Abstract 16819: Activation of Mitochondrial Calpain 1 Contributes to Complex I Damage During ER Stress

Abstract

Introduction: ER (Endoplasmic reticulum) stress is increased in failing hearts. The increased ER stress impairs complex I in cardiac mitochondria (MITO). Calpain 1 (CPN1) and calpain 2 (CPN2) are calcium-dependent cysteine proteases located in cytosol and MITO (mCPN1 and mCPN2). Activation of mCPN1 contributes to complex I damage during ischemia-reperfusion (IR) and aging. Activation of the mCPN2 causes complex I damage following IR. However. the role of mCPN1 and mCPN2 activation in ER stress-mediated complex I damage remains unclear. Hypothesis: ER stress leads to complex I damage by activating mCPN1 and mCPN2. Methods: Wild type (WT) and conditional cardiomyocyte specific calpain 4 (CPN4) knockout (KO) mice were used. CPN4 is a small regulatory subunit of CPN1 and 2, and KO of CPN4 eliminates CPN1 and CPN2 activities. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in both WT and KO mice through one time IP injection. DMSO was used as vehicle treatment. Mice were sacrificed for cardiac mitochondrial isolation after 72 hours of vehicle or TUNI treatment. Results: ER stress was increased in both WT and KO mice, as shown by increased BIP in TUNI-treated mice (Figure, Panel A). Compared to vehicle, the rate of oxidative phosphorylation (OXPHOS) was decreased in TUNI-treated WT when glutamate + malate was used as complex I substrate (Panel B). In contrast, TUNI treatment did not alter OXPHOS with complex I substrate in KO (Panel B). Direct measurement of complex I activity showed that TUNI treatment only decreased complex I in MITO from WT but not from KO (Panel C). Proteomic study showed that the contents of complex I subunits including NDUFV2 and ND5 were decreased in WT but not in KO mice (Panel D); confirmed by western blotting (data not shown). These results indicate that activation of mCPN1 and mCPN2 contributes to the degradation of complex I subunits during ER stress. Conclusion: Induction of acute ER stress using TUNI damages complex I by activating mCPN1 and mCPN2.