Abstract MP124: De-palmitoylation of Cysteine 202 of Cyclophilin-D by Calcium is Important for the Activation of the Permeability Transition Pore

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Georgios Amanakis ◽  
Junhui Sun ◽  
Maria Fergusson ◽  
Chengyu Liu ◽  
Jeff D Molkentin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Calcium Overload ◽  
Mitochondrial Calcium ◽  
Cyclophilin D ◽  
Perfused Heart ◽  
Knock Out

Cyclophilin-D (CypD) is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischemia/reperfusion (I/R) injury characterized by oxidative stress and calcium overload. However, the mechanism by which CypD activates PTP is poorly understood. Cysteine 202 of CypD (C202) is highly conserved across species and can undergo redox-sensitive post-translational modifications, such as S-nitrosylation and oxidation. To study the importance of C202, we developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). Hearts from these mice are protected against I/R injury. We found C202 to be abundantly S-palmitoylated under baseline conditions while C202 was de-palmitoylated during ischemia in WT hearts. To further investigate the mechanism of de-palmitoylation during ischemia, we considered the increase of matrix calcium, oxidative stress and uncoupling of ATP synthesis from the electron transport chain. We tested the effects of these conditions on the palmitoylation of CypD in isolated cardiac mitochondria. The palmitoylation of CypD was assessed using a resin-assisted capture (Acyl-RAC). We report that oxidative stress (phenylarsenide) and uncoupling (CCCP) had no effect on CypD palmitoylation (p>0.05, n=3 and n=7 respectively). However, calcium overload led to de-palmitoylation of CypD to the level observed at the end ischemia (1±0.10 vs 0.63±0.09, p=0.012, n=9). To further test the hypothesis that calcium regulates S-palmitoylation of CypD we measured S-palmitoylation of CypD in non-perfused heart lysates from global germline mitochondrial calcium uniporter knock-out mice (MCU-KO), which have reduced mitochondrial calcium and we found an increase in S-palmitoylation of CypD (WT 1±0.04 vs MCU-KO 1.603±0.11, p<0.001, n=6). The data are consistent with the hypothesis that C202 is important for the CypD mediated activation of PTP. Ischemia leads to increased matrix calcium which in turn promotes the de-palmitoylation of CypD on C202. The now free C202 can further be oxidized during reperfusion leading to the activation of PTP. Thus, S-palmitoylation and oxidation of CypD-C202 possibly target CypD to the PTP, making them potent regulators of cardiac I/R injury.

scholarly journals The P66Shc/Mitochondrial Permeability Transition Pore Pathway Determines Neurodegeneration

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Costanza Savino ◽  
PierGiuseppe Pelicci ◽  
Marco Giorgio
Keyword(s):  
Oxidative Stress ◽  
Knockout Mice ◽  
Mitochondrial Permeability Transition ◽  
Neuronal Damage ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Swelling ◽  
Cyclophilin D ◽  
Knock Out ◽  
Mitochondrial Permeability

Mitochondrial-mediated oxidative stress and apoptosis play a crucial role in neurodegenerative disease and aging. Both mitochondrial permeability transition (PT) and swelling of mitochondria have been involved in neurodegeneration. Indeed, knockout mice for cyclophilin-D (Cyc-D), a key regulatory component of the PT pore (PTP) that triggers mitochondrial swelling, resulted to be protected in preclinical models of multiple sclerosis (MS), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). However, how neuronal stress is transduced into mitochondrial oxidative stress and swelling is unclear. Recently, the aging determinant p66Shc that generates H2O2reacting with cytochrome c and induces oxidation of PTP and mitochondrial swelling was found to be involved in MS and ALS. To investigate the role of p66Shc/PTP pathway in neurodegeneration, we performed experimental autoimmune encephalomyelitis (EAE) experiments in p66Shc knockout mice (p66Shc−/−), knock out mice for cyclophilin-D (Cyc-D−/−), and p66Shc Cyc-D double knock out (p66Shc/Cyc-D−/−) mice. Results confirm that deletion of p66Shc protects from EAE without affecting immune response, whereas it is not epistatic to the Cyc-D mutation. These findings demonstrate that p66Shc contributes to EAE induced neuronal damage most likely through the opening of PTP suggesting that p66Shc/PTP pathway transduces neurodegenerative stresses.

Chronic Tempol treatment restores pharmacological preconditioning in the senescent rat heart

2013 ◽  
Vol 304 (5) ◽  
pp. H649-H659 ◽  
Author(s):  
Jiang Zhu ◽  
Mario J. Rebecchi ◽  
Qiang Wang ◽  
Peter S. A. Glass ◽  
Peter R. Brink ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Mitochondrial Permeability ◽  
Anesthetic Preconditioning ◽  
Permeability Transition Pore Opening ◽  
Pore Opening

Cardioprotective effects of anesthetic preconditioning and cyclosporine A (CsA) are lost with aging. To extend our previous work and address a possible mechanism underlying age-related differences, we investigated the role of oxidative stress in the aging heart by treating senescent animals with the oxygen free radical scavenger Tempol. Old male Fischer 344 rats (22–24 mo) were randomly assigned to control or Tempol treatment groups for 2 or 4 wk (T×2wk and T×4wk, respectively). Rats received isoflurane 30 min before ischemia-reperfusion injury or CsA just before reperfusion. Myocardial infarction sizes were significantly reduced by isoflurane or CsA in the aged rats treated with Tempol (T×4wk) compared with old control rats. In other experiments, young (4–6 mo) and old rats underwent either chronic Tempol or vehicle treatment, and the levels of myocardial protein oxidative damage, antioxidant enzymes, mitochondrial Ca2+ uptake, cyclophilin D protein, and mitochondrial permeability transition pore opening times were measured. T×4wk significantly increased MnSOD enzyme activity, GSH-to-GSSH ratios, MnSOD protein level, mitochondrial Ca2+ uptake capacity, reduced protein nitrotyrosine levels, and normalized cyclophilin D protein expression in the aged rat heart. T×4wk also significantly prolonged mitochondrial permeability transition pore opening times induced by reactive oxygen species in old cardiomyocytes. Our studies demonstrate that 4 wk of Tempol pretreatment restores anesthetic preconditioning and cardioprotection by CsA in the old rat and that this is associated with decreased oxidative stress and improved mitochondrial function. Our results point to a new protective strategy for the ischemic myocardium in the high-risk older population.

scholarly journals Cyclophilin D as a potential target for antioxidants in neurodegeneration: the X-ALD case

2013 ◽  
Vol 394 (5) ◽  
pp. 621-629 ◽  
Author(s):  
Jone López-Erauskin ◽  
Isidre Ferrer ◽  
Elena Galea ◽  
Aurora Pujol
Keyword(s):  
Oxidative Stress ◽  
Axonal Degeneration ◽  
Mitochondrial Permeability Transition ◽  
Neurodegenerative Disorder ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Loss Of Function ◽  
Locomotor Impairment ◽  
Fatty Acid Transporter

Abstract X-linked adrenoleukodystrophy (X-ALD) is a severe inherited neurodegenerative disorder characterized by adrenal insufficiency and graded damage in the nervous system. Loss of function of the peroxisomal ABCD1 fatty-acid transporter, resulting in the accumulation of very long-chain fatty acids in organs and plasma, is the genetic cause. Treatment with a combination of antioxidants halts the axonal degeneration and locomotor impairment displayed by the animal model of X-ALD, and is a proof of concept that oxidative stress contributes to axonal damage. New evidence demonstrates that metabolic failure and the opening of the mitochondrial permeability transition pore orchestrated by cyclophilin D underlies oxidative stress-induced axonal degeneration. Thus, cyclophilin D could serve as a therapeutic target for the treatment of X-ALD and cyclophilin D-dependent neurodegenerative and non-neurodegenerative diseases.

scholarly journals Mitochondria, Amyloid β, and Alzheimer's Disease

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Ryan D. Readnower ◽  
Andrew D. Sauerbeck ◽  
Patrick G. Sullivan
Keyword(s):  
Oxidative Stress ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mitochondrial Permeability Transition ◽  
Amyloid Β ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Enzymes ◽  
Cyclophilin D ◽  
Ample Evidence

Hypometabolism is a hallmark of Alzheimer's disease (AD) and implicates a mitochondrial role in the neuropathology associated with AD. Mitochondrial amyloid-beta (Aβ) accumulation precedes extracellular Aβdeposition. In addition to increasing oxidative stress, Aβhas been shown to directly inhibit mitochondrial enzymes. Inhibition of mitochondrial enzymes as a result of oxidative damage or Aβinteraction perpetuates oxidative stress and leads to a hypometabolic state. Additionally, Aβhas also been shown to interact with cyclophilin D, a component of the mitochondrial permeability transition pore, which may promote cell death. Therefore, ample evidence exists indicating that the mitochondrion plays a vital role in the pathophysiology observed in AD.

scholarly journals Melatonin Attenuates Ischemia/Reperfusion-Induced Oxidative Stress by Activating Mitochondrial Fusion in Cardiomyocytes

2022 ◽  
Vol 2022 ◽  
pp. 1-8
Author(s):  
Xiaoling Ma ◽  
Shengchi Wang ◽  
Hui Cheng ◽  
Haichun Ouyang ◽  
Xiaoning Ma
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Permeability Transition ◽  
Troponin T ◽  
Ischemia Reperfusion ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Redox Balance ◽  
Mitochondrial Fusion ◽  
Dependent Manner ◽  
Mitochondrial Oxidative Stress

Myocardial ischemia/reperfusion (I/R) injury can stimulate mitochondrial reactive oxygen species production. Optic atrophy 1- (OPA1-) induced mitochondrial fusion is an endogenous antioxidative mechanism that preserves the mitochondrial function. In our study, we investigated whether melatonin augments OPA1-dependent mitochondrial fusion and thus maintains redox balance during myocardial I/R injury. In hypoxia/reoxygenation- (H/R-) treated H9C2 cardiomyocytes, melatonin treatment upregulated OPA1 mRNA and protein expression, thereby enhancing mitochondrial fusion. Melatonin also suppressed apoptosis in H/R-treated cardiomyocytes, as evidenced by increased cell viability, diminished caspase-3 activity, and reduced Troponin T secretion; however, silencing OPA1 abolished these effects. H/R treatment augmented mitochondrial ROS production and repressed antioxidative molecule levels, while melatonin reversed these changes in an OPA1-dependent manner. Melatonin also inhibited mitochondrial permeability transition pore opening and maintained the mitochondrial membrane potential, but OPA1 silencing prevented these outcomes. These results illustrate that melatonin administration alleviates cardiomyocyte I/R injury by activating OPA1-induced mitochondrial fusion and inhibiting mitochondrial oxidative stress.

scholarly journals Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D

2021 ◽  
Author(s):  
Jing Zhao ◽  
Xinyue Liu ◽  
Alan Blayney ◽  
Lauren Gandy ◽  
Fuming Zhang ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Biophysical Characterization ◽  
Intrinsically Disordered ◽  
Dynamic Interface

Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that full-length p53 (FLp53) binds to CypD with KD of ~1 μM; while both NTD-DBD and NTD bind to CypD at ~10 μM KD (Fig. 1C and 1D). Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases.

Abstract 92: The Mitochondrial Calcium Uniporter is Necessary for Metabolic Matching of Contractile Demand During Acute Sympathetic Stress

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Timothy S Luongo ◽  
Jonathan P Lambert ◽  
Ancai Yuan ◽  
Xueqian Zhang ◽  
Santhanam Shanmughapriya ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Adult Mice ◽  
Calcium Uniporter ◽  
Myocardial Ischemia Reperfusion ◽  
Sympathetic Stress

Contractility is mediated by a variable flux in intracellular calcium (Ca 2+ ), which is proposed to be integrated into mitochondria to regulate cardiac energetics. Moreover, m Ca 2+ -overload is known to activate the mitochondrial permeability transition pore (MPTP) and induce cell death. Recent studies have reported that the Mcu gene encodes the channel-forming portion of the mitochondrial calcium uniporter (MCU) and is required for m Ca 2+ uptake. To examine the role of m Ca 2+ in the heart, we generated a conditional, cardiac-specific knockout model and deleted Mcu in adult mice ( Mcu-cKO ). Loss of Mcu protected against myocardial ischemia-reperfusion (IR) injury by preventing the activation of the MPTP. In addition while we found no baseline phenotype, Mcu -cKO mice lacked contractile responsiveness to beta-adrenergic receptor stimulation as assessed by invasive hemodynamics (Fig 1) and in parallel were unable to activate mitochondrial dehydrogenases and increase cardiac NADH levels. Further experimental analyses in isolated adult cardiomyocytes confirmed a lack of energetic responsiveness to acute sympathetic stress (isoproterenol failure to mediate an increase in NADH, Fig 2), supporting the hypothesis that the physiological function of the MCU in the heart is to modulate Ca 2+ -dependent metabolism during the ‘fight or flight’ response.

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