NEK7 suppresses inflammation in the aging heart through regulation of RNA binding proteins - SUMMARY/ABSTRACT Aging is a major contributing factor to the development of heart failure (HF). Inflammation is now considered to be an important driver of the adverse cardiac remodeling and chronic low-grade inflammation is a hallmark of aging (inflammaging). NIMA-related kinase-7 (NEK7) is a serine/threonine kinase that was originally discovered to play a critical role in cell-cycle progression. The functional role of NEK7 in the heart has not been determined although NEK7 is abundantly expressed in cardiomyocytes. Our preliminary results demonstrated that NEK7 activity is decreased in ventricular lysates from old mouse hearts and that NEK7 has an ability to suppress inflammatory gene expression. Our data further suggests a possibility that the anti-inflammatory effect of NEK7 is due to enhanced mRNA degradation. RNA-binding proteins (RBPs) regulate RNA metabolism including mRNA stability and play a key role in inflammatory disease. However, how RBPs are regulated in the heart and how such regulation alters the process of inflammaging are elusive. Aim 1 determines whether NEK7 inhibits inflammation and adverse cardiac remodeling induced by aging. Aim 1A examines if NEK7 activity is decreased in purified adult mouse ventricular myocytes isolated from young and old mouse hearts. Aim 1B determines whether NEK7 activity suppresses inflammaging, using AAV9 vectors encoding constitutively active (CA) and kinase-dead (KD) dominant negative NEK7. Inflammation will be assessed by cytokines and chemokines qPCR, protein arrays, immunohistochemistry and flow cytometry. NLRP3 inflammasome priming and activation will also be examined. Aim 1C determines if NEK7 activity provides salutary effects against aging in the heart using AAV9-CA-NEK7 and AAV9-KD-NEK7. Cardiac dimensions, systolic and diastolic function, hypertrophy, fibrosis and cell death will be assessed during the course of aging. Aim 2 seeks to identify the RBPs that are phosphorylated and regulated by NEK7 to suppress inflammatory gene expression in the aging heart. Aim 2A determines if NEK7 phosphorylates and regulates the RBPs that have been established to regulate inflammatory responses. Phosphorylation of the RBPs by NEK7, effects of NEK7 on their protein stability, subcellular localization and affinity for RNA, and their contributions to the NEK7-mediated suppression of inflammatory gene expression will be examined. In Aim 2B, to systematically identify the RBPs that are phosphorylated by NEK7 in the aging heart, we will employ phosphoproteomic analysis in control, CA-NEK7 and KD-NEK7 mice at the age of 24 months. Phosphorylation of the RBPs by NEK7 will be confirmed by in vitro experiments and their contributions to the NEK7-mediated anti-inflammatory responses will be examined in cardiomyocytes. Successful completion of this R21 exploratory/developmental grant will provide evidence that NEK7 provides salutary effects against aging in the heart and identify RBPs that are phosphorylated by NEK7 to suppress inflammatory responses. Our long-term goal is to understand how the NEK7/RBPs signaling network negatively regulates inflammatory responses and adverse remodeling in the aging heart.
