Abstract. In adult regenerating cardiomyocytes in culture, in contrast to fetal cells, mitochondrial creatine kinase (Mi-CK) was expressed. In the same cell, two populations of mitochondria, differing in shape, in distribution within the cell and in content of Mi-CK, could be distinguished. Immunofluorescence studies using antibodies against Mi-CK revealed a characteristic staining pattern for the two types of mitochondria: giant, mostly cylindrically shaped, and, as shown by confocal laser light microscopy, randomly distributed mitochondria exhibited a strong signal for Mi-CK, whereas small, "normal" mitochondria, localized in rows between myofibrils, gave a much weaker signal. Transmission EM of the giant mitochondria demonstrated paracrystalline inclusions located between cristae membranes. Immunogold labeling with anti-Mi-CK antibodies revealed a specific decoration of these inclusions for Mi-CK. Addition of 20 mM creatine, the substrate of Mi-CK, to the essentially creatine-free culture medium caused the disappearance of the giant cylindrically shaped mitochondria as well as of the paracrystalline inclusions, accompanied by an increase of the intracellular level of total creatine. Replacement of creatine in the medium by the creatine analogue and competitor B-guanidinopropionic acid caused the reappearance of the enlarged mitochondria. It is believed that the accumulation of Mi-CK within the paracrystalline inclusions, similar to those observed in certain myopathies, represents a compensatory effect of the cardiomyocytes to cope with a metabolic stress situation caused by low intracellular total creatine levels. IT was shown in preceding publications (3,5,9,11,17,29) that adult rat cardiomyocytes (ARCs) t in culture represent a suitable in vitro system for the investigation of cardiac differentiation. There it was demonstrated that ARCs in long term cultures in many ways repeat embryonic and fetal differentiation steps. Fully differentiated heart cells express mitochondrial creatine kinase (Mi-CK) in conjunction with the cytosolic creatine kinase isoforms MM-, MB-, or BB-CK, with a significant fraction of MM-CK being specifically associated subcellularly in a compartmented fashion at intracellular sites of high energy turnover, e.g., at the myofibriUar M-band, the sarcoplasmic reticulum, etc. (39, 59), which has led us to the postulation of the phosphocreatine-circuit model (60, 61). Expression and appearance of Mi-CK in rat heart in vivo, however, occurs only during postnatal development paralleling more or less the appearance of MM-CK within the M-line structure of myofibrils which does not occur before 2 wk after birth (35). Concomitantly, fetal cardiomyocytes in culture do not express and accumulate Mi-CK. ARCs in culture though accumulate Mi-CK 1. Abbreviations used in this paper: ARC, adult rat cardiomyoeyte; Mi-CK, mitochondrial creatine kinase.in their mitochondria, a feature characteristic for adult heart tissue and freshly isolated rod-shaped ARC. This is converse to contractile structures in cult...

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning followed by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.

Background: The oncoprotein c-Myc has been intensely studied in breast cancer and mouse mammary tumor models, but relatively little is known about the normal physiological role of c-Myc in the mammary gland. Here we investigated functions of c-Myc during mouse mammary gland development using a conditional knockout approach.

Nowadays, deconvolution in cell and tissue imaging has matured into a standard restoration technique that is accessible to large fraction of the microscopy community thanks to steadily improving algorithms. Still, deconvolution is often the ratelimiting step in the analysis of the acquired data, even at today's computer performance. Here, we present the Huygens Remote Manager, an opensource, efficient, multi-user web-based interface for parallel batch deconvolutions.

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