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 α-tropomyosin into mutant hearts in organ culture with subsequent formation of organized myofibrils. This study examines the predominant embryonic axolotl TPM-4 type tropomyosin (TPM4α), containing a conservative replacement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4α) 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 tropomyosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4α cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4α cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4α type or TPM1α (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.