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 mut...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
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2000
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| _version_ | 1797084806222184448 |
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| author | Redwood, C Lohmann, K Bing, W Esposito, G Elliott, K Abdulrazzak, H Knott, A Purcell, I Marston, S Watkins, H |
| author_facet | Redwood, C Lohmann, K Bing, W Esposito, G Elliott, K Abdulrazzak, H Knott, A Purcell, I Marston, S Watkins, H |
| author_sort | Redwood, C |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T02:00:13Z |
| format | Journal article |
| id | oxford-uuid:9d1dc14e-e1c6-4b52-9dd6-8768213e9a81 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T02:00:13Z |
| publishDate | 2000 |
| record_format | dspace |
| spelling | oxford-uuid:9d1dc14e-e1c6-4b52-9dd6-8768213e9a812022-03-27T00:40:39ZInvestigation 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.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:9d1dc14e-e1c6-4b52-9dd6-8768213e9a81EnglishSymplectic Elements at Oxford2000Redwood, CLohmann, KBing, WEsposito, GElliott, KAbdulrazzak, HKnott, APurcell, IMarston, SWatkins, HFamilial 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. |
| spellingShingle | Redwood, C Lohmann, K Bing, W Esposito, G Elliott, K Abdulrazzak, H Knott, A Purcell, I Marston, S Watkins, H 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. |
| title | 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. |
| title_full | 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. |
| title_fullStr | 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. |
| title_full_unstemmed | 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. |
| title_short | 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. |
| title_sort | 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 |
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