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For cell and molecular biological studies of heart formation and function cell cultures of embryonal, neonatal or adult hearts of various vertebrates, notably rat and chicken, have been widely used. As the myocardium-specific cell-cell junctions, the intercalated disks (ID), have recently been found to be particularly sensitive to losses of - or mutations in - certain cytoskeletal proteins, resulting in cardiac damages, we have examined the ID organization in primary cultures of cardiomyocytes obtained from neonatal rats. Using immunofluorescence and immunoelectron microscopy, we have studied the major ID components for up to 2 weeks in culture, paying special attention to spontaneously beating, individual cardiomyocytes and myocardial cell colonies. While our results demonstrate the formation of some ID-like cardiomyocyte-connecting junction arrays, they also reveal a variety of structural disorders such as rather extended, junction-free ID regions, sac-like invaginations and endocytotic blebs as well as accumulations of intracytoplasmic structures suggestive of endocytosed forms of junction-derived vesicles or of junction fragments resembling fascia adhaerens elements. Moreover, we have noticed a novel type of small, obviously plaque-free cytoplasmic vesicles containing one or both of the desmosomal cadherins, desmocollin Dsc2 and desmoglein Dsg2. We conclude that cardiomyocyte cultures are useful model systems for studies of certain aspects of myocardiac differentiation and functions but, on the other hand, show progressive disintegration and deterioration. The potential value of molecular markers and reagents in studies of myocardial pathology as well as in the monitoring of myocardial differentiation of so-called stem cells is discussed.

Desmosomes represent a special type of the plaque-bearing adhering junctions, characteristic of certain pathways of cell differentiation, which compositionally are not identical in the various kinds of desmosome-forming cells. While all desmosomes contain the cytoplasmic plaque proteins desmoplakin I and plakoglobin, they can vary in their specific complement of desmosomal cadherins and by the presence of additional plaque proteins. We have raised monoclonal antibodies recognizing one such 'accessory' plaque protein, the cytokeratin-binding, basic protein plakophilin 1, originally introduced as 'band 6 protein' or 'polypeptide D6', which is an abundant desmosomal component in certain epithelia. Using such antibodies, we have isolated cDNA clones encoding the bovine and the human protein and determined their complete amino acid sequences. The mRNAs, which on Northern blot tests appear as two bands corresponding to approximately 4 and 2.4 kb (bovine) or 5 and 2.6 kb (human), code for 727 amino acids (calculated mol. wt. 80,180; IEP 9.25) in bovine and 726 amino acids (mol. wt. 80,496; IEP 9.34) in human plakophilin. Sequence analyses have revealed the presence of 9.2 repeated units of the arm-motif sequence, confirming our previous conclusion that this protein is a member of a larger family of proteins including, inter alia, several membrane-associated plaque proteins such as vertebrate plakoglobin and beta-catenin as well as the product of the armadillo gene of Drosophila. The plakophilin antibodies and cDNA probes have also allowed us to examine its synthesis in various tissues and cell cultures. While we confirm the occurrence of the protein in cytoskeletal fractions from various stratified squamous, complex, glandular duct and bladder epithelia, where it can be localized to desmosomes, we have, surprisingly, also identified the protein, although at lower amounts, in cytoskeletal fractions from several cultured cell lines in which the protein has not been consistently localized to desmosomes by immunofluorescence microscopy. Examples include cultured cells derived from certain simple epithelia such as the kidney-derived line MDBK and cultured calf lens cells. We have also found that, in all plakophilin 1-positive cells examined, a pool of diffusible ('soluble') cytoplasmic plakophilin exists, including cell lines such as human mammary carcinoma MCF-7 cells in which this soluble plakophilin seems to be the only detectable form. In addition, we have identified some soluble proteins conspicuously cross-reacting with plakophilin 1. Possible functions of plakophilin and its potential value as a marker for specific states of cell differentiation are discussed, particularly with respect to tumor diagnosis.

Using immunofluorescence microscopy, we observed that in several established cell culture lines derived from different nonepithelial tissues and species, cells spontaneously emerge, usually at low frequencies, which contain cytoplasmic structures decorated by antibodies specific for cytokeratins 8 and 18. This phenomenon was further examined at both the protein (gel electrophoreses of cytoskeletal proteins, followed by immunoblotting) and the RNA (Northern blots, "nuclear run-on" analysis, in situ hybridization) level. Positive cell lines included simian virus (SV40)-transformed human fibroblasts (HF-SV80, WI-38 VA13), human astrocytic glioma cells (U333 CG/343MG), rat (RVF-SMC) and hamster (BHK-21/13) cells derived from vascular smooth muscle and murine sarcoma MS-180 cells. In two cell lines (HF-SV80 and BHK-21/13), the frequency of the cytokeratin-containing cells and of the cytokeratin fibril arrays per cell was drastically increased upon treatment with 5-azacytidine. The structural appearance of the cytokeratins was variable in the different cell lines but could also differ among cells of the same culture: While small granular or comma-shaped structures or bizarrely shaped filament arrays prevailed in WI-38, RVF and normally grown BHK-21 cells, most of the other lines revealed extended normal-looking, fibrillar arrays. In one line (MS-180), the appearance of cytokeratins was associated with a morphological change, as it was only found in a subpopulation of cells that had lost their typical elongated and spindle-shaped phenotype and assumed a rounded ("coccoid") shape. Our results show that the expression of the genes encoding cytokeratins 8 and 18 is not necessarily restricted to programs of epithelial differentiation and that factors stochastically effective appear in cultured cell lines that allow the synthesis of these cytoskeletal components. Mechanisms possibly involved in this spontaneous and selective advent of cytokeratins 8 and 18 and implications for tumor diagnosis are discussed.