scholarly journals Blockade of Electron Transport at the Onset of Reperfusion Decreases Cardiac Injury in Aged Hearts by Protecting the Inner Mitochondrial Membrane

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Qun Chen ◽  
Thomas Ross ◽  
Ying Hu ◽  
Edward J. Lesnefsky
Keyword(s):  
Membrane Potential ◽  
Electron Transport ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Membrane Integrity ◽  
Reversible Inhibitor ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Fischer 344

Myocardial injury is increased in the aged heart following ischemia-reperfusion (ISC-REP) compared to adult hearts. Intervention at REP with ischemic postconditioning decreases injury in the adult heart by attenuating mitochondrial driven cell injury. Unfortunately, postconditioning is ineffective in aged hearts. Blockade of electron transport at the onset of REP with the reversible inhibitor amobarbital (AMO) decreases injury in adult hearts. We tested if AMO treatment at REP protects the aged heart via preservation of mitochondrial integrity. Buffer-perfused elderly Fischer 344 24 mo. rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM) or vehicle was given for 3 min at the onset of REP. Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated after REP. Oxidative phosphorylation (OXPHOS) and mitochondrial inner membrane potential were measured. AMO treatment at REP decreased cardiac injury. Compared to untreated ISC-REP, AMO improved inner membrane potential in SSM and IFM during REP, indicating preserved inner membrane integrity. Thus, direct pharmacologic modulation of electron transport at REP protects mitochondria and decreases cardiac injury in the aged heart, even when signaling-induced pathways of postconditioning that are upstream of mitochondria are ineffective.

Abstract 13879: Ischemic Postconditioning Confers Cardioprotection through Adiponectin-dependent Mitochondrial STAT3 Activation in Mice

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Haobo Li ◽  
Michael G Irwin ◽  
Zhengyuan Xia
Keyword(s):  
Cell Injury ◽  
Troponin I ◽  
Ischemia Reperfusion Injury ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Systolic Pressure ◽  
Gene Knockdown ◽  
Area At Risk ◽  
In Cells

Introduction: Signal transducer and activator of transcription 3 (STAT3) plays a key role in postconditioning (IPo) mediated protection against myocardial ischemia reperfusion injury, but the mechanism by which IPo activates STAT3 is unknown. Adiponectin (APN), a protein with anti-ischemic properties, activates STAT3. We hypothesized that IPo activates mitochondrial STAT3 (MitoSTAT3) via APN signaling. Methods and Results: Wild type (WT) and APN knockout (KO) mice were either sham operated or subjected to 30 min of coronary artery occlusion followed by 2 hours of reperfusion with or without IPo (3 cycles of 10 seconds reperfusion and 10 seconds reocclusion; n=8/group). At the end of reperfusion, KO mice exhibited more severe myocardial injury evidenced as increased infarct size (% of area at risk) 49.2±2.0 vs WT 39.4±3.5, P <0.01; plasma troponin I (ng/ml): KO 72.8±7.6 vs WT 45.7±4.0, P <0.01; worse cardiac function (lower dP/dt max and end-systolic pressure-volume relation, P <0.05); more severely impaired mitochondrial function (reductions in complex IV and complex V protein expression) and more severe reduction of MitoSTAT3 phosphorylation (activation) at site Ser727, P <0.01. IPo significantly attenuated post-ischemic cardiac injury and dysfunction with a concomitant increase in phosphorylated MitoSTAT3 and attenuation of mitochondrial dysfunction in WT (all P <0.05) but not in KO mice. In cultured cardiac H9C2 cells, hypoxic postconditioning (HPo, 3 cycles of 5 min hypoxia and 5 min reoxygenation) significantly attenuated hypoxia/reoxygenation (HR, 3 hours hypoxia/3 hours reoxygenation) induced cell injury (increased apoptotic cell death as % of HR): HR 100.2±0.4 vs HPo 78.2±4.8, P <0.05) and reduced mitochondrial transmembrane potential (% total cells, HR 37.2±4.9 vs HPo 23.5±3.7, P <0.01). APN, adiponectin receptor 1 (AdipoR1), or STAT3 gene knockdown but not AdipoR2 gene knockdown, respectively, abolished HPo cellular protection (all P <0.05 vs. HPo). APN supplementation (10μg/ml) restored HPo protection in cells with APN knockdown but not in cells with AdipoR1or STAT3 gene knockdown. Conclusion: Adiponectin and AdipoR1 signaling are required for IPo to activate myocardial mitochondrial STAT3 to confer cardioprotection.

scholarly journals The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

2005 ◽  
Vol 187 (2) ◽  
pp. 729-738 ◽  
Author(s):  
Elizabeth A. Marcus ◽  
Amiel P. Moshfegh ◽  
George Sachs ◽  
David R. Scott
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Potential ◽  
Carbonic Anhydrase ◽  
Membrane Integrity ◽  
Carbonic Anhydrase Activity ◽  
Knockout Mutant ◽  
Inner Membrane ◽  
Acid Acclimation

ABSTRACT The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3 −, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3 − by the periplasmic α-CA.

Abstract 332: Mitochondrial Inner-Membrane Potential Instability Promotes Sarcolemmal Electrical Instability after Ischemia-Reperfusion in Monolayers of Cardiac Myocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Soroosh Solhjoo ◽  
Brian O’Rourke
Keyword(s):  
Membrane Potential ◽  
Cardiac Myocytes ◽  
Mitochondrial Function ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Partial Recovery ◽  
Mitochondrial Network ◽  
Electrical Excitability ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

Mitochondrial uncoupling due to oxidative stress can, through opening of sarcolemmal KATP channels, alter cellular electrical excitability, and it has been proposed that mitochondrial function is a major factor in arrhythmogenesis during ischemia-reperfusion. Here, we examine the effects of ischemia-reperfusion on mitochondrial inner membrane potential (ΔΨm) and corresponding changes in electrical excitability and wave propagation in monolayer cultures of neonatal rat ventricular myocytes. Changes in ΔΨm were observed using TMRM and changes in the sarcolemmal voltage were recorded with a 464-element photodiode array using di-4-ANEPPS. Ischemia was induced by covering the center part of the monolayer (D = 22 mm) with a coverslip (D = 15 mm). Cell contractions ceased after approximately 6 min of ischemia; however, electrical activity continued for 11.3 ± 4.2 min (N = 5). Amplitude and conduction velocity of the action potentials in the ischemic region decreased over the same time period. ΔΨm was lost in two phases: a reversible phase of partial depolarization, after 11.2 ± 1.3 min of ischemia, and a nonreversible phase, which happened after 30 ± 6 min of ischemia, during which the whole mitochondrial network of the myocyte became depolarized and the cells underwent contracture (N = 4). Reperfusion after the long ischemia resulted in only partial recovery and the observance of oscillations of ΔΨm in the mitochondrial network or rapid flickering of individual mitochondrial clusters and was associated with heterogeneous electrical recovery, and formation of wavelets and reentry (4/5 monolayers). In contrast, mitochondria fully recovered and reentry was rare (1/5 monolayers) for reperfusion after the short ischemia (10-12 min). 4’-chlorodiazepam, an inhibitor of inner membrane anion channels, stabilized mitochondrial function after the long ischemia, and prevented wavelets (5/5 monolayers) and reentry (4/5 monolayers). In conclusion, incomplete or unstable recovery of mitochondrial function after ischemia correlates with reentrant arrhythmias in monolayers of cardiac myocytes. Our findings suggest that stabilization of mitochondrial network dynamics is an important strategy for preventing ischemia/reperfusion-related arrhythmias.

scholarly journals Transient complex I inhibition at the onset of reperfusion by extracellular acidification decreases cardiac injury

2014 ◽  
Vol 306 (12) ◽  
pp. C1142-C1153 ◽  
Author(s):  
Aijun Xu ◽  
Karol Szczepanek ◽  
Michael W. Maceyka ◽  
Thomas Ross ◽  
Elizabeth Bowler ◽  
...  
Keyword(s):  
Complex I ◽  
Mitochondrial Permeability Transition ◽  
Cardiac Injury ◽  
Membrane Integrity ◽  
Mouse Heart ◽  
Acidic Environment ◽  
Inner Mitochondrial Membrane ◽  
Extracellular Acidification ◽  
Early Reperfusion ◽  
Reversible Inhibition

A reversible inhibition of mitochondrial respiration by complex I inhibition at the onset of reperfusion decreases injury in buffer-perfused hearts. Administration of acidic reperfusate for a brief period at reperfusion decreases cardiac injury. We asked if acidification treatment decreased cardiac injury during reperfusion by inhibiting complex I. Exposure of isolated mouse heart mitochondria to acidic buffer decreased the complex I substrate-stimulated respiration, whereas respiration with complex II substrates was unaltered. Evidence of the rapid and reversible inhibition of complex I by an acidic environment was obtained at the level of isolated complex, intact mitochondria and in situ mitochondria in digitonin-permeabilized cardiac myocytes. Moreover, ischemia-damaged complex I was also reversibly inhibited by an acidic environment. In the buffer-perfused mouse heart, reperfusion with pH 6.6 buffer for the initial 5 min decreased infarction. Compared with untreated hearts, acidification treatment markedly decreased the mitochondrial generation of reactive oxygen species and improved mitochondrial calcium retention capacity and inner mitochondrial membrane integrity. The decrease in infarct size achieved by acidic reperfusion approximates the reduction obtained by a reversible, partial blockade of complex I at reperfusion. Extracellular acidification decreases cardiac injury during reperfusion in part via the transient and reversible inhibition of complex I, leading to a reduction of oxyradical generation accompanied by a decreased susceptibility to mitochondrial permeability transition during early reperfusion.

Mitochondrial KATP channel activation reduces anoxic injury by restoring mitochondrial membrane potential

2001 ◽  
Vol 281 (3) ◽  
pp. H1295-H1303 ◽  
Author(s):  
Meifeng Xu ◽  
Yigang Wang ◽  
Ahmar Ayub ◽  
Muhammad Ashraf
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cell Injury ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Selective Inhibitor ◽  
Atp Depletion ◽  
Channel Activation

Mitochondrial membrane potential (ΔΨm) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive K+ (mitoKATP) channel activation prevents the collapse of ΔΨm in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed ΔΨm, as monitored by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome cimmunostaining. Activation of mitoKATP channel with diazoxide (100 μmol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized ΔΨm. This protection was blocked by 5-hydroxydecanoate (500 μmol/l), a mitoKATPchannel-selective inhibitor, but not by HMR-1098 (30 μmol/l), a putative sarcolemmal KATP channel-selective inhibitor. Dissipation of ΔΨm also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts ΔΨm and induces apoptosis, which are prevented by the activation of the mitoKATP channel. This further emphasizes the therapeutic significance of mitoKATP channel agonists in the prevention of ischemia-reperfusion cell injury.

scholarly journals Intermediary metabolism and fatty acid oxidation: novel targets of electron transport chain-driven injury during ischemia and reperfusion

2018 ◽  
Vol 314 (4) ◽  
pp. H787-H795 ◽  
Author(s):  
Qun Chen ◽  
Masood Younus ◽  
Jeremy Thompson ◽  
Ying Hu ◽  
John M. Hollander ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Electron Transport ◽  
Fatty Acid Oxidation ◽  
Electron Transport Chain ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Absolute Quantification ◽  
Intermediary Metabolism ◽  
Acid Oxidation ◽  
Transport Chain

Cardiac ischemia-reperfusion (I/R) damages the electron transport chain (ETC), causing mitochondrial and cardiomyocyte injury. Reversible blockade of the ETC at complex I during ischemia protects the ETC and decreases cardiac injury. In the present study, we used an unbiased proteomic approach to analyze the extent of ETC-driven mitochondrial injury during I/R. Isolated-perfused mouse (C57BL/6) hearts underwent 25-min global ischemia (37°C) and 30-min reperfusion. In treated hearts, amobarbital (2 mM) was given for 1 min before ischemia to rapidly and reversibly block the ETC at complex I. Mitochondria were isolated at the end of reperfusion and subjected to unbiased proteomic analysis using tryptic digestion followed by liquid chromatography-mass spectrometry with isotope tags for relative and absolute quantification. Amobarbital treatment decreased cardiac injury and protected respiration. I/R decreased the content ( P < 0.05) of multiple mitochondrial matrix enzymes involved in intermediary metabolism compared with the time control. The contents of several enzymes in fatty acid oxidation were decreased compared with the time control. Blockade of ETC during ischemia largely prevented the decreases. Thus, after I/R, not only the ETC but also multiple pathways of intermediary metabolism sustain damage initiated by the ETC. If these damaged mitochondria persist in the myocyte, they remain a potent stimulus for ongoing injury and the transition to cardiomyopathy during prolonged reperfusion. Modulation of ETC function during early reperfusion is a key strategy to preserve mitochondrial metabolism and to decrease persistent mitochondria-driven injury during longer periods of reperfusion that predispose to ventricular dysfunction and heart failure. NEW & NOTEWORTHY Ischemia-reperfusion (I/R) damages mitochondria, which could be protected by reversible blockade of the electron transport chain (ETC). Unbiased proteomics with isotope tags for relative and absolute quantification analyzed mitochondrial damage during I/R and found that multiple enzymes in the tricarboxylic acid cycle, fatty acid oxidation, and ETC decreased, which could be prevented by ETC blockade. Strategic ETC modulation can reduce mitochondrial damage and cardiac injury.

Protein import into mitochondria

2005 ◽  
Vol 33 (5) ◽  
pp. 1019-1023 ◽  
Author(s):  
D. Mokranjac ◽  
W. Neupert
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane ◽  
Protein Import ◽  
Molecular Machines ◽  
Energy Sources ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Preprotein Translocation ◽  
Almost All

Mitochondria comprise approx. 1000–3000 different proteins, almost all of which must be imported from the cytosol into the organelle. So far, six complex molecular machines, protein translocases, were identified that mediate this process. The TIM23 complex is a major translocase in the inner mitochondrial membrane. It uses two energy sources, namely membrane potential and ATP, to facilitate preprotein translocation across the inner membrane and insertion into the inner membrane. Recent research has led to the discovery of a number of new constituents of the TIM23 complex and to the unravelling of the mechanisms of preprotein translocation.

Abstract 995: Blockade Of Electron Transport Preserves The Contents Of Bcl-2 And Cytochrome c In Subsarcolemmal Mitochondria During Ischemia

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Qun Chen ◽  
Edward J Lesnefsky
Keyword(s):  
Electron Transport ◽  
Outer Membrane ◽  
Rabbit Heart ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Complex Iv ◽  
Inner Membrane ◽  
Transport Chain ◽  
Subsarcolemmal Mitochondria

Cardiac ischemia decreases the rate of oxidative phosphorylation (OXPHOS) and the contents of cardiolipin and cytochrome c (CYTc) in subsarcolemmal mitochondria (SSM) in the isolated rabbit heart. CYTc release first requires damage to the inner mitochondrial membrane to delocalize CYTc to the intermembrane space, followed by breach of the outer membrane. The decrease in cardiolipin content allows CYTc detachment from the inner membrane. It is still unclear how CYTc passes the outer membrane for release into cytosol. We propose that ischemia increases outer-membrane leakage by depletion of bcl-2 content, and that oxidants generated by the electron transport chain (ETC) during ischemia favor bcl-2 depletion. We used blockade of the proximal ETC at complex I during ischemia with amobarbital (AMO) to test the role of ETC during ischemia. Langendorff perfused rabbit hearts were treated with AMO (2.5 mM for 1 min) or vehicle immediately before 30 min global ischemia (37°C). Time controls were perfused for 45 min. SSM were isolated at the end of ischemia. CYTc content (reduced minus oxidized spectra), OXPHOS and bcl-2 (western blotting) were measured. Ischemia decreased OXPHOS with TMPD-ascorbate as substrate (electron donor to complex IV via CYTc) and the contents of CYTc and bcl-2. In contrast, AMO preserves OXPHOS, CYTc and bcl-2. Thus, blockade of electron transport preserves bcl-2 content during ischemia with enhanced CYTc retention by SSM. The ETC contributes to mitochondrial damage during ischemia, depleting cardiolipin in the inner membrane and bcl-2 in the outer membrane favoring the two steps required for release of CYTc from mitochondria during ischemia and reperfusion.

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Small Molecule, Permeable, Internally Redistributing for Inner Membrane Targeting Rhodamine Voltage Reporter

2020 ◽  
Author(s):  
Pavel Klier ◽  
Julia Martin ◽  
Evan Miller
Keyword(s):  
Membrane Potential ◽  
Small Molecule ◽  
Mitochondrial Membrane ◽  
Membrane Targeting ◽  
Inner Mitochondrial Membrane ◽  
Intact Cells ◽  
Inner Membrane ◽  
Biophysical Parameter ◽  
Negative Membrane Potential ◽  
Time Observation

<p>Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨ<sub>m</sub>) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨ<sub>m</sub> indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨ<sub>m</sub> dynamics. Here, we report the first example of a fluorescent ΔΨ<sub>m</sub> reporter that does not rely on ΔΨ<sub>m</sub>-dependent accumulation. We re-directed the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxy methyl (AM) ester. Once within mitochondria, esterases remove the AM-ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨ<sub>m</sub>. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨ<sub>m</sub> induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca<sup>2+</sup>, plasma membrane potential, and reversible ΔΨ<sub>m</sub> dynamics.</p>

Sign in / Sign up

Export Citation Format

Share Document