Analysis of actin and tropomyosin in hearts of cardiac mutant axolotls by two-dimensional gel electrophoresis, western blots, and immunofluorescent microscopy

When homozygous, recessive mutant gene c in Ambystoma mexicanum results in a failure of embryonic heart function. This failure is apparently due to abnormal inductive influences from the anterior endoderm resulting in an absence of normal sarcomeric myofibril formation. Biochemical and immunofluorescent studies were undertaken to evaluate the contractile proteins actin and tropomyosin in normal and mutant hearts. For the immunofluorescent studies, cardiac tissues were fixed in periodate-lysine-paraformaldehyde, frozen sectioned, and immunostained by an indirect method with monospecific polyclonal antibodies produced against highly purified chicken heart actin and tropomyosin. In normal hearts, both antiactin and antitropomyosin stained the myofibrillar I-bands intensely. In mutant hearts, intensity of staining with antiactin antibody was similar to normal, although sarcomeric patterns were not observed. Staining intensity for tropomyosin with antitropomyosin antibody was significantly reduced in mutant hearts when compared to normal. Biochemical studies were used to evaluate antibody specificity, antigenic variability, and relative protein concentrations of actin and tropomyosin in normal and mutant cardiac tissues. Tissue homogenates were electrophoresed in two dimensions, and second-dimension slab gels were either Coomassie Blue silver-stained or transblotted onto nitrocellulose and the proteins stained with antibodies. Stained gels and immunoblots of cardiac proteins reveal that the amounts of actin isoforms are identical in normal and mutant hearts. However, these methods demonstrate a significantly reduced amount of tropomyosin in mutant tissue. This confirms earlier studies suggesting reduced amounts of tropomyosin in mutant hearts based upon immunological assays. Thus, failure of normal myofibrillogenesis in gene c mutant hearts does not appear to result from a change in actin isoform composition but may be related to a deficiency in tropomyosin.