MitoWave: Spatio-temporal analysis of mitochondrial membrane potential fluctuations during ischemia-reperfusion

Ischemic heart diseases are the major reasons for disability and mortality in elderly individuals. In this study, we tried to examine the combined effects of nicotinamide mononucleotide (NMN) preconditioning and melatonin postconditioning on cardioprotection and mitochondrial function in ischemia/reperfusion (I/R) injury of aged male rats. Sixty aged Wistar rats were randomly allocated to 5 groups, including sham, control, NMN-receiving, melatonin-receiving, and combined therapy (NMN+melatonin). Isolated hearts were mounted on Langendorff apparatus and then underwent 30-minue ligation of left anterior descending coronary artery to induce regional ischemic insult, followed by 60 minutes of reperfusion. Nicotinamide mononucleotide (100 mg/kg/d intraperitoneally) was administered for every other day for 28 days before I/R. Melatonin added to perfusion solution, 5 minutes prior to the reperfusion up to 15 minutes early reperfusion. Myocardial hemodynamic and infarct size (IS) were measured, and the left ventricles samples were obtained to evaluate cardiac mitochondrial function and oxidative stress markers. Melatonin postconditioning and NMN had significant cardioprotective effects in aged rats; they could improve hemodynamic parameters and reduce IS and lactate dehydrogenase release compared to those of control group. Moreover, pretreatment with NMN increased the cardioprotection by melatonin. All treatments reduced oxidative stress and mitochondrial reactive oxygen species (ROS) levels and improved mitochondrial membrane potential and restored NAD+/NADH ratio. The effects of combined therapy on reduction of mitochondrial ROS and oxidative status and improvement of mitochondrial membrane potential were greater than those of alone treatments. Combination of melatonin and NMN can be a promising strategy to attenuate myocardial I/R damages in aged hearts. Restoration of mitochondrial function may substantially contribute to this cardioprotection.

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The spatio-temporal dynamics of mitochondrial membrane potential during oocyte maturation

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz055 ◽

2019 ◽

Vol 25 (11) ◽

pp. 695-705 ◽

Cited By ~ 5

Author(s):

Usama AL-Zubaidi ◽

Jun Liu ◽

Ozgur Cinar ◽

Rebecca L Robker ◽

Deepak Adhikari ◽

...

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Oocyte Maturation ◽

Mitochondrial Function ◽

Mitochondrial Membrane ◽

Local Level ◽

Meiotic Spindle ◽

Mitochondrial Activity ◽

Wide Range ◽

Spatio Temporal

Abstract Mitochondria are highly dynamic organelles and their distribution, structure and activity affect a wide range of cellular functions. Mitochondrial membrane potential (∆Ψm) is an indicator of mitochondrial activity and plays a major role in ATP production, redox balance, signaling and metabolism. Despite the absolute reliance of oocyte and early embryo development on mitochondrial function, there is little known about the spatial and temporal aspects of ΔΨm during oocyte maturation. The one exception is that previous findings using a ΔΨm indicator, JC-1, report that mitochondria in the cortex show a preferentially increased ΔΨm, relative to the rest of the cytoplasm. Using live-cell imaging and a new ratiometric approach for measuring ΔΨm in mouse oocytes, we find that ΔΨm increases through the time course of oocyte maturation and that mitochondria in the vicinity of the first meiotic spindle show an increase in ΔΨm, compared to other regions of the cytoplasm. We find no evidence for an elevated ΔΨm in the oocyte cortex. These findings suggest that mitochondrial activity is adaptive and responsive to the events of oocyte maturation at both a global and local level. In conclusion, we have provided a new approach to reliably measure ΔΨm that has shed new light onto the spatio-temporal regulation of mitochondrial function in oocytes and early embryos.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1295 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1295-H1303 ◽

Cited By ~ 62

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.

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Restored microRNA-326-5p Inhibits Neuronal Apoptosis and Attenuates Mitochondrial Damage via Suppressing STAT3 in Cerebral Ischemia/Reperfusion Injury

Nanoscale Research Letters ◽

10.1186/s11671-021-03520-3 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Yumin Huang ◽

Yingge Wang ◽

Zuowei Duan ◽

Jingyan Liang ◽

Yijun Xu ◽

...

Keyword(s):

Oxidative Stress ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Pathological Changes ◽

Cerebral Ischemia Reperfusion ◽

Cerebral Ischemia Reperfusion Injury ◽

Brain Tissues

AbstractStudies have greatly explored the role of microRNAs (miRNAs) in cerebral ischemia/reperfusion injury (CI/RI). But the specific mechanism of miR-326-5p in CI/RI is still elusive. Hence, this study was to unmask the mechanism of miR-326-5p/signal transducer and activator of transcription-3 (STAT3) axis in CI/RI. Two models (oxygen and glucose deprivation [OGD] in primary rat cortical neurons and middle cerebral artery occlusion [MCAO] in Sprague–Dawley rats) were established to mimic CI/RI in vitro and in vivo, respectively. Loss- and gain-of function assays were performed with OGD-treated neurons and with MCAO rats. Afterward, viability, apoptosis, oxidative stress and mitochondrial membrane potential in OGD-treated neurons were tested, as well as pathological changes, apoptosis and mitochondrial membrane potential in brain tissues of MCAO rats. Mitofusin-2 (Mfn2), miR-326-5p and STAT3 expression in OGD-treated neurons and in brain tissues of MCAO rats were detected. Mfn2 and miR-326-5p were reduced, and STAT3 was elevated in OGD-treated neurons and brain tissues of MCAO rats. miR-326-5p targeted and negatively regulated STAT3 expression. Restoring miR-326-5p or reducing STAT3 reinforced viability, inhibited apoptosis and oxidative stress, increased mitochondrial membrane potential and increased Mfn2 expression in OGD-treated neurons. Up-regulating miR-326-5p or down-regulating STAT3 relieved pathological changes, inhibited apoptosis and elevated mitochondrial membrane potential and Mfn2 expression in brain tissues of rats with MCAO. This study elucidates that up-regulated miR-326-5p or down-regulated STAT3 protects against CI/RI by elevating Mfn2 expression.

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MitoWave: Spatio-temporal analysis of mitochondrial membrane potential fluctuations during ischemia-reperfusion

10.1101/2020.05.21.108670 ◽

2020 ◽

Author(s):

D. Ashok ◽

B. O’Rourke

Keyword(s):

Membrane Potential ◽

Ventricular Myocytes ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Temporal Analysis ◽

Time Resolved ◽

Glass Coverslip ◽

Spatio Temporal ◽

Microscopy Data

AbstractMitochondria exhibit non-stationary unstable membrane potential (ΔΨm) when subjected to stress, such as during Ischemia/Reperfusion (I/R). Understanding the mechanism of ΔΨm instability involves characterizing and quantifying this phenomenon in response to I/R stress in an unbiased and reproducible manner. We designed a simple ImageJ-MATLAB-based workflow called ‘MitoWave’ to unravel dynamic mitochondrial ΔΨm changes that occur during ischemia and reperfusion. MitoWave employs MATLAB’s wavelet transform toolbox. In-vitro Ischemia was effected by placing a glass coverslip for 60 minutes on a monolayer of neonatal mouse ventricular myocytes (NMVMs). Removal of the coverslip allowed for reperfusion. ΔΨm response to I/R was recorded on a confocal microscope using TMRM as the indicator. As proof-of-principle, we used MitoWave analysis on ten invitro I/R experiments. Visual observations corroborated quantitative MitoWave analysis results in classifying the ten I/R experiments into five outcomes that were observed based on the oscillatory state of ΔΨm throughout the reperfusion time period. Statistical analysis of the distribution of oscillating mitochondrial clusters during reperfusion shows significant differences between five different outcomes (p< 0.001). Features such as time-points of ΔΨm depolarization during I/R, area of mitochondrial clusters and time-resolved frequency components during reperfusion were determined per cell and per mitochondrial cluster. We found that mitochondria from NMVMs subjected to I/R oscillate in the frequency range of 8.6-45mHz, with a mean of 8.73±4.35mHz. Oscillating clusters had smaller areas ranging from 49.78±40.64 μm2 while non-oscillating clusters had larger areas 65.97±42.07μm2. A negative correlation between frequency and mitochondrial cluster area was seen. We also observed that late ΔΨm loss during ischemia correlated with early ΔΨm stabilization after oscillation on reperfusion. Thus, MitoWave analysis provides a way to quantify complex time-resolved mitochondrial behavior. It provides an easy to follow workflow to automate microscopy analysis and allows for unbiased, reproducible quantitation of complex nonstationary cellular phenomena.Statement of SignificanceUnderstanding mitochondrial instability in Ischemia Reperfusion injury is key to determining efficacy of interventions. The MitoWave analysis is a powerful yet simple tool that enables even beginner MATALAB-Image J users to automate analysis of time-series from microscopy data. While we used it to detect ΔΨm changes during I/R, it can be adapted to detect any such spatio-temporal changes. It standardizes the quantitative analysis of complex biological signals, opens the door to in-depth screening of the genes, proteins and mechanisms underlying metabolic recovery after ischemia-reperfusion.

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Reduced β2-glycoproteinI Alleviated Rat Myocardial Ischemia/Reperfusion Injury via Mitochondrial Protection and Inhibition of Apoptosis

10.21203/rs.3.rs-87087/v1 ◽

2020 ◽

Author(s):

Saijun Zhou ◽

Zhenxing Meng ◽

Shumin Xiao ◽

Ting Cheng ◽

Shuai Huang ◽

...

Keyword(s):

Membrane Potential ◽

Myocardial Ischemia ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Ischemia Reperfusion ◽

Ros Generation ◽

H9c2 Cells ◽

Myocardial Ischemia Reperfusion ◽

Gsk 3Β

Abstract BackgroundMyocardial ischemia/reperfusion (I/R) injury is one of the most important reasons for death of coronary heart disease after vascular recanalization. New evidences have shown that β2-glycoprotein I (β2GPI) plays a protective role in cardiovascular diseases. This study aims to evaluate the effects of reduced β2GPI (R-β2GPI), one form of β2GPI, on myocardial I/R injury, and to explore related mechanisms. MethodsThe in vivo myocardial I/R models of Sprague Dawley rats and in vitro hypoxia/reoxygenation(H/R) models of H9c2 cells were established. The myocardial infarction and morphological changes in SD rats were measured by the TTC staining and HE staining. Creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) levels in plasma were detected by ELISA Assay kit. Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method and caspase-3 colorimetric assay kit were used to determine myocardial apoptosis. Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential of H9c2 cells were measured by fluorescent probe DCFH-DA and JC-1 fluorescent staining respectively. To evaluate cell damage, cell viability was assessed by determining the release of lactate dehydrogenase (LDH). The ratio of Bcl-2/Bax at mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis was used to detect the expression levels of total Akt and phosphorylated Akt as well as the expression levels of total GSK-3βand phosphorylated GSK-3β in H9c2 cells. ResultsOur results suggested that R-β2GPI improved I/R model rats’ heart function, decreased infarct size, reduced serum CK-MB, cTnI levels, cell apoptosis and caspase3 activity. In vitro, R-β2GPI decreased LDH leakage, reduced ROS generation, maintained mitochondrial membrane potential and increased bcl-2/bax mRNA ratio; increased phosphorylation of Akt and GSK-3β in H9c2 cells following Hypoxia/Reoxygenation (H/R) jnjury. ConclusionR-β2GPI alleviated myocardial I/R (or H/R) injury by reducing oxidative stress and inhibiting mitochondrial apoptotic pathway via increasing the phosphorylation of Akt/GSK-3β.

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