Activation of Mitochondrial Calpains Contributes to the Selective Degradation of Specific Mitochondrial Proteins

Abstract

INTRODUCTIONCalpain 1 (CPN1) and 2 (CPN2) are ubiquitous calpains that exist in both cytosol and mitochondria. Calpains are calcium‐activated proteases. Pharmacologic inhibition of CPN1 and CPN2 decreases cardiac injury by improving mitochondrial function during ischemia‐reperfusion. Since both CPN1 and CPN2 include one large subunit and a small regulatory subunit (calpain 4, CPN4), genetic removal of CPN4 eliminates the activities of CPN1 and CPN2. CPN4 knockout mice were used in this study to test if activation of mitochondrial calpains leads to mitochondrial damage by proteolysis of mitochondrial proteins.HYPOTHESISWe hypothesize that exogenous calcium‐mediated activation of mitochondrial CPN1 and CPN2 impairs mitochondrial function through degradation of mitochondrial proteins.METHODSPurified cardiac mitochondria were isolated from wild type and CPN4 knockout mice. Exogenous calcium (25 uM) was used to activate mitochondrial CPN1 and CPN2. Mitochondria were subjected to proteomic analysis using tryptic digestion, liquid chromatography, and mass spectrometry with iTRAQ quantification.RESULTSWe focused on the proteins that were decreased by exogenous calcium exposure in mitochondria from wild type but not in CPN4 knockout mice as specific targets of mitochondrial CPN1 and CPN2. In wild type mice, calcium treatment significantly decreased the contents of multiple metabolic enzymes involving fatty acid oxidation and oxidative phosphorylation (electron transport chain components and ATP synthase) compared to non‐calcium treated mitochondria. Knockout of CPN4 preserved these enzyme contents in calcium‐treated mitochondria (Table). Decreases in ATPase factor 6 and very long chain acyl‐coA dehydrogenase were confirmed by Western blot.CONCLUSIONActivation of mitochondrial calpains leads to decreased mitochondrial function by impairing metabolic enzyme activity. Prevention of CPN1 and CPN2 activation provides an attractive approach to protect mitochondria during pathologic conditions including ischemia‐reperfusion and heart failure.Support or Funding InformationThis work was supported by the Office of Research and Development, Medical Research Service Merit Review Award (2IO1BX001355‐01A2) and Department of Veterans Affairs (EJL); National Institutes of Health NIH R21AG054975‐01 (QC).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.