Cardiomyopathy-Linked Mutation K15N in Tropomyosin Alters Calcium-Dependent Regulation of Reconstituted Cardiac Thin Filaments

Abstract

In striated muscle, actin-based thin filaments and myosin-based thick filaments are organized into basic contractile units called sarcomeres. Tropomyosin (Tpm), a coiled-coil dimer, polymerizes head-to-tail along the thin filament, stabilizes the filament and, together with the troponin complex, regulates Ca2+-dependent actomyosin interactions which are molecular prerequisites for muscle contraction. Troponin, consisting of troponin I (TnI), troponin C (TnC), and troponin T (TnT), initiates muscle contraction through a series of structural changes within the complex, which is triggered by the binding of Ca2+ to TnC. The structural changes spread through TnT which in turn move Tpm's position on the thin filament, enabling actomyosin interactions. In this work, we studied two missense mutations in striated muscle tropomyosin (Tpm1.1), K15N and R21H, which are associated with dilated cardiomyopathy and hypertrophic cardiomyopathy, respectively. We investigated the mutations’ effects on Tpm1.1's affinity to F-actin and on the Ca2+-dependent regulation of reconstituted cardiac thin filaments using Fluorescence Resonance Energy Transfer (FRET). We labeled residues 151 and 167 of cardiac TnI with IAEDANS as FRET donors, and residue 89 of cardiac TnC with DDPM as FRET acceptor. We found that while both mutations reduced Tpm1.1's affinity to F-actin, the K15N mutation decreased Ca2+-sensitivity and Ca2+-dissociation-induced kinetics of reconstituted cardiac thin filaments. In the presence of the K15N mutant, the interprobe distances were greater than in the presence of wild type Tpm1.1, indicating that the mutation affected cation-induced conformational changes within troponin. Our data demonstrated that the K15N mutation in Tpm1.1 affected Ca2+-dependent thin filament regulation while the R21H mutation had almost no effect.