scholarly journals Disease Outcome and Brain Metabolomics of Cyclophilin-D Knockout Mice in Sepsis

2022 ◽  
Vol 23 (2) ◽  
pp. 961
Author(s):  
Takayuki Kobayashi ◽  
Hiroyuki Uchino ◽  
Eskil Elmér ◽  
Yukihiko Ogihara ◽  
Hidetoshi Fujita ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Reduced Glutathione ◽  
Cecal Ligation ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Brain Dysfunction ◽  
Cyclophilin D ◽  
Wild Type ◽  
Histological Changes ◽  
Diffuse Brain

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction resulting from a systemic inflammatory response to infection, but the mechanism remains unclear. The mitochondrial permeability transition pore (MPTP) could play a central role in the neuronal dysfunction, induction of apoptosis, and cell death in SAE. The mitochondrial isomerase cyclophilin D (CypD) is known to control the sensitivity of MPTP induction. We, therefore, established a cecal ligation and puncture (CLP) model, which is the gold standard in sepsis research, using CypD knockout (CypD KO) mice, and analyzed the disease phenotype and the possible molecular mechanism of SAE through metabolomic analyses of brain tissue. A comparison of adult, male wild-type, and CypD KO mice demonstrated statistically significant differences in body temperature, mortality, and histological changes. In the metabolomic analysis, the main finding was the maintenance of reduced glutathione (GSH) levels and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio in the KO animals following CLP. In conclusion, we demonstrate that CypD is implicated in the pathogenesis of SAE, possibly related to the inhibition of MPTP induction and, as a consequence, the decreased production of ROS and other free radicals, thereby protecting mitochondrial and cellular function.

scholarly journals Phosphate is not an absolute requirement for the inhibitory effects of cyclosporin A or cyclophilin D deletion on mitochondrial permeability transition

2012 ◽  
Vol 443 (1) ◽  
pp. 185-191 ◽  
Author(s):  
Allison M. McGee ◽  
Christopher P. Baines
Keyword(s):  
Cyclosporin A ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Cyclophilin D ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Mitochondrial Permeability ◽  
Pre Treatment

CypD (cyclophilin D) has been established as a critical regulator of the MPT (mitochondrial permeability transition) pore, and pharmacological or genetic inhibition of CypD attenuates MPT in numerous systems. However, it has recently been suggested that the inhibitory effects of CypD inhibition only manifest when Pi (inorganic phosphate) is present, and that inhibition is lost when Pi is replaced by Asi (inorganic arsenate) or Vi (inorganic vanadate). To test this, liver mitochondria were isolated from wild-type and CypD-deficient (Ppif−/−) mice and then incubated in buffer containing Pi, Asi or Vi. MPT was induced under both energized and de-energized conditions by the addition of Ca2+, and the resultant mitochondrial swelling was measured spectrophotometrically. For pharmacological inhibition of CypD, wild-type mitochondria were pre-incubated with CsA (cyclosporin A) before the addition of Ca2+. In energized and de-energized mitochondria, Ca2+ induced MPT regardless of the anion present, although the magnitude differed between Pi, Asi and Vi. However, in all cases, pre-treatment with CsA significantly inhibited MPT. Moreover, these effects were independent of mouse strain, organ type and rodent species. Similarly, attenuation of Ca2+-induced MPT in the Ppif−/− mitochondria was still observed irrespective of whether Pi, Asi or Vi was present. We conclude that the pharmacological and genetic inhibition of CypD is still able to attenuate MPT even in the absence of Pi.

Abstract 168: Resveratrol Translocates Gsk-3β To Mitochondria And Protects The Heart At Reperfusion By Targeting The Mitochondrial Permeability Transition Pore

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Jinkun Xi ◽  
Huihua Wang ◽  
Guillaume Chanoit ◽  
Guang Cheng ◽  
Robert A Mueller ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Risk Zone ◽  
Mitochondrial Permeability ◽  
Mptp Opening ◽  
Gsk 3Β

Although resveratrol has been demonstrated to be cardioprotective, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. We aimed to determine if resveratrol protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β). Resveratrol (10μM) given at reperfusion reduced infarct size (12.2 ± 2.5 % of risk zone vs. 37.9 ± 3.1 % of risk zone in control, n = 6) in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion, an effect that was abrogated by the mPTP opener atractyloside (30.9 ± 8.1 % of risk zone), implying that resveratrol may protect the heart at reperfusion by modulating the mPTP opening. To define the signaling mechanism underlying the action of resveratrol, we determined GSK-3β activity by measuring its phosphorylation at Ser 9 . Resveratrol significantly enhanced GSK-3β phosphorylation upon reperfusion (225.2 ± 30.0 % of control at 5 min of reperfusion). Further experiments showed that resveratrol induces translocation of GSK-3β to mitochondria and translocated GSK-3β interacts with the mPTP component cyclophilin D but not VDAC (the voltage-dependent anion channel) or ANT (the adenine nucleotide translocator) in cardiac mitochondria. Taken together, these data suggest that resveratrol prevents myocardial reperfusion injury by targeting the mPTP opening via GSK-3β. Translocation of GSK-3β to mitochondria and its interaction with the mPTP component cyclophilin D may serve as an essential mechanism that mediates the protective effect of resveratrol on reperfusion injury.

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.

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 SPG7 targets the m-AAA protease complex to process MCU for uniporter assembly, Ca2+ influx, and regulation of mitochondrial permeability transition pore opening

2019 ◽  
Vol 294 (28) ◽  
pp. 10807-10818 ◽  
Author(s):  
Stephen Hurst ◽  
Ariele Baggett ◽  
Gyorgy Csordas ◽  
Shey-Shing Sheu
Keyword(s):  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Spastic Paraplegia ◽  
Mitochondrial Permeability ◽  
Mptp Opening ◽  
Permeability Transition Pore Opening ◽  
Pore Opening

The mitochondrial matrix ATPase associated with diverse cellular activities (m-AAA) protease spastic paraplegia 7 (SPG7) has been recently implicated as either a negative or positive regulatory component of the mitochondrial permeability transition pore (mPTP) by two research groups. To address this controversy, we investigated possible mechanisms that explain the discrepancies between these two studies. We found that loss of the SPG7 gene increased resistance to Ca2+-induced mPTP opening. However, this occurs independently of cyclophilin D (cyclosporine A insensitive) rather it is through decreased mitochondrial Ca2+ concentrations and subsequent adaptations mediated by impaired formation of functional mitochondrial Ca2+ uniporter complexes. We found that SPG7 directs the m-AAA complex to favor association with the mitochondrial Ca2+ uniporter (MCU) and MCU processing regulates higher order MCU-complex formation. The results suggest that SPG7 does not constitute a core component of the mPTP but can modulate mPTP through regulation of the basal mitochondrial Ca2+ concentration.

Mitochondria and cell death

2000 ◽  
Vol 28 (2) ◽  
pp. 170-177 ◽  
Author(s):  
A. P. Halestrap ◽  
E. Doran ◽  
J. P. Gillespie ◽  
A. O'Toole
Keyword(s):  
Cell Death ◽  
Outer Membrane ◽  
Mitochondrial Permeability Transition ◽  
Adenine Nucleotide ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Ischaemia Reperfusion Injury ◽  
Mptp Opening ◽  
Cyt C

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca2+-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apototic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-xL. Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.

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