Abstract
In an attempt to uncover the linkage between hemodynamic load and cardiac hypertrophy, we focused on the cytoskeletal (CSK) assembly of signaling proteins in pressure-overloaded feline myocardium. Analysis showed CSK association of c-Src, β3-integrin, FAK, PTP-1B, and p130Cas in 4 to 48 hour pressure overloaded (PO) myocardium. This assembly was accompanied by increased amounts of both total fibronectin (FN) and vitronectin (VN) and their attachment to the cardiocyte sarcolemma, indicating that a change in extracellular matrix (ECM) composition might be responsible for the CSK assembly of these signaling proteins. Furthermore, analysis with adult feline cardiocytes cultured in a three-dimensional (3-D) gel matrix made of either collagen containing FN and VN or of agarose alone for 12 hours revealed a CSK assembly of some of the signaling proteins only in collagen matrix, an event that could be blocked with RGD peptide. To investigate the role of CSK-assembled signaling proteins, activation of the two S6 kinase (S6K) isoforms, p70S6K and p85S6K, that are involved in translational and transcriptional activation was measured in PO myocardium. While both isoforms are activated substantially, p70S6K activation occurred maximally at 1 hour of pressure overload prior to the CSK assembly of signaling proteins. The activation of both isoforms of S6K was found to be mediated by a PI 3-kinase-independent pathway with the possible involvement of protein kinase C. Furthermore, studies performed in vitro by embedding adult cardiocytes either in collagen or in agarose gels and stimulating them electrically to contract showed S6K activation in both types of gels, although CSK association of signaling proteins was present only in the collagen gel matrix. Therefore, these studies demonstrate that pressure overload activates both integrin-dependent and -independent signaling, which results, respectively, in cytoskeletal recruitment of signaling proteins and in S6K activation. Both may play critical roles in hypertrophic growth regulation.
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