Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

The mitochondrial ATP synthase is a negative regulator of the mitochondrial permeability transition pore.

The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.

Icariin improves oxidative stress injury during ischemic stroke via inhibiting mPTP opening.

BACKGROUND: Ischemic stroke presents a significant threat to human health due to its high disability rate and mortality. Currently, the clinical treatment drug, rt-PA, has a narrow therapeutic window and carries a high risk of bleeding. There is an urgent need to find new effective therapeutic drugs for ischemic stroke. Icariin (ICA), a key ingredient in the traditional Chinese medicine Epimedium, undergoes metabolism in vivo to produce Icaritin (ICT). While ICA has been reported to inhibit neuronal apoptosis after cerebral ischemia-reperfusion (I/R), yet its underlying mechanism remains unclear. METHODS: PC-12 cells were treated with 200 µM H2O2 for 8 h to establish a vitro model of oxidative damage. After administration of ICT, cell viability was detected by Thiazolyl blue tetrazolium Bromide (MTT) assay, reactive oxygen species (ROS) and apoptosis level, mPTP status and mitochondrial membrane potential (MMP) were detected by flow cytometry and immunofluorescence. Apoptosis and mitochondrial permeability transition pore (mPTP) related proteins were assessed by Western blotting. Middle cerebral artery occlusion (MCAO) model was used to establish I/R injury in vivo. After the treatment of ICA, the neurological function was scored by ZeaLonga socres; the infarct volume was observed by 2,3,5-Triphenyltetrazolium chloride (TTC) staining; HE and Nissl staining were used to detect the pathological state of the ischemic cortex; the expression changes of mPTP and apoptosis related proteins were detected by Western blotting. RESULTS: In vitro: ICT effectively improved H2O2-induced oxidative injury through decreasing the ROS level, inhibiting mPTP opening and apoptosis. In addition, the protective effects of ICT were not enhanced when it was co-treated with mPTP inhibitor Cyclosporin A (CsA), but reversed when combined with mPTP activator Lonidamine (LND). In vivo: Rats after MCAO shown cortical infarct volume of 32-40%, severe neurological impairment, while mPTP opening and apoptosis were obviously increased. Those damage caused was improved by the administration of ICA and CsA. CONCLUSIONS: ICA improves cerebral ischemia-reperfusion injury by inhibiting mPTP opening, making it a potential candidate drug for the treatment of ischemic stroke.

An ex vivo screening using mouse brain mitochondria identified seco-cycline D as an inhibitor of mitochondrial permeability transition pore.

Mitochondrial dysfunction is implicated in neuropsychiatric disorders. Inhibition of mitochondrial permeability transition pore (mPTP) and thereby enhancement of mitochondrial Ca2+ retention capacity (CRC) is a promising treatment strategy. Here, we screened 1718 compounds to search for drug candidates inhibiting mPTP by measuring their effects on CRC in mitochondria isolated from mouse brains. We identified seco-cycline D (SCD) as an active compound. SCD and its derivative were more potent than a known mPTP inhibitor, cyclosporine A (CsA). The mechanism of action of SCD was suggested likely to be different from CsA that acts on cyclophilin D. Repeated administration of SCD decreased ischemic area in a middle cerebral artery occlusion model in mice. These results suggest that SCD is a useful probe to explore mPTP function.

Gasotransmitters and noble gases in cardioprotection: unraveling molecular pathways for future therapeutic strategies.

Despite recent progress, ischemic heart disease poses a persistent global challenge, driving significant morbidity and mortality. The pursuit of therapeutic solutions has led to the emergence of strategies such as ischemic preconditioning, postconditioning, and remote conditioning to shield the heart from myocardial ischemia/reperfusion injury (MIRI). These ischemic conditioning approaches, applied before, after, or at a distance from the affected organ, inspire future therapeutic strategies, including pharmacological conditioning. Gasotransmitters, comprising nitric oxide, hydrogen sulfide, sulfur dioxide, and carbon monoxide, play pivotal roles in physiological and pathological processes, exhibiting shared features such as smooth muscle relaxation, antiapoptotic effects, and anti-inflammatory properties. Despite potential risks at high concentrations, physiological levels of gasotransmitters induce vasorelaxation and promote cardioprotective effects. Noble gases, notably argon, helium, and xenon, exhibit organ-protective properties by reducing cell death, minimizing infarct size, and enhancing functional recovery in post-ischemic organs. The protective role of noble gases appears to hinge on their modulation of molecular pathways governing cell survival, leading to both pro- and antiapoptotic effects. Among noble gases, helium and xenon emerge as particularly promising in the field of cardioprotection. This overview synthesizes our current understanding of the roles played by gasotransmitters and noble gases in the context of MIRI and cardioprotection. In addition, we underscore potential future developments involving the utilization of noble gases and gasotransmitter donor molecules in advancing cardioprotective strategies.

Nitric Oxide/Glucose Transporter Type 4 Pathway Mediates Cardioprotection against Ischemia/Reperfusion Injury under Hyperglycemic and Diabetic Conditions in Rats.

INTRODUCTION: The comorbidities of ischemic heart disease (IHD) and diabetes mellitus (DM) compromise the protection of the diabetic heart from ischemia/reperfusion (I/R) injury. We hypothesized that manipulation of reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways might protect the diabetic heart, and intervention of these pathways could be a new avenue for potentially protecting the diabetic heart. METHODS: All hearts were subjected to 30-min ischemia and 30-min reperfusion. During reperfusion, hearts were exposed to molecules proven to protect the heart from I/R injury. The hemodynamic data were collected using suitable software. The infarct size, troponin T levels, and protein levels in hearts were evaluated. RESULTS: Both cyclosporine-A and nitric oxide donor (SNAP) infusion at reperfusion protected 4-week diabetic hearts from I/R injury. However, 6-week diabetic hearts were protected only by SNAP, but not cyclosporin-A. These treatments significantly (p < 0.05) improved cardiac hemodynamics and decreased infarct size. CONCLUSIONS: The administration of SNAP to diabetic hearts protected both 4- and 6-week diabetic hearts; however, cyclosporine-A protected only the 4-week diabetic hearts. The eNOS/GLUT-4 pathway executed the SNAP-mediated cardioprotection.

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice.

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.

Imeglimin attenuates NLRP3 inflammasome activation by restoring mitochondrial functions in macrophages.

Imeglimin is a novel oral antidiabetic drug for treating type 2 diabetes. However, the effect of imeglimin on NLRP3 inflammasome activation has not been investigated yet. Here, we aimed to investigate whether imeglimin reduces LPS-induced NLRP3 inflammasome activation in THP-1 macrophages and examine the associated underlying mechanisms. We analyzed the mRNA and protein expression levels of NLRP3 inflammasome components and IL-1ß secretion. Additionally, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening were measured by flow cytometry. Imeglimin inhibited NLRP3 inflammasome-mediated IL-1ß production in LPS-stimulated THP-1-derived macrophages. In addition, imeglimin reduced LPS-induced mitochondrial ROS production and mitogen-activated protein kinase phosphorylation. Furthermore, imeglimin restored the mitochondrial function by modulating mitochondrial membrane depolarization and mPTP opening. We demonstrated for the first time that imeglimin reduces LPS-induced NLRP3 inflammasome activation by inhibiting mPTP opening in THP-1 macrophages. These results suggest that imeglimin could be a promising new anti-inflammatory agent for treating diabetic complications.

Cyclosporine A (CsA) prevents synaptic impairment caused by truncated tau by caspase-3.

During Alzheimer's (AD), tau protein suffers from abnormal post-translational modifications, including cleaving by caspase-3. These tau forms affect synaptic plasticity contributing to the cognitive decline observed in the early stages of AD. In addition, caspase-3 cleaved tau (TauC3) impairs mitochondrial dynamics and organelles transport, which are both relevant processes for synapse. We recently showed that the absence of tau expression reverts age-associated cognitive and mitochondrial failure by blocking the mitochondrial permeability transition pore (mPTP). mPTP is a mitochondrial complex involved in calcium regulation and apoptosis. Therefore, we studied the effects of TauC3 against the dendritic spine and synaptic vesicle formation and the possible role of mPTP in these alterations. We used mature hippocampal mice neurons to express a reporter protein (GFP, mCherry), coupled to full-length human tau protein (GFP-T4, mCherry-T4), and coupled to human tau protein cleaved at D421 by caspase-3 (GFP-T4C3, mCherry-T4C3) and synaptic elements were evaluated. Treatment with cyclosporine A (CsA), an immunosuppressive drug with inhibitory activity on mPTP, prevented ROS increase and mitochondrial depolarization induced by TauC3 in hippocampal neurons. These results were corroborated with immortalized cortical neurons in which ROS increase and ATP loss induced by this tau form were prevented by CsA. Interestingly, TauC3 expression significantly reduced dendritic spine density (filopodia type) and synaptic vesicle number in hippocampal neurons. Also, neurons transfected with TauC3 showed a significant accumulation of synaptophysin protein in their soma. More importantly, all these synaptic alterations were prevented by CsA, suggesting an mPTP role in these negative changes derived from TauC3 expression.

Enhanced mitochondrial buffering prevents Ca2+ overload in naked mole-rat brain.

Deleterious Ca2+ accumulation is central to hypoxic cell death in the brain of most mammals. Conversely, hypoxia-mediated increases in cytosolic Ca2+ are retarded in hypoxia-tolerant naked mole-rat brain. We hypothesized that naked mole-rat brain mitochondria have an enhanced capacity to buffer exogenous Ca2+ and examined Ca2+ handling in naked mole-rat cortical tissue. We report that naked mole-rat brain mitochondria buffer >2-fold more exogenous Ca2+ than mouse brain mitochondria, and that the half-maximal inhibitory concentration (IC50 ) at which Ca2+ inhibits aerobic oxidative phosphorylation is >2-fold higher in naked mole-rat brain. The primary driving force of Ca2+ uptake is the mitochondrial membrane potential (Δψm ), and the IC50 at which Ca2+ decreases Δψm is ∼4-fold higher in naked mole-rat than mouse brain. The ability of naked mole-rat brain mitochondria to safely retain large volumes of Ca2+ may be due to ultrastructural differences that support the uptake and physical storage of Ca2+ in mitochondria. Specifically, and relative to mouse brain, naked mole-rat brain mitochondria are larger and have higher crista density and increased physical interactions between adjacent mitochondrial membranes, all of which are associated with improved energetic homeostasis and Ca2+ management. We propose that excessive Ca2+ influx into naked mole-rat brain is buffered by physical storage in large mitochondria, which would reduce deleterious Ca2+ overload and may thus contribute to the hypoxia and ischaemia-tolerance of naked mole-rat brain. KEY POINTS: Unregulated Ca2+ influx is a hallmark of hypoxic brain death; however, hypoxia-mediated Ca2+ influx into naked mole-rat brain is markedly reduced relative to mice. This is important because naked mole-rat brain is robustly tolerant against in vitro hypoxia, and because Ca2+ is a key driver of hypoxic cell death in brain. We show that in hypoxic naked mole-rat brain, oxidative capacity and mitochondrial membrane integrity are better preserved following exogenous Ca2+ stress. This is due to mitochondrial buffering of exogenous Ca2+ and is driven by a mitochondrial membrane potential-dependant mechanism. The unique ultrastructure of naked mole-rat brain mitochondria, as a large physical storage space, may support increased Ca2+ buffering and thus hypoxia-tolerance.

A new circular RNA-encoded protein BIRC6-236aa inhibits transmissible gastroenteritis virus (TGEV)-induced mitochondrial dysfunction.

Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen responsible for transmissible gastroenteritis, which results in mitochondrial dysfunction in host cells. Previously, we identified 123 differentially expressed circular RNAs (cRNA)from the TGEV-infected porcine intestinal epithelial cell line jejunum 2 (IPEC-J2). Previous bioinformatics analysis suggested that, of these, circBIRC6 had the potential to regulate mitochondrial function. Furthermore, mitochondrial permeability transition, a key step in the process of mitochondrial dysfunction, is known to be caused by abnormal opening of mitochondrial permeability transition pores (mPTPs) regulated by the voltage-dependent anion-selective channel protein 1 (VDAC)-Cyclophilin D (CypD) complex. Therefore, in the present study, we investigated the effects of circBIRC6-2 on mitochondrial dysfunction and opening of mPTPs. We found that TGEV infection reduced circBIRC6-2 levels, which in turn reduced mitochondrial calcium (Ca2+) levels, the decrease of mitochondrial membrane potential, and opening of mPTPs. In addition, we also identified ORFs and internal ribosomal entrance sites within the circBIRC6-2 RNA. We demonstrate circBIRC6-2 encodes a novel protein, BIRC6-236aa, which we show inhibits TGEV-induced opening of mPTPs during TGEV infection. Mechanistically, we identified an interaction between BIRC6-236aa and VDAC1, suggesting that BIRC6-236aa destabilizes the VDAC1-CypD complex. Taken together, the results suggest that the novel protein BIRC6-236aa encoded by cRNA circBIRC6-2 inhibits mPTP opening and subsequent mitochondrial dysfunction by interacting with VDAC1.

Influence of sex on global myocardial ischemia tolerance and mitochondrial function in circulatory death donor hearts.

Donation after circulatory death (DCD) donor hearts are not routinely used for heart transplantation (HTx) because of ischemic damage, which is inherent to the DCD process. HTx outcomes are suboptimal in males who received female donor hearts. The exact mechanism for suboptimal outcomes from female donor hearts has not been defined. Differential susceptibility to ischemia tolerance, which would play a significant role in DCD donation, could be a reason but has not been studied. We studied the influence of sex on global myocardial ischemia tolerance and mitochondrial function. Sprague-Dawley rats of both sexes were assigned to DCD (n = 32) or control beating-heart donor (CBD, n = 28) groups. DCD hearts underwent 25 min of in vivo global myocardial ischemia and 90 min of ex vivo Krebs-Henseleit buffer perfusion at 37°C. CBD hearts were procured without ischemia. Infarct size was determined in hearts following 90 min of reperfusion, and in another set of hearts, mitochondrial function (oxidative-phosphorylation) was studied following 60 min of reperfusion. Infarct size was increased 3.3-fold in male and 3.1-fold in female DCD hearts compared with CBD hearts. However, infarct size (%) was comparable in female and male DCD hearts (male: 25.4 ± 3.7 vs. female 19.0 ± 3.3, P = NS). Oxidative phosphorylation was similarly decreased in male and female DCD hearts' mitochondria compared with CBD hearts' mitochondria. Thus, neither infarct size nor mitochondrial dysfunction was higher in female DCD hearts. These results suggest that the susceptibility to ischemia is not the reason for suboptimal HTx outcomes with female donor hearts.NEW & NOTEWORTHY The current study shows cardiac injury is not increased in female DCD hearts following global ischemia-reperfusion compared with male DCD hearts. In addition, mitochondrial dysfunction with DCD ischemia-reperfusion is comparable in both sexes. Sex-specific immune responses and hormone receptor modulation may contribute to suboptimal outcomes in male HTx recipients with female donor hearts.

Inorganic polyphosphate's role in energy production and mitochondrial permeability transition pore opening in tick mitochondria.

Inorganic polyphosphate (polyP) is a biopolymer composed of phosphoanhydride-linked orthophosphate molecules. PolyP is engaged in a variety of cellular functions, including mitochondrial metabolism. Here, we examined the effects of polyP on electron transport chain enzymes and F1 Fo ATP synthase in tick embryos during embryonic development. The study found that polyPs containing medium and long chains (polyP15 and polyP65 ) enhanced the activity of complex I, complex II, complex III, and F1 Fo ATP synthase, while short polyP chains (polyP3 ) had no effect. The study also examined the activity of exopolyphosphatases (PPX) in various energy-demand situations. PPX activity was stimulated when ADP concentrations are high, characterizing a low-energy context. When complexes I-III and F1 Fo ATP synthase inhibitors were added in energized mitochondria, PPX activity decreased, whereas the mitochondrial uncoupler FCCP had no impact on PPX activity. Additionally, the study investigated the effect of polyP on mitochondrial swelling, finding that polyP causes mitochondrial swelling by increasing calcium effects on the mitochondrial permeability transition pore. The findings presented here to increase our understanding of the function of polyP in mitochondrial metabolism and its relationship to mitochondrial permeability transition pore opening in an arthropod model.

Mitochondrial Permeability Transition in Stem Cells, Development, and Disease.

The mitochondrial permeability transition (mPT) is a process that permits rapid exchange of small molecules across the inner mitochondrial membrane (IMM) and thus plays a vital role in mitochondrial function and cellular signaling. Formation of the pore that mediates this flux is well-documented in injury and disease but its regulation has also emerged as critical to the fate of stem cells during embryonic development. The precise molecular composition of the mPTP has been enigmatic, with far more genetic studies eliminating molecular candidates than confirming them. Rigorous studies in the recent decade have implicated central involvement of the F1Fo ATP synthase, or complex V of the electron transport chain, and continue to confirm a regulatory role for Cyclophilin D (CypD), encoded by Ppif, in modulating the sensitivity of the pore to opening. A host of endogenous molecules have been shown to trigger flux characteristic of mPT, including positive regulators such as calcium ions, reactive oxygen species, inorganic phosphate, and fatty acids. Conductance of the pore has been described as low or high, and reversibility of pore opening appears to correspond with the relative abundance of negative regulators of mPT such as adenine nucleotides, hydrogen ion, and divalent cations that compete for calcium-binding sites in the mPTP. Current models suggest that distinct pores could be responsible for differing reversibility and conductance depending upon cellular context. Indeed, irreversible propagation of mPT inevitably leads to collapse of transmembrane potential, arrest of ATP synthesis, mitochondrial swelling, and cell death. Future studies should clarify ambiguities in mPTP structure and reveal new roles for mPT in dictating specialized cellular functions beyond cell survival that are tied to mitochondrial fitness including stem cell self-renewal and fate. The focus of this review is to describe contemporary models of the mPTP and highlight how pore activity impacts stem cells and development.

A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl+-Induced Mitochondrial Permeability Transition Pore.

Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl+-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3UDNP (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl+-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.

Heat Shock Protein 70 Is Involved in the Efficiency of Preconditioning with Cyclosporine A in Renal Ischemia Reperfusion Injury by Modulating Mitochondrial Functions.

Cyclosporine A (CsA) preconditioning is known to target mitochondrial permeability transition pore and protect renal function after ischemia reperfusion (IR). The upregulation of heat-shock protein 70 (Hsp70) expression after CsA injection is thought to be associated with renal protection. The aim of this study was to test the effect of Hsp70 expression on kidney and mitochondria functions after IR. Mice underwent a right unilateral nephrectomy and 30 min of left renal artery clamping, performed after CsA injection and/or administration of the Hsp70 inhibitor. Histological score, plasma creatinine, mitochondrial calcium retention capacity, and oxidative phosphorylation were assessed after 24 h of reperfusion. In parallel, we used a model of hypoxia reoxygenation on HK2 cells to modulate Hsp70 expression using an SiRNA or a plasmid. We assessed cell death after 18 h of hypoxia and 4 h of reoxygenation. CsA significantly improved renal function, histological score, and mitochondrial functions compared to the ischemic group but the inhibition of Hsp70 repealed the protection afforded by CsA injection. In vitro, Hsp70 inhibition by SiRNA increased cell death. Conversely, Hsp70 overexpression protected cells from the hypoxic condition, as well as the CsA injection. We did not find a synergic association between Hsp70 expression and CsA use. We demonstrated Hsp70 could modulate mitochondrial functions to protect kidneys from IR. This pathway may be targeted by drugs to provide new therapeutics to improve renal function after IR.

Mitochondrial Oxidative Stress Is the General Reason for Apoptosis Induced by Different-Valence Heavy Metals in Cells and Mitochondria.

This review analyzes the causes and consequences of apoptosis resulting from oxidative stress that occurs in mitochondria and cells exposed to the toxic effects of different-valence heavy metals (Ag+, Tl+, Hg2+, Cd2+, Pb2+, Al3+, Ga3+, In3+, As3+, Sb3+, Cr6+, and U6+). The problems of the relationship between the integration of these toxic metals into molecular mechanisms with the subsequent development of pathophysiological processes and the appearance of diseases caused by the accumulation of these metals in the body are also addressed in this review. Such apoptosis is characterized by a reduction in cell viability, the activation of caspase-3 and caspase-9, the expression of pro-apoptotic genes (Bax and Bcl-2), and the activation of protein kinases (ERK, JNK, p53, and p38) by mitogens. Moreover, the oxidative stress manifests as the mitochondrial permeability transition pore (MPTP) opening, mitochondrial swelling, an increase in the production of reactive oxygen species (ROS) and H2O2, lipid peroxidation, cytochrome c release, a decline in the inner mitochondrial membrane potential (ΔΨmito), a decrease in ATP synthesis, and reduced glutathione and oxygen consumption as well as cytoplasm and matrix calcium overload due to Ca2+ release from the endoplasmic reticulum (ER). The apoptosis and respiratory dysfunction induced by these metals are discussed regarding their interaction with cellular and mitochondrial thiol groups and Fe2+ metabolism disturbance. Similarities and differences in the toxic effects of Tl+ from those of other heavy metals under review are discussed. Similarities may be due to the increase in the cytoplasmic calcium concentration induced by Tl+ and these metals. One difference discussed is the failure to decrease Tl+ toxicity through metallothionein-dependent mechanisms. Another difference could be the decrease in reduced glutathione in the matrix due to the reversible oxidation of Tl+ to Tl3+ near the centers of ROS generation in the respiratory chain. The latter may explain why thallium toxicity to humans turned out to be higher than the toxicity of mercury, lead, cadmium, copper, and zinc.

[Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells].

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

A Lethal Channel between the ATP Synthase Monomers.

The molecular structure of the transmembrane domain of ATP synthases is responsible for the inner mitochondrial membrane bending. According to the hypothesized mechanism, ATP synthase dissociation from dimers to monomers, triggered by Ca2+ binding to F1, allows the mitochondrial permeability transition pore formation at the interface between the detached monomers.

Small-molecule inhibitors of cyclophilin D as potential therapeutics in mitochondria-related diseases.

Cyclophilin D (CypD) is a key regulator of mitochondrial permeability transition pore (mPTP) opening. This pathophysiological phenomenon is associated with the development of several human diseases, including ischemia-reperfusion injury and neurodegeneration. Blocking mPTP opening through CypD inhibition could be a novel and promising therapeutic approach for these conditions. While numerous CypD inhibitors have been discovered to date, none have been introduced into clinical practice, mostly owing to their high toxicity, unfavorable pharmacokinetics, and low selectivity for CypD over other cyclophilins. This review summarizes current knowledge of CypD inhibitors, with a particular focus on small-molecule compounds with regard to their in vitro activity, their selectivity for CypD, and their binding mode within the enzyme's active site. Finally, approaches for improving the molecular design of CypD inhibitors are discussed.