Sigmar1's Subcellular Localization and Function in the Heart

Abstract

Background Sigma-1 receptor (Sigmar1) is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of Sigmar1 targeted ligands in clinical and preclinical rodent models of heart failure. However, Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite extensive studies, Sigmar1's subcellular localization and transmembrane topology in cardiomyocytes remain elusive. Objective We determined Sigmar1's subcellular localization, transmembrane topology, and functionality using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Methods and Results Subcellular fractionation of heart cell lysates showed Sigmar1's localization in purified mitochondria fraction. Sigmar1's mitochondrial localization in isolated adult mouse cardiomyocytes by immunocytochemistry was confirmed by Sigmar1 co-localization with the mitochondrial indicator OXPHOS. We performed quantum dots in transmission electron microscopy and observed Sigmar1 leveled quantum dots on the mitochondrial membranes. We also confirmed Sigmar1 localization confocal microscopy using isolated mitochondria stained with Mito-Tracker red and Sigmar1 with the anti-Sigmar1 antibody. We confirmed Sigmar1 is an integral mitochondrial membrane protein by performing a series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria. We determined the Sigmar1's structural requirement for mitochondrial localization by expressing Sigmar1 fragments in cells. Immunostaining with anti-FLAG antibody showed that full-length Sigmar1 and Sigmar1's C terminal-deletion mutants (amino acid (aa) 1-223 and aa1-101) is localized in the mitochondrial membrane (mitochondria stained with Mito-Tracker red) whereas N- terminal deletion mutants (aa 102-223) were unable to incorporate into the mitochondria. Finally, we determined the functional role of Sigmar1 in cardiomyocytes using high-resolution Respirometry (Oroboros O2K) in intact cardiomyocytes. Genetic loss-of-function studies demonstrated that siRNA knockdown of Sigmar1 causes a significant reduction of oxygen flux in the presence of Complex I substrates and mitochondrial respiration un-coupler, under ADP supported OXPHOS state in isolated neonatal cardiomyocytes. Conclusions Overall, our studies revealed that Sigmar1 localizes to mitochondrial membranes and indispensable for maintaining mitochondrial respiratory homeostasis in cardiac myocytes.