TNNT2 Restrictive and Hypertrophic Cardiomyopathy Mutations Depress the Inhibitory Properties of the Troponin-T1 Fragment, In Vitro

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Restrictive (RCM) and hypertrophic (HCM) cardiomyopathies are heterogeneous diseases of the heart, characterized by impaired relaxation and diastolic dysfunction, without/with ventricular wall thickening. The pathogenesis can be attributed to hyperdynamic contractile function of mutant sarcomeres, which is considered the most proximal effect of disease-causing mutations. ∼100 causative thin filament mutations have been identified, of which 15% occur in cardiac troponin-T (cTnT/TNNT2), the tropomyosin-binding subunit of the regulatory troponin complex. Of these, 70% are clustered in the N-terminal cTnT1 tail, which contains a highly conserved tropomyosin binding element spanning residues 112-136. At low Ca2+ concentrations, troponin constrains tropomyosin to the blocked/B-state, occluding acto-myosin interactions. cTnT1 contributes to B-state formation and, thus, contractile inhibition. Here, we investigated three charge-altering mutations in invariant cTnT1 residues: E136K (RCM-causing), K124N, R130C (HCM-causing). We hypothesize that these mutations weaken cTnT1's inherent inhibitory properties via altered cTnT1-tropomyosin binding, leading to destabilization of the blocked/B-state and excessive acto-myosin cycling. Human cTnT1 was cloned, mutagenized, expressed, purified, and reconstituted with vertebrate F-actin-tropomyosin to determine the effects of the mutations on in vitro motility. We verified the inhibitory effect of WT-TnT1 on F-actin-tropomyosin sliding speed. All 3 mutant-cTnT1-actin-tropomyosin filaments showed significantly higher sliding speeds versus control: E136K (1.15±0.22 vs. 1.00±0.24μm/s), K124N (1.21±0.21 vs. 1.00±0.18μm/s) and R130C (1.63±0.51 vs. 1.00±0.18μm/s). We also examined sliding speeds as a function of myosin concentration, and observed differences between WT and mutant filaments to be amplified at 50-75μg/ml. Additionally, there was a 6-12% increase in the number of moving versus non-moving mutant filaments. These data suggest a destabilized cTnT1-mediated B-state, which could contribute to the cardiac hypercontractility and, importantly, impaired relaxation observed in patients. We are currently investigating the effects of these mutations on cTnT1-tropomyosin binding affinities.