MOLECULAR MECHANISMS OF MYOFIBRIL ASSEMBLY IN HEART

`We investigated the assembly of the first sarcomeres in chicken embry os by confocal microscopy of immunofluorescently stained whole mount r udiments of early chicken hearts isolated around the onset of beating. In embryos with merely 9 somites, myomesin was found to be present in a cross striated pattern, indicating that myomesin is expressed rathe r early during development. RNA studies confirmed these findings and R T-PCR revealed the presence of myomesin mRNA already in the 7 somite e mbryo. The expression of myomesin mRNA coding for the skeletal isoform preceded the heart specific transcript. In the adult heart, however, only the heart isoform was detectable. The interaction of myomesin dom ains with the sarcomere was investigated by transfection of epitope ta gged constructs into cultured cardiomyocytes. The second domain of myo mesin, an immunoglobulin-like domain, was found to specifically bind t o the M-band region of chicken cardiomyocytes. All constructs containi ng this domain also showed M-band localization. Additionally, construc ts consisting of either the second domain of myomesin or the myosin li ght chain isoform MLC 3f fused to the green fluorescent protein (eGFP) were expressed in rat cardiomyocytes and were found to be distributed in the same manner as the expression constructs tagged only with the much shorter VSV epitope. Transfected cells did not show any alteratio n in beating activity and no alteration of myofibrillar structure, as judged by simultaneous staining for the Z-disc protein alpha-actinin a nd other sarcomeric markers. Apparently, the addition of eGFP did not disturb the assembly properties or the function of the two proteins an d therefore allowed the easy visualization of assembly and contraction processes directly in the living cardiomyocyte.