Abstract P3040: Mitochondrial Membrane Protein Sigmar1 Regulates Mitochondrial Ultrastructure And Respiratory Functions In The Heart

Background: Sigma-1 receptor (Sigmar1) is multifunctional, ubiquitously expressed chaperone protein in mammalian cell systems. Earlier biochemical and immuno-electron microscopic analysis revealed Sigmar1’s cellular localization depends on cell and tissue systems, explaining its involvement in regulating diverse cellular and molecular functions. However, Sigmar1’s localization at organelle levels, membrane tropism, and direct molecular functions in cardiomyocytes remain elusive. Objective: We conducted systematic biochemical, microscopic, and functional assays to define Sigmar1’s subcellular localization, membrane topology, and functions in the heart. Methods and Results: Immuno-labeled quantum dots (QDs) in transmission electron microscopy revealed anti-Sigmar1 labeled QDs on the mitochondrial membranes, lysosomes, and at the sarcoplasmic reticulum-mitochondria interface. Subcellular fractionation of heart lysates also confirmed Sigmar1’s distribution in purified mitochondrial and lysosomal fractions. We further confirmed Sigmar1’s mitochondrial localization by Sigmar1’s colocalization with Mito-Tracker Red labeled isolated cardiac mitochondria. Biochemical experiments constituting alkaline extraction, detergent solubilization, and dose-dependent Proteinase K digestion on isolated heart mitochondria demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1’s structural requirement for mitochondrial localization by expressing Flag-tagged Sigmar1 fragments demonstrated N-terminal required for mitochondrial localization. High-resolution mitochondrial respirometry studies showed that genetic loss of Sigmar1 in primary cardiomyocytes reduces mitochondrial respiration. Sigmar1 knock-out mice hearts also showed the accumulation of abnormal, irregular-shaped giant mitochondria and defects in mitochondrial respiration. Conclusions: Overall, our studies demonstrated that Sigmar1 is an integral mitochondrial membrane protein and is essential in maintaining mitochondrial ultrastructure and respiratory functions in the heart.