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

ABSTRACT

We investigated dynamic events during the formation of intercalated disc-like structures of adult rat cardiomyocytes (ARC) in long-term culture. Given the complexity of ARC cytoIarchitecture after de- and re-differentiation, and the non-uniform morphological development of individual cells, green fluorescent protein (GFP) technology was used to track N-cadherin in living cells. Sorting and functionality of the GFP fusion protein was tested in ARC. Isolated ARC were micro-injected with the expression construct at the onset of spreading in culture, and the fluorescence signals were tracked during contact formation and in fully redifferentiated living cells. The first contact sites were found to be established by cellular protrusions, which were marked by an ultrastructure similar to microspikes and probably have a role as exploratory units in the spreading phase. Subsequently, initial contact sites served as anchorage for the most prominent stress fibre-like structures. The fusion protein appeared before connexin-43 at newly established cell-cell contacts. Membrane invaginations at the sarcolemma facing the substratum of cultured ARC may be responsible for the appearance of a striped pattern of N-cadherin and other adherens junction proteins away from intercalated 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 micro m/min. These results contribute to the understanding of the mechanisms of cell-cell contact formation of adult cardiomyocytes, which is a prerequisite for any future implantation technology.