Immunofluorescent, immunogold, and electrophoretic studies for desmin in embryonic hearts of normal and cardiac mutant Mexican axolotls, Ambystoma mexicanum

Recessive mutant gene c for "cardiac nonfunction" in axolotls results in an absence of normal heart contractions in affected embryos due to a failure of myofibril formation. In the present study, the intermediate filament protein, desmin, is compared in developing normal and mutant hearts by means of two-dimensional gel electrophoresis, immunofluorescent microscopy, and immunoelectron microscopy. Tissues were fixed in periodate-lysine-paraformaldehyde or paraformaldehyde-glutaraldehyde solutions and rapidly frozen or embedded in Lowicryl resin. Frozen sections stained with FITC-conjugated antibodies by an indirect approach revealed that desmin is localized in the I-band regions of adult cardiac myofibrils. In normal embryonic hearts at stage 32 (preheartbeat) desmin is localized as "spots" or amorphous collections in the cells. As development progresses to stage 35, staining for desmin in normal hearts becomes more intense with localization being most pronounced at the cell peripheries. By stage 41 most of the desmin in normal hearts is localized in the I band areas of the organized myofibrils and the staining of amorphous areas is much less prominent. During early development, the distribution of desmin in mutant hearts is similar to normal. However, while most of the desmin in normal organs at stage 41 is associated with myofibrils, the staining remains diffuse in mutants. Two-dimensional gel electrophoresis reveals comparable patterns for desmin from normal and mutant hearts. Immunogold staining shows desmin localization to be between the myofibrils and around the I-band regions in adult cardiac muscle and in stage 41 normal embryonic hearts. Immunogold staining confirms a diffuse distribution of desmin in mutant hearts.