Abstract P2023: Mitogen-activated Protein Kinase-directed Modulation Of The Non-sarcomeric Actin Cytoskeleton In Cardiomyocytes

Abstract

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the widening and lengthening of cardiomyocytes, respectively. Mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinases 1/2 (ERK1/2) are known to control this directional growth of cardiomyocytes, such that activated MEK1 causes concentric hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 MEK1 inhibitor, or control GFP adenovirus. The resulting data pinpointed over 2700 sites on 324 proteins as changing their phosphorylation status with modulation of this signaling cascade. Gene ontology analysis found that cytoskeletal-associated proteins were the largest subset of proteins with phosphorylation changes. Traditional and PhosTag western blots verified that many of these cytoskeletal proteins change expression as well as their phosphorylation status in NRVMs. In vivo experiments showed that cytoplasmic actin expression was upregulated in MEK1 transgenic or ERK1/2 gene-deleted hearts. Furthermore, MEK1 transgenic adult cardiomyocytes had enhanced cytoplasmic actin at lateral membranes. Palladin, an actin nucleating protein, changed both phosphorylation status and isoform expression in response to modulation of MEK1-ERK1/2 signaling. Adeno-associated viruses were used to overexpress different palladin isoforms. Immunostaining for these proteins showed that localization of palladin isoforms moved from the subcortical compartment to the sarcomere, suggesting these isoforms preferentially associate with either cytoskeletal or sarcomeric actin. Future studies will further examine the signaling roles that cytoplasmic actin may play in healthy and diseased cardiomyocytes and delineate preferential binding of actin associated proteins between cytoplasmic and sarcomeric actin in cardiomyocytes.