The role of β₁ integrin in spreading and myofibrillogenesis in neonatal rat cardiomyocytes in vitro

Abstract

The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I+III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN greater than LN greater than C I+III greater than plastic greater than BSA. Addition of anti-beta 1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration. Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of alpha-actinin aggregates into Z bands. At later times, vinculin at 8 hours and beta 1 integrin at 22 hours became co-localized with alpha-actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM-integrin-cytoskeleton associations.

Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes

“…Ubiquitination is the first step in non-lysosomal protein degradation (Hershko and Ciechanover, 1992) and we do indeed find an upregulation of ubiquitin expression in dividing cardiomyocytes. While in interphase cardiomyocytes the signal for the ubiquitin antibody is rather weak and mainly associated with the nuclei as reported previously (Hilenski et al, 1992) (Fig. 6B, cells in top left corner of E); once cardiomyocytes enter mitosis, ubiquitin starts to be spread throughout the cytoplasm (Fig.…”

Differentiation of human adipose-derived stem cells towards cardiomyocytes is facilitated by laminin

“…For example, Foster et al (1987) have found that a laminin substrate promotes myogenesis in rat skeletal muscle cultures and Hilenski et al (1992) have reported that laminin influences cytoskeletal and myofibrillar organization in vitro in neonatal rat cardiac myocytes. Further, mutations in the genes coding for the alpha-2 chain of two laminin isoforms have been shown to cause a severe form of congenital muscular dystrophy in humans and mice (Kuang et al 1998).…”

Cadherin-mediated adhesion is essential for myofibril continuity across the plasma membrane but not for assembly of the contractile apparatus

“…Previous studies have indicated that ECM components play an important role in assembly and/or maintenance of myocyte cytoarchitecture (Hilenski et al, 1992; Imanaka-Yoshida et al, 1999; Shiraishi et al, 1997). In addition, it has been proposed that focal adhesions might serve as nucleation sites for the assembly of myofibrils (Lin et al, 1989).…”

“…Similar to Ncadherin, β1 integrin is expressed in the developing and mature myocardium (Carver et al, 1994;Terracio et al, 1991). In the presence of anti-β1 integrin antibodies, normal cell spreading and myofibril organization was perturbed in cultured neonatal rat cardiomyocytes (Hilenski et al, 1992). Myocytes derived from β1 integrin double-knockout embryonic stem (ES) cells exhibited altered in vitro differentiation and abnormal sarcomeric architecture (Fassler et al, 1996).…”