Diminished myofibril organization in mutant axolotl hearts transfected with site-directed mutants of sarcomeric tropomyosins.
We used a model lacking endogenous sarcomeric tropomyosin, the cardiac mutant of the Mexican axolotl, to examine the effect of mutant tropomyosins on sarcomeric myofibril formation. Previous studies have introduced wild-type mouse alpha-tropomyosin into mutant hearts in organ culture with subsequent for-mation of organized myofibrils. This study examines the predominant embry-onic axolotl TPM-4 type tropomyosin (TPM4alpha), containing a conservative re- placement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4alpha) promoted formation of organized myofibrils in hearts without endogenous tropomyosin. Site-directed mutagenesis of 175 glutamic acid with 175 glutamine or 175 lysine was toxic to the formation of organized myofibrils in mutant hearts in the absence of endogenous tropo-myosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4alpha cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4alpha cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4alpha type or TPM1alpha (striated muscle isoform of the TPM1 gene) was sufficient to alter the protein such that organized myofibrils would not form in ventricles of mutant hearts without endogenous tropomyosin.
Zajdel
Robert W
RW
Department of Cell and Developmental Biology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.
Denz
Christopher R
CR
McLean
Matthew D
MD
Dube
Syamalima
S
Muthuchamy
Mariappan
M
Poiesz
Bernard J
BJ
Wieczorek
David F
DF
Dube
Dipak K
DK
D000557:Ambystoma; D000818:Animals; D004625:Embryo, Nonmammalian; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000473:pathology; D009210:Myofibrils / Q000235:genetics* / Q000473:pathology; D009924:Organ Culture Techniques; D012518:Sarcomeres / Q000235:genetics* / Q000473:pathology; D014162:Transfection / Q000379:methods*; D014335:Tropomyosin / Q000235:genetics*
D014335:Tropomyosin