De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy

Figure 1. Pedigree of the family SD5. The affected family member is shown with closed and crossed-out symbols, and the unaffected parents with open symbols. Proband is marked by the black arrow. Fragments of the Sanger electropherograms of the TPM1 gene are shown near the symbol of a family member. The letters in the upper row do not designate amino acids but nucleotides (A—adenine, green peaks; C—cytosine, blue peaks; G—guanine, black peaks; and T—thymine, red peaks). The heterozygous mutation c.656A>T (p.D219V) is marked by the red arrow.

Figure 2. Temperature dependences of the excess heat capacity (Cp) monitored by DSC and deconvolution analysis of the heat sorption curves for WT Tpm (A) and Tpm with mutation D219V (B). Solid lines represent the experimental curves after the subtraction of instrumental and chemical baselines, and dotted red lines represent the individual thermal transitions (calorimetric domains) obtained from fitting the non-two-state model [25] to the data.

Figure 3. The effect of the D219V Tpm mutation on the Tpm affinity for F-actin was estimated by the co-sedimentation assay (A), and the thermal stability of the Tpm-actin complex was determined by light scattering (B).

Figure 4. The effects of the D219V Tpm mutation on the dependence of the sliding velocity of the F-actin–Tpm filament on the concentration of ventricular (A) and atrial (B) myosin in the in vitro motility assay. Each data point represents the Mean ± SD from three experiments. The data are fitted with the Hill equation. The parameters of the Hill equation are presented in Table 2.

Figure 5. The effects of the D219V Tpm mutation on the Ca2+-dependent sliding velocity of regulated thin filaments moving over ventricular (A) and atrial (B) myosin in the in vitro motility assay. Each data point represents the mean ± SD from three experiments. The data are fitted with the Hill equation, and the parameters of the pCa-velocity relationships are presented in Table 3.

Figure 6. The dependence of the maximal sliding velocity of thin filaments with WT and D219V Tpm at saturated Ca2+ concentration on LV (A) and LA (B) myosin concentrations added to the flow cell. C50 for LV myosin with WT Tpm was 30.1 ± 0.9 µg/mL and 25.9 ± 3.1 µg/mL with D219V Tpm. The C50 for LA myosin with WT Tpm was 45.3 ± 1.5 µg/mL and 47.2 ± 1.1 µg/mL with D219V Tpm.

Figure 7. Location of the D219 Tpm residues in the structure of the regulatory unit of the thin filament of cardiac muscle ([31]; PDB code 6KN7). Actin atoms are shown by space-filling (CPK) representation (cyan); the Tpm molecules are shown by red ribbons; Tn-Cs are magenta ribbons; Tn-Is are orange ribbons; and Tn-Ts are blue ribbons. An h-bond between the D219 residue of Tpm and the K326 residue of actin is shown in the inset. Neighbor E218 residue of Tpm and the K328 residue of actin are also shown in CPK.

Figure 8. The time course of the existence of h-bonds between E218 and D (or V)219 Tpm residues and K326 and K328 residues of neighboring actin monomers during a 200 ns-long MD trajectory with WT Tpm (A,C) and D219V Tpm (B,D); vertical axis: one means at least one h-bond between the residues, zero otherwise; (A–D) correspond to two different Tpm strands. The h-bond between E218 of Tpm and K326 of actin is red; the h-bond between E218 of Tpm and K328 of actin is blue; and the h-bond between D (or V)219 Tpm residue and K326 of actin is green.