Evy Lundgren

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

Recognition of extracellular matrix (ECM) components by isolated cardiac myocytes from neonatal (4-5 days postpartum) and adult rats was determined by measuring cell attachment to substrates made of ECM components. The substrates were petri dishes coated with either fibronectin, laminin, native monomers of collagen types I, II, III, IV, and V, denatured collagen, or gels containing reconstituted collagen fibers. Adult myocytes attached efficiently to laminin and type IV collagen, weakly to fibronectin, but not at all to the other types of collagen. Neonatal myocytes attached well to all types of collagen and to fibronectin and laminin. Antibodies raised against surface membranes of neonatal myocytes, adult myocytes, or adult hepatocytes were assayed for their ability to inhibit cell attachment to the various ECM substrates. Antibodies against the surface of neonatal myocytes as well as antibodies against the hepatocyte cell surface inhibited the attachment of neonatal myocytes and hepatocytes to collagen but not to fibronectin. Antibodies against the adult myocyte cell surface did not inhibit the attachment of neonatal myocytes or hepatocytes to ECM components. These results indicate the presence of binding molecules on the surface of neonatal myocytes that are involved in the recognition of collagen at a time when collagen is being secreted and formed into a three-dimensional network that attaches to the cell surface of the myocytes. This recognition and adhesion to collagen occurs by a mechanism independent of fibronectin. The binding molecules for collagen could not be detected on normal adult myocytes isolated at a time when the formation of the collagen network has already been completed.

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro.

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.

Isolation, characterization and adhesion of calcium-tolerant myocytes from the adult rat heart

High yields of Ca2+ - stable myocytes were obtained by perfusion of adult rat heart with a buffered collagenase medium followed by mincing and three additional digestion periods. Release of lactate dehydrogenase, respiratory control, content of ATP and creatine phosphate, electrical stimulation and attachment to extracellular matrix components indicated that the sarcolemma of the isolated myocytes remained intact and that the cells maintained some of the most basic physiological functions. The myocytes maintained their rod-shape in a medium containing 2.5 mM of Ca2+ and their release of LDH was slow. Some of the myocytes were contracting spontaneously, at a low rate, in an abrupt end-to-end contraction. Other cells appeared quiescent but they were all able to respond to external electrical stimulus. The oxygen consumption was measured by a perifusion method. In different preparations the basal consumption was 14-26 nmol O2/min X 10(5) rod-shaped myocytes. Freshly isolated rod-shaped heart cells attached in 30 minutes to dishes coated with collagen type IV, laminin or fibronectin but did not attach to dishes coated with collagen type I or III or to collagen gels. Attachment occurred at the ends of the cells.

Adhesion of cardiac myocytes to extracellular matrix components

The interaction of ECM components with adult cardiac myocytes is not well understood, but is of physiological importance. Most physiological studies are conducted on myocytes in suspension yet in vivo the cells are attached to each other and to the ECM. In this paper, we further define the interaction of isolated adult myocytes with the ECM substrates. Of interest is not only the short-term attachment of cells to ECM substrates but also the ability of ECM components to support the long-term maintenance of cardiac myocytes in cultures.

Modulation of β-receptors as adult and neonatal cardiac myocytes progress into culture

SummaryModulation of β-adrenergic receptors and their ability to respond to β-receptor stimulation was studied in cultures of adult and neonatal rat cardiac myocytes. The radioligand iodocyanopindolol (125I-CYP) was used to identify β-adrenoceptors on the intact cells.125I-CYP was found to bind to the receptors in a stereospecific and saturable manner. Freshly isolated neonatal and adult myocytes both had a receptor density of approximately 50 fmol/mg protein. The number of β-receptors per milligram protein was similar during a 10-d culture period for adult myocytes but increased after a 5-d culture period for neonatal myocytes. Both cell types responded to β-receptor stimulation with isoproterenol by a twofold increase in the concentration of cAMP and this response increased with time in culture. The number of receptors as well as the response to isoproterenol was similar for neonatal myocytes cultured on laminin, collagen type I, or on uncoated culture dishes. From these data we conclude that cultured cardiac myocytes maintain functional β-receptors as they progress into culture, and the expression of β-receptors is not influenced by culture substrates.