Spectrin in developing normal and cardiomyopathic hamster heart.

In the present study, we compare fetal, newborn and adult normal and cardiomyopathic hearts for the presence of spectrin. Spectrin is a cytoskeletal-membrane protein found associated with the membranes and cortical regions of a variety of cell types. Its function in non-erythroid tissue is not fully understood. We determined previously that spectrin is associated with the membranes, myofibrils, intercalated discs and possibly the T-tubules of developing normal Syrian hamsters. Thus, it is a good candidate for comparison with cardiomyopathic (CM) Syrian hamster hearts since this model system exhibits myofibril and membrane abnormalities. Normal and CM hamsters were analysed at three stages of fetal development, as well as newborn, and adult stages using immunohistochemical, electron microscopic, and electrophoretic techniques. The data presented here indicate that no obvious difference exists in the distribution of spectrin between normal and CM hearts. Thus, the isoform of spectrin recognized by our antibody with the present protocols is not affected by the cardiomyopathic condition and can be ruled out as a contributor to the myofibril disarray and membrane-associated defects seen in these animals.

Studies of hamster cardiac myofibrillogenesis in vivo with antibodies to spectrin, desmin, and alpha-actinin.

The spectrins are a family of cytoskeletal-membrane proteins that have generated much interest in the past decade. In the present study, we utilized immunohistochemical, morphological, and electrophoretic techniques to assess the possible function(s) of spectrin in mammalian cardiac tissue during development. Antibodies generated against alpha-actinin and desmin were also employed to identify myofibrils and intermediate filaments in relation to changes in the distribution of spectrin. Spectrin is localized along the sarcolemma of pre-myofibrillar hamster cardiac myocytes (day 8, postcoitum) and remains associated with the cell membrane throughout development. The staining pattern is somewhat diffuse at first, but eventually the cell margin becomes clearly defined by day 13 postcoitum. A second, more profound change in the distribution of spectrin occurs during the newborn stage, when spectrin begins to appear in the sarcoplasm. It appears as regularly spaced invaginations that are diffuse at first, eventually attaining a position around the Z-bands of adult muscle. The change in the distribution of spectrin coincides temporally with the appearance of T-tubules, which are sarcolemmal invaginations that reside at the Z-bands of adult heart. Thus, spectrin may act as a guidance mechanism for the proper positioning of T-tubules around the Z-discs of mammalian cardiac tissue. Although spectrin does not appear to interact directly with early myofibrils it may assist in the proper alignment of T-tubules and, in doing so, act to stabilize the entire contractile apparatus by enveloping it and attaching it to the sarcolemma.

Immunocytochemical studies of spectrin in hamster cardiac tissue.

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.