Allosteric effects of cardiac troponin TNT1 mutations on actomyosin binding: A novel pathogenic mechanism for hypertrophic cardiomyopathy

Abstract

The majority of hypertrophic cardiomyopathy mutations in (cTnT) occur within the alpha-helical tropomyosin binding TNT1 domain. A highly charged region at the C-terminal end of TNT1 unwinds to create a flexible “hinge”. While this region has not been structurally resolved, it likely acts as an extended linker between the two cTnT functional domains. Mutations in this region cause phenotypically diverse and often severe forms of HCM. Mechanistic insight, however, has been limited by the lack of structural information. To overcome this limitation, we evaluated the effects of cTnT 160–163 mutations using regulated in vitro motility (R-IVM) assays and transgenic mouse models. R-IVM revealed that cTnT mutations Δ160E, E163R and E163K disrupted weak electrostatic actomyosin binding. Reducing the ionic strength or decreasing Brownian motion rescued function. This is the first observation of HCM-linked mutations in cTnT disrupting weak interactions between the thin filament and myosin. To evaluate the in vivo effects of altering weak actomyosin binding we generated transgenic mice expressing Δ160E and E163R mutant cTnT and observed severe cardiac remodeling and profound myofilament disarray. The functional changes observed in vitro may contribute to the structural impairment seen in vivo by destabilizing myofilament structure and acting as a constant pathophysiologic stress.

Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models

“…This behavior, together with the increase in resting tension, suggests that in E163R sarcomeres, the thin filament may not turn off properly upon Ca 2+ removal and may be unable to fully maintain its switched‐off state during diastole, in analogy with previously described HCM‐related tropomyosin mutations . The modest increase in myofilament Ca 2+ sensitivity in E163R myocardium is in line with previous findings and may be a consequence of the impaired thin filament blocked state that allows recruitment of some force‐generating crossbridges at low Ca 2+ . We excluded that the increase of myofilament Ca 2+ sensitivity depended on a reduction of troponin I phosphorylation at the protein kinase A site (Figure ), although we have not explored other possible posttranslational modifications of sarcomere proteins (eg, MyPBC3).…”

“…All experimental protocols were performed in agreement with current Italian and European regulations and were approved by the local institutional review board and the animal‐welfare committee of the Italian Ministry of Health. We used a total of sixty‐seven 6‐ to 8‐month‐old male C57BL/6N transgenic mice carrying the R92Q or E163R mutation in the TNNT2 gene, as well as wild‐type (WT) littermates: 22 R92Q, 24 E163R, and 21 WT mice were used for the experiments described below. The mouse colonies were housed in the animal facility of the University of Florence and all experiments were conducted locally.…”

“…The mouse colonies were housed in the animal facility of the University of Florence and all experiments were conducted locally. The 2 transgenic lines were generated in laboratories as previously described …”

The Genetic Landscape of Cardiomyopathy and Its Role in Heart Failure

“…Recently, Moore and colleagues examined the tropomyosin-binding region of cardiac troponin T, a region that harbors severe and phenotypically diverse HCM mutations (Moore et al, 2014). In vitro motility assays showed that specific mutations in cardiac troponin T disrupted weak electrostatic interactions between the thin filament and myosin.…”

“…In vitro motility assays showed that specific mutations in cardiac troponin T disrupted weak electrostatic interactions between the thin filament and myosin. Complementary in vivo data indicates that these same mutations cause cardiac remodeling and disarray of the myofiber, suggesting that the weak cross-bridge formation causes destabilization of the myofilament structure ultimately resulting in disease (Moore et al, 2014). …”