Muscle Hypertrophy is Regulated by Acetylation and Phosphorylation of the Actin Capping Protein CapZ

This goal of this thesis is to investigate the assembly of the actin thin filament in exercise or disease because this is a major component of the contractile mass of the heart cell. The focus is the actin capping protein, CapZ, which binds to the barbed ends of the actin filaments and regulates their assembly. The approach is to use cultured myocytes stimulated for growth or tissue from the failing human heart. First, to address physiologic short-term processes, neonatal rat ventricular myocytes in culture were subjected to cyclic 10% strain at 1 Hz for 1 hour to mimic increased mechanical loading during a normal bout of exercise. CapZ and actin dynamics were analyzed by fluorescence recovery after photobleaching (FRAP). After cyclic strain, CapZ dynamics were increased and but then abated 2~3 hours after cessation of the stimulation. Similarly, actin dynamics initially increased, and then abated 1.5~2 hours after the end of stimulation. Also, the deletion of the CapZβ1 C-terminus mimicked mechanical stimulation that altered CapZ and actin dynamics, showing its importance in actin capping. The very rapid increases in the dynamics of both actin and CapZ suggest post-translational regulations (PTMs) might be the underlying mechanism since there is insufficient time for transcriptional regulation. Chronic stimulation was mimicked by neurohormonal stimulation to achieve a hypertrophic state by 24 hours treatment with 10μM phenylephrine. After treatment, more spots were seen with 2D electrophoresis for CapZβ1 with more negative charges, suggesting that PTMs of CapZβ1 were up-regulated. Mass spectrometry showed the increased PTM spots included the acetylation of K199 and phosphorylation of S204, which are both close to the actin-binding region of CapZ. CapZ dynamics and acetylation were increased with HDAC inhibitors (trichostatin A 5μM/5 hours and MGCD0103 500nM / 24 hours). The effect of phenylephrine on CapZ dynamics was diminished by HDAC activator (10μM theophylline / 24 hours), suggesting CapZ dynamics were regulated via CapZ acetylation by altered HDAC1-3 activities. HDAC3 was decreased in the myofibrillar compartment of myocytes but increased in the membrane fraction, suggesting the diminished HDAC activities in hypertrophic myocytes were due to HDAC translocation. Also, CapZ dynamics and PTMs were increased with constitutively active PKCɛ (caPKCɛ) expression, but were diminished with dominant negative PKCɛ (dnPKCɛ), suggesting that CapZ dynamics can be regulated by PKCɛ and CapZ phosphorylation. Finally, the inhibitory effects on PTMs and dynamics of CapZ by the dominant negative PKCɛ were counteracted by MGCD0103, suggesting acetylation overrode the increased CapZ dynamics. Interestingly, PTMs of endogenous CapZβ1 were also elevated in the failing human heart compared to normal human ventricular tissue, suggestion the involvement of PTMs in human heart disease. Better understanding of the differences of specific PTMs in exercise or chronic activation may provide a therapeutic target for the future treatment of hypertrophic heart diseases.