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

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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 ≈2-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–β1 and anti–β3 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 stretch-conditioned 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[3,4-d]pyrimidine blocked stretch-induced upregulation of mechanical junction proteins but not Cx43. Thus, stretch upregulates expression of both electrical and mechanical junction proteins via integrin-dependent activation of FAK. Stretch-induced upregulation of Cx43 expression is mediated by FAK-dependent secretion of VEGF. In contrast, stretch-induced upregulation of adhesion junction proteins involves intracellular mechanotransduction pathways initiated via integrin signaling and acting downstream of src kinase.