Abstract P2009: MPTM1: A Cardiac-Enriched Microprotein That Is Upregulated During Heart Failure

Abstract

Although they were originally misclassified as lncRNAs due to oversights in genome annotation methods, microproteins—proteins with open reading frames (ORFs) of 150 amino acids or less—execute critical roles in heart and skeletal muscle. We have identified a novel microprotein, MPTM1, that is enriched in cardiac and skeletal muscle tissues. The MPTM1 amino acid sequence is strongly conserved across multiple species, which indicates that it is a functional microprotein that has been maintained in response to evolutionary pressure. Genome-wide association studies (GWAS) of patients have demonstrated that MPTM1 is upregulated during heart failure. Additionally, MPTM1 expression strongly increases during skeletal muscle differentiation and maturation. In the present study, we assessed the hypothesis that MPTM1 promotes heart and skeletal muscle homeostasis using both in vitro and in vivo gain- and loss-of-function approaches. Furthermore, we generated C2C12 myoblast and NIH3T3 fibroblast cell lines null for MPTM1 using CRISPR/Cas9 gene editing methods. MPTM1 depletion in knockout cell lines resulted in striking membrane and nuclear abnormalities. Additionally, MPTM1 null C2C12 cells exhibited defects in myoblast differentiation characterized by premature myosin expression, altered cellular architecture, abnormal nuclear distribution, and the accumulation of intracellular vacuoles. We further evaluated MPTM1 cellular function using CRISPR/Cas9 homology-directed repair (HDR) to insert tandem affinity tags (3XHA-V5 or 3X FLAG) into the MPTM1 locus to assess binding partners in vitro using immunoprecipitation and mass spectrometry. Our results provide strong evidence that MPTM1 is a multi-localizing protein that is differentially recruited to the cytosol, nucleolus, and mitochondria, indicating it may act as a key intracellular signaling effector in response to stress. Additional studies in MPTM1 null mice are ongoing and will provide critical insight into MPTM1 function in the heart at under normal physiologic conditions and in response to pathological stress.