Changes in the chemical and dynamic properties of cardiac troponin T cause discrete cardiomyopathies in transgenic mice

Abstract

Cardiac troponin T (cTnT) is a central component of the regulatory thin filament. Mutations in cTnT have been linked to severe forms of familial hypertrophic cardiomyopathy. A mutational "hotspot" that leads to distinct clinical phenotypes has been identified at codon 92. Although the basic functional and structural roles of cTnT in modulating contractility are relatively well understood, the mechanisms that link point mutations in cTnT to the development of this complex cardiomyopathy are unknown. To address this question, we have taken a highly interdisciplinary approach by first determining the effects of the residue 92 mutations on the molecular flexibility and stability of cTnT by means of molecular dynamics simulations. To test whether the predicted alterations in thin filament structure could lead to distinct cardiomyopathies in vivo, we developed transgenic mouse models expressing either the Arg-92-Trp or Arg-92-Leu cTnT proteins in the heart. Characterization of these models at the cellular and whole-heart levels has revealed mutation-specific early alterations in transcriptional activation that result in distinct pathways of ventricular remodeling and contractile performance. Thus, our computational and experimental results show that changes in thin filament structure caused by single amino acid substitutions lead to differences in the biophysical properties of cTnT and alter disease pathogenesis.

Fig. 1.

Structural modeling of murine cTnT and generation of residue 92 transgenic mice. Mutation-specific alterations in the secondary structure of the 101-aa sequence of mouse cTnT corresponding to amino acids 70–170. “Hinge” residues 104–108 are shown in green, and residue R92 is shown in blue. The rightmost panel shows the radius of gyration as a function of time during the simulation. Black, WT; red, R92L; green, R92W.

Fig. 2.

Generation and characterization of mutant residue 92 mice. (A) Transgenic construct. (B) Identical Western blots of whole-heart homogenates were probed with c-myc (Tg) or cTnT mAb (Tg and Endo). Arrows correspond to the specific lines used in this study. (C) Immunostaining of adult ventricular myocytes isolated from all four lines and probed with either cTnT (NT) or c-myc (WT, R92W, and R92L) mAbs. (Scale bars: C,20 μm; Insets,4 μm.) (D) Isolated myofilament lysis and discontinuity (black arrows) were found in both R92W and R92L sections (R92W >> R92L). Dilated sarcomeric reticulum was found in the R92W sections (white arrows). Histopathology: R92W hearts exhibit a more severe cardiomyopathy. (Magnification: Top, ×30,000; Middle, ×120,000.) (Scale bar, 10 μm.)

Fig. 3.

Mutation-specific alterations in ventricular mass and myocyte size. (A) Heart weight/body weight measurements. n = 9, 6, 6, and 8 hearts, respectively. (B) Isolated adult cardiac myocyte surface area. n = 71, 52, 29, and 89 cells, respectively. All values are expressed as mean ± SEM. *, P < 0.01; **, P < 0.005.

Fig. 4.

cTnT R92 mutant hearts exhibit systolic and diastolic dysfunction. (A and B) Systolic performance: LVSP and the rate of pressure development (+dP/dt) were abnormally low for hearts with either R92 mutation in response to increasing [Ca²⁺]; the extent of dysfunction for R92W hearts was greater. Both R92W and R92L hearts developed less LVSP at low [Ca²⁺]. Note that R92L hearts reached a similar LVSP as NT hearts only at high [Ca²⁺]. (C and D) Diastolic performance: Hearts with either R92 mutation showed increased end-diastolic pressure (EDP) at high perfusate [Ca²⁺]. Hearts with a mutation also had slower rates of relaxation (-dP/dt), with R92W << R92L. NT, circles; R92W, squares; R92L, triangles; n = 4, for all lines studied.

Fig. 5.

Mutation-specific temporal activation of cardiomyopathic markers and representative gross hearts. (A) Male WT, R92W (RW), R92L (RL), and NT mice were killed at 2-, 6-, and 12-month time points; representative hearts from each time point are shown immediately above the appropriate lane on the Northern blot. Probes were ANF,αSK, MCIP1, sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), phospholamban (PLN), and GAPDH. Each experiment was performed on three independent hearts. (B) Quantitation of ANF and MCIP1 transcript levels. Values are shown as ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.