Our long-term objectives are to elucidate the cellular, molecular and genetic mechanisms that regulate early myocardial cell differentiation and myofibrillogenesis in developing hearts. A naturally occurring recessive lethal mutation in axolotls (salamanders), Ambystoma mexicanum, is an intriguing model for studying early heart development, because homozygous embryos (c/c) form hearts that are deficient in tropomyosin, lack organized myofibrils, and fail to beat. The defect can be corrected by culturing hearts with normal anterior endoderm tissue, in medium conditioned by the anterior endoderm, or in total RNA isolated from endoderm or conditioned medium. In addition, we have identified a novel Clone (#4) from a cDNA library constructed from conditioned medium RNA which acts as a template for a bioactive RNA capable of correcting the heart defect. The RNA can bind at least two proteins in the embryos. It is possible that this RNA is a regulatory molecule which upregulates tropomyosin synthesis in mutant hearts and promotes myofibrillogenesis either directly or in complex with its binding protein(s). The following specific aims have been developed to address our hypothesis that this RNA, most likely in conjunction with RNA binding proteins, promotes myocardial cell differentiation and myofibrillogenesis in developing heart cells: 1) The role of the Clone #4 RNA in promoting myofibrillogenesis in embryonic hearts will be determined; 2) Transgenic axolotls will be used to test the ability of Clone #4 to rescue mutant hearts in vivo; 3) Expression of the bioactive Clone #4 RNA will be analyzed in developing embryos by in situ hybridization and quantitative RT-PCR; 4) The structural-functional relationships of the bioactive Clone #4 RNA will be evaluated by in vitro mutagenesis; 5) The proteins that bind to the bioactive RNA will be purified and characterized at the cellular, biochemical and molecular levels; 6) Potential homolog(s) of the bioactive Clone #4 RNA will be examined in other animal systems. This research will provide new basic information on the molecular mechanisms Of myofibrillogenesis and myocardial cell rescue in vertebrate hearts. The health relevance of understanding and being able to turn defective, non-beating cardiac tissue into contracting muscle could be tremendous; if this can be applied in humans, patients who have damaged heart tissue might be able to have that tissue redifferentiate into functional muscle again.
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