TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction

Abstract

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca²⁺ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

Keywords: Drosophila; cardiomyopathy; diastolic dysfunction; tropomyosin; troponin T.

Fig. 1.

In vivo effects of transgenic overexpression of mutant TnTs in the Drosophila heart. (A) Fluorescent micrograph (40×) of a tdtK-expressing heart tube imaged through the cuticle of a live animal. O2 and O3 denote ostia 2 and 3, which served as positional markers for consistent measurements of chamber areas from multiple flies. Dotted rectangular box depicts the region imaged in B. (Scale bar, 50 µm.) (B) Confocal micrographs of a dissected Hand > TnT^WT.His fly heart, taken at 100× magnification, show colocalization of actin and transgenic His-TnT along cardiac thin filaments. (Scale bar, 5 μm.) (C) In vivo analysis of beating hearts from 3-wk-old Hand > TnT flies revealed significantly reduced diastolic chamber areas in all mutants, and reduced systolic chamber areas in the R81C and E87K mutants, relative to control. Data are presented as mean ± SEM (n = 26; *P ≤ 0.05; ***P ≤ 0.001). (D) M-mode kymograms generated from high-speed videos of beating hearts overexpressing WT or mutant TnT and the fluorescent reporter, tdtK. Vertical red lines terminate at opposing edges of the heart wall, with the left line indicating diastolic and the right line indicating the systolic diameter.

Fig. 2.

In situ effects of transgenic overexpression of mutant TnTs on Drosophila cardiac function. (A) In situ analysis of 3-wk-old Hand > TnT hearts resolved cardiac restriction, decreased cardiac outputs, and reduced rates of relaxation in mutants relative to control. Data are presented as mean ± SEM (n = 24 to 36; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (B) M-mode kymograms generated from high-speed videos of beating hearts that overexpressed WT or mutant TnT. Vertical red lines terminate at opposing edges of the heart wall, with the left line indicating diastolic and the right line indicating systolic diameter.

Fig. 3.

Ca²⁺-dependent and Ca²⁺-independent processes contribute to impaired myocardial relaxation in mutant flies. (A) Small-molecule compounds elicited significant, incremental increases in the diameters of 3-wk-old fly hearts. Incubation with EGTA/EGTA,AM-containing AHL chelated extracellular and intracellular Ca²⁺, halted contraction, and prompted increases in heart tube diameters from baseline (i.e., diastole). Subsequent incubation with blebbistatin inhibited acto-myosin attachments, leading to further increases in cardiac diameters. (B) Ca²⁺ chelation prompted a significantly greater increase in the diameter of hearts overexpressing the TnT variants vs. WT. (C) Blebbistatin treatment caused an exaggerated response across the wall of all mutant hearts, relative to control. Data are presented as mean ± SEM (n = 20; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig. 4.

Effects of the hcTnT RCM mutation on IFM myofibrillar properties. (A) Fluorescence (10×) and confocal micrographs (63×, 100×) show no gross structural differences in IFMs (Top) or myofibrils (Middle and Bottom) between control and up^E87K Drosophila. Top (10×) depicts hemithoraces that were flash-frozen and sagittally bisected to reveal IFM fiber morphology. Preparations were stained with Alexa-568 phalloidin to label actin. Z discs were distinguished by the expression of GFP-tagged Zasp52, a protein that binds to α-actinin, thereby restricting GFP fluorescence to Z discs. Dotted rectangular boxes highlight the regions imaged below. Sarcomeres were visualized along myofibrils of the hemithoraces, in situ, by confocal microscopy (Middle, 63×). Myofibrils were also isolated for ex vivo imaging (Bottom, 100×). (Scale bars, 5 μm.) (B) up^E87K sarcomeres, measured from hemithoraces (in situ) or along isolated myofibrils (ex vivo), were significantly shorter than control (n = 220 to 240). Hence, myofibril removal and isolation from IFMs did not affect mutant sarcomere length. (C) Representative image of an IFM myofibril held between a glass microtool and cantilevered force probe for mechanical assessment. (D) up^E87K IFM myofibrils exhibited significantly higher resting tension relative to control, which was restored to WT values postincubation with BDM. Control myofibrils showed no difference in stiffness pre- and post-BDM incubation (n = 7 to 16). Data are presented as mean ± SEM (ns > 0.05; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig. 5.

Effects of the hcTnT RCM mutation on rat cardiac myofibrillar properties. (A) Myofibrils isolated from rat ventricles were incubated with myc-cTnT^WT− or myc-cTnT^E138K−containing Tn to replace endogenous Tn complexes. Western blotting was performed with an anti-TnT antibody to quantify the extent of Tn exchange. The first two lanes of the blot show mobility differences between purified recombinant rat myc-cTnT and endogenous rat cTnT, followed by rat myofibrillar cTnT composition after exchange with either myc-cTnT^WT− or myc-cTnT^E138K−containing Tn. (B) Tn exchange was quantified by dividing the signal intensity of the myc-tagged protein by the total intensity of cTnT (myc-cTnT + cTnT), which confirmed a similar extent of exchange for both myc-cTnT^WT− and myc-cTnT^E138K−containing Tn, within myofibrils (n = 4 biological replicates, 9 or 10 technical replicates each). (C) No difference was observed in sarcomere lengths along myofibrils exchanged with either myc-cTnT^WT− or myc-cTnT^E138K−containing Tn. (D) Analysis of mechanical properties of rat ventricular myofibrils exchanged with myc-cTnT^WT− vs. myc-cTnT^E138K−containing Tn revealed no difference in maximum tension generated. Significant differences were observed in myofibrillar resting tension and in the duration of the linear phase of relaxation (t_REL,LIN). However, the rate constant of this phase (k_REL,LIN) was not affected (n = 4; six to eight myofibrils/replicate). Data are presented as mean ± SEM (*P ≤ 0.05).

Fig. 6.

In vitro effects of mutant hcTNT1 peptides on F-actin−Tpm sliding velocity. (A) IVM assays were performed over a range of myosin concentrations. The addition of Tpm to F-actin (dark gray line) reduced sliding velocities relative to F-actin alone (light gray line), at all myosin concentrations. This inhibitory effect was more pronounced upon addition of Tpm−hcTNT1^WT (black line). Myosin concentration and the filament type (i.e., F-actin−Tpm ± hcTNT1^WT) had significant effects on F-actin sliding velocity as determined by two-way ANOVA. (B–D) Filaments containing the three mutant hcTNT1 peptides (blue lines) showed higher sliding velocities relative to internal F-actin−Tpm−hcTNT1^WT controls (black lines), at all myosin concentrations tested. Two-way ANOVA confirmed significant effects of filament type (WT vs. mutant hcTNT1-containing filaments) as well as myosin concentration, on F-actin−Tpm sliding velocity. Note, motility of the internal F-actin−Tpm−hcTNT1^WT controls, from each experiment, did not significantly differ (SI Appendix, Fig. S8). Data are presented as mean ± SEM (n = 2 to 7 replicates; 20 to 30 filaments/replicate).