Identification and localization of caldesmon in cardiac muscle

Abstract

Caldesmon has been detected in smooth muscle and in a number of non-muscle cells. It binds both actin and myosin and may act as a regulator of contraction or a structural element in smooth muscle. The presence of caldesmon in striated muscle has not been well established. To address this issue, polyclonal antibodies and a panel of monoclonal antibodies were raised against chicken gizzard smooth muscle caldesmon and used to demonstrate that caldesmon is present in adult cardiac muscle of a variety of mammalian species. Western-blot analysis revealed the presence of caldesmon in ventricular myocytes isolated from rat heart. The epitopes for the individual monoclonal antibodies were mapped to the caldesmon primary structure using chymotryptic and 2-nitro-5-thiocyanatobenzoic acid fragments. Bovine and rat cardiac caldesmons were recognized only by a subset of these monoclonal antibodies, indicating primary sequence differences from the chicken smooth muscle protein. Immunofluorescence labelling of isolated myocytes from rat, rabbit and guinea pig cardiac muscle revealed a striated pattern of fluorescence labelling. Dual labelling of caldesmon and myosin or caldesmon and α-actinin demonstrated that caldesmon was present at the centre of the I-band rather than in the A-band, as might have been expected from the myosin binding properties of the smooth muscle protein. These results suggest a structural role for caldesmon in cardiac muscle cells.