Unique Structural and Functional Effects of Alpha-Tropomyosin Mutations in HCM and DCM

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Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are diseases of the cardiac sarcomere with complex phenotypical heterogeneity. Although the genetic basis of HCM and DCM is widely recognized, our understanding of the precise mechanisms underlying these diseases remains unclear. Cardiac contraction is regulated by the thin filament (TF) composed of actin, tropomyosin (Tm), and the troponin complex (Tn). The “gate-keeper” protein, α-Tm, is a highly conserved α-helical, coiled-coil dimer that spans actin and regulates myosin-actin interactions. Two DCM causing mutations in Tm (D84N and D230N) were compared with two HCM causing mutations (E62Q, D219N) to study how primary alterations in protein structure can cause functional deficits. We hypothesize that structural changes from point mutations propagate through Tm, and affect the regulatory function of the TF via distinct perturbations of structure and protein-protein interactions. Differential scanning calorimetry (DSC) was employed to determine mutational effects on the structure of the Tm dimer and TF. All mutants in the Tm dimer system showed an increase in thermal stability at the proximal terminus, suggesting proximity had a dominant effect on Tm stability. DSC of mutated TF revealed corresponding shifts in Tm C- and N-termini thermal stability. The second domain, however, reflected the TF interaction strength and cooperativity, consistent with phenotype-concordant trends. DCM causative-mutations exhibited increases in interaction strength, while HCM mutations resulted in a decrease. These data suggest that the structural alterations of each mutation are unique, and pathogenesis of HCM and DCM is both mutation and location specific. To characterize the resultant functional effects of these discrete changes, in-vitro motility is in progress to assess myofilament activation. Together, these data will provide a molecular level understanding of the primary deficits caused by these mutations and help elucidate the differential primary disease mechanisms.