Morphological, Biochemical and Immunohistochemical Studies on Heart Development in Cardiac Mutant Axolotls,Ambystoma mexicanum

Abstract

SYNOPSIS. A naturally-occurring genetic mutation, designated c for “cardiac lethal” in axolotls, Ambystoma mexicanum , is proving to be a useful model for studying myofibrillogenesis in differentiating heart cells. In this paper I describe morphological, biochemical and immunofluorescence studies which compare the contractile proteins in normal and mutant hearts. In addition, morphological studies on anterior endoderm, an important heart inductor tissue in salamanders, are reviewed. Detailed electron microscopic studies show that normal heart myocytes contain numerous well-organized myofibrils. Although mutant heart cells contain a few myosin and actin filaments, there are no organized myofibrils. Instead, amorphous proteinaceous collections are prominent in the peripheral cytoplasm of the cell where myofibrils would be expected to first form. SDS-polyacrylamide gel electrophoresis shows that actin is present in almost normal amounts in mutant hearts, myosin heavy chain is reduced and tropomyosin is virtually absent. Immunofluorescence studies reveal that myosin, α-actinin and tropomyosin are located prominently in theorganized myofibrils of normal heart cells. In mutant hearts myosin is localized almost exclusively in the amorphous collections at the cell peripheries, α-actinin also is distributed mainly in the peripheral cell cytoplasm. There is almost no staining for tropomyosin. Heavy meromyosin (HMM) binding experiments demonstrate that the actin in mutant heart cells is contained within the amorphous collections in a non-filamentous state and the addition of HMM causes its polymerization into filaments. In view of these findings, we undertook studies to determine whether there might be a causal relationship between theabsence of tropomyosin in mutants and the failure of actin to form into filaments. Our results indeed show that addition of tropomyosin to glycerinated mutant hearts or homogenates of mutant hearts causes the amorphous actin to form into filaments. Thus, this single gene mutation results in mutant heart cells having reduced, but significant, amounts of myosin and actin, even though non-filamentous, and substantial amounts of α-actinin. There is almost no tropomyosin. It is implied that the drastic reduction of tropomyosin in mutant cells is somehow related to the failure of normal myofilament formation, which in turn would seem to be an essential step in the normal organization of myofibrils.