MitoSOX Red Research Articles

Urechistachykinin I triggers mitochondrial dysfunction leading to a ferroptosis-like response in Saccharomyces cerevisiae.

The purpose of this paper was to demonstrate the antimicrobial activity of urechistachykinin I (LRQSQFVGSR-NH2) extracted from Urechis unicinctus,and its mode of action dependent on mitochondrial dysfunction. The antifungal activity of urechistachykinin I generated reactive oxygen species (ROS), as demonstrated with MitoSOX Red and hydroxyphenyl fluorescein (HPF). Overaccumulation of ROS caused oxidative damage to cells by inducing mitochondrial dysfunction. Mitochondrial disruption resulted in cell death, creating several hallmarks that included lipid peroxidation, glutathione oxidation, and depolarization. Moreover, the loss of mitochondria changed the calcium ion imbalance by depolarization of the mitochondrial membrane. In particular, iron accumulation and DNA fragmentation measurement determined the type of cell death. Our results indicate that urechistachykinin I treatment induced ferroptosis-like death in Saccharomyces cerevisiae via mitochondrial dysfunction. Urechistachykinin I treatment induced mitochondrial dysfunction in S. cerevisiae by generating ROS, and the subsequent oxidative damage caused the ferroptosis-like cell death.

Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

Mitochondrial damage and the resultant oxidative stress are associated with neurodegenerative diseases, ageing, and cancer. However, the triggers of mitochondrial damage remain unclear. We previously reported that cell-free chromatin particles (cfChPs) released from the billions of cells that die in the body every day can readily enter healthy cells and damage their DNA. Here, we show that cfChPs isolated from the sera of healthy individuals, when applied to NIH3T3 mouse fibroblast cells, cause physical damage to mitochondrial DNA (mtDNA). cfChPs also induce ultrastructural changes, increase mitochondrial mass, alter mitochondrial shape, upregulate mitochondrial outer membrane protein translocase of the outer membrane 20, and change mitochondrial membrane potential. Furthermore, a marked increase was observed in mitochondrial superoxide (ROS) production, as detected by MitoSOX Red, and intracellular superoxide dismutase-1 activation. ROS production was also activated when a conditioned medium containing cfChPs released from hypoxia-induced dying NIH3T3 cells was applied to healthy NIH3T3 cells. ROS activation was significantly reduced when the conditioned medium was pre-treated with three different cfChP-deactivating agents: anti-histone antibody-complexed nanoparticles, DNase I, and the novel pro-oxidant combination of the nutraceuticals resveratrol and copper. Given that 1 × 109–1 × 1012 cells die in the body every day, we hypothesise that cfChPs from dying cells are the major physiological triggers for mtDNA damage and ROS production. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions linked to oxidative stress.

Inhibition of the HMGB1/RAGE axis protects against cisplatin-induced ototoxicity via suppression of inflammation and oxidative stress.

As an anti-tumor drug widely used in the clinic, cisplatin is limited by its ototoxic side effects associated with various factors, including inflammatory responses. Receptor for Advanced Glycation Endproducts (RAGE) recognizes damage-associated molecular patterns (DAMPs) and promotes stress and inflammation. This study intended to determine the potential behavior of the HMGB1/RAGE axis after cisplatin injury and whether it has a protective effect after inhibiting this pathway. We used FPS-ZM1, a RAGE inhibitor, to modulate the axis of HMGB1/RAGE in neonatal mouse cochlear explants and C57BL/6 mice in vivo. Apoptosis was identified by Annexin V-FITC/PI assay, Cleaved Caspase-3, and TUNEL staining. Reactive oxygen species (ROS) level was assessed by MitoSOX Red and CellROX Green assay. The expression of proteins associated with the HMGB1/RAGE axis and apoptosis was observed by western blotting. The expression of inflammatory cytokines was evaluated by qPCR. The protective effect of HMGB1/RAGE knockdown was also assessed on cisplatin-induced ototoxicity. These results demonstrated that cisplatin could activate the HMGB1/RAGE pathway in cochlear hair cells and release inflammatory factors. Pretreatment with FPS-ZM1 alleviated cisplatin-induced ototoxicity in vivo and in vitro. Knocking down HMGB1 and RAGE achieved specific protective effects. Altogether, inhibiting HMGB1/RAGE axis can reverse the increase of ROS accumulation, the activation of apoptosis, and the production of inflammatory reactions after cisplatin injury. FPS-ZM1 could resist the ototoxicity of cisplatin by suppressing the HMGB1/RAGE signal pathway, and it may be considered the new otoprotective potential strategy for hearing loss.

Detection of Induction of Mitochondrial Oxidative Stress by Nanoparticles in T Cells Using MitoSOX Red Dye.

The induction of oxidative stress by engineered nanomaterials has been associated with cytotoxic and inflammatory responses, damaging healthy cells and tissues. In contrast, when directed against cancer and autoinflammatory diseases, some nanomaterials inducing oxidative stress have also been reported as potential therapies for these disorders. Therefore, studying oxidative stress has become a popular tool not only in toxicology and immunotoxicology but in other areas of biology as well, including those related to developing novel therapies. Total oxidative stress may result from multiple cellular organelles. The protocol described herein allows for the analysis of oxidative stress in mitochondria.

Fluoxetine exerts anti-inflammatory effects on human epidermal keratinocytes and suppresses their endothelin release.

Fluoxetine is a safe antidepressant with remarkable anti-inflammatory actions; therefore, we aimed to investigate its effects on immortalized (HaCaT) as well as primary human epidermal keratinocytes in a polyinosinic-polycytidylic acid (p(I:C))-induced inflammatory model. We found that a non-cytotoxic concentration (MTT-assay, CyQUANT-assay) of fluoxetine significantly suppressed p(I:C)-induced expression and release of several pro-inflammatory cytokines (Q-PCR, cytokine array, ELISA), and it decreased the release of the itch mediator endothelins (ELISA). These effects were not mediated by the inhibition of the NF-κB or p38 MAPK pathways (western blot), or by the suppression of the p(I:C)-induced elevation of mitochondrial ROS production (MitoSOX Red labeling). Instead, unbiased activity profiling revealed that they were most likely mediated via the inhibition of the phosphoinositide 3-kinase (PI3K) pathway. Importantly, the PI3K-inhibitor GDC0941 fully mimicked the effects of fluoxetine (Q-PCR, ELISA). Although fluoxetine was able to occupy the binding site of GDC0941 (in silico molecular docking), and exerted direct inhibitory effect on PI3K (cell-free PI3K activity assay), it exhibited much lower potency and efficacy as compared to GDC0941. Finally, RNA-Seq analysis revealed that fluoxetine deeply influenced the transcriptional alterations induced by p(I:C)-treatment, and exerted an overall anti-inflammatory activity. Collectively, our findings demonstrate that fluoxetine exerts potent anti-inflammatory effects, and suppresses the release of the endogenous itch mediator endothelins in human keratinocytes, most likely via interfering with the PI3K pathway. Thus, clinical studies are encouraged to explore whether the currently reported beneficial effects translate invivo following its topical administration in inflammatory and pruritic dermatoses.

The role of the intrinsic pathway of apoptosis in human ejaculated sperm damage under a state of scrotal heat stress

PurposeThe study aimed to determine the associations among standard sperm characteristics and oxidative/apoptotic markers in ejaculated sperm of men exposed to prolonged scrotal hyperthermia of either environmental or clinical origin.MethodsThe original study design included four research groups: professional drivers (n = 54), infertile men with varicocele (n = 78), infertile men not exposed to prolonged genital heat stress (n = 37), and fertile individuals serving as the control group (n = 29). Standard semen analysis was performed according to the 5th WHO laboratory manual. The following oxidative and apoptotic parameters of sperm were investigated: mitochondrial superoxide anion generation (MitoSOX Red dye), phosphatidylserine externalization (Annexin V binding assay), mitochondrial membrane potential (JC-1 dye), DNA fragmentation (TUNEL/PI assay), and membrane fluidity (merocyanine 540 dye).ResultsAll the studied groups presented a strong deterioration in routine sperm parameters and a strongly apoptotic phenotype in sperm, characterized by both decreased mitochondrial membrane potential and enhanced DNA fragmentation, regardless of the thermal insult. Significant induction of mitochondrial superoxide anion generation was noted only in the groups exposed to genital heat stress. A positive correlation between the production of superoxide anion in the mitochondrial chain and the level of DNA fragmentation in drivers was also noted.ConclusionLong-term exposure to scrotal hyperthermia in real-life situations is sufficient to reduce sperm quality in humans. The thermal stress directly induces the oxidative stress cascade in ejaculated sperm, affecting the plasma membrane fluidity, mitochondrial homeostasis, and sperm DNA integrity.

Hexavalent Chromium Induces Neurotoxicity by Triggering Mitochondrial Dysfunction and ROS-Mediated Signals.

Hexavalent chromium (Cr (VI)), one of the most detrimental pollutants, has been ubiquitously present in the environment and causes serious toxicity to humans, such as hepatotoxicity, nephrotoxicity, pulmonary toxicity, and cardiotoxicity. However, Cr (VI)-induced neurotoxicity in primary neuron level has not been well explored yet. Herein, potassium dichromate (K2Cr2O7) was employed to examine the neurotoxicity of Cr (VI) in rat primary hippocampal neurons. MTT test was used to examine the neural viability. Mitochondrial dysfunction was assessed by the JC-1 probe and Mito-Tracker probe. DCFH-DA and Mito-SOX Red were utilized to evaluate the oxidative status. Bcl-2 family and MAPKs expression were investigated using Western blotting. The results demonstrated that Cr (VI) treatment dose- and time-dependently inhibited neural viability. Mechanism investigation found that Cr (VI) treatment causes mitochondrial dysfunction by affecting Bcl-2 family expression. Moreover, Cr (VI) treatment also induces intracellular reactive oxygen species (ROS) generation, DNA damage, and MAPKs activation in neurons. However, inhibition of ROS by glutathione (GSH) effectually balanced Bcl-2 family expression, attenuated DNA damage and the MAPKs activation, and eventually improved neural viability neurons. Collectively, these above results above suggest that Cr (VI) causes significant neurotoxicity by triggering mitochondrial dysfunction, ROS-mediated oxidative damage and MAKPs activation.

Myrislignan targets extracellular signal-regulated kinase (ERK) and modulates mitochondrial function to dampen osteoclastogenesis and ovariectomy-induced osteoporosis

BackgroundActivated osteoclasts cause excessive bone resorption, and disrupt bone homeostasis, leading to osteoporosis. The extracellular signal-regulated kinase (ERK) signaling is the classical pathway related to osteoclast differentiation, and mitochondrial reactive oxygen species are closely associated with the differentiation of osteoclasts. Myrislignan (MRL), a natural product derived from nutmeg, has multiple pharmacological activities; however, its therapeutic effect on osteoporosis is unclear. Here, we investigated whether MRL could inhibit osteoclastogenesis and bone mass loss in an ovariectomy mouse model by suppressing mitochondrial function and ERK signaling.MethodsTartrate-resistant and phosphatase (TRAP) and bone resorption assays were performed to observe the effect of MRL on osteoclastogenesis of bone marrow macrophages. MitoSOX RED and tetramethyl rhodamine methyl ester (TMRM) staining was performed to evaluate the inhibitory effect of MRL on mitochondria. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed to detect whether MRL suppressed the expression of osteoclast-specific genes. The impact of MRL on the protein involved in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB pathways was evaluated using western blotting. In addition, a specific ERK agonist LM22B-10, was used to revalidate the inhibitory effect of MRL on ERK. Finally, we established an ovariectomy mouse model to assess the therapeutic effect of MRL on osteoporosis in vivo.ResultsMRL inhibited osteoclast differentiation and the associated bone resorption, by significantly decreasing osteoclastic gene expression. Mechanistically, MRL inhibited the phosphorylation of ERK by suppressing the mitochondrial function, thereby downregulating the nuclear factor of activated T cells 1 (NFATc1) signaling. LM22B-10 treatment further verified the targeted inhibition effect of MRL on ERK. Microscopic computed tomographic and histologic analyses of the tibial tissue sections indicated that ovariectomized mice had lower bone mass and higher expression of ERK compared with normal controls. However, MRL treatment significantly reversed these effects, indicating the anti-osteoporosis effect of MRL.ConclusionWe report for the first time that MRL inhibits ERK signaling by suppressing mitochondrial function, thereby ameliorating ovariectomy-induced osteoporosis. Our findings can provide a basis for the development of a novel therapeutic strategy for osteoporosis.

Deficiency of connexin43 increases arrhythmias due to increases in Ca2+ and ROS within the mitochondria

Abstract Introduction Connexin43 (Cx43) is a major connexin that forms gap junctions in the hearts and plays important roles in the occurrence of reentrant arrhythmias. Cx43 also exits as hemichannels in the inner mitochondrial membrane (mCx43), but it has not yet been established whether mCx43 are involved in the occurrence of arrhythmias. Purpose To examine what roles mCx43 plays in the occurrence of arrhythmias and whether mitochondrial Ca2+ and ROS are involved in their occurrence. Methods To generate cardiac-specific Cx43-deficient (Cx43-/-) mice, Cx43flox/flox mice were crossed with α-myosin heavy chain cre+/- mice. The resulting offspring, Cx43-/- mice and their littermates (Cx43+/+ mice) were used. Trabeculae were dissected from right ventricles of mouse hearts. Force was measured with a strain gauge, mitochondrial Ca2+ with rhod-2, and mitochondrial ROS with MitoSox Red. The spatial distribution of rhod-2 and MitoSox Red was imaged using confocal microscopy, and it was similar to that of MitoTracker Green within trabeculae, meaning that rhod-2 and MitoSox Red are mainly loaded within the mitochondria. To determine the amount of ROS production, MitoSox Red fluorescence was measured before (Fl) and after the addition of 10 mM H2O2 (FlH2O2), and the ratio of Fl to FlH2O2 was calculated. To estimate arrhythmia susceptibility, the minimal extracellular Ca2+ concentration (Cao), at which arrhythmias were induced by electrical stimulation, was determined (Caomin). Arrhythmias were induced by electrical stimulation with 0.3-s stimulus intervals for 30 s. Results Most of Cx43-/- mice died within 8 weeks (p<0.01). Body and heart weights and tibial length of Cx43-/- mice were similar to those of Cx43+/+ mice. Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice but not in Cx43-/- mice. Developed force of Cx43-/- mice was similar to that of Cx43+/+ mice at Cao ranging from 0.2 to 6 mM. Rhod-2 fluorescence in Cx43-/- mice was higher than that of Cx43+/+ mice, especially at higher Cao such as 4 and 6 mM (n=7, p<0.01), and the Caomin in Cx43-/- mice was lower than that in Cx43+/+ mice (n=5, p<0.01), suggesting that Cx43-/- mice shows higher arrhythmia susceptibility with higher mitochondrial Ca2+. In Cx43-/- mice, Ru360 (5 μM), a mitochondrial calcium uniporter inhibitor, increased the Caomin, that is, decreased arrhythmia susceptibility with decreases in rhod-2 and MitoSox Red fluorescence (n=6, p<0.05). Additionally, antioxidant peptide SS-31 (50 µM) and N-acetyl-L-cysteine (1 mM) increased the Caomin in Cx43-/- mice (n=5, p<0.05), and the ratio of Fl to FlH2O2 in Cx43-/- mice was larger that that in Cx43+/+ mice, suggesting that an increase in ROS production plays important roles in the occurrence of arrhythmias in Cx43-/- mice. Conclusions Deficiency of Cx43 increases arrhythmia susceptibility with increases in mitochondrial Ca2+ and ROS, providing an attractive therapeutic target for improving arrhythmias in diseased hearts.

Abstract 15068: Connexin43 Concerns Arrhythmias Through Changes in Mitochondrial Ca 2+ and ROS

Introduction: Connexin43 (Cx43) is a connexin that forms gap junctions in hearts and exists as hemichannels in the inner mitochondrial membrane (mCx43). It is still unknown whether mCx43 is involved in the occurrence of arrhythmias. Thus, we examined whether deficiency of Cx43 increases arrhythmias and what roles mitochondrial Ca 2+ and ROS play in the occurrence of arrhythmias. Methods: Cardiac-specific Cx43-deficient ( Cx43 -/- ) mice were generated by crossing Cx43 flox/flox mice with α-myosin heavy chain cre +/- mice. The resulting offspring, Cx43 -/- mice and their littermates ( Cx43 +/+ mice), were used. Trabeculae were dissected from mouse hearts. Force was measured with a strain gauge, mitochondrial Ca 2+ with rhod-2, and mitochondrial ROS with MitoSox Red. The spatial distribution of rhod-2 and MitoSox Red imaged with confocal microscopy was similar to that of MitoTracker Green within trabeculae, meaning that rhod-2 and MitoSox Red are loaded within mitochondria. MitoSox Red fluorescence was measured before (Fl) and after the addition of 10 mM H 2 O 2 (FlH 2 O 2 ), and the ratio of Fl to FlH 2 O 2 was calculated. To estimate arrhythmogenesis, the minimal extracellular Ca 2+ concentration (Cao), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals for 30 s), was determined (Cao min ). Results: Cx43 -/- mice died within 8 weeks (p<0.01). Cx43 was present in the inner mitochondrial membrane in Cx43 +/+ mice but not in Cx43 -/- mice. In Cx43 -/- mice, rhod-2 fluorescence was higher than that of Cx43 +/+ mice (n=7, p<0.01), while developed force of Cx43 -/- mice was similar to that of Cx43 +/+ mice. The Cao min in Cx43 -/- mice was lower than that in Cx43 +/+ mice (n=5, p<0.01), suggesting that Cx43 -/- mice shows increased arrhythmogenesis. In Cx43 -/- mice, Ru360 (5 μM), a mitochondrial calcium uniporter inhibitor, increased the Cao min , that is, decreased arrhythmogenesis, with decreases in rhod-2 and MitoSox Red fluorescence (n=6, p<0.05). Antioxidant peptide SS-31 (50 μM) and N-acetyl-L-cysteine (1 mM) increased the Cao min in Cx43 -/- mice (n=5, p<0.05), and the ratio of Fl to FlH 2 O 2 in Cx43 -/- mice was larger than that in Cx43 +/+ mice. Conclusions: Deficiency of Cx43 increases arrhythmogenesis with increases in mitochondrial Ca 2+ and ROS.

Abstract 14255: Roles of Mitochondrial Ca 2+ in Contractile Properties in Diabetic Rat Myocardium

Introduction: Diabetes mellitus is an important risk factor for heart failure with reduced relaxation. It has not yet been established how mitochondrial Ca 2+ is involved in the reduced relaxation in diabetic myocardium. Thus, using trabeculae from diabetic hearts, we examined whether mitochondrial Ca 2+ affects contractile properties in diabetic myocardium. Methods: Rats were given a subcutaneous injection of 55 mg/kg streptozotocin (STZ-rats) or solvent (Ctr-rats). Eight weeks after the injection, trabeculae were dissected from rat hearts. Force was measured with a strain gauge, intracellular Ca 2+ (Ca i ) with fura-2, mitochondrial Ca 2+ with rhod-2, and mitochondrial ROS with MitoSox Red (24°C). To evaluate contractile properties of cardiac muscle, the velocities of contraction (dF/dt) and relaxation (-dF/dt) were calculated at 0.7 and 2.0 mM extracellular Ca 2+ . To eliminate the effect of developed force (F dev ), the dF/dt and -dF/dt were normalized by dividing them by their F dev . Results: Blood glucose of STZ-rats was higher than that of Ctr-rats (p<0.01). STZ-rats showed smaller F dev , smaller dF/dt max /F dev , and smaller -dF/dt min /F dev (n=9, p<0.05), suggesting that muscle contraction and relaxation are impaired in STZ-rats. STZ-rats showed lower peak and slower decline of Ca i transients compared to Ctr-rats (n=9, p<0.01). In addition, Ru360 (5 μM), a mitochondrial calcium uniporter (MCU) inhibitor, decreased rhod-2 fluorescence and increased dF/dt max /F dev and -dF/dt min /F dev in STZ-rats (n=7, p<0.05), suggesting that a decrease in mitochondrial Ca 2+ improves muscle contraction and relaxation. On the contrary, spermine (150 μM), a MCU activator, increased rhod-2 fluorescence and decreased dF/dt max /F dev and -dF/dt min /F dev (n=5, p<0.05). In STZ-rats, Ru360 increased MitoSox Red fluorescence (n=5, p<0.05). The spatial distribution of rhod-2 and MitoSox Red imaged with confocal microscopy was similar to that of MitoTracker Green within trabeculae, meaning that rhod-2 and MitoSoxRed are loaded within mitochondria. Conclusions: These results suggest that in diabetic hearts, mitochondrial Ca 2+ is involved in the improvement of muscle contraction and relaxation, probably through changes in ROS production.

Circulating Platelets in Thrombotic Antiphospholipid Syndrome Display High Procoagulant Activity and Surface Complement Deposition: Correlation with Thrombotic Risk

Background Antiphospholipid syndrome (APS) is characterized by circulating antiphospholipid antibodies (aPL) accompanied by arterial and/or venous thrombosis, and/or recurrent pregnancy loss. It commonly affects young women in whom it dramatically increases their risk of myocardial infarction, ischemic stroke, deep vein thrombosis and pulmonary embolism. Although the thrombotic risk associated with aPL is established, the underlying mechanisms are still incompletely defined and there are no definitive predictive biomarkers. As a result the management of APS involves indefinite anticoagulation. Increased activation of vascular cells, including platelets is considered to underlie the pathogenesis of APS, and may be mediated in some cases by complement. There is a considerable crosstalk between platelets and complement at various levels during thrombosis. First, activated complement proteins stimulate platelets through their cognate receptors on the platelet surface, and genetic deficiency of complement proteins in mice leads to decreased platelet responsiveness. Second, platelets secrete complement proteins into the medium upon activation. Third, agonist-stimulated platelets contribute to activation of both the classical and alternative pathways of the complement cascade. Hence, we sought to investigate the association between the complement-platelet axis and the prothrombotic pathology observed in APS. Methods Venous blood was drawn from patients with APS and healthy controls, and washed human platelets were prepared by differential centrifugation. Platelets either remained unstimulated or were treated with thrombin (0.1, 0.25, 0.5 U/ml) for 5 min and then labelled with FITC-PAC1, PE-anti-CD62P antibody, Alexa Fluor 488-anti-C4d conjugate, PE-Annexin V, Mitotracker Red, MitoSOX Red, Fluo4-AM, Rhod2-AM, Cell Event Green Caspase 3/7 detection reagent and FITC-IETD-FMK for 30 min in the dark, and then analyzed by flow cytometry to measure integrin activation, P-selectin expression (α-granule secretion), complement C4d binding, phosphatidylserine (PS) exposure, mitochondrial membrane potential, mitochondrial ROS, cytosolic calcium, mitochondrial calcium, caspase 3/7 activity (apoptosis) and caspase 8 activity (extrinsic apoptosis pathway), respectively. Platelet markers were correlated with complement binding and clinical characteristics of APS patients including number of thrombotic events and circulating levels of aPL. Results Thrombin-induced platelet integrin activation and P-selectin expression were identical in APS patients and healthy controls. However, platelets isolated from APS patients had significantly higher procoagulant activity (PS exposure) as well as complement C4d binding in both unstimulated and thrombin-stimulated states compared to healthy controls. There was a strong positive correlation between platelet PS exposure and C4d binding in APS patients, but not healthy individuals, suggesting a role for complement activation in formation of procoagulant platelets in APS. A subset of APS patients with high platelet procoagulant activity (PS exposure > mean + 2SD observed in healthy individuals) had lower mitochondrial membrane potential and increased mitochondrial calcium transients compared to healthy individuals, suggesting mitochondrial permeability transition pore (mPTP)-driven PS exposure. Another subset of APS patients with PS exposing platelets showed caspase activation indicating apoptosis. Platelet PS exposure and C4d binding correlated strongly with the number of thrombotic events as well as levels of circulating aPL, which identifies complement-induced procoagulant platelets as predictors and possible underlying mechanisms for thrombosis in APS. Conclusion Our preliminary studies suggest that PS-exposing platelets are generated in APS by multiple mechanisms including agonist-induced mPTP formation and apoptosis-associated caspase activation. Platelet PS exposure in APS is positively correlated with C4d binding, suggesting a role for complement activation in driving platelet procoagulant activity. Complement activation and PS exposure on platelets are strong predictors of thrombotic risk in APS and could serve as promising targets for novel antithrombotic agents in APS management.

Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.

Mitochondrial ROS-dependent CD4+PD-1+T cells are pathological expansion in patients with primary immune thrombocytopenia.

Aberrant-activated T cells, especially CD4+T cells, play a crucial part in the pathogenetic progress of immune thrombocytopenia (ITP). PD-1-mediated signals play a negative part in the activation of CD4+T cells. However, knowledge is limited on the pathogenic characteristics and function of CD4+PD-1+T cells in ITP. The frequency and phenotype including cell activation, apoptosis, and cytokine production of CD4+PD-1+T cells were evaluated by flow cytometry. PD-1 Ligation Assay was performed to assess the function of PD-1 pathway in CD4+T cells. Mitochondrial reactive oxygen species (mtROS) were detected by MitoSOX Red probe. Compared with healthy controls (HC), the frequencies of CD4+PD-1+T cells were significantly increased in ITP patients. However, these cells are not exhausted despite PD-1 expression. Besides retaining cytokine-producing potential, these CD4+PD-1+T cells also had a possible B-cell helper function including expressing ICOS, CD84, and CD40L. Moreover, the CD4+PD-1+T cell subset contained higher levels of mitochondrial ROS than CD4+PD-1-T cell subset in patients with ITP. And mtROS inhibition could reduce the secretion of the inflammatory cytokines and regulate the function of CD4+PD-1+T cells. Upon in-vitro T cell receptor (TCR) stimulation of CD4+T cells in the presence of plate-bound PD-L1 fusion protein (PD-L1-Ig), CD4+T cells from ITP patients appeared resistant to such PD-1-mediated inhibition of interferon (IFN)-γ secretion. The CD4+PD-1+T cells were more abundant in patients with ITP. Additionally, this CD4+PD-1+T cell subset may be a potential etiology of ITP and a potential immune therapeutic target for ITP patients in the future.

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) protects hair cells from cisplatin-induced ototoxicity in vitro: possible relation to the activities of p38 MAPK signaling pathway.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) gene encodes rate-limiting enzyme in cholesterol biosynthesis, which is related to cell proliferation and mitochondrial function. The present study was designed to explore the expression of HMGCR in murine cochlear hair cells and HEI-OC1 cells and the possible mechanisms underpinning the actions of HMGCR in cisplatin-induced ototoxicity, with special attention given to p38 mitogen-activated protein kinase (MAPK) activities in vitro. The expressions of HMGCR, p-p38, cleaved caspase-3 and LC3B was measured by immunofluorescence and western blot. JC-1 staining and MitoSOX Red were used to detect mitochondria membrane potential (MMP) and reactive oxygen species (ROS) levels respectively. The apoptosis of auditory cells was assessed by TUNEL staining and flow cytometry. Protein levels of bcl2/bax and beclin1 were examined by western blot. We found that HMGCR was widely expressed in the auditory cells, of both neonatal mice and 2-month-old mice, in cytoplasm, nucleus and stereocilia. Moreover, 30μM cisplatin elicited the formation of ROS, which, in turn, led to HMGCR reduction, activating p38 kinase-related apoptosis and autophagy in auditory cells. Meanwhile, co-treatment with ROS scavenger at a concentration of 2mM, N-acetyl-L-cysteine (NAC), could alleviate the aforementioned changes. In addition, HMGCR silencing resulted in higher p38 MAPK-mediated apoptosis and autophagy under cisplatin injury. Taken together, we demonstrate that, for the first time, that HMGCR is expressed in the cochlear. Furthermore, HMGCR exerts protective benefit on auditory cells against cisplatin-mediated injury stimulated by ROS, culminating in regulation of p38 MAPK-dependent apoptosis and autophagy.

Cinchonine and cinchonidine alleviate cisplatin-induced ototoxicity by regulating PI3K-AKT signaling.

Cinchonine (CN) and its isomer cinchonidine (CD), two of the common cinchona alkaloids, are wildly used as antimalarial drugs. However, the effects of CN and CD on the auditory system are unknown. Molecular docking and molecular dynamics (MD) simulation were used for predicting effective drugs. The CCK-8 assay was conducted for assessing cell viability in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. MitoSox Red staining revealed reactive oxygen species (ROS) amounts. TMRM staining was used to assess the mitochondrial membrane potential (ΔΨm). Immunofluorescence staining of myosin 7a was used to examine hair cells (HCs) in cisplatin-treated neonatal mouse cochlear explants, while TUJ-1 immunostaining was used for the detection of spiral ganglion neurons (SGNs). Cleaved caspase-3 and TUNEL immunostaining were utilized for apoptosis assessment. Immunoblot was carried out to detect PI3K-AKT signaling effectors. Pretreatment with CN or CD significantly increased cell viability and reduced mitochondrial dysfunction and ROS accumulation in cisplatin-treated HEI-OC1 cells. Immunofluorescent staining of cochlear explants showed that CN and CD attenuated cisplatin-induced damage to SGNs and HCs. Immunoblot revealed that CN and CD downregulated the expression of cleaved caspase-3 and activated PI3K-AKT signaling in cisplatin-injured HEI-OC1 cells. CD and CN can reduce ototoxicity caused by cisplatin and might help treat cisplatin-associated hearing loss.

Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation

BackgroundLeber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation.MethodsNucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays.ResultsThe transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function.ConclusionWe demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases.

Frontotemporal Dementia-Related V57E Mutation Impairs Mitochondrial Function and Alters the Structural Properties of CHCHD10.

The V57E pathological variant of the mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) plays a role in frontotemporal dementia. The wild-type and V57E mutant CHCHD10 proteins contain intrinsically disordered regions, and therefore, these regions hampered structural characterization of these proteins using conventional experimental tools. For the first time in the literature, we represent that the V57E mutation is pathogenic to mitochondria as it increases mitochondrial superoxide and impairs mitochondrial respiration. In addition, we represent here the structural ensemble properties of the V57E mutant CHCHD10 and describe the impacts of V57E mutation on the structural ensembles of wild-type CHCHD10 in aqueous solution. We conducted experimental and computational studies for this research. Namely, MitoSOX Red staining and Seahorse Mito Stress experiments, atomic force microscopy measurements, bioinformatics, homology modeling, and multiple-run molecular dynamics simulation computational studies were conducted. Our experiments show that the V57E mutation results in mitochondrial dysfunction, and our computational studies present that the structural ensemble properties of wild-type CHCHD10 are impacted by the frontotemporal dementia-associated V57E genetic mutation.

NQO1 alleviates renal fibrosis by inhibiting the TLR4/NF-κB and TGF-β/Smad signaling pathways in diabetic nephropathy

ObjectiveDiabetic nephropathy (DN) is one of the main complications of diabetes, and inflammation and fibrosis play an important role in its progression. NAD(P)H: quinone oxidoreductase 1 (NQO1) protects cells from oxidative stress and damage caused by toxic quinones. In the present study, we aimed to investigate the protective effects of NQO1 against diabetes-induced renal inflammation and fibrosis and the underlying mechanisms. MethodsIn vivo, the kidneys of type 2 diabetes model db/db mice were infected with adeno-associated virus vectors to induce NQO1 overexpression. In vitro, human renal tubular epithelial (HK-2) cells transfected with NQO1 pcDNA3.1(+) were cultured under high-glucose (HG) conditions. Gene and protein expression was assessed by quantitative real-time PCR, Western blotting, immunofluorescence, and immunohistochemical staining. Mitochondrial reactive oxygen species (ROS) were detected with MitoSOX Red. ResultOur study revealed that the expression of NQO1 was markedly downregulated and that Toll-like receptor (TLR)4 and TGF-β1 expression was upregulated in vivo and in vitro under diabetic conditions. Overexpression of NQO1 suppressed proinflammatory cytokine (IL-6, TNF-α, MCP-1) secretion, extracellular matrix (ECM) (collagen IV, fibronectin) accumulation and epithelial-mesenchymal transition (EMT) (α-SMA, E-cadherin) in the db/db mouse kidneys and HG-cultured HK-2 cells. Furthermore, NQO1 overexpression ameliorated HG-induced TLR4/NF-κB and TGF-β/Smad pathways activation. Mechanistic studies demonstrated that a TLR4 inhibitor (TAK-242) suppressed the TLR4/NF-κB signaling pathway, proinflammatory cytokine secretion, EMT and ECM-related protein expression in HG-exposed HK-2 cells. In addition, we found that the antioxidants N-acetylcysteine (NAC) and tempol increased the expression of NQO1 and decreased the expression of TLR4, TGF-β1, Nox1, and Nox4 and ROS production in HK-2 cells cultured under HG conditions. ConclusionsThese data suggest that NQO1 alleviates diabetes-induced renal inflammation and fibrosis by regulating the TLR4/NF-κB and TGF-β/Smad signaling pathways.

Acellular hemoglobin impairs calcium release and contractility in cardiomyocytes from healthy animals

Hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOC) have been proposed as an alternative for blood transfusion. Phase III clinical trials of previous HBOC generations presented issues, such as vasoconstriction and myocardial infarction, that were not detected in pre-clinical trials. These problems have been attributed to extravasation of the low molecular weight (LMW) Hb species, nitric oxide (NO) scavenging and O 2 oversupply to the vasculature. However, the mechanisms responsible for the cardiac toxicity of HBOCs remain unclear. To explore the effects of acellular (acell) Hb and HBOC in cardiac tissues, we exposed cardiomyocytes (CMCs) to highly purified stroma-free acell bovine Hb (bHb) or human Hb (hHb). CMCs were loaded with Ca2+ fluorescent indicator (fluo-3) and put on laminin-coated coverslips. Cells were superfused with standard Tyrode solution with and without acell Hb. CMCs were stimulated with current pulses given at 1 Hz, and free cytosolic Ca2+ concentration was measured. To eliminate the effects of NO scavenging on CMCs, L-Name was added to the superfused with bHb and hHb. To study the effect of acell Hb MW on CMCs, LMW polymerized hHb (PolyhHb) was used to challenge CMCs. To explore the effect of Hb oxidation on CMCs, LMW MetHb was used to challenge the CMCs and compared to LMW PolyhMetHb. To explore the effects of antioxidants and heme toxicity, the studies with hHb and MethHb were repeated in the presence of the antioxidant N-acetylcysteine (NAC) or purified human Haptoglobin (Hp). Mitochondrial reactive oxygen species (ROS) production was measured using the fluorescent indicator MitoSOX Red after removing the Hb by washing the cells. After ROS determination, the maximal ROS production by CMCs was measured by challenging the cells with a high concentration of H 2 O 2 . Acell Hb increased the Ca 2+ transient decay time constant (tau) relative to control. Removing the effects of NO production with L-Name, increased the tau of the Ca 2+ transient in control and acell Hb challenge cells. Challenging CMCs with PolyhHb reduced the effects observed with acell Hb in Ca 2+ transients. MetHb appears to be worse than reduced Hb in terms of the changes induced to CMCs Ca 2+ transients. The antioxidant NAC mitigated the effects of acell Hb and MetHb but did not eliminate the changes on increased Ca2+ transients. The Hb scavenger, Hp, mitigated the effects of acell hHb and MethHb on Ca 2+ transients. The MitoSOX Red fluorescence was higher for CMCs exposed to acell Hb and MetHb compared to the control. PolyhHb and PolyMetHb challenge decreased the MitoSOX compared to acell hHb and MethHb. Cells with higher levels of ROS showed lower changes in fluorescence when exposed to H 2 O 2 . Here, we show that the toxicity of acell Hb is impart determined by facilitated diffusion, NO scavenging, heme release, and ROS, potentially affecting the energy supply and metabolism of CMCs. This study was supported by National Institutes of Health grants R01HL162120, R01HL159862 and the Department of Defense under grants W81XWH1810059. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.