Characterization of Nrac, a Novel Cardiac and Adipose Enriched Microprotein

PROJECT SUMMARY Obesity is a widespread health concern impacting millions of individuals and is strongly associated with increased risk of cardiovascular disease (CVD). Despite advancements in understanding the metabolic implications of obesity, the connection between lipid droplet (LD) dynamics and CVD remains relatively unexplored. This project aims to address this gap by focusing on the functional characterization of a novel microprotein, Nutritionally regulated adipose and cardiac-enriched gene (Nrac), and its involvement in LD biogenesis and lipid metabolism within the cardiovascular system. Microproteins, a class of proteins often overlooked due to their small size, are increasingly recognized for their crucial roles in cellular functions. Nrac, encoded by the previously annotated noncoding RNA A530016L24Rik, has emerged as a significant player in lipid metabolism. Prior studies have shown that Nrac is highly expressed in cardiac and adipose tissues, with dynamic regulation in response to nutritional states. Our preliminary data indicates that a global gene deletion of Nrac leads to cardiac stress and altered lipid metabolism, emphasizing its potential significance in cardiovascular health. Furthermore, our data suggests Nrac localizes to the sarcoplasmic reticulum (SR) membrane, the primary site of intracellular LD biogenesis, and protein interaction data predicts its direct interaction with proteins involved in this process. Therefore, we hypothesize that Nrac localizes to the SR membrane in cardiomyocytes to coordinate LD biogenesis and subsequently contribute to lipid metabolism. Through two specific aims, this research seeks to understand the mechanisms by which Nrac influences LD dynamics and lipid metabolism. Aim 1 will investigate Nrac's localization and interaction with proteins involved in LD biogenesis using human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (hiPSC-CMs). This aim will examine the endogenous expression dynamics and localization of Nrac, along with functional assays to assess its potential role in LD biogenesis. Aim 2 will evaluate Nrac's impact on lipid metabolism in a diet-induced obesity model utilizing Nrac knockout mice. By subjecting these mice to a high-fat diet challenge, this research aims to determine whether Nrac plays a regulatory role in lipid metabolism, contributing to obesity progression. Echocardiography, metabolic assessments, lipidomic analyses, and functional studies will be conducted to evaluate the effects of Nrac deficiency and overexpression on adiposity and cardiac health. Upon completion of these aims, the project aims to provide a comprehensive understanding of Nrac's role in LD dynamics and lipid metabolism, particularly in cardiomyocytes. This knowledge could potentially identify Nrac as a therapeutic target for mitigating lipid abnormalities associated with obesity and reducing the risk of CVD.