Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin

Abstract

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin–myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.

Structural and Functional Properties of Kappa Tropomyosin

“…TnT1 suppressed the sliding velocity of F-actin-Tpm filaments containing ααTpm and κκTpm but did not affect the velocity of the filaments with ακTpm (Figure 10c,d). The formation of the overlap junction between the N-and C-ends of the Tpm molecule was shown to be important for the interaction of TnT1 with tropomyosin [23][24][25][26] and the Tpm regulatory function [14,21,22,[27][28][29]. The ability of the N-and C-ends of ααTpm and κκTpm to interact was determined by measuring the viscosity of the solution.…”

“…We hypothesized that Tn can interact differently with ααTpm, κκTpm, and ακTpm. We previously found that the troponin T1 fragment (TnT1; residues 1-158 for rabbit fast skeletal troponin T, P02641) containing 1 site interaction with Tpm [19] enhances the affinity of ααTpm to F-actin [22]. TnT1 decreased the Tpm concentration at which half of the actin was saturated, K 50% , from 3 ± 0.2 µM to 1.25 ± 0.11 µM [22].…”

“…We previously found that the troponin T1 fragment (TnT1; residues 1-158 for rabbit fast skeletal troponin T, P02641) containing 1 site interaction with Tpm [19] enhances the affinity of ααTpm to F-actin [22]. TnT1 decreased the Tpm concentration at which half of the actin was saturated, K 50% , from 3 ± 0.2 µM to 1.25 ± 0.11 µM [22]. Here, we analyzed the effect of TnT1 on the affinity of κκTpm and ακTpm to F-actin.…”

“…The cardiac Tn complex was extracted from the left ventricle of the pig [43]. Human adult cardiac troponin T (TNT3, or isoform 6 of P45379) fragment TnT1 was obtained as previously described [22]. TnT1 concentration was determined spectrophotometrically at 280 nm using an E 1% of 0.71 cm −1 .…”

SNHG 12 and hsa-miR-140-5P may play an important role in the ceRNA network related to hypertrophic cardiomyopathy

“…Genetic testing panels have shown vast heterogeneity and diverse molecular pathways, with more than 2,000 identified sarcomere mutations. Some of the mutations are pathogenic, but most of them are benign ( 3 , 12 ). Furthermore, more evidence regarding the genotype–phenotype correlations is needed to enable the development of genetic tests or other laboratory tests for diagnosis and prediction.…”