L.F.Lemanski
(Department of Medical Physiology, Texas A&M University System Health ScienceCenter,College Station, Texas 77843)
( Department of Biology, Texas A&M University, College Station, Texas 77843;)
X.Huang
(Department of Medical Physiology, Texas A&M University System Health ScienceCenter,College Station, Texas 77843)
R.W.Zajdel
( Department of Biology, Texas A&M University, College Station, Texas 77843;)
S.L.Lemanski
(Department of Medical Physiology, Texas A&M University System Health ScienceCenter,College Station, Texas 77843)
C.Zhang
( Department of Biology, Texas A&M University, College Station, Texas 77843;)
F.Meng
(Department of Medical Physiology, Texas A&M University System Health ScienceCenter,College Station, Texas 77843)
D.Foster
(Department of Medical Physiology, Texas A&M University System Health ScienceCenter,College Station, Texas 77843)
Q.Li
(Department of Medical Physiology, Texas A&M University System Health ScienceCenter,College Station, Texas 77843)
N.Erginel-Unaltuna
D.K.Dube
(Department of Anatomy and Cell Biology, State University of New York Health
Science Center, Syracuse, New York 13210)

The Mexican axolotl (Ambystoma mexicanum) providesand excellent model for studying heart development since it carries a simple recessivecardiac lethal mutation that results in a failure of mutant embryonic myocardium tocontract. In cardiac mutant axolotls the hearts do not beat, apparently due to an absenceof organized myofibrils. The mutant hearts can be rescued by coculturing them with thenormal anterior endoderm/mesoderm tissue, by medium conditioned with normal anteriorendoderm/mesoderm or by RNA isolated from the conditioned medium. We have previouslyisolated a single cDNA clone from a library prepared with total RNA from conditionedmedium; this 166 nt long in vitro synthesized RNA, directed by the unique cDNA clone(Clone#4), has the ability to correct the heart defect and promote myofibrillogenesis in mutanthearts. The criteria for rescue include contraction of mutant hearts throughout theirlengths, an increase in sarcomeric tropomyosin arrays as shown by immunofluorescentconfocal microscopy, and the ultrastructural appearance of organized sarcomericmyofibrils. Quantitative PT-PCR has shown that Clone #4 RNA is specifically expressed inaxolotl heart, not in other tissues or organs. Moreover, the peak expression of Clone #4is from stage 15 to 30, corresponding to the heart induction and tube formation stages.These data indicate that this bioactive RNA plays an important role in heart developmentin axolotls. More recently, a point mutation (G-T) was found in Clone #4 RNA, which wasderived from mutant axolotl embryos at stages 20 and 30. At present, we are searching forthe full-length Clone #4 gene in an axolotl genomic library. Primer extension yields aproduct of ～500 bp at the 5' -end of the Clone #4 gene. The nucleotide sequence of theextended Clone #4(Ext-Clone #4) was determined and found to be unique showing nosignificant homology with other known sequences available from the gene databases.Interestingly, T7 sense RNA from the Ext-Clone # 4 (～500 nt RNA) shows a higherefficiency in rescuing mutant hearts than the T7 RNA from original Clone #4 (166 nt RNA).Our working hypothesis is that the bioactive RNA is a regulatory RNA that may upregulatedirectly or indirectly tropomyosin production in axolotl heart resulting in organized andfunctional myofibrils.

Supported by NIH Grants HL-58435 andHL-061246 (L.F.Lemanski X.Huang R.W.Zajdel S.L.Lemanski C.Zhang F.Meng D.Foster Q.Li N.Erginel-Unaltu)