Molecular Basis of Human Cardiac Troponin T Isoforms Expressed in the Developing, Adult, and Failing Heart

Abstract

Abstract Cardiac troponin T (cTnT), a protein essential for calcium-regulated myofibrillar ATPase activity, is expressed in the human heart as four isoforms (cTnT 1 through cTnT 4 , numbered in the order of decreasing molecular size). The expression of these isoforms at the protein level has previously been found by us to differ in the normal and failing adult and fetal human heart. In the present study, we have cloned and sequenced four full-length cDNAs corresponding to the four native cTnT protein isoforms and have expressed these cDNAs in an in vitro transcription and translation system. The cDNAs differ by the variable inclusion of a 15- and a 30-nt exon in the 5′ half of the coding region. These cDNAs yielded proteins that comigrate with the native isoforms, cTnT 1 through cTnT 4 . Polyclonal antisera, raised against a synthetic peptide corresponding to the 10-residue peptide encoded by the 30-nt exon, reacted with the two human isoforms largest in molecular size (cTnT 1 and cTnT 2 ) and the two largest cTnT isoforms of the rabbit and rat. The isoforms cTnT 1 and cTnT 2 , containing either both peptides encoded by the 30- and 15-nt exons or the peptide encoded by the 30-nt exon alone, are expressed in the fetal heart, with cTnT 2 being expressed at a very low level. cTnT 4 , lacking both of these sequences, is expressed in the fetal heart and is reexpressed in the failing adult heart, whereas cTnT 3 , containing the 5-residue peptide, is the dominant isoform in the adult heart. The identification and acquisition of these full-length clones that encode the four human cTnT isoforms should prove valuable in analyzing genomic DNA, identifying cTnT mutations in these alternatively spliced coding sequences and their splice sites, and studying the functional role of these isoform sequence differences in the normal and the diseased human heart.