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 alpha-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.

Postnatal changes in caldesmon expression and localization in cardiac myocytes

“…: +1 403 220 3873;fax: +1 403 270 2211;e-mail: kargacin@ucalgary.ca filaments (see Kargacin & Walsh, 1995;Marston & Huber, 1996;Morgan & Gangopadhyay, 2001). In previous work (Scott-Woo et al 1998), using both polyclonal and monoclonal antibodies to smooth muscle hand l -caldesmon along with antibodies to α -actinin and myosin, we demonstrated the presence of caldesmon at the Z-lines in adult cardiac myocytes. We did not detect caldesmon in the A-band in cardiac myocytes.…”

“…Myocytes from hearts of newborn to 10-day-old Sprague-Dawley rats were isolated using a neonatal cardiomyocyte isolation kit purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA) following the instructions provided by the manufacturer. Myocytes from cardiac ventricles of day 10 and older Sprague-Dawley rats were isolated as described previously (Scott-Woo et al 1998;Phillips et al 1999) using the method of Rodrigues et al (1992) with the following modification. Instead of removing the heart prior to perfusion with digesting enzymes, the heart was kept in situ in the dead animal and perfused through the aorta with the enzyme solutions.…”

“…SDS-PAGE of isolated cell homogenates was carried out as described previously (Scott-Woo et al 1998) in 10% or 5-15% gradient polyacrylamide gels using the discontinuous buffer system of Laemmli (1970). Separated proteins were transferred to nitrocellulose membranes in the presence of transfer buffer with SDS as described previously (Scott-Woo et al 1998;Phillips et al 1999).…”

“…Caldesmon is known to be present in non-muscle cells as well as in cardiac (Scott-Woo et al 1998;Phillips et al 1999) and smooth muscle cells (for recent reviews see Kargacin & Walsh, 1995;Marston & Huber, 1996;Morgan & Gangopadhyay, 2001). Two molecular weight forms (120 -150 kDa and 70 -80 kDa on SDS-PAGE) are known to exist.…”

Inhibition of SERCA2 Ca2+-ATPases by Cs+

“…Cardiac ventricular tissue was harvested and cardiac myocytes isolated enzymatically from ventricular muscle as described previously [15,21] using the procedure of Rodrigues et al [20]. The plasma membrane was then permeabilized with saponin using the method described previously for the permeabilization of isolated smooth muscle cells [6].…”