Mechanical Strain Regulates Contractile Protein Dynamics Related to Heart Remodeling

The mechanisms of sarcomere remodeling in cardiomyocyte hypertrophy are complex, and our previous studies have identified an important role for protein kinase C-ε (PKCε) and the f-actin capping protein CapZ in this process. We now hypothesize that CapZ undergoes PKCε-dependent post-translational modification, which affects its binding kinetics in response to cyclic mechanical strain (CS). Neonatal rat ventricular myocytes were subjected to 10% CS at 1Hz for 1h. CapZ dissociation rates were analyzed by fluorescence recovery after photobleaching (FRAP) using adenoviruses expressing wildtype (wt) GFP-CapZβ1 or wt GFP-CapZβ2. To examine the role of PKCε-dependent post-translational modification of CapZ, cells were infected (100 moi, 24h) with adenoviruses expressing dominant-negative (dn) or constitutively active (ca) PKCε prior to CS. CS enhanced dissociation rates for CapZβ1 (196%, P<0.05) above resting controls, but CapZβ2 was unchanged. caPKCε significantly enhanced dissociation rates for CapZβ1 above controls (176%, P<0.05). Additionally, CapZ post-translational modifications were investigated with quantitative two-dimensional gel electrophoresis (2DGE). 2DGE western blotting identified two post-translational modifications of CapZβ1 and one of CapZβ2. Cyclic strain deceased the unmodified form of CapZβ1 (P<0.05) and increased the doubly modified form (P<0.05) compared to controls, while the addition of dnPKCε with CS reversed this effect (P>0.05). Treatment of caPKCε on unstretched cells increased the singly modified form compared to controls (P<0.05). Neither CS nor either PKCε adenovirus had any effect on CapZβ2 dynamics or post-translational profile. Taken together, our data strongly suggests that CS produces post-translational modification of CapZβ1 but not of CapZβ2. Furthermore, since caPKCε modifies the post-translational profile of CapZβ1 differently from mechanical stress, other mechanisms must also be operative. It seems likely that PKCε post-translational modification and other signaling pathways are both needed for CapZ-actin capping regulation necessary for cell remodeling and hypertrophy.