The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes

Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1 & PRIME;s sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1 & PRIME;s direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1 & PRIME;s subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1 & PRIME;s localiza-tion in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 coloc-alization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1 & PRIME;s mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mito-chondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of pu-rified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1 & PRIME;s structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1 & PRIME;s C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mito-chondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respi-ratory homeostasis in cardiomyocytes.

Automated summary

This study reveals the localization of Sigmar1 on mitochondrial membranes in cardiomyocytes and its essential role in maintaining mitochondrial respiratory homeostasis.