scholarly journals NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

2021 ◽  
Vol 22 (16) ◽  
pp. 8560
Author(s):  
Ekaterina Kharechkina ◽  
Anna Nikiforova ◽  
Alexey Kruglov
Keyword(s):  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Brain Mitochondria ◽  
Mitochondrial Swelling ◽  
Mitochondrial Depolarization ◽  
Retention Capacity ◽  
The Matrix ◽  
External Site ◽  
Dependent Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.

Abstract 1783: Pro-Apoptotic Bnip3 Mediates Permeabilization of Mitochondria and Release of Cytocrome c via a Novel Mechanism

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Melissa N Quinsay ◽  
Shivaji Rikka ◽  
M Richard Sayen ◽  
Jeffery D Molkentin ◽  
Roberta A Gottlieb ◽  
...  
Keyword(s):  
Cell Death ◽  
Mitochondrial Dysfunction ◽  
Matrix Protein ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Heart Association ◽  
Permeability Transition ◽  
Mitochondrial Swelling ◽  
Cyclophilin D ◽  
The Matrix

Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with mitochondrial dysfunction and cell death in the myocardium. The pro-apoptotic Bcl-2 proteins mediate mitochondrial dysfunction independent of the mitochondrial permeability transition pore (mPTP). However, Bnip3 has been reported to mediate cell death via the mPTP. In this study, we investigated the mechanism(s) by which Bnip3 causes mitochondrial dysfunction. Using a mitochondrial swelling assay to assess pore opening, we found that addition of 200 microM Ca2+ to mitochondria isolated from rat hearts induced rapid swelling of mitochondria and release of cytochrome c (cyto c). Bnip3 also induced mitochondrial swelling and cyto c release, but always at a slower rate and to a greater degree, suggesting that Bnip3 mediates swelling via a different mechanism. Cyclosporin A (CsA), an inhibitor of mPTP opening, prevented Ca2+-induced swelling and cyto c release, but had no effect on Bnip3. Another BH3-only protein, tBid, caused release of cyto c but failed to induce swelling of mitochondria. Interestingly, Bnip3, but not Ca2+ and tBid, induced release of the matrix protein MnSOD. Cyclophilin D (cycD) is an essential component of the mPTP and heart mitochondria isolated from cycD−/− mice were resistant to Ca2+-, but not to Bnip3-induced swelling and cyto c release. Also, tBid caused cyto c release without mitochondrial swelling in the absence of cycD. To further explore the mPTP as a downstream effector of Bnip3-mediated cell death, we assessed cell death in mouse embryonic fibroblasts (MEFs) isolated from wild type (wt) and cycD−/− mice. Infection with an adenovirus expressing Bnip3 caused significant cell death in wt (52.8±1.8%) and cycD−/− (61.8±6.1%) MEFs as measured by LDH release. In addition, both Bnip3 and opening of the mPTP have been reported to initiate upregulation of autophagy. Monitoring of GFP-LC3 incorporation into autophagosomes by fluorescence microscopy revealed that Bnip3 infection induced autophagy in wt (86.5±6.6%) and cycD−/− (96.4±1.4%) MEFs (n=3, p<0.05). Thus, these studies suggest that Bnip3 mediates permeabilization of the inner and outer mitochondrial membranes via a novel mechanism that is different from other BH3-only proteins. This research has received full or partial funding support from the American Heart Association, AHA National Center.

The mitochondrial permeability transition: the brain's point of view

1999 ◽  
Vol 66 ◽  
pp. 75-84 ◽  
Author(s):  
Janet M. Dubinsky ◽  
Nickolay Brustovetsky ◽  
Vsevolod Pinelis ◽  
Bruce S. Kristal ◽  
Carter Herman ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Hippocampal Neurons ◽  
Mitochondrial Permeability Transition ◽  
Calcium Ionophore ◽  
Permeability Transition ◽  
Brain Mitochondria ◽  
Mitochondrial Swelling ◽  
Mitochondrial Depolarization ◽  
Mitochondrial Permeability ◽  
Calcium Accumulation

The mitochondrial permeability transition (mPT) has been implicated in both central nervous system ischaemia/reperfusion injury and excitotoxic neuronal death. To characterize the mPT of brain mitochondria, fluorescent mitochondrial dyes were applied to cultured neurons and astrocytes and isolated brain mitochondria were prepared. In astrocytes, mPT induction was observed as calcium-induced mitochondrial swelling following permeabilization by digitonin or introduction of a calcium ionophore. In hippocampal neurons, mPT induction was observed upon introduction of calcium and ionophore or application of toxic doses of glutamate. In isolated brain mitochondria, calcium dose-dependently produced calcium accumulation and mitochondrial swelling that was prevented by pretreatment with ADP or cyclosporin A. Additionally, when mitochondrial substrates were limited, calcium dose-dependently produced mitochondrial depolarization without swelling or calcium accumulation that was reversed by ADP, cyclosporin A or Ruthenium Red. The degree of mitochondrial depolarization was modulated by free fatty acids, magnesium, calcium concentration and protonophore Repolarization of mitochondria and closure of this low-conductance manifestation of the mPT pore by cyclosporin A was modulated by the degree of depolarization.

scholarly journals Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering

2012 ◽  
Vol 139 (6) ◽  
pp. 465-478 ◽  
Author(s):  
An-Chi Wei ◽  
Ting Liu ◽  
Raimond L. Winslow ◽  
Brian O'Rourke
Keyword(s):  
Mitochondrial Permeability Transition ◽  
High Capacity ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cardiac Mitochondria ◽  
The Matrix ◽  
The Relationship ◽  
Matrix Free ◽  
Trigger Cell Death

Mitochondrial Ca2+ uptake is thought to provide an important signal to increase energy production to meet demand but, in excess, can also trigger cell death. The mechanisms defining the relationship between total Ca2+ uptake, changes in mitochondrial matrix free Ca2+, and the activation of the mitochondrial permeability transition pore (PTP) are not well understood. We quantitatively measure changes in [Ca2+]out and [Ca2+]mito during Ca2+ uptake in isolated cardiac mitochondria and identify two components of Ca2+ influx. [Ca2+]mito recordings revealed that the first, MCUmode1, required at least 1 µM Ru360 to be completely inhibited, and responded to small Ca2+ additions in the range of 0.1 to 2 µM with rapid and large changes in [Ca2+]mito. The second component, MCUmode2, was blocked by 100 nM Ru360 and was responsible for the bulk of total Ca2+ uptake for large Ca2+ additions in the range of 2 to 10 µM; however, it had little effect on steady-state [Ca2+]mito. MCUmode1 mediates changes in [Ca2+]mito of 10s of μM, even in the presence of 100 nM Ru360, indicating that there is a finite degree of Ca2+ buffering in the matrix associated with this pathway. In contrast, the much higher Ca2+ loads evoked by MCUmode2 activate a secondary dynamic Ca2+ buffering system consistent with calcium-phosphate complex formation. Increasing Pi potentiated [Ca2+]mito increases via MCUmode1 but suppressed [Ca2+]mito changes via MCUmode2. The results suggest that the role of MCUmode1 might be to modulate oxidative phosphorylation in response to intracellular Ca2+ signaling, whereas MCUmode2 and the dynamic high-capacity Ca2+ buffering system constitute a Ca2+ sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca2+]mito itself but rather a downstream byproduct of total mitochondrial Ca2+ loading.

Role of cyclophilin D in the resistance of brain mitochondria to the permeability transition

2007 ◽  
Vol 28 (10) ◽  
pp. 1532-1542 ◽  
Author(s):  
Roman A. Eliseev ◽  
Gleb Filippov ◽  
Janice Velos ◽  
Beth VanWinkle ◽  
Aaron Goldman ◽  
...  
Keyword(s):  
Permeability Transition ◽  
Brain Mitochondria ◽  
Cyclophilin D

Abstract 3782: β2-Adrenergic Receptor Signaling Positively Modulates Pro-Survival Kinases and Ameliorates Mitochondrial Dysfunction during Doxorubicin Cardiotoxicity

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Giovanni Fajardo ◽  
Mingming Zhao ◽  
Gerald Berry ◽  
Daria Mochly-Rosen ◽  
Daniel Bernstein
Keyword(s):  
Mitochondrial Dysfunction ◽  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Surface Receptors ◽  
Β2 Adrenergic Receptor ◽  
Improvement In Survival ◽  
Complex I Activity

β2-adrenergic receptors (β2-ARs) modulate cardioprotection through crosstalk with multiple pathways. We have previously shown that β2-ARs are cardioprotective during acute exposure to Doxorubicin (DOX). DOX cardiotoxicity is mediated through a Ca 2+ -dependent opening of the mitochondrial permeability transition pore (MPT) and mitochondrial dysfunction, however the upstream signals linking cell surface receptors and the MPT are not clear. The purpose of this study was to assess crosstalk between β2-AR signaling and mitochondrial function in DOX toxicity. DOX 10 mg/kg was administered to β2−/− and WT mice. Whereas there was no mortality in WT, 85% of β2−/− mice died within 30 min (n=20). Pro- and anti-survival kinases were assessed by immunobloting. At baseline, β2−/− showed normal levels of ϵPKC, but a 16% increase in δPKC compared to WT (p<0.05). After DOX, β2−/− showed a 64% decrease in ϵPKC (p<0.01) and 22% increase in δPKC (p<0.01). The ϵPKC activator ΨϵRACK decreased mortality by 40% in β2−/− mice receiving DOX; there was no improvement in survival with the δPKC inhibitor δV1–1. After DOX, AKT activity was decreased by 76% (p<0.01) in β2−/− but not in WT. The α1-AR blocker prazosin, inhibiting signaling through Gαq, restored AKT activity and reduced DOX mortality by 47%. We next assessed the role of mitochondrial dysfunction in β2−/− mediated DOX toxicity. DOX treated β2−/− mice, but not WT, show marked vacuolization of mitochondrial cristae. Complex I activity decreased 31% in β2−/− mice with DOX; but not in WT. Baseline rate of Ca2+ release and peak [Ca2+]i ratio were increased 85% and 17% respectively in β2−/− myocytes compared to WT. Verapamil decreased mortality by 27% in DOX treated β2−/− mice. Cyclosporine, a blocker of both MPT and calcineurin, reduced DOX mortality to 50%. In contrast, FK506, a blocker of calcineurin but not the MPT, did not reduce DOX mortality. Cyclosporine prevented the decrease in AKT activity in β2−/− whereas FK506 did not. These findings suggest that β2-ARs modulate pro-survival kinases and attenuate mitochondrial dysfunction during DOX cardiotoxicity; absence of β2-ARs enhances DOX toxicity via negative regulation of survival kinases and enhancement of intracellular Ca2+, sensitizing mitochondria to opening of the MPT.

scholarly journals Altered skeletal muscle microtubule-mitochondrial VDAC2 binding is related to bioenergetic impairments after paclitaxel but not vinblastine chemotherapies

2019 ◽  
Vol 316 (3) ◽  
pp. C449-C455 ◽  
Author(s):  
Sofhia V. Ramos ◽  
Meghan C. Hughes ◽  
Christopher G. R. Perry
Keyword(s):  
Skeletal Muscle ◽  
Direct Interaction ◽  
Permeability Transition Pore ◽  
Anion Channel ◽  
Permeability Transition ◽  
Mitochondrial Bioenergetics ◽  
Voltage Dependent Anion Channel ◽  
Retention Capacity ◽  
Microtubule Stabilization

Microtubule-targeting chemotherapies are linked to impaired cellular metabolism, which may contribute to skeletal muscle dysfunction. However, the mechanisms by which metabolic homeostasis is perturbed remains unknown. Tubulin, the fundamental unit of microtubules, has been implicated in the regulation of mitochondrial-cytosolic ADP/ATP exchange through its interaction with the outer membrane voltage-dependent anion channel (VDAC). Based on this model, we predicted that disrupting microtubule architecture with the stabilizer paclitaxel and destabilizer vinblastine would impair skeletal muscle mitochondrial bioenergetics. Here, we provide in vitro evidence of a direct interaction between both α-tubulin and βII-tubulin with VDAC2 in untreated single extensor digitorum longus (EDL) fibers. Paclitaxel increased both α- and βII-tubulin-VDAC2 interactions, whereas vinblastine had no effect. Utilizing a permeabilized muscle fiber bundle preparation that retains the cytoskeleton, paclitaxel treatment impaired the ability of ADP to attenuate H2O2 emission, resulting in greater H2O2 emission kinetics. Despite no effect on tubulin-VDAC2 binding, vinblastine still altered mitochondrial bioenergetics through a surprising increase in ADP-stimulated respiration while also impairing ADP suppression of H2O2 and increasing mitochondrial susceptibility to calcium-induced formation of the proapoptotic permeability transition pore. Collectively, these results demonstrate that altering microtubule architecture with chemotherapeutics disrupts mitochondrial bioenergetics in EDL skeletal muscle. Specifically, microtubule stabilization increases H2O2 emission by impairing ADP sensitivity in association with greater tubulin-VDAC binding. In contrast, decreasing microtubule abundance triggers a broad impairment of ADP’s governance of respiration and H2O2 emission as well as calcium retention capacity, albeit through an unknown mechanism.

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening

2011 ◽  
Vol 300 (4) ◽  
pp. H1237-H1251 ◽  
Author(s):  
María C. Villa-Abrille ◽  
Eugenio Cingolani ◽  
Horacio E. Cingolani ◽  
Bernardo V. Alvarez
Keyword(s):  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Mitochondrial Swelling ◽  
Heart Mitochondria ◽  
Rat Cardiomyocytes ◽  
Mitochondrial Permeability ◽  
Mptp Opening

Inhibition of Na+/H+ exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ∼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl2 to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl2-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca2+-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.

Sign in / Sign up

Export Citation Format

Share Document