Distinct Pathways Regulate Expression of Cardiac Electrical and Mechanical Junction Proteins in Response to Stretch

Abstract

Abstract-To define mechanisms regulating expression of cell-cell junction proteins, we have developed an in vitro system in which neonatal rat ventricular myocytes were subjected to pulsatile stretch. Previously, we showed that expression of the gap junction protein, connexin (Cx) 43, is increased by Ϸ2-fold after 1 hour of stretch, and this response is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF). Here, we report that the mechanical junction proteins plakoglobin, desmoplakin, and N-cadherin are also upregulated by pulsatile stretch but by a mechanism independent of VEGF or other secreted chemical signals. Stretch-induced upregulation of mechanical junction proteins was blocked by anti-␤ 1 and anti-␤ 3 integrin antibodies. Transfection of cells with adenovirus expressing GFP-FRNK, a dominant-negative inhibitor of focal adhesion kinase (FAK)-dependent signaling, blocked stretch-induced upregulation of Cx43 and mechanical junction proteins but did not block the ability of exogenous VEGF to upregulate Cx43 expression. Conditioned medium removed from uninfected cells after stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretchconditioned medium from GFP-FRNK cells had no effect on Cx43 expression. The src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

“…Partial knockdown of FAK, as our siRNA achieved, might for example disrupt fibronectinactivated signaling pathways that are more sensitive to total FAK levels than collagenstimulated pathways. In other cells, these FAK complexes are sensitive to a wide variety of extracellular stimuli beyond ECM binding, including glucocorticoids [36], peptide growth factors [37], and mechanical stress [38]. These same stimuli contribute to osteogenic differentiation on hMSC [5,39,40], and our data support the idea that FAK mediates this response.…”

Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip

“…We fabricated stretchable muscular thin film (MTF) substrates by selectively coating silicone membranes with a temperature-sensitive polymer and a thin layer of polydimethylsiloxane (PDMS; Fig. 1A) (23)(24)(25), building on previous efforts of Zhuang et al (26) and Yamada et al (27). Substrates micropatterned with fibronectin (FN) in a "brick wall" pattern ( Fig.…”

Cell cultures as models of cardiac mechanoelectric feedback

“…Several groups have shown that cyclic stretch upregulates the expression levels of Cx43 in cultured monolayers of neonatal rat ventricular myocytes (NRVMs) grown on silicone membranes (Wang et al, 2000;Zhuang et al, 2000;Shyu et al, 2001;Pimental et al, 2002;Shanker et al, 2005;Yamada et al, 2005). The stretch-induced upregulation of Cx43 has been related to activation of the Na-H exchanger (Wang et al, 2000), increased secretion of angiotensin II that acts on AT1 receptors (Shyu et al, 2001), secretion of vascular endothelial growth factor (VEGF) acting downstream of TGFβ and FAK in an autocrine fashion (Pimental et al, 2002;Yamada et al, 2005), and type of extracellular matrix protein on which the cells are grown (Shanker et al, 2005).…”

“…The stretch-induced upregulation of Cx43 has been related to activation of the Na-H exchanger (Wang et al, 2000), increased secretion of angiotensin II that acts on AT1 receptors (Shyu et al, 2001), secretion of vascular endothelial growth factor (VEGF) acting downstream of TGFβ and FAK in an autocrine fashion (Pimental et al, 2002;Yamada et al, 2005), and type of extracellular matrix protein on which the cells are grown (Shanker et al, 2005). Uniaxial cyclic stretch induces cell alignment of NRVMs (Terracio et al, 1988;Vandenburgh et al, 1995;Matsuda et al, 2005) and localization of Cx43 at the longitudinal cell termini that is regulated by the Rac1 pathway downstream of N-cadherin (Matsuda et al, 2006).…”