Role of the Fetal and α/β Exons in the Function of Fast Skeletal Troponin T Isoforms: Correlation with Altered Ca2+ Regulation Associated with Development

In mammalian fast skeletal muscle, constitutive and alternative splicing from a single troponin T (TnT) gene produce multiple developmentally regulated and tissue specific TnT isoforms. Two exons, α (exon 16) and β (exon 17), located near the 3′ end of the gene and coding for two different 14 amino acid residue peptides are spliced in a mutually exclusive manner giving rise to the adult TnTα and the fetal TnTβ isoforms. In addition, an acidic peptide coded by a fetal (f) exon located between exons 8 and 9 near the 5′ end of the gene, is specifically present in TnTβ and absent in the adult isoforms. To define the functional role of the f and α/β exons, we constructed combinations of TnT cDNAs from a single human fetal fast skeletal TnTβ cDNA clone in order to circumvent the problem of N-terminal sequence heterogeneity present in wild-type TnT isoforms, irrespective of the stage of development. Nucleotide sequences of these constructs, viz. TnTα, TnTα+f, TnTβ-f and TnTβ are identical, except for the presence or absence of the α or β and f exons. Our results, using the recombinant TnT isoforms in different functional in vitro assays, show that the presence of the f peptide in the N-terminal T1 region of TnT, has a strong inhibitory effect on binary interactions between TnT and other thin filament proteins, TnI, TnC and Tm. The presence of the f peptide led to reduced Ca2+-dependent ATPase activity in a reconstituted thin filament, whereas the contribution of the α and β peptides in the biological activity of TnT was primarily modulatory. These results indicate that the f peptide confers an inhibitory effect on the biological function of fast skeletal TnT and this can be correlated with changes in the Ca2+ regulation associated with development in fast skeletal muscle.