Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca(2+) 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 least 8 contractile protein genes, most commonly beta myosin heavy chain, myosin binding protein C, and cardiac troponin T. Affected individuals are heterozygous for a particular mutation, and most evidence suggests that the mutant protein acts in a dominant-negative fashion. To investigate the functional 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 reconstitute human cardiac troponin. Significant differences were found between the regulatory properties of wild-type and mutant troponin in vitro, as follows. (1) In actin-tropomyosin-activated myosin ATPase assays at pCa 9, wild-type troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition. (2) Similarly, in the in vitro motility assay, mutant troponin failed to decrease both the proportion of actin-tropomyosin filaments 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 velocity of actin-tropomyosin filaments than wild-type troponin (12.3%). These data suggest that the truncated troponin T prevents switching off of the thin filament at low Ca(2+). However, the study of thin filaments containing varying ratios of wild-type and mutant troponin T at low Ca(2+) indicated an opposite 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%). These multiple effects need to be taken into account to explain the physiological consequences of this mutation in HCM. Further, these findings underscore the importance of studying mixed mutant:wild-type preparations to faithfully model this autosomal-dominant disease.
Redwood
C
C
Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK. credwood@molbiol.ox.ac.uk
Lohmann
K
K
Bing
W
W
Esposito
G M
GM
Elliott
K
K
Abdulrazzak
H
H
Knott
A
A
Purcell
I
I
Marston
S
S
Watkins
H
H
D000199:Actins / Q000502:physiology; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics*; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000502:physiology; D014336:Troponin / Q000235:genetics / Q000502:physiology; D020107:Troponin T / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D020107:Troponin T; D009218:Myosins; D002118:Calcium