Heart development will be studied in normal and cardiac mutant axolotls and in normal and cardiomyopathic hamsters. Our objectives are to elucidate the sequence of events and mechanism(s) of myofibrillogenesis and to determine the roles of embryonic inductive interactions regulating and directing heart differentiation at the cellular and molecular levels. Developing heart cells will be examined by immunohistochemical methods to establish the appearance and precise locations of the various contractile/cytoskeletal proteins. Deep freeze etching methods in combination with immunoelectron microscopy will permit three-dimensional analyses of the protein distributions during myofibril formation. Two-dimensional gel electrophoresis studies and Western blots will aid in determining the early appearance and relative concentrations of the proteins in the differentiating cells. """"""""New"""""""" contractile/cytoskeletal proteins will be probed by preparing specific polyclonal and monoclonal antibodies against unknown proteins detected by two-dimensional gel electrophoresis. Micro-injection of fluorescently-tagged specific proteins into cultured heart cells will be employed to examine their incorporation into the myofibrils and/or cytoskeletal elements of the living differentiating cells. In addition, antibodies against the various proteins will be injected into cultured cells to help determine roles that specific proteins play in myofibril organization. Heart inductive processes which regulate normal myofibrillogenesis will be studied at the cellular and molecular levels. To accopmlish this, gene c in axolotls will be employed as a """"""""tool"""""""" in the studies. Since a diffusible RNA from normal anterior endoderm corrects the defect in cardiac mutant heart cells by promoting myofibrillogenesis, a variety of biochemical experiments will be performed to determine the mechanism(s) of this inductive relationship at the cellular and molecular levels. The use o these two unique genetic mutations should provide the biological """"""""tools"""""""" necessary for determining the sequence of events and mechanism(s) of normal myofibrillogenesis and heart induction. By understanding the basic mechanism(s) of how heart cells form into normal functional units, the various diseases which alter these processes can be understood, and, perhaps, reversed. In a broader sense, the proposed research may help to answer one of the major unsolved problems in modern biology: the control of gene expression during development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037702-02
Application #
3353614
Study Section
(SRC)
Project Start
1986-09-30
Project End
1991-09-29
Budget Start
1987-09-30
Budget End
1988-09-29
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Upstate Medical University
Department
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
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Spinner, Belinda J; Zajdel, Robert W; McLean, Matthew D et al. (2002) Characterization of a TM-4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl. J Cell Biochem 85:747-61
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Gaur, A; Bhatia, R; Spring-Mills, E et al. (1998) The heart of metamorphosing Mexican axolotl but not that of the cardiac mutant is associated with the upregulation of Hox A5. Biochem Biophys Res Commun 245:746-51
Bhatia, R; Gaur, A; Lemanski, L F et al. (1998) Cloning and sequencing of the cDNA for an RNA-binding protein from the Mexican axolotl: binding affinity of the in vitro synthesized protein. Biochim Biophys Acta 1398:265-74
Zajdel, R W; McLean, M D; Lemanski, S L et al. (1998) Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts. Dev Dyn 213:412-20
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Lemanski, S F; Kovacs, C P; Lemanski, L F (1997) Analysis of the three-dimensional distributions of alpha-actinin, ankyrin, and filamin in developing hearts of normal and cardiac mutant axolotls (Ambystoma mexicanum). Anat Embryol (Berl) 195:155-63
Luque, E A; Spinner, B J; Dube, S et al. (1997) Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum). Gene 185:175-80

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