Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants

FIGURE 1. (A,B) Echocardiography images and (C) electrocardiogram. Still images obtained from echocardiogram of the proband at ages 2 weeks (A) and 12 months (B). Note particularly the progressive dilation and prominent rounding of the left ventricle (LV) in the later image. (C) Electrocardiogram at 19 months of age demonstrating sinus rhythm and left ventricular enlargement.

FIGURE 2. (A) Pedigree and (B) Sanger sequencing. (A) Family pedigree showing the proband and similarly affected brother (black shading). There was a prior loss of a severely premature live birth and three prior failed IVF pregnancies. (B) Sanger sequencing data from the four available family members. Red arrows indicate residue of interest. The left side shows residues around the c.394G>A change that results in D132N in one of the two alleles for TNNC1 for three of four individuals (mother is reference, other three are heterozygous for the variant). The right side shows residues around the c.435C>A change that results in D145E in one of the two alleles for TNNC1 for three of four individuals (father is reference, other three are heterozygous for the variant).

FIGURE 3. (A–D) Sequence and structure analyses of cTnC surrounding divalent cation-binding site IV. (A) Primary sequence of human cTnC (NP_003271.1). Divalent cation-coordinating regions (sites II, III, and IV) are highlighted in bold, and amino acids that comprise α-helical regions (N-helix and α-helices A–H) are underlined [annotated according to Li and Hwang (2015)]. Site II [EF-hand comprised of a-helices C–D and joining residues] is in the N-domain of cTnC and is the regulatory “trigger” site that activates contraction when Ca2+ binds during the systolic Ca2+ transient. Sites III and IV (EF-hands comprised of α-helices E–F or G–H, respectively, along with the respective residues that join each pair of α-helices) are in the C-domain and can bind Mg2+ or Ca2+. Note that all 161 amino acids of the primary sequence are intentionally shown as a single row to illustrate the relative sizes and locations of annotated regions. (B,C) Ribbon structures of the Ca2+-saturated C-domain of human cTnC in complex with a cardiac troponin I peptide (cTnI128–147), illustrating the locations of D132 and D145 (red arrows). NMR structure adapted from PDB 1OZS model 1 (Lindhout and Sykes, 2003) using MATLAB…

FIGURE 4. (A,B) Brightfield microscopy and (C,D) Optical Coherence Tomography (OCT) imaging of Control and TNNC1 F0 knockout tadpoles. As compared to control tadpoles (A), TNNC1 knockout tadpoles (B) show normal gross morphology, but significant total body edema (white arrows) secondary to severe dilated cardiomyopathy leading to congestive heart failure. Red arrows indicate plane of OCT images in (C,D). OCT images demonstrate normal size and wall thickness of cardiac chambers in control tadpole (C), with severe dilation and enlargement of the ventricle in TNNC1 knockout tadpole (D), indicated with white arrowheads. V = ventricle, LA = left atrium, RA = right atrium.

FIGURE 5. Ca2+ dependence of steady-state isometric tension in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) Relative steady-state isometric force as a function of pCa. The force values were normalized to the maximal steady-state isometric force in the same preparation. (B) Normalized steady-state isometric force as a function of pCa. The force values were normalized to the maximal steady-state isometric force generated by WT. Data are shown as mean ± S.E. and best fit parameter estimates from non-linear least squares regression on the Hill equation are summarized in Table 2 (n = 4–8).

FIGURE 6. Sinusoidal stiffness analysis in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) Ca2+ dependence of steady-state sinusoidal stiffness. (B) Normalized force vs. steady-state sinusoidal stiffness. The force values were normalized to the maximal steady-state isometric force generated by WT. (B) Dashed lines were drawn to connect the points. Data are shown as mean ± S.E. and maximum SS values are summarized in Table 3.

FIGURE 7. Rate of tension redevelopment (kTR) analysis in porcine CMPs reconstituted with exogenous cTnC WT (100% WT) or variants (50% WT/50% D132N, 50% WT/50% D145E, or 50% D132N/50% D145E). (A) kTR as a function of increasing Ca2+ concentration. Solid lines were drawn to connect the points. (B) Relative force vs. kTR. The force values were normalized to the maximal steady-state isometric force for each preparation. Data are shown as mean ± S.E. and maximum kTR values are summarized in Table 3. Dashed lines were obtained from fits to the 3-state model, which take into account the changes in Ca2+ sensitivity (Table 2 and Figures 5A,B) as described in section “Experimental Procedures”; best fit parameter estimates are summarized in Table 4.