Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy - Ca2+ regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein

Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in at lea st 8 contractile protein genes, most commonly beta myosin heavy chain, myos in binding protein C, and cardiac troponin T. Affected individuals are hete rozygous for a particular mutation, and most evidence suggests that the mut ant protein acts in a dominant-negative fashion. To investigate the functio nal properties of a truncated troponin T shown to cause HCM, both wild-type and mutant human cardiac troponin T were overexpressed in Escherichia coli , purified, and combined with human cardiac troponins I and C to reconstitu te human cardiac troponin. Significant differences were found between the r egulatory properties of wild-type and mutant troponin in vitro, as follows. (1) In actin-tropomyosin-activated myosin ATPase assays at pCa 9, wild-typ e troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition. (2) Similarly, in the in vitro motility assay, muta nt troponin failed to decrease both the proportion of actin-tropomyosin fil aments motile and the velocity of motile filaments at pCa 9. (3) At pCa 5, the addition of mutant complex caused a greater increase (21.7%) in velocit y of actin-tropomyosin filaments than wild-type troponin (12.3%). These dat a suggest that the truncated troponin T prevents switching off of the thin filament st low Ca2+. However, the study of thin filaments containing varyi ng ratios of wild-type and mutant troponin T at low Ca2+ indicated an oppos ite effect of mutant troponin, causing enhancement of the inhibitory effect of wild-type complex, when it is present in a low ratio (10% to 50%). Thes e multiple effects need to be taken into account to explain the physiologic al consequences of this mutation in HCM. Further, these findings underscore the importance of studying mixed mutant wild-type preparations to faithful ly model this autosomal-dominant disease.