When isolated beating ventricular heart cells from newborn rats were grown in tissue culture on untreated polystyrene surfaces, they showed a striking tendency to grow focally in three dimensions from the single layer cell sheets which were formed early in growth. During this process, they frequently formed miniature spherical heart-like masses, which continued to beat and grow in size. These often were somewhat lobulated in appearance, and grew up to 2 mm in diameter. Histological sections of such structures sometimes revealed evidence of appreciable orientation of the cells to each other, in fiber-like units. Electron microscope sections of such mini-hearts showed structures resembling intercalated discs between myocardial cells. The precise factors which induced the cardiac cells to apparently organize into these heart-like structures are not presently known.

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Using rabbit anti-rabbit heart auto-antibodies, cultures of dissociated beating rat heart cells were examined for the presence of heart auto-antigens by immunofluorescence. Several patterns of staining of myocardial cells were observed. One type of auto-antibody revealed crisp myofibrillar cross-striations, consisting of wide bright segments often split by a thin dark band. Auto-antibody producing this pattern was potent for auto-antigen B. Rabbit anti-rabbit heart auto-antibodies rich in the auto-antigen D systems produced a granular staining in the myocardial cells, while an auto-antibody reactive with all five of the cardiac auto-antigens showed both granular and myofibrillar cross-striations. All the heart auto-antibody preparations reacted with cultured neonatal rat skeletal muscle cells to show diffuse cytoplasmic fluorescence and fine myofibrils. In all cases, rabbit anti-rabbit heart auto-antibodies failed to stain significantly the non-contractile cells present in the tissue cultures (‘endotheliod’, fibroblast, etc.). Sarcolemmal staining was not a prominent feature of these reactions. When the rabbit anti-rabbit heart auto-antibodies were exposed to living beating rat heart cells, no staining was observed, indicating the absence of these antigens on the cell surface.