Effect of hypertrophic cardiomyopathy mutations in human cardiac muscle alpha-tropomyosin (Asp175Asn and Glu180Gly) on the regulatory properties of human cardiac troponin determined by in vitro motility assay

The properties of mutant contractile proteins that cause hypertrophic cardi omyopathy (HCM) have been investigated in expression studies and in mouse m odels. There is growing evidence that the precise isoforms of both the muta ted protein and its interacting partners can qualitatively influence the ef fects 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 w ith an N-terminal alanine-serine extension (AStm) to mimic acetylation in v ivo, 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 vel ocity, between wildtype and mutant filaments. However, we observed increase d Ca2+ sensitivity of fraction of filaments motile using the mutant tropomy osin compared to wild-type (Delta EC50 + 0.082 +/- 0.019 pCa units for Asp1 75Asn and + 0.115 +/- 0.021 for Glu180Gly). Indirect measurements using imm obilized alpha-actinin to retard filament movement showed that filaments re constituted with mutant AStm produced the same force as wild-type filaments . The results using human cardiac regulatory proteins reveal different effe cts of the HCM mutations in tropomyosin compared to studies using heterolog ous 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 functio nal changes when interacting with cardiac troponin. (C) 2000 Academic Press .