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.
Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse ␣-tropomyosin cDNA under the control of a cardiac-specific ␣-myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well-organized sarcomeric myofibrils. Transfection of a -tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a -galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of ␣-tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412-420.1998 Wiley-Liss, Inc.
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