Identification and Characterization of the Role of REEP5 in Sarco-Endoplasmic Reticulum Formation, Maintenance, and Function in Cardiac Muscle

Abstract

Heart failure (HF) remains the most rapidly rising cardiovascular disease and the leading cause of inpatient hospitalization worldwide, with costs exceeding $30 billion dollars annually in North America. While many key regulators of heart function have been identified, yet effective therapies aimed at healing or reversing the progression of HF remain restricted due to the complex nature of the disease and a lack of understanding of the functional membrane proteome of the heart. Here, we created a blueprint of all critical membrane and membrane-associated proteins in the heart by mapping several transcriptomic- and proteomic-based datasets against our mass spectrometry dataset of membrane-enriched protein clusters from human fetal and mouse neonatal cardiomyocytes. We identified 173 membrane-associated proteins that are conserved among eukaryotic species, cardiac-enriched, and have not been previously linked to a cardiac phenotype. These poorly annotated cardiac-enriched membrane proteins represent excellent candidates in follow-up studies aimed at elucidating the underlying molecular mechanisms of cardiomyopathies and HF. One of the highly ranked, poorly annotated, and cardiac-enriched membrane proteins was REEP5, a sarco-endoplasmic reticulum (SR/ER) membrane protein. In a follow-up study we tested the hypothesis that physiological REEP5 expression is important for cardiac SR/ER organization and heart function. In vitro REEP5 depletion in isolated functional adult mouse cardiomyocytes resulted in SR/ER membrane vacuolization and impaired cellular processes including activated cardiac ER stress pathways and dysregulated Ca2+ cycles. Subsequent in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction upon pharmacological HF induction. Similarly, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse resulted in lethal diastolic cardiac dysfunction with dilated cardiac chambers and reduced ejection fraction. Altogether, these results demonstrated 1. Our cardiomyocyte membrane proteome dataset proves instrumental to studies aimed at characterizing novel regulators of heart function and identifying potential heart disease markers and/or therapeutic targets, 2. The critical role of REEP5 in cardiac SR/ER organization, embryonic heart development, and heart function.