Functional analysis of human cardiac troponin by the in vitro motility assay: comparison of adult, foetal and failing hearts.
OBJECTIVE
Human cardiac development and heart failure are associated with altered troponin isoform expression and phosphorylation. As the functional effects of these changes in troponin are unknown, we isolated troponin from human foetal, normal adult and failing adult hearts and investigated their regulatory function.


METHODS
Human cardiac troponin was assayed for regulatory function by in vitro motility assay and for protein content by SDS PAGE and immunoblotting.


RESULTS
Human cardiac troponin regulated movement of actin-tropomyosin filaments over a bed of immobilised heavy meromyosin. At pCa 9, troponin from foetal and adult hearts reduced the fraction of filaments moving from 90% to less than 15% with a modest (25-30%) decrease in velocity. At pCa 5, troponin from normal adult hearts increased filament velocity by up to 47 +/- 3% with no change in the fraction of filaments moving. Foetal troponin increased velocity by only 4 +/- 6% and the effect of troponin from failing hearts was between these values at 31 +/- 5%. Foetal hearts showed different troponin I and T isoform expression compared with adult hearts. No differences in troponin isoform expression were demonstrated between normal and failing adult hearts.


CONCLUSIONS
Functioning troponin and tropomyosin may be isolated from human heart and their properties investigated by in vitro motility assay. Both functional and isoform expression differences exist between foetal and adult cardiac troponin. The regulatory function of troponin from adults with end stage heart failure is different from normal adult troponin. These data suggest a role for altered troponin function in human cardiac development and heart failure.
Purcell
I F
IF
Imperial College School of Medicine, National Heart and Lung Institute, London, UK. i.purcell@ic.ac.uk
Bing
W
W
Marston
S B
SB
D000199:Actins; D000328:Adult; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005318:Fetal Heart / Q000187:drug effects*; D006333:Heart Failure / Q000503:physiopathology*; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000187:drug effects; D009200:Myocardial Contraction / Q000187:drug effects*; D011817:Rabbits; D014335:Tropomyosin / Q000302:isolation & purification; D014336:Troponin / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D019209:Troponin C / Q000032:analysis; D019210:Troponin I / Q000032:analysis; D020107:Troponin T / Q000032:analysis
D000199:Actins; D014335:Tropomyosin; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D002118:Calcium