Identification, characterization, and expression of sarcomeric tropomyosin isoforms in zebrafish

Tropomyosin is a component of thin filaments that constitute myofibrils, the contractile apparatus of striated muscles. In vertebrates, except for fish, four TPM genes TPM1, TPM2, TPM3, and TPM4 are known. In zebrafish, there are six TPM genes that include the paralogs of the TPM1 (TPM1-1 and TPM1-2), the paralogs of the TPM4 gene (TPM4-1 and TPM4-2), and the two single copy genes TPM2 and TPM3. In this study, we have identified, cloned, and sequenced the TPM1-1κ isoform of the TPM1-1 gene and also discovered a new isoform TPM1-2ν of the TPM1-2. Further, we have cloned and sequenced the sarcomeric isoform of the TPM4-2 gene designated as TPM4-2α. Using conventional RT-PCR, we have shown the expression of the sarcomeric isoforms of TPM1-1, TPM1-2, TPM2, TPM3, TPM4-1, and TPM4-2 in heart and skeletal muscles. By qRT-PCR using both relative expression as well as the absolute copy number, we have shown that TPM1-1α, TPM1-2α, and TPM1-2ν are expressed mostly in skeletal muscle; the level of expression of TPM1-1κ is significantly lower compared to TPM1-1α in skeletal muscle. In addition, both TPM4-1α and TPM4-2α are predominantly expressed in heart. 2D Western blot analyses using anti-TPM antibody followed by Mass Spectrometry of the proteins from the antibody-stained spots show that TPM1-1α and TPM3α are expressed in skeletal muscle whereas TPM4-1α and TPM3α are expressed in zebrafish heart. To the best of our knowledge, this is by far the most comprehensive analysis of tropomyosin expression in zebrafish, one of the most popular animal models for gene expression study.