... (more)

nalysis of the dynamics of the actin capping protein, CapZ, and its post-translational modifications (PTM). CapZ binding rates were assessed after strain by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) expressed by a GFP-CapZbeta1 adenovirus. To assess the role of the protein kinase C epsilon isoform (PKCepsilon), rest or cyclic strain were combined with specific PKCepsilon activation by constitutively active PKCepsilon, or by inhibition with dominant negative PKCepsilon (dnPKCepsilon) expression. Significant increases of CapZ FRAP kinetics with strain were blunted by dnPKCepsilon, suggesting that PKCepsilon is involved in mechano-transduction signaling. Similar combinations of strain and PKC regulation in NRVMs were studied by PTM profiles of CapZbeta1 using quantitative two-dimensional gel electrophoresis. The significantly increased charge on CapZ seen with mechanical strain was reversed by the addition of dnPKCepsilon. Potential clinical relevance was confirmed in vivo by PTMs of CapZ in the failing heart of one-year old transgenic mice over-expressing PKCepsilon. Furthermore, with strain there was significant PKCepsilon translocation to the Z-disc and co-localization with CapZbeta1 or alpha-actinin, which was quantified on confocal images. A hypothetical model is presented proposing that one destination of the mechanotransduction signaling pathways might be for PTMs of CapZ thereby regulating actin capping and filament assembly.