Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum).

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.

A specific synthetic RNA promotes cardiac myofibrillogenesis in the Mexican axolotl.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. However, the defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or RNA from endoderm mesoderm-conditioned medium. We constructed a cDNA library from total conditioned medium RNA in a pcDNAII expression vector. We screened the cDNA library by an organ culture bioassay and isolated a single clone (Cl#4), the synthetic RNA from which corrects the heart defect by promoting myofibrillogenesis. The insert size of the active clone is 166 nt in length with a unique nucleotide sequence. The anti-sense RNA from Cl#4 using SP6 RNA polymerase failed to rescue mutant hearts. The ability of this small RNA to correct the mutant heart defect suggests that the RNA probably does not act as an mRNA. While the precise mechanism of action is not yet known, on the basis of our studies to date it is very clear that the sense strand of Cl#4 RNA has the ability to promote myofibrillogenesis and rescue the mutant hearts both in vitro and in vivo.

Expression of myosin heavy chain transcripts in normal and cardiac mutant Mexican axolotls.

In this study, we have cloned a 1.0 kb myosin heavy chain (MHC) cDNA by screening an axolotl heart cDNA library with the monoclonal antibody MF20 against a light meromyosin (LMM) region of MHC. The nucleotide sequence analysis shows 85-86% homology at the amino acid and 78-81% homology at the nucleic acid level with MHC from other vertebrates. Phylogenetic analyses suggest that axolotl beta-MHC forms a cluster with the myosin II group of vertebrate striated muscles. Within the myosin II cluster, axolotl beta-MHC forms a distinct subclade from avian MHC and is instead closer to mammalian MHC. RT-PCR analyses show that transcripts of beta-MHC are present at stage 2 and the onset of the MHC gene expression is at stage 8-10 (gastrulation). Expression increases with embryonic development and reaches a maximum at stage 20. Beyond stage 35, the heart-beat initiation stage, the expression level of beta-MHC is higher in cardiac muscle than in skeletal muscle. We could not detect significant differences in the levels of expression of MHC transcripts in normal and cardiac lethal mutant (c/c) axolotls (Ambystoma mexicanum).