taken over 10 ns in a molecular dynamic simulation, (Williams et al. 2016). Grey – filamentous actin (F-actin), Green/Orange – adjacent Tm dimers, Yellow – cardiac Troponin T (cTnT), Red – cardiac Troponin C (cTnC), Blue – cardiac Troponin I (cTnI). Red balls indicate the position of the D230N-Tm mutation. (B) Representative helical wheel of two interacting Tm monomers, monomer one contains a seven heptad repeat (positions A–G) and monomer 2 contains the same repeat (positions A’–G’). Residues in positions A and D are typically hydrophobic (dashed line) and stabilize the core of the dimer. Residues in positions E and G typically salt bridges (dotted line) to further stabilize the dimer. Residues B, C, and F are solvent exposed and can interact with neighboring proteins. The helical position of D230N is marked in red on both monomers.

Figure 2

Cardiac Morphology of hearts taken from 6 month old Non-Tg (NT), D230N-low, and D230N-high hearts. (A) Top - whole hearts, Middle - transverse section, Bottom - sagittal section. Line represents 5 mm. (B–C) Heart weight/body weight (HW/BW) measurements of 2 and 6 month old, Non-tg, D230N-low, and D230N-high mice. Values are expressed as mean ± S.E.M. One-way ANOVA and Newman Keuls post hoc analysis was used for statistical comparison to Non-tg. * p < 0.05, ** p < 0.01, *** p < 0.001, NS not significant.

Figure 3

Left Ventricular Histology. (A–C) Hematoxylin and eosin stain of cardiac left ventricular fibers under polarized light. (A) 6 month old Non-tg, (B) D230N-low, and (C) D230N-high mice. Line represents 25 µm. (D–F) Masson’s trichrome stain of sections from cardiac left ventricles. (D) Non-tg, (E) D230N-low, and (F) D230N-high mice. Line represents 50 µm.

Figure 4

Baseline and Occlusion Pressure-Volume Loops for Non-tg (Blue) and D230N-Tm (Red) mice. (A–B) male, (C–D) female aged 3 months. N = 3–4 per group. Values are summarized in Table 2.

Figure 5

Myocellular mechanics and Intracellular Calcium Transients from field stimulated ventricular myocytes (A) Representative contraction/relaxation recordings from 4–6 month old male Non-Tg and D230N-high transgenic myocytes. (B–C) Cardiac myocyte mechanical measurements. (D) Representative calcium transient recordings from 4–6 month old Non-Tg and D230N-high transgenic myocytes. (E–F) Cardiac myocyte calcium transient measurements. (G) Baseline sarcomere length and (H) peak calcium transient amplitude of D230N-Tm myocytes vs. WT-Tm myocytes. An n = 4–5 animals were used for each group with at least 30 cells analyzed. Values expressed as mean ± S.E.M. Two-way ANOVA was used for statistical analysis with a Bonferonni post test. ** p < 0.01 in comparison to NT, # p < 0.05 baseline versus isoproterenol (ISO).

Figure 6

Regulated-In Vitro Motility assays of filaments containing WT (blue) or D230N-Tm (red) at varying calcium concentrations showing the (top) normalized velocity of filament sliding as a function of pCa (bottom) percent uniformly motile filaments. V_max values are reported as means of the filament sliding velocity at pCa 5. The EC₅₀ and slope values were obtained from normalized fits of the Hill equation to mean filament speed at each pCa; 150 filaments per condition were analyzed. Values are expressed as mean ± S.E.M and summarized in Table 3. A student’s t test was used for statistical comparison. *** p < 0.001 relative to WT filaments.

Figure 7

Thermal Stability and Structure of human WT and D230N-Tm assessed via CD and DSC. (A) The mean residue elipticity of 0.3 mg/mL WT (blue) and D230N (red) at 222 nm is graphed as a function of temperature. Inset: Spectra from 200 to 260 nm of WT and D230N-Tm at 20°C. n=3, each an average of 3–5 scans. Extra sum of squares F test and least squares fit analysis were used for statistical comparison of WT to D230N-Tm. (B) Thermal Stability of WT and D230N-Tm assessed via DSC. The heat capacity (kJ/mol*K) of 1.8 mg/mL WT (blue) and D230N (red) Tm is graphed as a function of temperature. Solid lines represent experimental fit after subtraction of baseline and instrumental background. The heating rate was 1°C/min from 20–70°C. Reported values were determined from the fit of the two curves (Van’t Hoff two-state model), one-way ANOVA was used to determine statistical significance. Values in Table 4 are reported as mean ± S.E.M **p < 0.01, ****p < 0.0001 compared to WT-Tm.