Mitochondrial Permeability Transition Pore Opening Research Articles

Knockout of VDAC1 in H9c2 Cells Promotes tBHP‐induced Cell Apoptosis Through Decreased Mitochondrial HK II Binding and Enhanced Glycolytic Stress

VDAC1, the most abundant mitochondrial outer membrane protein, transports ions and metabolites between the cytoplasm and mitochondria. It is also involved in cellular energy production and apoptosis, but the role of VDAC1 in cell death remains controversial. Silencing or upregulation of VDAC1 may stimulate or inhibit cell apoptosis, growth and survival depending on cell types and stimuli. Using VDAC1 knockout (Vdac1−/−) embryonic stem cells (ES) and mouse embryonic fibroblast (MEF), it was revealed that VDAC1 was dispensable for both mitochondrial membrane permeability transition pore (mPTP) opening and Bax and Bid‐driven cell death. The effect of Vdac1−/− on oxidative stress‐induced cell death in a cardiac cell line has not been reported. We hypothesized that although VDAC1 is dispensable for mPTP and is not involved in mPTP‐mediated cell death, VDAC1 is an essential regulator of oxidative stress‐induced cell apoptosis in cardiac cells. To this end, we knocked out VDAC1 in the cardiac fibroblast cell line, H9c2 (Vdac1−/− H9c2) by the CRISPR‐cas9 technique and examined whether VDAC1 plays a role in cell death via oxidative stress induced by tert‐butylhydroperoxide (tBHP), an organic peroxide. We found that under physiologic conditions, Vdac1−/− did not affect H9c2 cell proliferation, and mitochondrial respiration. However, tBHP treatment enhanced the extracellular acidification rate (ECAR) and proton production rate (PPR) produced from glycolysis and promoted cell apoptosis in Vdac1−/− H9c2 cells compared to the wildtype. Vdac1−/− H9c2 cells also showed markedly reduced binding of mitochondria hexokinase II (HK II), a key glycolytic enzyme. Rescue of VDAC1 in Vdac1−/− H9c2 cells reinstated mitochondria HK II binding and concomitantly decreased tBHP‐induced cell death. Mitochondrial respiration remained the same after tBHP treatment in Vdac1−/− and wild type H9c2 cells. Our results suggest that Vdac1−/− in H9c2 cells enhances tBHP‐induced oxidative stress‐mediated cell apoptosis by reducing mitochondria HK II binding and by increasing ECAR and PPR produced from glycolytic stress.Support or Funding InformationNational Institutes of Health (R01 HL‐131673‐01A1), Veterans Administration (BX‐002539‐01), United States.

INHIBITION OF FERROPTOSIS BY LIPROXSTATIN‐1 COMPOUND PROTECTS THE MYOCARDIUM AGAINST ISCHEMIA/REPERFUSION INJURY BY DECREASING VDAC1 LEVELS AND INCREASING IN GPX4 ACTIVITY

Ferroptosis is a non‐apoptotic form of cell death characterized by iron‐dependent accumulation of lethal lipid reactive oxygen species (ROS) and liproxstatin‐1 is a potent inhibitor of ferroptosis. We investigated whether Liproxstatin‐1 can protect heart against ischemia/reperfusion (I/R) injury and determine the mechanism underlying cardioprotection. Isolated C57BL/6J mice hearts were subjected to 35 min ischemia followed 120 min reperfusion and treated with Liproxstatin‐1 (200nM). Myocardial infarct size was assessed at the end of the reperfusion, mitochondrial integrity was measured by analyzing cristae morphology and ROS production was assessed using amplex red method. The calcium retention capacity (CRC) required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green dye. The levels of mitochondrial proteins cyclophilin D (CypD), Glutathione peroxidase 4 (GPX4), VDAC1, 2 and 3 were assessed by Western blot; and GPX4 activity was measured using a kit. We found that liproxstatin‐1 treatment decreased myocardial infarction size, ROS production, and VDAC1 expression as well as preserved mitochondrial structural integrity by protecting cristae from damage. These mitochondrial effects of liproxstatin‐1 were associated with the increase in the GPX4 levels and activity. But, liproxstatin‐1 did not affect the CypD, VDAC2, and VDAC3 expression as well as mitochondrial CRC. Together, these result indicate that liproxstatin‐1 treatment protects myocardium against I/R injury by increasing GPX4 activity resulting in the decrease in mitochondrial ROS production; preserving mitochondrial structure as well as decrease in VDAC1 (but not VDAC 2 and 3) expression. Liproxstain‐1 cardioprotective action does not involve the regulation of the mPTP opening. These results point to the cardioprotective role of anti‐ferroptosis compound after I/R.Support or Funding Information[NIH RO1 grant HL138093 (JCB)]

Effects of Atrial Natriuretic Peptide knockout on renal mitochondrial bioenergetics in salt‐sensitive hypertension

BackgroundAtrial Natriuretic Peptide (ANP) encoded by Nppa, is a hormone known to promote salt excretion and BP reduction; there are clinical data implicating inherently low levels of ANP in the development of salt‐sensitive (SS) hypertension. In the heart and fat tissue, ANP is known to affect lipid peroxidation, mitochondrial bioenergetics and biogenesis. However, there is a gap in knowledge regarding the effects of ANP on mitochondria in the kidney. We hypothesized here that in SS hypertension ANP deficiency causes renal mitochondrial dysfunction.MethodsSS hypertension was induced in SSNPPA−/− (ANP knockout in Dahl SS background) and SSWT (wild type Dahl SS) rats by placing them on a high salt diet (HS, 4% NaCl) or a normal salt diet (control, NS, 0.4% NaCl) for 21 days. A combination of in vivo techniques with studies performed on isolated renal mitochondria (seahorse respiration and spectrofluorimetry assays) were used to test the role of ANP knockout in the mitochondria bioenergetics.ResultsOur previous studies demonstrated that during a HS challenge SSNPPA−/− rats exhibit exacerbated salt‐sensitivity of blood pressure, reduced sodium excretion, decreased urine flow, and aggravated kidney injury. In addition, there is a significant reduction in renal cortical cGMP production in the HS fed SSNPPA−/− rats vs SSWT. cGMP is known to exert protective effects in mitochondria; in order to test mitochondrial function in our model, we measured membrane potential and levels of superoxide and hydrogen peroxide (H2O2) in mitochondria isolated from kidney cortex. Specifically, TMRM, Amplex Red and MCLA were used to detect mitochondrial membrane potential, H2O2 and superoxide, respectively. We report a decrease in mitochondrial membrane potential in the SSNPPA−/− rats vs SSWT (both on NS and HS diets), and an increase in mitochondrial H2O2 and superoxide levels in the same groups. Swelling assay revealed a trend for an increased mitochondria swelling rate in the SSNPPA−/− rats. Pilot calcium uptake studies implicated calcium influx via MCU (mitochondrial calcium uniporter) and mitochondrial permeability transition pore (mPTP) opening in this process. Interestingly, seahorse respirometry performed on cortical mitochondria revealed elevation of the oxygen consumption rate (OCR) in the knockout rats on HS, while in SSWT rats HS diet feeding reduced OCR (both vs NS). In addition, we observed activation of the antioxidant system in the SSNPPA−/− rats as opposed to SSWT (on HS diet, assessed in a Trolox‐based assay).ConclusionsTaken together, our data indicate that lack of ANP, via disruption of protective cGMP‐mediated pathways, results in significant alterations in mitochondrial bioenergetics in the renal tissue. By establishing the mechanistic link between ANP and mitochondrial function, further studies will advance the understanding of the mitochondria‐mediated mechanisms affecting renal disease progression.Support or Funding InformationThe study was supported by NIH R00 DK105160 and Dialysis Clinic Inc Reserve Funds.

EP4 activation ameliorates liver ischemia/reperfusion injury via ERK1/2‑GSK3β‑dependent MPTP inhibition.

Prostaglandin E receptor subtype 4 (EP4) is widely distributed in the heart, but its role in hepatic ischemia/reperfusion (I/R), particularly in mitochondrial permeability transition pore (MPTP) modulation, is yet to be elucidated. In the present study, an EP4 agonist (CAY10598) was used in a rat model to evaluate the effects of EP4 activation on liver I/R and the mechanisms underlying this. I/R insult upregulated hepatic EP4 expression during early reperfusion. In addition, subcutaneous CAY10598 injection prior to the onset of reperfusion significantly increased hepatocyte cAMP concentrations and decreased serum ALT and AST levels and necrotic and apoptotic cell percentages, after 6 h of reperfusion. Moreover, CAY10598 protected mitochondrial morphology, markedly inhibited mitochondrial permeability transition pore (MPTP) opening and decreased liver reactive oxygen species levels. This occurred via activation of the ERK1/2-GSK3β pathway rather than the janus kinase (JAK)2-signal transducers and activators of transcription (STAT)3 pathway, and resulted in prevention of mitochondria-associated cell injury. The MPTP opener carboxyatractyloside (CATR) and the ERK1/2 inhibitor PD98059 also partially reversed the protective effects of CAY10598 on the liver and mitochondria. The current findings indicate that EP4 activation induces ERK1/2-GSK3β signaling and subsequent MPTP inhibition to provide hepatoprotection, and these observations are informative for developing new molecular targets and preventative therapies for I/R in a clinical setting.

Hibernator-Derived Cells Show Superior Protection and Survival in Hypothermia Compared to Non-Hibernator Cells.

Mitochondrial failure is recognized to play an important role in a variety of diseases. We previously showed hibernating species to have cell-autonomous protective mechanisms to resist cellular stress and sustain mitochondrial function. Here, we set out to detail these mitochondrial features of hibernators. We compared two hibernator-derived cell lines (HaK and DDT1MF2) with two non-hibernating cell lines (HEK293 and NRK) during hypothermia (4 °C) and rewarming (37 °C). Although all cell lines showed a strong decrease in oxygen consumption upon cooling, hibernator cells maintained functional mitochondria during hypothermia, without mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential decline or decreased adenosine triphosphate (ATP) levels, which were all observed in both non-hibernator cell lines. In addition, hibernator cells survived hypothermia in the absence of extracellular energy sources, suggesting their use of an endogenous substrate to maintain ATP levels. Moreover, hibernator-derived cells did not accumulate reactive oxygen species (ROS) damage and showed normal cell viability even after 48 h of cold-exposure. In contrast, non-hibernator cells accumulated ROS and showed extensive cell death through ferroptosis. Understanding the mechanisms that hibernators use to sustain mitochondrial activity and counteract damage in hypothermic circumstances may help to define novel preservation techniques with relevance to a variety of fields, such as organ transplantation and cardiac arrest.

Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes.

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease worldwide. Renal tubular epithelial cell apoptosis and tubular atrophy have been recognized as indicators of the severity and progression of DKD, while the mechanism remains elusive. Tumor necrosis factor receptor-associated protein 1 (TRAP1) plays critical roles in apoptosis. The aim of this study was to investigate the protective role TRAP1 plays in DKD and to study the potential underlying mechanisms. TRAP1 expression was decreased, and mitochondria were injured in NRK-52e cells under high-glucose (HG) conditions. The overexpression of TRAP1 ameliorated HG-induced apoptosis, increased cell viability, maintained mitochondrial morphology, adenosine triphosphate (ATP) levels, and mitochondrial membrane potential (MMP), and buffered oxidative stress, whereas TRAP1 knockdown aggravated these effects. The protective effects of TRAP1 may be exerted via the inhibition of mitochondrial permeability transition pore (mPTP) opening, and the damage caused by TRAP1 knockdown can be partially reversed by treatment with the mPTP opening inhibitor cyclosporin A (CsA). In vivo, TRAP1 expression upregulation by AAV2/9 injection prevented renal dysfunction, ameliorated histopathological changes, maintained mitochondrial morphology and function, and reduced apoptosis and reactive oxygen species (ROS) in STZ-treated DKD rats. Thus, our results suggest that TRAP1 ameliorates diabetes-induced renal injury by preventing abnormal mPTP opening and maintaining mitochondrial structure and function, which may be treated as a potential target for DKD treatment.

Dual dynamics of mitochondrial permeability transition pore opening

Mitochondria play an essential role in bioenergetics and cellular Ca{}^{2+} handling. The mitochondrial permeability transition pore (mPTP) is a non-specific channel located in the inner mitochondrial membrane. Long-lasting openings of the pore allow the rapid passage of ions and large molecules, which can result in cell death. The mPTP also exhibits transient, low conductance openings that contribute to Ca{}^{2+} homeostasis. Although many regulators of the pore have been identified, none of them uniquely governs the passage between the two operating modes, which thus probably relies on a still unidentified network of interactions. By developing a core computational model for mPTP opening under the control of mitochondrial voltage and Ca{}^{2+}, we uncovered the existence of a positive feedback loop leading to bistability. The characteristics of the two stable steady-states correspond to those of the two opening states. When inserted in a full model of Ca{}^{2+} handling by mitochondria, our description of the pore reproduces observations in mitochondrial suspensions. Moreover, the model predicted the occurrence of hysteresis in the switching between the two modes, upon addition and removal of free Ca{}^{2+} in the extra-mitochondrial medium. Stochastic simulations then confirmed that the pore can undergo transient openings resembling those observed in intact cells.

PD98059 Protects Cerebral Cortex Mitochondrial Structure and Function at 48 h Post-Resuscitation in a Rat Model of Cardiac Arrest.

BackgroundMitochondria play a critical role as effectors and targets of brain injury in the post-resuscitation period. Although we found previously that the extracellular signal-regulated kinase (ERK)1/2 inhibitor PD98059 (PD) protects the brain against mitochondrial-mediated cell death at 24 h post-resuscitation in rats subjected to cardiac arrest/cardiopulmonary resuscitation (CA/CPR), it is not clear whether PD also exerts mitochondrial protective effect for a lasting time. Therefore, we examined the effect of PD on brain mitochondria at 48 h post-resuscitation to evaluate the time-effect of PD in the current study.MethodsExperimental rats were divided randomly into 5 groups: Sham, CA, dimethylsulfoxide (DMSO), 0.15mg/kg PD and 0.3mg/kg PD. Rats except for sham group were subjected to CA for 6 min followed by CPR. We detected survival rates and neurologic deficit scores, cerebral cortex mitochondrial function by evaluating adenosine triphosphate (ATP) levels, mitochondrial permeability transition pore (MPTP) opening, and the expression of mitofusin2 (Mfn2) and observing the ultrastructure by electron microscopy at 48 h post-resuscitation in a 6-min CA rat model.ResultsPD improved survival rates and neurologic deficit scores, alleviated cerebral cortex mitochondrial damage by reducing MPTP opening and increasing Mfn2 production at 48 h post-resuscitation in a 6-min CA rat model.ConclusionA single dose of PD improved 48 h post-resuscitation outcome and mitochondrial function, indicating the potential of the use of ERK inhibitors for the treatment of brain injury resulting from CA in the future.

Curculigoside attenuates myocardial ischemia‑reperfusion injury by inhibiting the opening of themitochondrial permeability transition pore.

The aim of the present study was to determine whether curculigoside protects against myocardial ischemia-reperfusion injury (MIRI) and to investigate the underlying mechanisms. An in vitro model of hypoxia/reoxygenation (H/R) was established by culturing H9c2 cells under hypoxic conditions for 12 h, followed by reoxygenation for 1 h. Cell Counting kit-8 and lactate dehydrogenase (LDH) assays were subsequently used to examine cell viability and the degree of cell injury. In addition, isolated rat hearts were subjected to 30 min of ischemia followed by 1 h of reperfusion to establish a MIRI model. Triphenyltetrazolium chloride (TTC) staining was performed to measure the infarct size. Furthermore, TUNEL staining and flow cytometry were employed to evaluate cell apoptosis. The opening of the mitochondrial permeability transition pore (MPTP) and changes in the mitochondrial membrane potential (ΔΨm) were assessed. Reverse transcription-quantitative PCR and western blot analysis were performed to investigate the expression levels of mitochondrial apoptosis-related proteins. Curculigoside pre-treatment significantly improved cell viability, decreased cell apoptosis and LDH activity, and reduced the infarct size and myocardial apoptosis in vitro and ex vivo, respectively. Moreover, curculigoside markedly inhibited MPTP opening and preserved the ΔΨm. In addition, curculigoside significantly decreased the expression of cytochrome c, apoptotic protease activating factor-1, cleaved caspase-9 and cleaved caspase-3. Notably, atractyloside, a known MPTP opener, abrogated the protective effects of curculigoside. On the whole, the present study demonstrated that curculigoside protected against MIRI, potentially by decreasing the levels of mitochondria-mediated apoptosis via the inhibition of MPTP opening. Therefore, the results obtained in the present study may provide the theoretical basis for the future clinical application of curculigoside.

Sappanone A alleviates hypoxia/reoxygenation-induced cardiomyocytes injury through inhibition of mitochondrial apoptosis and activation of PI3K-Akt-Gsk-3β pathway.

Myocardial ischemia reperfusion injury (MIRI) is a complex pathophysiological process involved with the activation of oxidative stress, inflammation and apoptosis. Sappanone A (SA), a homoisoflavanone isolated from the heartwood of Caesalpinia sappan L., could exhibit antioxidant, anti-inflammatory and anti-apoptotic activities. Therefore, we assumed that SA has a potential use for preventing against MIRI. The present study aimed to investigate the effect of SA treatment on MIRI and its mechanism. Cardiomyocytes (H9c2 cells) were treated with SA for 1 h, followed by 6 h of hypoxia/3 h of reoxygenation. Cell viability assay was detected by CCK-8 assay. Apoptosis was measured by flow cytometry and Hoechst staining. Mitochondrial permeability transition pore (mPTP) opening and mitochondrial transmembrane potential (ΔΨm) were measured by spectrophotometry and JC-1 staining. The changes of mitochondrial apoptosis-related proteins and PI3K–Akt–Gsk-3β signaling pathway were evaluated by Western blotting. The results showed that SA pretreatment enhanced the cell viability and decreased the activity of myocardial enzyme in a dose-dependent manner. Moreover, SA pretreatment significantly inhibited apoptosis, blocked mPTP opening, suppressed the release of ΔΨm, prevented the cytochrome c releasing from mitochondria into cytoplasm, and repressed the cleavage of caspase-9 and caspase-3. Furthermore, SA pretreatment increased the phosphorylation levels of Akt and Gsk-3β but not of Stat-3. Meanwhile, the protective effect of SA was abrogated by PI3K inhibitor (LY294002). In conclusion, our results demonstrate that SA could prevent hypoxia/reoxygenation-induced cardiomyocytes injury through inhibition of mitochondrial apoptosis and activation of PI3K–Akt–Gsk-3β pathway. Thus, SA may have a potential use for the prevention of MIRI.

MPTP opening caused by Cdk5 loss is due to increased mitochondrial Ca2+ uptake

We previously demonstrated that loss of Cdk5 in breast cancer cells promotes ROS-mediated cell death by inducing mitochondrial permeability transition pore (mPTP) opening (Oncogene 37, 1788–1804). However, the molecular mechanism by which Cdk5 loss causes mPTP opening remains to be investigated. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5−/− mouse embryos, we show that absence of Cdk5 causes a significant increase in both mPTP opening and mitochondrial Ca2+ level. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and Cdk5 loss in MAMs causes increased ER-mitochondria tethering, a process required for Ca2+ transfer from the ER to the mitochondria. Loss of Cdk5 also causes increased ATP-mediated mitochondrial Ca2+ uptake from the ER. Inhibition of ER Ca2+ release or mitochondrial Ca2+ uptake in Cdk5−/− MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5−/− MEFs is due to increased Ca2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 in MAMs regulates mitochondrial Ca2+ homeostasis that is disturbed upon Cdk5 loss, which leads to mPTP opening.

Methanol extract and fraction of Anchomanes difformis root tuber modulate liver mitochondrial membrane permeability transition pore opening in rats.

Extracts of Anchomanes difformis (AD) are used in folkloric medicine to treat several diseases and infections. However, their roles in mitochondrial permeability transition pore opening are not known. The viability of mitochondria isolated from Wistar rat liver used in this experiment, was assessed by monitoring their swelling amplitude in the absence of calcium and reversal of calcium-induced pore opening by spermine. The effects of methanol extract and fraction of A. difformis (MEAD and MFAD, respectively) on Mitochondrial Membrane Permeability Transition (MMPT) pore opening, ATPase activity, cytochrome c release and ferrous-induced lipid peroxidation were assessed spectrophotometrically. Phytochemical constituents of MEAD and MFAD were assessed using Gas Chromatography- Mass Spectrometry (GC-MS). The MEAD (10, 20, 40 and 80 μg/ ml) had no effect on MMPT pore opening in the absence of Ca2+, whereas MFAD at 80 μg/ml had a large amplitude pore opening effect. Both MEAD and MFAD reversed Ca2+‌‌-induced swelling with inhibition values of 18, 21, 24, 23% (for MEAD) and 41, 36, 35, and 26% (for MFAD) at 10, 20, 40 and 80 μg/ml, respectively. MFAD significantly enhanced F1F0 ATPase activity and caused cytochrome c release. Both MEAD and MFAD significantly inhibited ferrous-induced lipid peroxidation by 33.0, 64.0, 66, and 75% (for MEAD) and 24, 25, 30, and 45% (for MFAD), respectively. The GC-MS results revealed the presence of squalene as one of the major constituents of MEAD. These findings suggest that MFAD can be used to induce cell death via mitochondrial permeability transition in isolated rat liver. Inhibition of lipid peroxidation by MEAD and MFAD showed that the pore opening effect of the extract and fraction was not mediated via peroxidation of mitochondrial membrane lipids.

Identification of Role of Mitochondrial Chloride Intracellular Channel (CLIC) Protein, CLIC4 and CLIC5 in Cardioprotection from IR Injury via Probably Modulating the Opening of MPTP Pore

Chloride intracellular channels (CLICs) are widely distributed across species and play a pivotal role in many physiological and pathophysiological process such as cell cycle regulation, tumor progression, metastasis and pulmonary hypertension. They are unique class of dimorphic ion channel proteins existing in soluble and integral membrane form. Recently, we demonstrated that indanyloxyacetic acid 94 (IAA-94), a known CLIC blocker, increased myocardial infarction (MI) by 25±8% (p=0.01, n=5) upon in vivo ischemia-reperfusion (IR) injury. Further, the calcium retention capacity (CRC) of mitochondria upon IR injury in IAA-94 treated groups were reduced by 21±1.5% (p<0.05, n>3) in comparison to controls. Thus, suggesting the targets of IAA-94 such as CLICs are important mediators of cardioprotection from IR injury. Interestingly, we observed that amongst the six mammalian paralogs of CLICs, CLIC4 (49±7%) and CLIC5 (69±12%) localize to Percoll-purified ultra-pure cardiac mitochondria. We also detected IAA-94 sensitive CLIC like channel activity in cardiac mitoplast further indicating their functional activity. Cardiac mitochondria from clic5-/- mice exhibited increased ROS (p< 0.05, n=3) production. Moreover, we demonstrate that lack of CLIC4 and CLIC5 reduce the mitochondrial CRC by ∼17% and ∼20% (p=0.01, n≥7 for each) respectively, signifying their role in mitochondrial permeability transition pore (mPTP) opening. In support, pull down assays and mass spectrometry analysis reveal the interaction of CLIC4 and CLIC5 to each other as well as Cyclophilin D (CypD) and ATP synthase, the probable component of mPTP pore. To conclude, our results highlight the significance of mitochondrial chloride channels CLIC4 and CLIC5 in cardioprotection from IR injury probably by regulating the opening of mPTP via interacting with ATP synthase and Cyclophilin D.

Metformin rescues Parkin protein expression and mitophagy in high glucose-challenged human renal epithelial cells by inhibiting NF-κB via PP2A activation

Our preliminary research revealed that metformin, a classic anti-diabetic drug, could rescue Parkin protein expression and mitophagy in high glucose-challenged human renal epithelial cells in vitro, but the molecular mechanism remains to be explored. In the study, Human Renal Cortical Epithelial Cells (HRCEpiC) and Human Renal Proximal Tubular Epithelial Cells (HRPTEpic) were challenged with high glucose with or without metformin pre-treatment to monitor Parkin mRNA and protein expression level change. PRKN gene knockdown was performed by lentiviral-based shRNA delivery. Cell viability, apoptosis and mitophagy were monitored after treatment. Mitochondrial damage was evaluated by analyzing mitochondrial permeability transition pore opening, membrane potential change, mitochondrial superoxide accumulation and cytochrome C release. Protein levels of activating transcription factor 4 (ATF4), p53 phospho-Ser15, IκBα phosphor-Ser32, IKKα phosphor-Ser176/180 in whole cell lysate and nuclear entry of p50/p65 were assessed by western blot. Okadaic acid was used to inhibit protein phosphatase 2A (PP2A). The data suggested high glucose challenge significantly reduced PRKN gene expression, mitophagy, mitochondria integrity and cell viability in vitro, which was rescued by metformin co-treatment. The effects of metformin were crippled by PRKN gene knockdown. Metformin increased PRKN gene transcription while reducednuclear factor kappa B (NF-κB) activation but not that of p53 or ATF4. Inhibiting PP2A weakened NF-κB inhibition and PRKN induction by metformin in high glucose-challenged cells, reducing its mitochondrial protective and cytoprotective effect. So, we concluded thatmetformin protects human renal epithelial cells from high glucose-induced apoptosis by restoring Parkin protein expression and mitophagy via PP2A activation and NF-κB inhibition.

Effects of Ca(NO3)2 Stress on Mitochondria and Nitrogen Metabolism in Roots of Cucumber Seedlings

Cucumber is a major vegetable crop grown in facilities, and its seedlings are sensitive to salinity. Thus, it is important for cucumber cultivation to research the effects of Ca(NO3)2. In this study, we investigated salt-sensitive Chunqiu (CQ) and salt-tolerant BoMei 49 (BM) seedlings, the changes to the enzymes involved in the mitochondria antioxidant system in the seedlings, and the changes in the MPTP (Mitochondrial Permeability Transition Pore) opening, mitochondrial membrane fluidity, mitochondrial membrane potential depolarization, mitochondrial electron transfer chain, and NO metabolism in response to Ca(NO3)2. Ca(NO3)2 stresses inhibited the growth of the cucumber seedlings, which showed a reduced root length, a decreased surface area and a decreased number of root tips, but a significantly increased root diameter. The mitochondrial malondialdehyde (MDA) content, H2O2 content, and O2− producing rate of the two cucumber roots were gradually increased in the Ca(NO3)2 treatment. The activity of superoxide dismutase (SOD) and peroxidation enzyme (POD) gradually increased, and catalase (CAT) activity gradually decreased. The electron transport chain activity of “BM” was higher than “CQ” under Ca(NO3)2 stress. Ca(NO3)2 stress destroyed the membrane structure of the mitochondria, so that the mitochondrial body tended to bend, causing double-membrane digestion and a hollow interior. Under the stress of Ca(NO3)2, the NO3− content of the seedlings increased significantly. The contents of NH4+ and NO, as well as the activities of nitrate reductase (NR), nitrite reductase (NIR), and nitric oxide synthase (NOS) decreased significantly. These findings provide physiological insights into root damage in response to salt stress for salt tolerance in cucumber.

Suppression of Superoxide-Hydrogen Peroxide Production at Site IQ of Mitochondrial Complex I Attenuates Myocardial Stunning and Improves Postcardiac Arrest Outcomes.

Cardiogenic shock following cardiopulmonary resuscitation for sudden cardiac arrest is common, occurring even in the absence of acute coronary artery occlusion, and contributes to high rates of postcardiopulmonary resuscitation mortality. The pathophysiology of this shock is unclear, and effective therapies for improving clinical outcomes are lacking. Laboratory investigation. University laboratory. C57BL/6 adult female mice. Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 4, 8, 12, or 16-minute potassium chloride-induced cardiac arrest followed by 90 seconds of cardiopulmonary resuscitation. Mice were then blindly randomized to a single IV injection of vehicle (phosphate-buffered saline) or suppressor of site IQ electron leak, an inhibitor of superoxide production by complex I of the mitochondrial electron transport chain. Suppressor of site IQ electron leak and vehicle were administered during cardiopulmonary resuscitation. Using a murine model of asystolic cardiac arrest, we discovered that duration of cardiac arrest prior to cardiopulmonary resuscitation determined postresuscitation success rates, degree of neurologic injury, and severity of myocardial dysfunction. Post-cardiopulmonary resuscitation cardiac dysfunction was not associated with myocardial necrosis, apoptosis, inflammation, or mitochondrial permeability transition pore opening. Furthermore, left ventricular function recovered within 72 hours of cardiopulmonary resuscitation, indicative of myocardial stunning. Postcardiopulmonary resuscitation, the myocardium exhibited increased reactive oxygen species and evidence of mitochondrial injury, specifically reperfusion-induced reactive oxygen species generation at electron transport chain complex I. Suppressor of site IQ electron leak, which inhibits complex I-dependent reactive oxygen species generation by suppression of site IQ electron leak, decreased myocardial reactive oxygen species generation and improved postcardiopulmonary resuscitation myocardial function, neurologic outcomes, and survival. The severity of cardiogenic shock following asystolic cardiac arrest is dependent on the length of cardiac arrest prior to cardiopulmonary resuscitation and is mediated by myocardial stunning resulting from mitochondrial electron transport chain complex I dysfunction. A novel pharmacologic agent targeting this mechanism, suppressor of site IQ electron leak, represents a potential, practical therapy for improving sudden cardiac arrest resuscitation outcomes.

Neuroprotective effects of leptin on cerebral ischemia through JAK2/STAT3/PGC-1-mediated mitochondrial function modulation

Neuroprotective effects of leptin have been shown in mouse model of cerebral ischemia/reperfusion injury and primary cortical neuronal culture with oxygen-glucose deprivation (OGD), while the underlying mechanisms are less understood. In the present study, we investigated whether leptin modulated mitochondrial function through JAK2/STAT3 in vivo mouse model of transient middle cerebral artery occlusion (MCAO) and in OGD-challenged primary neuronal cultures. JAK2/STAT3; mitochondrial biogenesis markers (PGC-1α); and apoptosis-associated proteins (caspase-3, BCL-2, BCL-XL, and cytochrome c) were detected by western blotting and reverse transcription-polymerase chain reaction at 1 h before and after ischemia/reperfusion. P-STAT3 and PGC-1α in neurons and astrocytes were detected. Moreover, mitochondrial morphology of the ischemic ipsilateral penumbra is examined using transmission electron microscopy. Primary cerebral cortical neurons were evaluated for viability, mitochondrial membrane potential (MMP), and apoptosis to assess whether dose-dependent neuroprotective effects of leptin during OGD were mitigated by the JAK2/STAT3 inhibitor AG490. Leptin activated JAK2/STAT3 signaling in neurons and astrocytes distributed in the ischemic ipsilateral penumbra, with peak p-STAT3 levels observed at 1 h after reperfusion. Leptin increased PGC-1α, BCL-2, and BCL-XL protein levels, cell viability, and MMP and decreased apoptosis both in vitro and in vivo; these effects were reversed by AG490 treatment. Our findings suggest that leptin-mediated neuroprotective effects in tMCAO may peak at 1 h to induce the transcription of its target gene PGC-1α, stabilization of MMP, inhibition of mitochondrial permeability transition pore opening, release of cytochrome c, and apoptosis.

Melatonin Inhibits Formation of Mitochondrial Permeability Transition Pores and Improves Oxidative Phosphorylation of Frozen-Thawed Ram Sperm.

Structural and functional damages to mitochondria of frozen-thawed sperm are a typical cryoinjury, with mitochondrial permeability transition pore (MPTP) formation being the hallmark change. Mitochondria are both a primary synthesis site and principle target for melatonin; this compound can directly inhibit MPTP formation and therefore confer protection at a mitochondrial level. The objective was to determine effects of melatonin on MPTP opening, viability, motility, and oxidative phosphorylation (OXPHOS) of frozen-thawed ram sperm. Ram semen was diluted in glucose-egg yolk buffer with 0 or 10−7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow frozen, with fresh semen as Control. In frozen-thawed sperm, melatonin inhibited MPTP opening and lactate concentrations and improved sperm viability, motility, acetyl-CoA concentration and adenosine triphosphate (ATP) production. With regard to the underlying physiological mechanism, melatonin suppressed movement of citrate synthase, isocitrate dehydrogenase, oxoglutarate dehydrogenase complex, and F0F1-ATP synthase permeability from mitochondrial to cytosolic fractions induced by MPTP opening; furthermore, it increased mRNA expressions of respiratory chain complex components and activities of complexes I, II, III, and IV and thereby improved oxygen consumption capacity in frozen-thawed sperm. In conclusion, melatonin improved OXPHOS of frozen-thawed ram sperm, attributed to inhibition of cryopreservation-induced MPTP opening.

Rosuvastatin protects isolated hearts against ischemia-reperfusion injury: role of Akt-GSK-3β, metabolic environment, and mitochondrial permeability transition pore

The cardioprotective activity of rosuvastatin (R) is yet to be known. The objective of this study was to research whether R perfusion before global ischemia can mitigate myocardial ischemia-reperfusion damage, considering the metabolic condition in which these effects occur, and to contemplate potential mitochondrial benefits. Protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) and mitochondrial permeability transition pore (MPTP) are key elements in myocardial injury produced by ischemia-reperfusion. Isolated rat hearts were subjected to 25-min ischemia and 1-h reperfusion in the presence or absence of R, with or without Wortmannin (W), a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor. Akt and GSK-3β were measured by Western blot analysis; lactate, glycogen, and G6PDH were determined; and Ca2+-induced MPTP opening was evaluated using a spectrophotometric method. Contractility was assessed by left ventricular developed pressure (LVDP), and rate-pressure product (RPP), peak rate of contraction and peak rate of relaxation (± dP/dt), and left ventricular end-diastolic pressure (LVEDP) were determined. Tissue samples were extracted to evaluate mitochondrial damage by electron microscopy and to assess infarct size. Statistical analysis employed ANOVA (n = 6/per group). Myocardial infarct size was significantly reduced by R, which also improved cardiac function. MPTP opening was delayed to 300μM CaCl2, while use of W resulted in MPTP opening at 200μM CaCl2. Electron microscopy showed better mitochondrial preservation with R, which reduced lactic acid production, increased glycogen consumption and G6PDH activity, as well as phosphorylation of Akt and GSK-3β. R before ischemia is cardioprotective against ischemic and reperfusion damage, activating Akt and regulating GSK-3β negatively and attenuating the MPTP opening.

Shexiang Tongxin Dropping Pill () Reduces Coronary Microembolization in Rats via Regulation of Mitochondrial Permeability Transition Pore Opening and AKT-GSK3β Phosphorylation.

To investigate the protective effects of Shexiang Tongxin Dropping Pill (, STDP) following sodium laurate-induced coronary microembolization (CME) in rats. Forty rats were divided into 4 groups: the control (sham) group, CME group, low-dose STDP pretreatment group (20 mg·kg-1·d-1), and high-dose STDP pretreatment group (40 mg·kg-1·d-1). The rats were intragastric administrated with STDP 2 weeks before operation. Moreover, the histopathological alterations were observed using optical microscopy and transmission electron microscopy. Antioxidant biomarkers were analyzed by enzyme-linked immunosorbent assay. Mitochondrial functions including the mitochondrial permeability transition pore (mPTP) mtDNA copy number were determined and proteins of AKT/GSK3β were analyzed by Western blot. The rats in the CME group showed a significant increase in the fibrinogen-like protein 2 expression level and mitochondrial dysfunction and a decrease in the expression level of antioxidant biomarkers (superoxide dismutase and catalase, P<0.01 for all). In contrast, the rats in the low- and high-dose STDP pretreatment groups showed a significant decrease in coronary microthrombi (P<0.05); moreover, STDP restored the antioxidant-related protein activities and mitochondrial function, inhibited mPTP opening, decreased AKT-Ser473 phosphorylation, and increased GSK3β-Ser9 phosphorylation (P<0.05 or P<0.01). STDP may be useful for treatment of CME, possibly via regulation of mPTP opening and AKT/GSK3β phosphorylation.