Mitochondrial Calpain Research Articles

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

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.

Tunicamycin-Induced Endoplasmic Reticulum Stress Damages Complex I in Cardiac Mitochondria.

Background: Induction of acute ER (endoplasmic reticulum) stress using thapsigargin contributes to complex I damage in mouse hearts. Thapsigargin impairs complex I by increasing mitochondrial calcium through inhibition of Ca2+-ATPase in the ER. Tunicamycin (TUNI) is used to induce ER stress by inhibiting protein folding. We asked if TUNI-induced ER stress led to complex I damage. Methods: TUNI (0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 24 or 72 h following TUNI treatment for mitochondrial functional analysis. Results: ER stress was only increased in mice following 72 h of TUNI treatment. TUNI treatment decreased oxidative phosphorylation with complex I substrates compared to vehicle with a decrease in complex I activity. The contents of complex I subunits including NBUPL and NDUFS7 were decreased in TUNI-treated mice. TUNI treatment activated both cytosolic and mitochondrial calpain 1. Our results indicate that TUNI-induced ER stress damages complex I through degradation of its subunits including NDUFS7. Conclusion: Induction of the ER stress using TUNI contributes to complex I damage by activating calpain 1.

Mitochondrial localization of calpain-13 in mouse brain

Calpains are Ca2+-dependent cysteine proteases involved in various intercellular physiological functions. Although most calpains exist in the cytosol, four isoforms of calpain (calpains-1, -2, -5, -10) are also localized in the mitochondria. In the present study, we examined the mitochondrial localization of calpain-13, as a novel mitochondrial calpain, in C57BL/6J mice. The tissue distribution and mitochondrial subfractionation of calpain-13 were investigated using western blotting. Calpain-13 was present in both the mitochondrial membrane (outer membrane and inner membrane) and soluble (intermembrane space and matrix) fractions. Through immunohistochemistry, calpain-13 was found to be expressed in the cerebral cortex and hippocampus of the mouse brain. We further confirmed the localization of calpain-13 in the mitochondria of the mouse brain using immunoelectron microscopy. Our present study thus revealed that calpain-13 is localized in the mitochondria, in addition to the cytosol, in the mouse brain. Future studies investigating the enzymatic properties and physiological functions of both cytosolic and mitochondrial calpain-13 will shed light on the potential involvement of calpain-13 in neurodegenerative diseases including Parkinson's disease and Alzheimer's disease.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Calpain-mediated protein targets in cardiac mitochondria following ischemia\u2013reperfusion

Calpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)–reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC–REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC–REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC–REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia–reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H2O2 generation was also decreased in mitochondria from deletion mice following ISC–REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia–reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol.

Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast.

Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor factors induce the apoptosis of myoblasts to impair the regeneration of skeletal muscle. Cancer cachectic myoblast apoptosis is associated with mitochondria injury. It has been reported that activated mitochondrial calpain caused mitochondria injury in mouse cardiomyocytes and pulmonary smooth muscle. We wondered if mitochondrial calpains exist in skeletal myoblast and their potential role in myoblast apoptosis of cancer cachexia. We used a transwell to build a novel myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. Calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used during coculture. We found for the first time that two calpains (calpain-1 and calpain-2) and CAST were present in the mitochondria of myoblast. The activation of mitochondrial calpain decreased mitochondrial complex I activity, promoted mitochondrial permeability transition pore opening, and impaired mitochondrial membrane potential in myoblast during coculture, which induced myoblasts apoptosis. CAST and CAPT protected myoblasts from apoptosis by inhibiting mitochondrial calpain activity, which may attenuate or even reverse cancer cachectic muscle atrophy by improving muscle regeneration ability. Our study provides a new perspective for understanding the mechanism of cancer cachexia, and will further contribute to treat cancer cachexia by focusing on the mitochondrial calpain activity.

Mitochondrial calpain inhibition restores defective SR-mitochondrial crosstalk in CPVT rat myocytes

Cardiac RYR2-mediated sarcoplasmic Ca2+ (SR) release is essential for matching increased energy demand during fight-or-flight response with mitochondrial metabolic output by delivering Ca2+ into the mitochondrial matrix to activate Ca2+-dependent Krebs cycle dehydrogenases. RYR2 complex gain-of-function mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) have been linked to mitochondrial structural damage and enhanced production of reactive oxygen species (ROS). Despite being critical for arrhythmogenesis in CPVT, the exact causes of these phenomena remain undetermined. Taking advantage of a new rat model of CPVT induced by heterozygous RYR2 gain-of-function mutation S2222L, we tested how RYR2 overactivity alters mitochondrial Ca2+ and ROS handling, and how these changes cause mitochondrial structural defects. Injection of epinephrine (1 mg/kg) and caffeine (120 mg/kg) induced bigamy and bidirectional VT in vivo in 100% of CPVT rats. Simultaneous whole-cell patch clamp and confocal Ca2+-imaging demonstrated that under β-adrenergic stimulation with isoproterenol (50 nM), CPVT ventricular myocytes (VMs) exhibited severe Ca2+ mishandling and high propensity for generation of spontaneous Ca2+ waves (SCWs) that cause arrhythmogenic afterdepolarizations. Diminished Ca2+ transient amplitude in CPVT VMs resulted in a significant reduction in mitochondrial matrix-[Ca2+], and thereby a mito-ROS surge, visualized using matrix-targeted biosensors mtRCaMP1h and MLS-HyPer, respectively. Importantly, using novel Ca2+-biosensors targeted to intermembrane space (IMS-GECO), we uncovered that [Ca2+] in this compartment reaches 1 µM, sufficient for activation of Ca2+-dependent protease μ-calpain. Adenoviral overexpression of IMS-targeted calpastatin, an endogenous calpain inhibitor, reduced mito-ROS, restored cytosolic Ca2+ transient amplitude and SR Ca2+ content, and reduced RYR2-mediated SCWs in CPVT VMs. These changes were paralleled by restored expression levels of OPA1, a mitochondrial structural protein responsible for tight cristae organization. Our data suggest that enhanced mito-ROS due to matrix-[Ca2+] reduction in CPVT VMs and unexpectedly high IMS-[Ca2+] promotes IMS-calpain-mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling.

Calpain-Mediated Mitochondrial Damage: An Emerging Mechanism Contributing to Cardiac Disease.

Calpains belong to the family of calcium-dependent cysteine proteases expressed ubiquitously in mammals and many other organisms. Activation of calpain is observed in diseased hearts and is implicated in cardiac cell death, hypertrophy, fibrosis, and inflammation. However, the underlying mechanisms remain incompletely understood. Recent studies have revealed that calpains target and impair mitochondria in cardiac disease. The objective of this review is to discuss the role of calpains in mediating mitochondrial damage and the underlying mechanisms, and to evaluate whether targeted inhibition of mitochondrial calpain is a potential strategy in treating cardiac disease. We expect to describe the wealth of new evidence surrounding calpain-mediated mitochondrial damage to facilitate future mechanistic studies and therapy development for cardiac disease.

Inhibition of mitochondrial and cytosolic calpain attenuates atrophy in myotubes co-cultured with colon carcinoma cells.

Cancer cachexia is a life-threatening syndrome characterized by muscle atrophy. Cancer cachectic muscle atrophy (CCMA) is associated with mitochondrial injury. Mitochondrial calpains have been reported to induce mitochondrial injury in mouse cardiomyocytes and pulmonary smooth muscle. In the present study, the presence of calpain in the mitochondria of skeletal muscle and its potential role in CCMA were investigated. Transwell plates were used to develop a myotube-carcinoma cell co-culture model to simulate the cancer cachexia environment in vitro. The calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used to inhibit calpain activity in myotubes during co-culture. Calpain-1, calpain-2 and CAST were found to be present in mouse myotube mitochondria. Co-culture activated calpain in both cytoplasm and mitochondria, which caused myotube atrophy. CAST and CAPT treatment prevented calpain activation in both cytoplasm and mitochondria, which inhibited myotube atrophy during co-culture. Additionally, CAST and CAPT treatment increased mitochondrial complex I activity, decreased mitochondrial permeability transition pore opening and improved mitochondrial membrane potential in myotubes during co-culture. In addition, CAST and CAPT treatment increased AKT/mTOR activity, inhibited FoxO3a activity and decreased atrogin-1 content in myotubes during co-culture. The present findings provide new insights to understand the mechanism of CCMA and further help the development of focused approaches to treat CCMA by manipulating the mitochondrial and cytosolic calpain activity.

Endoplasmic reticulum stress-induced complex I defect: Central role of calcium overload

BackgroundER (endoplasmic reticulum) stress leads to decreased complex I activity in cardiac mitochondria. The aim of the current study is to explore the potential mechanisms by which ER stress leads to the complex I defect. ER stress contributes to intracellular calcium overload and oxidative stress that are two key factors to induce mitochondrial dysfunction. Since oxidative stress is often accompanied by intracellular calcium overload during ER stress in vivo, the role of oxidative stress and calcium overload in mitochondrial dysfunction was studied using in vitro models. ER stress results in intracellular calcium overload that favors activation of calcium-dependent calpains. The contribution of mitochondrial calpain activation in ER stress-mediated complex I damage was studied. MethodsThapsigargin (THAP) was used to induce acute ER stress in H9c2 cells and C57BL/6 mice. Exogenous calcium (25 μM) and H2O2 (100 μM) were used to induce modest calcium overload and oxidative stress in isolated mitochondria. Calpain small subunit 1 (CAPNS1) is essential to maintain calpain 1 and calpain 2 (CPN1/2) activities. Deletion of CAPNS1 eliminates the activities of CPN1/2. Wild type and cardiac-specific CAPNS1 deletion mice were used to explore the role of CPN1/2 activation in calcium-induced mitochondrial damage. ResultsIn isolated mitochondria, exogenous calcium but not H2O2 treatment led to decreased oxidative phosphorylation, supporting that calcium overload contributes a key role in the mitochondrial damage. THAP treatment of H9c2 cells decreased respiration selectively with complex I substrates. THAP treatment activated cytosolic and mitochondrial CPN1/2 in C57BL/6 mice and led to degradation of complex I subunits including NDUFS7. Calcium treatment decreased NDUFS7 content in wild type but not in CAPNS1 knockout mice. ConclusionER stress-mediated activation of mitochondria-localized CPN1/2 contributes to complex I damage by cleaving component subunits.

Inhibition of the ubiquitous calpains protects complex I activity and enables improved mitophagy in the heart following ischemia-reperfusion.

Activation of calpain 1 (CPN1) and calpain 2 (CPN2) contributes to cardiac injury during ischemia (ISC) and reperfusion (REP). Complex I activity is decreased in heart mitochondria following ISC-REP. CPN1 and CPN2 are ubiquitous calpains that exist in both cytosol (cs)-CPN1 and 2 and mitochondria (mit)-CPN1 and 2. Recent work shows that the complex I subunit (NDUFS7) is a potential substrate of the mit-CPN1. We asked whether ISC-REP led to decreased complex I activity via proteolysis of the NDUFS7 subunit via activation of mit-CPN1 and -2. Activation of cs-CPN1 and -2 decreases mitophagy in hepatocytes following ISC-REP. We asked whether activation of cs-CPN1 and -2 impaired mitophagy in the heart following ISC-REP. Buffer-perfused rat hearts underwent 25 min of global ISC and 30 min of REP. MDL-28170 (MDL; 10 µM) was used to inhibit CPN1 and -2. Cytosol, subsarcolemmal mitochondria (SSM), and interfibrillar mitochondria (IFM) were isolated at the end of heart perfusion. Cardiac ISC-REP led to decreased complex I activity with a decrease in the content of NDUFS7 in both SSM and IFM. ISC-REP also resulted in a decrease in cytosolic beclin-1 content, a key component of the autophagy pathway required to form autophagosomes. MDL treatment protected the contents of cytosolic beclin-1 and mitochondrial NDUFS7 in hearts following ISC-REP. These results support that activation of both cytosolic and mitochondrial calpains impairs mitochondria during cardiac ISC-REP. Mitochondria-localized calpains impair complex I via cleavage of a key subunit. Activation of cytosolic calpains contributes to mitochondrial dysfunction by impairing removal of the impaired mitochondria through depletion of a key component of the mitophagy process.

Impaired SIRT3 activity mediates cardiac dysfunction in endotoxemia by calpain-dependent disruption of ATP synthesis

BackgroundSepsis-induced cardiomyopathy contributes to the high mortality of septic shock in critically ill patients. Since the underlying mechanisms are incompletely understood, we hypothesized that sepsis-induced impairment of sirtuin 3 (SIRT3) activity contributes to the development of septic cardiomyopathy. Methods and resultsTreatment of mice with lipopolysaccharide (LPS) for 6 h resulted in myocardial NAD+ depletion and increased mitochondrial protein acetylation, indicating impaired myocardial SIRT3 activity due to NAD+ depletion. LPS treatment also resulted in impaired cardiac output in isolated working hearts, indicating endotoxemia-induced cardiomyopathy. Maintaining normal myocardial NAD+ levels in LPS-treated mice by Poly(ADP-ribose)polymerase 1 (PARP1) deletion prevented cardiac dysfunction, whereas additional SIRT3 deficiency blunted this beneficial effect, indicating that impaired SIRT3 activity contributes to cardiac dysfunction in endotoxemia. Measurements of mitochondrial ATP synthesis suggest that LPS-induced contractile dysfunction may result from cardiac energy depletion due to impaired SIRT3 activity. Pharmacological inhibition of mitochondrial calpains using MDL28170 normalized LPS-induced cleavage of the ATP5A1 subunit of ATP synthase and normalized contractile dysfunction, suggesting that cardiac energy depletion may result from calpain-mediated cleavage of ATP5A1. These beneficial effects were completely blunted by SIRT3 deficiency. Finally, a gene set enrichment analysis of hearts of patients with septic, ischemic or dilated cardiomyopathy revealed a sepsis-specific suppression of SIRT3 deacetylation targets, including ATP5A1, indicating a functional relevance of SIRT3-dependent pathways in human sepsis. ConclusionsImpaired SIRT3 activity may mediate cardiac dysfunction in endotoxemia by facilitating calpain-mediated disruption of ATP synthesis, suggesting SIRT3 activation as a potential therapeutic strategy to treat septic cardiomyopathy.

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

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.

Activation of Mitochondrial Calpain 1 Leads to Degradation of PDH

INTODUCTIONCalpain 1 (CPN1) is a ubiquitous calpain including one large subunit and a small regulatory subunit (calpain 4, CPN4). Genetic removal of CPN4 eliminates the CPN1 activity. CPN1 exists in both cytosol and mitochondria. Activation of CPN1 contributes to mitochondrial damage and cardiac injury during ischemia (ISC)‐reperfusion (REP). Pyruvate dehydrogenase (PDH) is degraded in mitochondria following ISC‐REP. Since mitochondrial CPN1 (mit‐CPN1) is found in the matrix, we propose that activation of mit‐CPN1 contributes to PDH degradation. Purified heart mitochondria were used to exclude potential cytosolic CPN1 contamination. CPN4 knockout mice were used to eliminate mit‐CPN1.HYPOTHESISWe hypothesize that activation of the mit‐CPN1 impairs oxidative phosphorylation (OXPHOS) through degradation of PDH in isolated mouse heart mitochondria.METHODSPurified cardiac mitochondria were isolated from wild‐type and CPN4 knockout mice. Exogenous calcium (25 uM) was used to activate mit‐CPN1. OXPHOS was determined in mitochondria with or without calcium treatment. Protein content of PDH E1 subunit was assessed by Western blotting.RESULTSIn wild type mice, calcium treatment significantly decreased the rate of OXPHOS using pyruvate + malate as complex I substrates compared to non‐calcium‐treated control. Succinate oxidation was also slightly decreased in calcium treated mitochondria from wild type hearts (Table). In contrast, calcium treatment did not affect OXPHOS in mitochondria from CPN4 knockout mice in both substrates. PDH E1 subunit content was decreased in calcium‐treated mitochondria from wild type mice but not from CPN4 knockout mice. A representative image is shown in Figure 1.CONCLUSIONKnockout of CPN4 prevents exogenous calcium‐mediated PDH degradation. Inhibition of CPN4 during calcium over load conditions including heart failure and endoplasmic reticulum stress may decrease cardiac injury by protecting mitochondria.Support or Funding InformationA Grant‐in‐aid (15GRNT24480123) from the American Heart Association (QC), R21AG054975‐01 (QC), and Merit Review Award (2IO1BX001355‐01, EL)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest.

Mitofilin is an inner membrane protein that has been defined as a mitochondria-shaping protein in controlling and maintaining mitochondrial cristae structure and remodeling. We determined the role of mitofilin in cell survival by investigating the mechanism underlying mitofilin knockdown-induced cell death by apoptosis. Cultured H9c2 myoblasts and HEK 293 cells were treated with mitofilin siRNA or scrambled siRNA for 24 h. Cell death (apoptosis), caspase 3 activity and cell cycle phases were assessed by flow cytometry, while cytochrome c release and intracellular ATP production were measured by ELISA. Mitofilin, apoptosis-inducing factor (AIF) and poly(ADP-ribose) polymerase (PARP) expression were measured by Western blot analysis and calpain activity was assessed using a calpain activity kit. Mitochondrial images were taken using electron microscopy. We found that mitofilin knockdown increases apoptosis mainly via activation of the AIF-PARP pathway leading to nuclear fragmentation that is correlated with S phase arrest of the cell cycle. Knockdown of mitofilin also led to mitochondrial swelling and damage of cristae that is associated with the increase in reactive oxygen species production and mitochondrial calpain activity, as well as a marked decrease in intracellular ATP production and mitochondrial membrane potential. Together, these results indicate that mitofilin knockdown by siRNA increases calpain activity that presumably leads to mitochondrial structural degradation resulting in a critical reduction of mitochondrial function that is responsible for the increase in cell death by apoptosis via an AIF-PARP mechanism and associated with nuclear fragmentation, and S phase arrest of the cell cycle.

The protection of rat retinal ganglion cells from ischemia/reperfusion injury by the inhibitory peptide of mitochondrial μ-calpain

Intracellular Ca2+-dependent cysteine proteases such as calpains have been suggested as critical factors in retinal ganglion cell (RGC) death. However, it is unknown whether mitochondrial calpains are involved in RGC death. The purpose of the present study was to determine whether the inhibition of mitochondrial μ-calpain activity protects against RGC death during ischemia/reperfusion (I/R) injury. This study used a well-established rat model of experimental acute glaucoma involving I/R injury. A specific peptide inhibitor of mitochondrial μ-calpain, Tat-μCL, was topically applied to rats via eye drops three times a day for 5 days after I/R. RGC death was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The truncation of apoptosis-inducing factor (AIF) was determined by western blot analyses. Retinal morphology was determined after staining with hematoxyline and eosin. In addition, the number of Fluoro Gold-labeled RGCs in flat-mounted retinas was used to determine the percentage of surviving RGCs after I/R injury. After 1 day of I/R, RGC death was observed in the ganglion cell layer. Treatment with Tat-μCL eye drops significantly prevented the death of RGCs and the truncation of AIF. After 5 days of I/R, RGC death decreased by approximately 40%. However, Tat-μCL significantly inhibited the decrease in the retinal sections and flat-mounted retinas. The results suggested that mitochondrial μ-calpain is associated with RGC death during I/R injury via truncation of AIF. In addition, the inhibition of mitochondrial μ-calpain activity by Tat-μCL had a neuroprotective effect against I/R-induced RGC death.

Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study

Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis.

Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release

Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I.

Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia–reperfusion

Background/objectives: Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia–reperfusion injury. Isoforms of Ca2+-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia–reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia–reperfusion. Methods and results: Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of >500μM of Ca2+ to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca2+-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca2+ to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30min of coronary occlusion followed by 30min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1. Conclusions: We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart.

Abstract 17153: Increased Atherosclerotic Plaque Instability in Advanced Atherosclerosis with Aging: the Role of Calpain 2 in SOD2-deficient ApoE-/- Mice

The prevalence of atherosclerosis and incidence of cardiovascular events increase with advancing age. Increased generation and/or decreased scavenging of reactive oxygen species (ROS) in the vascular wall are major determinants of vascular disease progression. We previously showed that SOD2 deficiency in aging increases vascular oxidative stress and reduces aortic compliance, while young ApoE-/-/SOD2+/- mice show increased mitochondrial DNA damage and accelerated atherogenesis. Here we investigated the effects of aging and SOD2 deficiency on atherosclerosis and vascular wall morphology in ApoE-/- mice. Compared with the control ApoE-/-, aged ApoE-/-/SOD2+/- mice had increased total vascular wall (P < 0.001) and mitochondrial ROS levels (P < 0.05) and 70% higher atherosclerotic lesion area (P < 0.001). Pulse wave velocity (P < 0.001) and aortic wall collagen content (P < 0.05) were significantly increased, while atherosclerotic plaque fibrous cap thickness (P < 0.01) and vascular smooth muscle cell (VSMC)-positive area (P < 0.01) were decreased in aged ApoE-/-/SOD2+/- mice. Immunofluorescence analysis of atherosclerotic lesions revealed higher levels of calpain 2, a cysteine protease implicated in apoptosis and cleaved caspase-3 localized in smooth muscle-positive areas of the plaque in aged ApoE-/-/SOD2+/- mice. VSMC isolated from aged SOD2+/- had higher levels of mitochondrial oxidative stress and dysfunction compared with VSMC from wild-type mice, as determined by increased superoxide generation and mitochondrial protein carbonylation and lower complex I and III activity. The calpain 2 protein levels were significantly higher in the SOD2+/- VSMCs compared with the wild-type cells and its expression and activity increased after TNF-α treatment, causing apoptosis. TNF-α increased MMP2 activity in SOD2+/- VSMC in Calpain 2-dependent manner. In conclusion, enhanced mitochondrial oxidative stress in aging increases aortic stiffening, atherosclerotic burden and plaque instability in advanced atherosclerosis, in part, via the activation of calpain 2 and MMP2. Targeting mitochondrial oxidative stress and calpain 2 activity may be a viable strategy to prevent aging-associated atherosclerosis complications.