Mechanisms Regulating Expression of Cell Cell Junction Proteins in Response to Mechanical Load

To elucidate mechanisms regulating expression of intercellular junction proteins, we developed an in vitro system in which monolayers of neonatal rat ven tricular myocytes are grown on silicone membranes and subjected to uniaxial pulsatile stretch [13]. Imposition of this mechanical load rapidly induces a hypertrophic response which can be rigorously quantified and characterized. An important feature of this response is a marked increase in Cx43 expression and enhanced intercellular coupling. After only 1h of stretch (110% of resting length at 3 Hz), expression of Cx43 is increased by approximately twofold, resulting in a significant increase in both the number of gap junctions and the velocity of impulse propagation [13]. We have shown previously that upregu-lation of Cx43 expression is mediated by stretch-induced secretion of vascular endothelial growth factor (VEGF) which acts in an autocrine fashion [14]. Incubation of cells with exogenous VEGF for 1h increases Cx43 expression by an amount roughly equal to that seen after 1h of pulsatile stretch. Moreover, stretch-induced upregulation of Cx43 expression can be blocked by stretching cells in the presence of anti-VEGF or anti-VEGF receptor antibodies.

To determine whether stretch-induced formation of new gap junctions requires concomitant assembly of new mechanical junctions, we measured changes in mechanical junction protein expression in cells subjected to stretch [15]. The amounts of plakoglobin, desmoplakin, and N-cadherin at cell-cell junctions all increased by at least twofold in myocytes subjected to 1h of pulsatile stretch (Fig. 5.5). However, VEGF secretion plays no role in this process. For example, addition of exogenous VEGF does not affect plakoglo-bin, desmoplakin, or N-cadherin expression, nor is stretch-induced upregulation of these proteins blocked by anti-VEGF antibodies. To further define the responsible mechanisms, we studied the role of focal adhesion kinase (FAK), which is phosphorylated in response to integrin engagement and activates

Fig.5.5 • Representative confo-cal immunofluorescence images (a) (top = control,bottom = stretch and quantitative confo-cal microscopy data (b) showing the effects of stretch on expression of Cx43 and the mechanical junction proteins, plakoglobin, desmoplakin, and N-cadherin.* p<0.05 compared with control

Fig.5.5 • Representative confo-cal immunofluorescence images (a) (top = control,bottom = stretch and quantitative confo-cal microscopy data (b) showing the effects of stretch on expression of Cx43 and the mechanical junction proteins, plakoglobin, desmoplakin, and N-cadherin.* p<0.05 compared with control multiple intracellular signaling molecules including src kinase. We infected cardiac myocytes with an adenovirus containing GFP-FRNK, a GFP-tagged dominant-negative inhibitor of FAK-dependant signaling, and then subjected cells to pulsatile stretch [15]. FRNK blocked stretch-induced upregulation of both electrical (Cx43) and mechanical (N-cadherin, desmoplakin, and plakoglobin) junction proteins. Infection of cells with virus expressing GFP alone had no effect. Addition of exogenous VEGF to FRNK-in-fected cells upregulated expression of Cx43 but not mechanical junction proteins. Conditioned medium removed from uninfected cells after 1h of stretch increased Cx43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF antibodies, but stretch-conditioned medium from FRNK-infected cells had no effect on Cx43 expression. Thus, secretion of VEGF in response to stretch requires activation of FAK. Finally, the src kinase inhibitor PP2 blocked stretch-induced upregulation of mechanical junction proteins but not Cx43 [15].These results indicate that mechanical load regulates expression of both electrical and mechanical junctions proteins, but by disparate mechanisms. Cx43 expression is regulated by autocrine actions of chemical mediators secreted during stretch, whereas adhesion junction proteins are regulated by intracellular mechanotrans-duction pathways initiated via FAK and dependent on downstream activation of src kinase.

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