Characterization of a TM‐4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl*

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Abstract
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 ∼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. J. Cell. Biochem. 85: 747–761, 2002.

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