Variant R94C in TNNT2 ‐Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction

Figures

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  Figure 1. Clinical evaluation of TNNT2‐R94C–positive kindred. (A) Representative ECG obtained from the proband demonstrating right and left atrial enlargement with nonspecific ST and T‐wave abnormalities and ST depression in the inferolateral leads. An enlarged representative ECG is shown. (B) Representative echocardiographic images in the parasternal long axis and apical 4‐chamber windows demonstrating severe left atrial enlargement. (C) Cardiac telemetry demonstrating ventricular tachycardia overtaking sinus rhythm. (D) Representative echocardiographic images from the father of the proband in the parasternal short‐axis view demonstrating normal LV size and systolic function. (E) Representative cardiac MRI images demonstrating right and left atrial enlargement with normal biventricular size and function. LV indicates left ventricular; MRI, magnetic resonance imaging; TNNT2, TNNT2‐encoded cardiac troponin T.

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  Figure 2. R94C localizes to a highly conserved region of TNNT2. (A) Sanger sequencing chromatograms of heterozygous mutant TNNT2‐R94C (obtained from subject III‐3) and wild‐type genotypes. (B) The topological map of TNNT2 with primary sequence alignment from multiple divergent species is shown. The variant localizes to the N‐terminal segment of TNNT2 in the tropomyosin binding domain I. This region is highly conserved across species. AH indicates actin helix; BD, binding domain; TnC, troponin C; TnI, troponin I; TNNT2, TNNT2‐encoded cardiac troponin T; WT, wild type.

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  Figure 3. Pedigree of TNNT‐R94C kindred. Four generations were evaluated (denoted by roman numerals). Clinical presentations are noted in red. The predominant phenotype is an RCM with mild hypertrophic features. Arrow denotes the proband (IV.4). Circles denote female, and squares denote male. Black fill denotes diagnosis of cardiomyopathy. Gray fill denotes fetal demise (IUFD). White fill denotes no cardiac diagnosis either following clinical evaluation or by reported history. +/− denotes heterozygosity for TNNT2‐R94C variant whereas −/− denotes wild type. Diagonal line denotes deceased. HCM indicates hypertrophic cardiomyopathy; IUFD, intrauterine fetal demise; LVH, left ventricular hypertrophy; RCM, restrictive cardiomyopathy; SCD, sudden cardiac death; TNNT2, TNNT2‐encoded cardiac troponin T; VT, ventricular tachycardia.

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  Figure 4. Measurement of the biochemical steps involved in thin filament activation. (A) Kinetic scheme for thin filament activation. Tropomyosin lies along the thin filament in 3 positions: blocked (red), closed (yellow), and open (green). Positioning of tropomyosin depends both on calcium and myosin binding. Myosin binds weakly to the thin filament before undergoing an isomerization to the strong‐binding state, where force is generated. (B) Measurement of KB, the equilibrium constant between the blocked and closed states, was measured by rapidly mixing myosin with pyrene‐labeled thin filaments at low (pCa 9) and high calcium (pCa 4). Myosin binding quenches fluorescence. The ratio of the rates of binding at high and low calcium can be used to calculate KB (see Methods). There is significantly less blocking in the R94C mutant than the WT at low calcium, as evidenced by the faster binding of the mutant at low calcium. (C and D) Equilibrium titrations of myosin with pyrene‐labeled regulated thin filaments enable the calculation of several equilibrium constants (see Methods). Experiments were conducted with thin filaments containing either (C) TNNT2WT or (D) TNNT2R94C. TNNT2 indicates…

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  Figure 5. (A) Simplified kinetic scheme for thin filament activation. (B) Equilibrium constants measured for the thin filaments containing TNNT2WT and TNNT2R94C. The mutant causes statistically significant increases in KB and K W. (C) Computational modeling of the effects of the variant on the force‐calcium relationship. The variant causes a leftward shift in the curve, signifying hypercontractility at lower calcium and a basal level of tension, even at resting calcium concentrations. K B indicates the equilibrium constant between the blocked and closed states; K W, the equilibrium constant between the closed and open states; TNNT2, TNNT2‐encoded cardiac troponin T; WT, wild type.