Fast Troponin T Research Articles

N-terminal amino acid sequences of three functionally different troponin T isoforms from rabbit fast skeletal muscle

The different isoforms of fast skeletal muscle troponin T (TnT) are generated by alternative splicing of several 5′ exons in the fast TnT gene. In rabbit skeletal muscle this process results in three major fast TnT species, TnT 1f, TnT 2f and TnT 3f, that differ in a region of 30 to 40 amino acid residues near the N terminus. Differential expression of these three isoforms modulates the activation of the thin filament by calcium. To establish a basis for further structure-function studies, we have sequenced the N-terminal region of these proteins. TnT 2f is the fast TnT sequenced by Pearlstone et al.. The larger species TnT 1f contains six additional amino acid residues identical in sequence and position to those encoded by exon 4 in the rat fast skeletal muscle TnT gene. TnT 3f also contains that sequence but lacks 17 amino acid residues spanning the region encoded by exons 6 and 7 of the rat gene. These three TnTs appear to be generated by discrete alternative splicing pathways, each differing by a single event. Comparison of these TnT sequences with those from chicken fast skeletal muscle and bovine heart shows that the splicing pattern resulting in the excision of exon 4 is evolutionarily conserved and leads to a more calcium-sensitive thin filament.

The multiplicity of troponin T isoforms. Distribution in normal rabbit muscles and effects of chronic stimulation.

Polyclonal antibodies were raised in guinea pigs against troponin-T (TnT) isoforms purified from fast- and slow-twitch rabbit muscles. With the use of these antibodies and immunoblots of one- and two-dimensional electrophoreses, the distribution of fast and slow TnT isoforms was investigated in normal and chronically stimulated hindlimb muscles of the rabbit. According to differences in their apparent molecular masses, six fast TnT isoforms (TnTcf, TnT1f, TnT2f, TnT3f, TnT4f, TnT5f) were distinguished in normal tibialis anterior and extensor digitorum longus muscles. These muscles also contained low amounts of TnT1s and TnT2s which were the predominant TnT isoforms in slow-twitch soleus muscle. Fast and slow TnT isoforms were found to exist in several charge variants, i.e. one for TnTcf, three different charge forms for TnT1f, seven for TnT2f, four for TnT3f, three for TnT4f, one for TnT5f, four for TnT1s, and three for TnT2s. Some charge variants were phosphorylated isoforms because treatment with alkaline phosphatase reduced the number of the 19 fast and 7 slow variants to 12 and 3, respectively. The stimulation-induced fast-to-slow transition caused progressive decreases in fast and increases in slow isoforms. The decrease and the disappearance of the major fast isoforms followed a sequence of TnT2f, TnTcf, TnT4f, TnT1f, and TnT3f. This decrease in fast isoforms fits well with the reduction of fast TnT mRNAs assessed by Northern blot analysis. Prolonged stimulation ultimately created a TnT isoform pattern similar to that found in normal slow-twitch muscle. Stimulation also induced changes in the tropomyosin subunit pattern with a decrease in the fast and an increase in the slow alpha-tropomyosin subunit without altering the alpha/beta-tropomyosin subunit ratio. Similar to slow-twitch soleus muscle, long-term stimulated muscles contained appreciable amounts of the fast alpha-tropomyosin subunit, but only traces of fast TnT isoforms. This combination indicated that the predominant slow TnT isoforms may be capable of interacting with fast tropomyosin in these muscles.

Alternative splicing generates variants in important functional domains of human slow skeletal troponin T.

We provide the first nucleotide sequence information for the slow isoform of troponin T (TnT). Sequence and hybridization analyses revealed that a single slow TnT gene present in the human genome gives rise to at least two different slow TnT variants by alternative splicing. The observed variations in slow TnT splicing generated major structural differences between the two corresponding slow TnT proteins in a domain that is likely to be involved in critical interactions with troponin C, troponin I, and tropomyosin in the thin filament. Corresponding variations have not been found for fast or for cardiac TnT. The comparison of splicing patterns for fast, cardiac, and slow TnT reveals that the splicing pattern for each isoform is unique. These features raise important questions of why and how all the individual members of the closely related TnT gene family developed such complex but different schemes of alternative splicing to create sets of variant proteins. This unusual familial trait is not known in any other muscle or nonmuscle multigene family.

Patterns of troponin T expression in mammalian fast, slow and promiscuous muscle fibres.

The distribution of troponin T (TnT) species in typed single muscle fibres was analysed using one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and a monoclonal antibody specific for fast TnT. Fibres taken from erector spinae (Es), plantaris (Plt), diaphragm (Dia) and soleus (Sol) muscles of adult rabbits were pretyped as fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), slow-twitch-oxidative (SO) or promiscuous (P) using a combination of histochemical staining and PAGE. Although none of the four size classes of TnT was either muscle or fibre type specific, their pattern of expression differed in each muscle and between the fibre types. FG fibres expressed TnT1f or TnT2f as predominant species, depending on the muscle; TnT3f and TnT4f were minor components. In contrast, all size classes of TnT were expressed in varying proportions in FOG fibres from Es and Plt, while those from Dia resembled FG fibres, expressing TnT1f as their major species. P fibres from Es, Plt, and Sol exhibited a distinctive pattern of fast TnT expression, TnT3f being the predominant species. Dia differed from the other muscles as TnT1f was the dominant fast TnT species in its P fibres as it is in the Dia fast fibres. Quantitative analysis of one- and two-dimensional gels revealed that the P fibres could be divided into two classes, those that exhibited discoordinate expression of fast and slow TnTs, myosin light chains and myosin heavy chains and those in which their expression was coordinate. In addition low levels of TnT4f were detected in SO fibres and of slow TnT in fast fibres.

The extent of amino-terminal heterogeneity in rabbit fast skeletal muscle troponin T

The extent and nature of fast troponin T (TnT) heterogeneity has been assessed in rabbit skeletal muscle. Previous studies identified two major fast TnT species (TnT1f and TnT2f), in the fast white muscle erector spinae, differing in their N-terminal cyanogen bromide (CNBr) fragments. Here a monoclonal antibody that recognizes a conserved region of TnT was used to characterize two additional TnT species (TnT3f and TnT4f) in the epaxial and limb musculature and a minor species (TnTcf) in craniofacial muscles. A combination of CNBr peptide mapping, immunoblotting and specific labelling of the N-terminus shows that these TnT species also differ in their N-terminal region. This observation is consistent with cDNA studies that predicted the N-terminal region is hypervariable. One additional species, a variant of TnT2f present in the tongue, was identified by two-dimensional gel electrophoresis. The limited number of TnT variants indicates that the full potential for heterogeneity inferred from the cDNA studies is not realized. This conclusion is supported by immunoblot analysis with a monoclonal antibody that recognizes an epitope in the hypervariable N-terminal region which is present in all variants of TnT1f and TnT2f but absent from the lower molecular weight species TnT3f and TnT4f.

Heterogeneity of contractile proteins. A continuum of troponin-tropomyosin expression in mammalian skeletal muscle.

Comparison of the myofibrillar proteins from several adult rabbit skeletal muscles has led to the identification of multiple forms of fast and slow troponin T. In Briggs et al. (Briggs, M. M., Klevit, R., and Schachat, F. H. (1984) J. Biol. Chem. 259, 10369-10375) two species of rabbit fast skeletal muscle troponin T (TnT), TnT1f and TnT2f, were characterized. Here, the distribution of these fast TnT species and the alpha- and beta- tropomyosin (Tm) subunits is characterized in fast muscles and in single muscle fibers. Evidence is also presented for two forms of slow skeletal muscle TnT. The presence of each fast TnT species is associated with the presence of a different Tm dimer: TnT1f with alpha beta-Tm and TnT2f with alpha 2-Tm. Histochemical analysis shows that expression of the fast TnT-Tm combinations is not due to differences in the distribution of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fiber types. The absence of a correlation between histochemical typing and the composition of the thin filament Ca2+-regulatory complex is more apparent in individual fast muscle fibers where both fast TnT-Tm combinations appear to be expressed in a continuum. The implications of these observations for mammalian skeletal muscle fiber diversity are discussed.