The Mechanistic Role of Tropomyosin Overlap Dysregulation in Early Cardiomyopathic Disease Progression

Abstract

An oft-noted component of sarcomeric dilated (DCM) and hypertrophic (HCM) cardiomyopathies is that primary mutations can exhibit significant phenotypic variability. This variability extends even to families carrying the same mutation suggesting that independently segregating genetic modifiers exist that differentially affect susceptible individuals. In addition, recent clinical studies have demonstrated the importance of early stage treatment, emphasizing the mechanistic role of the primary biophysical disruption “domain” on pathogenesis. One possible site of primary biophysical disruption is the crucial tropomyosin (Tm)-overlap which provides the stability and flexibility required during cross bridge cycling. We hypothesize that the cardiac thin filament (CTF) has an innate but finite capacity to tolerate changes in these parameters, beyond which cardiomyopathies result. Highly penetrant mutations throughout the CTF were studied using differential scanning calorimetry to assess changes in thermodynamic stability and flexibility of fully reconstituted CTF's. N-terminus proximal Tm mutations (E62Q, D84N) decreased Tm-cTn stability while increasing flexibility, where C-terminus proximal Tm mutations (D219N, D230N) do the opposite. Strikingly these proximal-terminal dependent effects occurred independent of late-stage pathology. Mutations in cardiac troponin T (cTnT), however, only increased thermal stability and flexibility when directly adjacent to the overlap. Furthermore, we investigated the role of developmental cTnT isoform switching on the Tm-overlap to assess its capacity as an independent modifier. Fetal-cTnT alone had little effect, however, when expressed with D230N-Tm, filaments exhibited an additive decrease in flexibility of the Tm-overlap. Fetal-cTnT did not cause additive changes when expressed with D84N-Tm and R92L-cTnT. Similarly, mice expressing D230N-Tm and fetal-cTnT exhibited additively reduced % fractional shortening whereas R92L-cTnT did not display additive changes, suggesting fetal-cTnT may be a mutation-specific modifier. Together these data suggest that the Tm overlap represents a sensitive “domain” of early biophysical dysregulation in cardiomyopathic disease progression.