RESUMO
A striated muscle isoform of a Tropomyosin (TM-4) gene was characterized and found to be necessary for contractile function in embryonic heart. The full-length clone of this isoform was isolated from the Mexican axolotl (Ambystoma mexicanum) and named Axolotl Tropomyosin Cardiac-3 (ATmC-3). The gene encoded a cardiac-specific tropomyosin protein with 284 amino acid residues that demonstrated high homology to the Xenopus cardiac TM-4 type tropomyosin. Northern blot analysis indicates a transcript of approximately 1.25 kb in size. RT-PCR and in situ hybridization demonstrated that this isoform is predominantly in cardiac tissue. Our laboratory uses an animal model that carries a cardiac lethal mutation (gene c), this mutation results in a greatly diminished level of tropomyosin protein in the ventricle. Transfection of ATmC-3 DNA into mutant hearts increased tropomyosin levels and promoted myofibrillogenesis. ATmC-3 expression was blocked in normal hearts by transfection of exon-specific anti-sense oligonucleotide (AS-ODN). RT-PCR confirmed lower transcript expression of ATmC-3 and in vitro analysis confirmed the specificity of the ATmC-3 exon 2 anti-sense oligonucleotide. These AS-ODN treated hearts also had a disruption of myofibril organization and disruption of synchronous contractions. These results demonstrated that a striated muscle isoform of the TM-4 gene was expressed embryonically and was necessary for normal structure and function of the ventricle.
