Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4αIsoform in Humans

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4 α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4 α and a novel isoform TPM4 δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4 α and TPM4 δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4 δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4 α along with TPM1 α protein in human adult heart.