Acetylation and Phosphorylation Post-Translational Modifications of the CapZ β1 Subunit Regulate FRAP Dynamics Leading to Myocyte Hypertrophy

Abstract

The mechanism by which more thin filaments are built during cardiac hypertrophy is not fully understood. Very rapid increases the dynamics both actin and the actin capping protein (CapZ) following mechanical flexing suggest that a post-translational regulation is the underlying mechanism. Neonatal rat ventricular myocytes in culture were stimulated to hypertrophy by a neurohormone (10μM phenylephrine, PE, for 24 hr). CapZ dynamics were analyzed by fluorescence recovery after photobleaching (FRAP) using CapZβ1-GFP. After PE treatment, CapZ dynamics increased above resting controls by ∼3.17 fold (p=0.0004). Post-translational modifications of CapZ were analyzed by 2D gel electrophoresis. After PE treatment, 2D spots of CapZβ1-GFP have an increased negative shift, suggesting that post-translational modification of CapZ is up regulated. To identify the types of post-translational modifications, 2D western blotting and mass spectrometry (MS) was applied. Increased post-translational spots included the acetylation of K199 and phosphorylation of S204, which are both close to the actin-binding region of CapZ. To test whether CapZ acetylation was mediated by HDAC3, located at the Z-disc of myocytes, the class I HDAC inhibitor (5μM trichostatin A / 5hr) and HDAC3 activator (10μM theophylline / 24hr) were applied. CapZ dynamics with trichostatin A increased by four-fold (p=0.01), and the effect of PE on CapZ dynamics was blunted by theophylline (p=0.09). Thus, the increased sarcomere remodeling during cardiac hypertrophy may be induced via altered acetylation of CapZ, which is mediated by HDAC3. In addition to MS identification of phosphorylation sites, post-translational modifications were reduced by dominant negative PKCɛ, suggesting a regulatory role. Together the acetyl and phospho posttranslational modifications of CapZ reduce the capping property and may increase thin filament assembly. NIH HL62426 (BR) and AHA 12PRE12050371 (Y-H L).