Adult rabbit cardiomyocytes undergo typical hibernation-like dedifferentiation when co-cultured with cardiac fibroblasts.

Objectives: Little is known about the causal factors which induce the typical structural changes accompanying cardiomyocyte dedifferentiation in vivo such as in chronic hibernating myocardium. For identifying important factors involved in cardiomyocyte dedifferentiation, as seen in chronic hibernation, an in vitro model mimicking those morphological changes, would be extremely helpful. Methods: Adult rabbit cardiomyocytes were co-cultured with cardiac fibroblasts. The typical changes induced by this culturing paradigm were investigated using morphometry, electron microscopy and immunocytochemical analysis of several structural proteins, which were used as dedifferentiation markers, i.e., titin, desmin, cardiotin and alpha -smooth muscle actin. Results: Close apposition of fibroblasts with adult rabbit cardiomyocytes induced hibernation-like dedifferentiation, similar to the typical changes seen in chronic hibernation in vivo. Both changes in ultrastructure and in the protein expression pattern of dedifferentiation markers as seen in chronic hibernating myocardium were seen in the co-cultured cardiomyocytes. Conclusion: Hibernation-like changes can be induced by co-culturing adult rabbit cardiomyocytes with fibroblasts. This cellular model can be a valuable tool in identifying and characterizing the pathways involved in the dedifferentiation phenotype in vivo, and already suggests that many of the structural changes accompanying dedifferentiation are not per se dependent on a decreased oxygen availability

Partial cell fusion: A newly recognized type of communication between dedifferentiating cardiomyocytes and fibroblasts

Objective: Fibroblasts have been shown to couple to neonatal cardiomyocytes in heterocellular cultures through functional gap junctions. Our objective was to provide evidence for an additional type of heterocellular communication between fibroblasts and adult cardiomyocytes in vitro and in vivo. Methods: The contact areas in heterocellular co-cultures were evaluated by specific labeling and the intercellular communication was studied using preloading of fibroblasts with tracer molecules. Heterocellular fibroblast-cardiomyocyte contacts present in the in vitro setting and in the border zone of a rabbit myocardial infarction in vivo were further examined by electron microscopy. Results: Addition of fibroblasts preloaded with the fluorescent low molecular weight tracer calcein-AM to cultured myocytes indicated early dye transfer via connexin 43 functional gap junctions. At a later time-period after co-culturing, dye transfer of fibroblasts preloaded with the high molecular weight tracer dextran 10,000 suggested partial cell fusion. The membrane continuity giving rise to this partial cell fusion was confirmed by electron microscopy, clearly showing areas of intercytoplasmic contacts between fibroblasts and phenotypically adapted (dedifferentiated) cardiomyocytes. Fluorescein-labeled annexin V affinity studies revealed transient exposure of phosphatidylserine at the contact sites, suggesting that phosphatidylserine mediates the fusion process. Close contacts between cardiac fibroblasts and dedifferentiated cardiomyocytes accompanied by disruption of the basal lamina were observed in the border zone of a rabbit myocardial infarction in vivo. Conclusion: Our results suggest that the partial cell fusion-type of heterocellular communication in our co-culture model and the contacts observed in vivo may lead to new insights in cardiovascular disease