Larry S. Tobacman, Cheryl Landis, David Lin, Carol A. Butters
The Univ. of Iowa College of Medicine, Iowa City IA 52242 , and Earl Homsher and Alyona Bobkova, UCLA School of Medicine, Los Angeles CA 90024.
In many kindreds with familial hypertrophic cardiomyopathy (FHC), the disease is caused by mutations in cardiac troponin T (TnT). To study these molecules, bovine cardiac TnT cDNAs were isolated from a bovine heart library, assembled into a pET vector for bacterial expression, and PCR-based mutagenesis was performed to create cDNAs corresponding to the defects in affected human kindreds. The expressed and purified TnTs were reconstituted with bovine cardiac TnI and TnC, and then compared to cardiac muscle whole troponin, troponin reconstituted with wt TnT, and to a previously described FHC-causing Ile to Asn mutant created within rat cardiac TnT cDNA. Major defects in troponin function were produced by the bovine TnT equivalent of a human TnT splice site mutation deleting 30 COOH-terminal residues. Troponin complex formation was dependent upon the presence of 0.1 mM CaCl2 for this truncation mutant, whereas it occurred without added Ca2+ for wt TnT. Troponin containing the TnT truncation inhibited the myosin S1-thin filament MgATPase rate, regardless whether Ca2+ was present, and the troponin-tropomyosin complex had diminished affinity for actin. In contrast to these results, two other FHC-causing mutations had relatively subtle effects on thin filament properties, and showed evidence of abnormal stimulation rather than abnormal inhibition. The bovine homologue of an Glu-160 point deletion in human cardiac TnT had no effect on troponin assembly, thin filament assembly, or the extent of Ca2+-sensitive MgATPase rate regulation. However the Kapp for MgATPase activation was increased by 50%, suggesting tighter Ca2+ binding. A homologue of an Ile79Asn point mutation had a third pattern of altered function, different from either of the other mutations. Apparent Ca2+ binding was normal, as was thin filament assembly, and regulation. However, in vitro motility experiments demonstrated 50-60% faster sliding speed than normal, and also suggested a small decrease in force in the presence of this TnT mutant. The sliding of filaments containing this mutant was stopped by lower concentrations of drag-force-inducing pPDM HMM than the concentrations required to stop movement of filaments containing wt TnT. We conclude that different FHC-causing TnT mutations produce effects that are unexpected, diverse in character, and diverse in the severity of dysfunction.