Morphological and biochemical abnormalities in hearts of cardiac mutant salamanders (Ambystoma mexicanum)

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Abstract
The effect of homozygosity for recessive gene c in Ambystoma mexicanum is the absence of a heartbeat even though initially heart development appears normal. Mutant embryos (c/c) are first distinguishable from their normal siblings (+/+; +/c) at stage 34 (7 days after fertilization) when the normals develop contracting hearts. The mutant hearts at this stage, upon gross examination, appear structurally normal but fail to beat. Nevertheless, the mutants survive through stage 41, which is about 20 days beyond the heartbeat stage, and they exhibit normal swimming movements, indicating that gene c does not affect skeletal muscle. Electron microscopic studies of normal hearts show some myofibrils to be present at stage 34; by stage 41, the normal myocardial cells have become highly differentiated muscle cells. Although some mutant heart cells contain a few thin 60 A and thick 150 A filaments, organized myofibrils are absent. Instead, amorphous proteinaceous collections are prominent. Heavy meromyosin (HMM) binding experiments were performed on mutant hearts to determine whether the myocardial cells contain actin. Mutant myocardial cells that are glycerinated but not treated with HMM contain intact amorphous bodies. After incubation in HMM, the amorphous collections are no longer present and large numbers of decorated actin filaments appear. The results suggest that the amorphous proteinaceous collections contain actin in a nonfilamentous form, and the addition of HMM induces this actin to polymerize into filaments. SDS-polyacrylamide gel electrophoresis of mutant heart tissue supports this conclusion by showing a prominent 43,000 dalton band suggestive of actin. The electrophoresis experiments also demonstrate a significant reduction of myosin heavy chain (200,000 daltons) in mutant hearts when compared to normal, and this latter observation is confirmed by radioimmunoassay experiments. Muscle tropomyosin (34,000 daltons), prominent in normal hearts, is virtually nonexistent in mutants. Thus, it appears that this single gene mutation affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.