Abstract
The troponin complex regulates the Ca2+ activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid-rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+ buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.
Highlights
The troponin complex regulates the Ca2؉ activation of myofilaments during striated muscle contraction and relaxation
Considering that avian leg muscle Troponin T (TnT) has a much shorter N-terminal variable region [18] and fast TnT of the emu, a flightless bird, has lost this structure (Fig. 1B), we propose that the long Glu-rich segments in insect TnT and avian pectoral muscle TnT may have both evolved from an analogous evolutionary selection for flight abilities
A novel splice form of Drosophila TnT with the exon 5-encoded segment in the N-terminal variable region excluded and exon 10A in the C-terminal mutually-exclusive splicing region was identified among the cloned cDNAs
Summary
Dept. of Physiology, Wayne State University School of Medicine, 5374 Scott Hall, 540 E. In the vertebrate skeletal and cardiac muscles, contraction is powered by actin-activated myosin ATPase under the regulation of intracellular Ca2ϩ via the troponin complex in the thin filament [1,2,3]. Genes encoding the subunits of troponin are found in all invertebrate animals higher than Cnidaria (jellyfish and sponges) [6]. This phylogenetic chronicle corresponds to the emergence of the central nervous system [7], implying an essential role of troponin in the more coordinated muscle contractions of higher animal species. Gene structure and molecular evolution studies identified the fast skeletal muscle TnT as the ancestral form of the three vertebrate muscle-type– specific isoforms [9]. The Drosophila TpnT locus on chromosome X encodes several physiological alternative splicing variants, whereas aberrant splicing or missense mutations cause muscle abnormalities such as the upheld and indented thorax pheno-
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