Dynamics of early contact formation in cultured adult rat cardiomyocytes studied by N-cadherin fused to green fluorescent protein

We investigated dynamic events during the formation of intercalated disc-li ke structures of adult rat cardiomyocytes (ARC) in long-term culture. Given the complexity of ARC cytoarchitecture after de- and re-differentiation, a nd the non-uniform morphological development of individual cells, green flu orescent protein (GFP) technology was used to track N-cadherin in living ce lls. Sorting and functionality of the GFP fusion protein was tested in ARC. Isolated ARC were micro-injected with the expression construct at the onse t of spreading in culture, and the fluorescence signals were tracked during contact formation and in fully redifferentiated living cells, The first co ntact sites were found to be established by cellular protrusions, which wer e marked by an ultrastructure similar to microspikes and probably have a ro le as exploratory units in the spreading phase. Subsequently, initial conta ct sites served as anchorage for the most prominent stress fibre-like struc tures. The fusion protein appeared before connexin-43 at newly established cell-cell contacts. Membrane invaginations at the sarcolemma facing the sub stratum of cultured ARC may be responsible for the appearance of a striped pattern of N-cadherin and other adherens junction proteins away from interc alated disc-like structures. The stripes were immobile in redifferentiated cells, while the distinct small fluorescent particles in the cell body were found to move directionally at speeds around 10 mu m/min. These results co ntribute to the understanding of the mechanisms of cell-cell contact format ion of adult cardiomyocytes, which is a prerequisite for any future implant ation technology. (C) 2000 Academic Press.