Lemanski, Larry F.

The Mexican axolotl (Ambystoma mexicanum) carries a cardiac lethal mutation
resulting in mutant embryos with no heartbeat. This homozygous recessive gene results
in tropomyosin deficiency and absence of organized myofibrils. Co-culturing mutant
hearts with bioactive RNA, termed myofibril-inducing RNA (MIR), from normal axolotl
embryonic anterior endoderm causes the mutant hearts to beat. It is hypothesized that the
secondary structure of the MIR binds a specific protein(s) and this is required to
synthesize tropomyosin and form organized myofibrils. In this study mutant hearts are
co-cultured with human fetal and adult heart total RNA to assess rescue of the mutant
hearts. Results show that both human fetal and adult heart total RNA rescue the mutant
condition in a manner similar to the MIR. Thus, the MIR human functional homologs
induce events leading to normal heart differentiation and function. This finding may help
people with heart muscle damage regain normal heart function again.

The Mexican axolotl, Ambystoma mexicanum, possesses a naturally-occurring
lethal mutation, designated gene "c", for cardiac non-function. Hearts form but fail to
beat, lack organized myofibrils, and are deficient in tropomyosin. Treatment with a noncoding
RNA MIR (Myofibril-Inducing RNA) rescues this defect in organ culture.
Rescued mutant hearts have restored tropomyosin, form organized myofibrils, and beat
vigorously. Studies to elucidate the mechanism of MIR heart rescue are underway.
Current evidence suggests that MIR acts by binding with at least two proteins. The yeast
three-hybrid system is being used to screen an axolotl eDNA library for these two
proteins and other possible MIR-binding candidates. This is a method utilizing two
hybrid proteins and a hybrid RNA. An interaction between these three components will
activate the expression of reporter genes, whose activity is assayed through phenotypical
and biochemical methods. In this study, the protocol for yeast three-hybrid technology is
being established for analyzing the MIR in the Mexican axolotl, cardiac mutant animal
model.

A naturally-occurring recessive lethal mutation in axolotls, Ambystoma mexicanum, is an intriguing model for studying tropomyosin expression regulation. Homozygous embryos(c/c) form hearts that are deficient in tropomyosin, lack organized myofibrils and fail to beat. Previous studies have shown that a non-coding RNA gene, MIR (Myofibril Inducing RNA), is sufficient to rescue the non-beating homozygous recessive mutant hearts by promoting sarcomeric tropomyosin expression that leads to formation of organized myofibrils and beating hearts. Real time RT-PCR reveals that mutant hearts express the same level mRNA of the alpha-tropomyosin and TM4 type tropomyosin (ATmC-3) gene as normal embryonic hearts. These genes show no differences with regard to the splicing patterns of normal and mutant. Using protease inhibitor LLnL and E-64d treatments and two-dimensional Western blots of normal and mutant hearts, it is found that mutant hearts express all tropomyosin protein isoforms as normal hearts but protein expression are at low levels. These studies suggest that there is a failure in the translational or posttranslational control mechanisms for tropomyosin protein synthesis in cardiac mutant axolotl hearts during development.

A recessive mutant gene, termed "c", for cardiac non-function in the Mexican axolotl, Ambystoma mexicanum, is responsible for the failure of myofibrillogenesis in cardiac nonfunction mutant embryonic hearts. Animals that are homozygous for the mutation (c/c) fail to develop beating hearts and consequently die. Thus, the Mexican axolotl has been a useful animal model to study embryonic heart development. Recently, the cardiac troponin T (cTnT) gene, along with three additional shorter isoforms of the gene, were cloned from normal embryonic hearts. These isoforms are believed to be the alternately spliced forms of the full length gene. One of the isoforms cloned is missing a cardiac-specific exon. Real-time PCR reveals that homozygous recessive mutant embryos (c/c) exhibit a lower transcription level of the cTnT gene than wildtype animals (+/+ or +/c). Expression levels of each of the isoforms are compared in normal and mutant hearts using quantitative real-time PCR.