The properties of mutant
contractile proteins that cause
hypertrophic cardiomyopathy (HCM) have been investigated in expression studies and in mouse models. There is growing evidence that the precise
isoforms of both the mutated
protein and its interacting partners can qualitatively influence the effects of the mutation. We therefore investigated the functional effects of two HCM mutations in
alpha -tropomyosin, Asp175Asn and Glu180Gly, in the in vitro motility assay using recombinant human
alpha -tropomyosin, expressed with an N-terminal
alanine-
serine extension (AStm) to mimic acetylation in vivo, and purified native human cardiac
troponin. The expected switching off of reconstituted filament movement at pCa9, and switching on at pCa5, was observed with no difference in fraction of filaments motile or filament velocity, between wild-type and mutant filaments. However, we observed increased Ca(2+)sensitivity of fraction of filaments motile using the mutant
tropomyosin compared to wild-type (DeltaEC(50)+0.082+/-0. 019 pCa units for Asp175Asn and +0.115+/-0.021 for Glu180Gly). Indirect measurements using immobilized
alpha -actinin to retard filament movement showed that filaments reconstituted with mutant AStm produced the same force as wild-type filaments. The results using human cardiac regulatory
proteins reveal different effects of the HCM mutations in
tropomyosin compared to studies using heterologous systems. By performing parallel experiments using either human cardiac or rabbit skeletal
troponin we show that the cardiac-specific phenotype of HCM mutations in
alpha -tropomyosin is not the result of more marked functional changes when interacting with cardiac
troponin.