scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Metformin Collaborates With PINK1/Mfn2 Overexpression Prevented Isoproterenol-Induced Cardiomyocyte Injury By Improving Mitochondrial Function

2021 ◽  
Author(s):  
Zhuang Ma ◽  
Zuheng Liu ◽  
Yuting Xue ◽  
Hao Zhang ◽  
Wenjun Xiong ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Energy Metabolism ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Combination Strategy ◽  
Mitochondrial Energy Metabolism ◽  
Atp Production

Abstract Background: Both mitochondrial quality control and energy metabolism are critical in maintaining the physiological function of cardiomyocytes. Previous studies indicated that PGC-1α is a transcription co-activator in promoting mitochondrial energy metabolism which would be beneficial for cardiomyocytes. However, PGC-1α overexpression in heart tissues could also result in the development of cardiomyopathy. This discrepancy in vivo and in vitro might be due to neglecting the elimination of damaged mitochondrial. Thus, an integration strategy of mitochondrial biogenesis and mitophagy might be beneficial.Methods: We studied the function of PINK1 in mitophagy in isoproterenol (Iso)-induced cardiomyocyte injury. Adenovirus was used to provoke an overexpression of the PINK1/Mfn2 protein. Mitochondrial morphology was examined via electron microscopy and confocal microscopy. Cardiomyocytes injury were measured by mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and apoptosis. Metformin was used to increase mitochondrial biogenesis, the level of which was detected via immunoblotting. Additionally, mitochondrial respiratory function was measured by ATP production and oxygen consumption rate (OCR). Results: Cardiomyocytes treated with Iso had high levels of PINK1 and low levels of Mfn2 in a time-dependent manner. PINK1 overexpression promoted mitophagy, alleviated Iso-induced reduction in MMP, reduced ROS production and the apoptotic rate. In addition to increasing mitophagy, metformin could promote mitochondrial biogenensis and the overexpression of Mfn2 induce mitochondrial fusion. Moreover, metformin treatment and PINK1/Mfn2 overexpression reduced the mitochondrial dysfunction by inhibiting the generation of ROS, and leading to an increase in both ATP production and mitochondrial membrane potential in Iso-induced cardiomyocytes injury. Conclusion: Our findings indicate that a combination strategy may help ameliorate myocardial injury through mitophagy and mitochondrial biogenesis.

Glutamine protects mitochondrial structure and function in oxygen toxicity

2001 ◽  
Vol 280 (4) ◽  
pp. L779-L791 ◽  
Author(s):  
Shama Ahmad ◽  
Carl W. White ◽  
Ling-Yi Chang ◽  
Barbara K. Schneider ◽  
Corrie B. Allen
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Tricarboxylic Acid Cycle ◽  
A549 Cells ◽  
Oxygen Toxicity ◽  
Cycle Enzyme ◽  
Glutamine Deprivation ◽  
And Function ◽  
Dose Dependent Increase

Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2 for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2 consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.

scholarly journals Manganese (II) chloride leads to dopaminergic neurotoxicity by promoting mitophagy through BNIP3-mediated oxidative stress in SH-SY5Y cells

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Yanning Huang ◽  
Qiaolin Wen ◽  
Jinfeng Huang ◽  
Man Luo ◽  
Yousheng Xiao ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Clinical Manifestations ◽  
Receptor Protein ◽  
Ros Generation ◽  
Mitochondrial Morphology ◽  
Human Neuroblastoma ◽  
Protein Levels ◽  
Mitochondrial Membrane Potential Loss

Abstract Background Manganese overexposure can induce neurotoxicity, lead to manganism and result in clinical manifestations similar to those of parkinsonism. However, the underlying molecular mechanism is still unclear. This study demonstrated that MnCl2 induces mitophagy and leads to neurotoxicity by promoting BNIP3-mediated reactive oxygen species (ROS) generation. Methods Human neuroblastoma SH-SY5Y cells were used throughout our experiments. Cell viability was detected by cell proliferation/toxicity test kits. Mitochondrial membrane potential was measured by flow cytometry. ROS generation was detected using a microplate reader. Protein levels were evaluated by Western blot. Transmission electron microscopy was used to evaluate mitochondrial morphology. Co-immunoprecipitation was used to verify the interaction between BNIP3 and LC3. Results MnCl2 led to loss of mitochondrial membrane potential and apoptosis of SH-SY5Y cells by enhancing expression of BNIP3 and conversion of LC3-I to LC3-II. Moreover, MnCl2 reduced expression of the mitochondrial marker protein TOMM20 and promoted interaction between BNIP3 and LC3. The results also indicated that a decrease in BNIP3 expression reduced the mitochondrial membrane potential loss, attenuated apoptosis and reduced mitochondrial autophagosome formation in SH-SY5Y cells after MnCl2 treatment. Finally, we found that manganese-induced ROS generation could be reversed by the antioxidant N-acetyl cysteine (NAC) or silencing BNIP3 expression. Conclusions BNIP3 mediates MnCl2-induced mitophagy and neurotoxicity in dopaminergic SH-SY5Y cells through ROS. Thus, BNIP3 contributes to manganese-induced neurotoxicity by functioning as a mitophagy receptor protein.

scholarly journals A Mandibular Advancement Device Attenuates the Abnormal Morphology and Function of Mitochondria from the Genioglossus in OSAHS Rabbits

2021 ◽  
Author(s):  
Chunyan Liu ◽  
Shilong Zhang ◽  
Dechao Zhu ◽  
Dengying Fan ◽  
Yahui Zhu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Complex I ◽  
Mitochondrial Membrane ◽  
Mandibular Advancement Device ◽  
Mandibular Advancement ◽  
Control Group ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex ◽  
And Function

Abstract Background: To examine the morphology and function of mitochondria from the genioglossus in a rabbit model of obstructive sleep apnea-hypopnea syndrome (OSAHS), as well as these factors after insertion of a mandibular advancement device (MAD). Methods: Thirty male New Zealand white rabbits were randomized into three groups: control, OSAHS and MAD, with 10 rabbits in each group. Animals in Group OSAHS and Group MAD were induced to develop OSAHS by injection of gel into the submucosal muscular layer of the soft palate. The rabbits in Group MAD were fitted with a MAD. The animals in the control group were not treated. Further, polysomnography (PSG) and CBCT scan were used to measure MAD effectiveness. CBCT of the upper airway and PSG suggested that MAD was effective. Rabbits in the three groups were induced to sleep for 4–6 hours per day for 8 consecutive weeks. The genioglossus was harvested and detected by optical microscopy and transmission electron microscopy. The mitochondrial membrane potential was determined by laser confocal microscopy and flow cytometry. Mitochondrial complex I and IV activities were detected by mitochondrial complex assay kits.Results: OSAHS-like symptoms were induced successfully in Group OSAHS and rescued by MAD treatment. The relative values of the mitochondrial membrane potential, mitochondrial complex I activity and complex IV activity were significantly lower in Group OSAHS than in the control group; however, there was no significant difference between Group MAD and the control group. The OSAHS-induced injury and the dysfunctional mitochondria of the genioglossus muscle were reduced by MAD treatment.Conclusion: Damaged mitochondrial structure and function were induced by OSAHS and could be attenuated by MAD treatment.

scholarly journals Regulation of mitochondrial morphology and function by O-GlcNAcylation in neonatal cardiac myocytes

2011 ◽  
Vol 300 (6) ◽  
pp. R1296-R1302 ◽  
Author(s):  
Ayako Makino ◽  
Jorge Suarez ◽  
Thomas Gawlowski ◽  
Wenlong Han ◽  
Hong Wang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cardiac Myocytes ◽  
Mitochondrial Membrane Potential ◽  
High Glucose ◽  
Mitochondrial Membrane ◽  
Control Level ◽  
Mitochondrial Morphology ◽  
Cell Physiology ◽  
Mitochondrial Fragmentation ◽  
Complex Iv

Mitochondria are crucial organelles in cell life serving as a source of energy production and as regulators of Ca2+ homeostasis, apoptosis, and development. Mitochondria frequently change their shape by fusion and fission, and recent research on these morphological dynamics of mitochondria has highlighted their role in normal cell physiology and disease. In this study, we investigated the effect of high glucose on mitochondrial dynamics in neonatal cardiac myocytes (NCMs). High-glucose treatment of NCMs significantly decreased the level of optical atrophy 1 (OPA1) (mitochondrial fusion-related protein) protein expression. NCMs exhibit two different kinds of mitochondrial structure: round shape around the nuclear area and elongated tubular structures in the pseudopod area. High-glucose-treated NCMs exhibited augmented mitochondrial fragmentation in the pseudopod area. This effect was significantly decreased by OPA1 overexpression. High-glucose exposure also led to increased O-GlcNAcylation of OPA1 in NCMs. GlcNAcase (GCA) overexpression in high-glucose-treated NCMs decreased OPA1 protein O-GlcNAcylation and significantly increased mitochondrial elongation. In addition to the morphological change caused by high glucose, we observed that high glucose decreased mitochondrial membrane potential and complex IV activity and that OPA1 overexpression increased both levels to the control level. These data suggest that decreased OPA1 protein level and increased O-GlcNAcylation of OPA1 protein by high glucose lead to mitochondrial dysfunction by increasing mitochondrial fragmentation, decreasing mitochondrial membrane potential, and attenuating the activity of mitochondrial complex IV, and that overexpression of OPA1 and GCA in cardiac myocytes may help improve the cardiac dysfunction in diabetes.

Mitochondrial Regulation of Calcium in the Avian Cochlear Nucleus

1997 ◽  
Vol 78 (4) ◽  
pp. 1928-1934 ◽  
Author(s):  
Sam P. Mostafapour ◽  
Edward A. Lachica ◽  
Edwin W Rubel
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cochlear Nucleus ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
Cytosolic Calcium ◽  
Mitochondrial Regulation ◽  
Mitochondrial Number ◽  
And Function

Mostafapour, Sam P., Edward A. Lachica, and Edwin W Rubel. Mitochondrial regulation of calcium in the avian choclear nucleus. J. Neurophysiol. 78: 1928–1934, 1997. The role of mitochondria and the endoplasmic reticulum in buffering [Ca2+]i in response to imposed calcium loads in neurons of the chick cochlear nucleus, nucleus magnocellularis (NM), was examined. Intracellular calcium concentrations were measured using fluorometric videomicroscopy. After depolarization with 125 mM KCl, NM neurons demonstrate an increase in [Ca2+]i that returns to near-basal levels within 6 min. Addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) dissipated the mitochondrial membrane potential, as evidenced by increased fluorescence when cells were loaded with rhodamine-123. Two micromolar CCCP had minimal effect on baseline [Ca2+]i. However, 2 or 10 μM CCCP interfered with the ability of NM cells to buffer [Ca2+]i in response to KCl depolarization without significantly affecting peak [Ca2+]i. Oligomycin also interfered with postdepolarization regulation of [Ca2+]i, but blocked late (7–8 min postdepolarization) increases in [Ca2+]i caused by CCCP. Thapsigargin had no effect on baseline, peak, or postdepolarization [Ca2+]i in NM cells. These results suggest that normal mitochondrial membrane potential and ATP synthesis play an important role in buffering [Ca2+]i in response to imposed calcium loads in NM neurons. Furthermore, the endoplasmic reticulum does not appear to play a significant role in either of these processes. Thus increases in mitochondrial number and function noted in NM cells after deafferentation may represent an adaptive response to an increased cytosolic calcium load.

scholarly journals Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ

1992 ◽  
Vol 284 (2) ◽  
pp. 463-467 ◽  
Author(s):  
A E Vercesi ◽  
R Docampo
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Leishmania Donovani ◽  
Initial Rate ◽  
Reaction Medium ◽  
Ruthenium Red ◽  
Permeabilized Cells ◽  
Safranine O

The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of Leishmania donovani promastigotes in situ using safranine O. L. donovani mitochondria were able to build up and retain a membrane potential of a value comparable with that of mammalian mitochondria. The response of promastigotes mitochondrial membrane potential to phosphate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), valinomycin and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria with regard to the properties of their electrochemical proton gradient. When L. donovani promastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM). The presence of 1 microM-FCCP decreased by about 75% the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably by the endoplasmic reticulum, was completely inhibited by 500 microM-vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased by about 25% and the Ca2+ set point was increased to 0.7 microM. The succinate-dependence and FCCP-and Ruthenium Red-sensitivity of the Ca2+ uptake detected in the presence of vanadate indicate that this uptake is probably by the mitochondria. This interpretation was further supported by the Ruthenium Red-sensitive decrease in the mitochondrial membrane potential caused by Ca2+ addition. The anti-leishmanial cationic drugs pentamidine and WR-6026 also induced a rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes.

scholarly journals Chemical-Free Extraction of Functional Mitochondria Using a Microfluidic Device

Inventions ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. 68 ◽  
Author(s):  
Yu-Han Hsiao ◽  
Ching-Wen Li ◽  
Jui-Chih Chang ◽  
Sung-Tzu Chen ◽  
Chin-San Liu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Microfluidic Device ◽  
Complex I ◽  
Structural Damage ◽  
Mitochondrial Membrane ◽  
Normal Activity ◽  
Mitochondrial Morphology ◽  
Transmission Electron ◽  
Device Extraction

This paper proposes the use of a chip-based microfluidic device to extract functional and chemical free mitochondria. A simple microfluidic device was designed and fabricated. An osteosarcoma cybrid cell line was employed to demonstrate the efficiency of the proposed microfluidic device. The membrane proteins (mitochondrial complex I-V and Tom20) and morphology of the extracted mitochondria were examined by Western blot and transmission electron microscopy (TEM), respectively. The purity and mitochondrial membrane potential of the extracted mitochondria were individually measured by 10-N-alkyl acridine orange and tetramethylrhodamine ethyl ester staining via flow cytometry. Experimental results revealed that expressed pattern of complex I–V in device-extracted mitochondria was close to that of mitochondria in total cell lysis and device extraction significantly prevented chemical modification of complex IV protein via a conventional kit, although device extract similar amounts of mitochondria to the conventional kit revealed by Tom20 expression. Furthermore, purity of device-extracted mitochondria was above 93.7% and mitochondria still retained normal activity after device extraction proven by expression of mitochondrial membrane potential as well as the entire mitochondrial morphology. These results confirmed that the proposed microfluidic device could obtain functional mitochondria without structural damage.

scholarly journals Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion

1999 ◽  
Vol 276 (3) ◽  
pp. C611-C620 ◽  
Author(s):  
Jorge Daniel Cortese
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Binding Proteins ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The variety of mitochondrial morphology in healthy and diseased cells can be explained by regulated mitochondrial fusion. Previously, a mitochondrial outer membrane fraction containing fusogenic, aluminum fluoride (AlF4)-sensitive GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese, Exp. Cell Res. 240: 122–133, 1998). Quantitative confocal microscopy now reveals that mtg transiently increases mitochondrial membrane potential (ΔΨ) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced ΔΨ increment is blocked by fusogenic GTPase-specific modulators such as guanosine 5′- O-(3-thiotriphosphate), excess GTP (>100 μM), and AlF4, suggesting a linkage between ΔΨ and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing ΔΨ or ATP synthesis with respiratory inhibitors limits mtg- and AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor ( Bordetella pertussis toxin) hyperpolarizes mitochondria and leads to a loss of AlF4-dependent mitochondrial fusion. These results place mtg-induced ΔΨ changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert ΔΨ-dependent control of the fusion process. Mammalian mitochondrial morphology thus can be modulated by cellular energetics.

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