Evaluation of cardiac histology and cell death markers during the progression of diabetes in a streptozotocin-induced diabetes rat model.

Oxidative stress, mitochondrial permeability transition pore opening and cell death during hypoxia–reoxygenation in adult cardiomyocytes

Oxidative stress (OS) and disrupted antioxidant defense mechanisms play a pivotal role in the etiology of male infertility. The alterations in reactive oxygen species (ROS) production and calcium (Ca2+) homeostasis are the main activators for the mitochondrial permeability transition pore (mPTP) opening. The mPTP opening is one of the main mechanisms involved in mitochondrial dysfunction in spermatozoa. This alteration in mitochondrial function adversely affects energy supply, sperm motility, and fertilizing capacity and contributes to the development of male infertility. In human spermatozoa, the mPTP opening has been associated with ionomycin-induced endogenous oxidative stress and peroxynitrite-induced nitrosative stress; however, the effect of exogenous oxidative stress on mPTP opening in sperm has not been evaluated. The aim of this study was to determine the effect of exogenous oxidative stress induced by hydrogen peroxide (H2O2) on mPTP opening, mitochondrial function, motility, and cell death markers in human spermatozoa. Human spermatozoa were incubated with 3 mmol/L of H2O2 for 60 min, and intracellular Ca2+ concentration, mPTP opening, mitochondrial membrane potential (ΔΨm), ATP levels, mitochondrial reactive oxygen species (mROS) production, phosphatidylserine (PS) externalization, DNA fragmentation, viability, and sperm motility were evaluated. H2O2-induced exogenous oxidative stress caused increased intracellular Ca2+, leading to subsequent mPTP opening and alteration of mitochondrial function, characterized by ΔΨm dissipation, decreased ATP levels, increased mROS production, and the subsequent alteration of sperm motility. Furthermore, H2O2-induced opening of mPTP was associated with the expression of apoptotic cell death markers including PS externalization and DNA fragmentation. These results highlight the role of exogenous oxidative stress in causing mitochondrial dysfunction, deterioration of sperm motility, and an increase in apoptotic cell death markers, including PS externalization and DNA fragmentation, through the mPTP opening. This study yielded new knowledge regarding the effects of this type of stress on mitochondrial function and specifically on mPTP opening, factors that can contribute to the development of male infertility, considering that the role of mPTP in mitochondrial dysfunction in human sperm is not completely elucidated. Therefore, these findings are relevant to understanding male infertility and may provide an in vitro model for further research aimed at improving human sperm quality.

To the Editor: A recent article in Circulation 1 proposes that prevention of myocardial damage by postconditioning involves the inhibition of mitochondrial permeability transition pore (mPTP) opening. We find the idea attractive and in keeping with studies implicating mPTP in postischemic damage2 but believe this study is too preliminary to prove this hypothesis. The authors present 2 main findings to support their claim. The first is that NIM811, an mPTP inhibitor, presents an effect similar to postconditioning. This finding indisputably shows that mPTP opening is involved in reperfusion injury. The results of NIM811 and postconditioning were both partial, however, and possible additive effects of both treatments were not determined. As a result, these experiments do not exclude that postconditioning can act on a pathway independent of mPTP opening. The second finding the authors present is an experiment with isolated mitochondria, which indicates that larger Ca2+ loads are required to induce Ca2+ release in …

Mitochondrial permeability transition pore (mPTP) opening is a key event in cell death during myocardial ischemia reperfusion. Inhibition of its modulator cyclophilin D (CypD) by cyclosporine A (CsA) reduces ischemia-reperfusion injury. The use of cyclosporine A in this indication is debated; however, targeting mPTP remains a major goal to achieve. We investigated the protective effects of a new original small-molecule cyclophilin inhibitor C31, which was specifically designed to target CypD. CypD peptidylprolyl cis-trans isomerase (PPIase) activity was assessed by the standard chemotrypsin-coupled assay. The effects of C31 on mPTP opening were investigated in isolated mouse cardiac mitochondria by measuring mitochondrial swelling and calcium retention capacity (CRC) in rat H9C2 cardiomyoblasts and in adult mouse cardiomyocytes by fluorescence microscopy in isolated perfused mouse hearts and ex vivo after drug infusion in mice. C31 potently inhibited CypD PPIase activity and mitochondrial swelling. C31 was more effective at increasing mitochondrial CRC than CsA and was still able to increase CRC in Ppif -/- (CypD-inactivated) cardiac mitochondria. C31 delayed both mPTP opening and cell death in cardiomyocytes subjected to hypoxia reoxygenation. However, high concentrations of both drugs were necessary to reduce mPTP opening in isolated perfused hearts, and neither CsA nor C31 inhibited mPTP opening in heart after in vivo infusion, underlying the importance of myocardial drug distribution for cardioprotection. C31 is an original inhibitor of mPTP opening involving both CypD-dependent and -independent mechanisms. It constitutes a promising new cytoprotective agent. Optimization of its pharmacokinetic properties is now required prior to its use against cardiac ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: This study demonstrates that the new cyclophilin inhibitor C31 potently inhibits cardiac mitochondrial permeability transition pore (mPTP) opening in vitro and ex vivo. The dual mechanism of action of C31 allows the prevention of mPTP opening beyond cyclophilin D inhibition. Further development of the compound might bring promising drug candidates for cardioprotection. However, the lack of effect of both C31 and cyclosporine A after systemic administration demonstrates the difficulties of targeting myocardial mitochondria in vivo and should be taken into account in cardioprotective strategies.

Mitochondrial permeability transition pore (mPTP) opening is critical for mediating cell death during hepatic ischaemia-reperfusion injury (IRI). Blocking mPTP opening by inhibiting cyclophilin D (CypD) is a promising pharmacological approach for the treatment of IRI. Here, we show that diastereoisomers of a new class of small-molecule cyclophilin inhibitors (SMCypIs) have properties that make them attractive candidates for the development of therapeutic agents against liver IRI. Derivatives of the parent SMCypI were synthesised and evaluated for their ability to inhibit CypD peptidyl-prolyl cis-trans isomerase (PPIase) activity and for their mitoprotective properties, evaluated by measuring mitochondrial swelling and calcium retention capacity in liver mitochondria. The ability of the selected compounds to inhibit mPTP opening was evaluated in cells subjected to hypoxia/reoxygenation using a calcein/cobalt assay. Their ability to inhibit cell death was evaluated in cells subjected to hypoxia/reoxygenation by measuring lactate dehydrogenase (LDH) release, propidium iodide staining, and cell viability. The compound performing best invitro was selected for invivo efficacy evaluation in a mouse model of hepatic IRI. The two compounds that showed the strongest inhibition of CypD PPIase activity and mPTP opening, C105 and C110, were selected. Their SR diastereoisomers carried the activity of the racemic mixture and exhibited mitoprotective properties superior to those of the known macrocyclic cyclophilin inhibitors cyclosporin A and alisporivir. C105SR was more potent than C110SR in inhibiting mPTP opening and prevented cell death in a model of hypoxia/reoxygenation. Finally, C105SR substantially protected against hepatic IRI invivo by reducing hepatocyte necrosis and apoptosis. We identified a novel cyclophilin inhibitor with strong mitoprotective properties both invitro and invivo that represents a promising candidate for cellular protection in hepatic IRI. Hepatic ischaemia-reperfusion injury (IRI) is one of the main causes of morbidity and mortality during or after liver surgery. However, no effective therapies are available to prevent or treat this devastating syndrome. An attractive strategy to prevent hepatic IRI aims at reducing cell death by targeting mitochondrial permeability transition pore opening, a phenomenon regulated by cyclophilin D. Here, we identified a new small-molecule cyclophilin inhibitor, and demonstrated the enhanced mitoprotective and hepatoprotective properties of one of its diastereoisomers both invitro and invivo, making it an attractive lead compound for subsequent clinical development.

Mitochondrial permeability transition pore (mPTP) opening is a key event in ischemia and reperfusion (I/R) injury. Several factors modulate mPTP opening, of which the increase in mitochondrial free Ca2+ plays a critical role. During I/R, there is a substrate switch in mitochondria and it is not known with certainty if/how substrate conditions influence mPTP opening. Hence, we investigated Ca2+‐induced mPTP opening by monitoring Ca2+ retention capacity (CRC) under different substrate conditions. Guinea pig heart mitochondria were suspended in respiration media containing rhodamine 123 or indo‐1 to monitor changes in ΔΨ or extra matrix [Ca2+], respectively. Either pyruvate or succinate was added first, followed by continuous infusion of CaCl2. ΔΨ became depolarized at 275±25 s or at 687±37 s with pyruvate or succinate, respectively. Indo‐1 experiments showed mitochondrial Ca2+ release in the buffer that paralleled ΔΨ depolarization. Adding cyclosporine A prolonged CRC with both substrates indicating that ΔΨ depolarization and Ca2+ release were due to mPTP opening. It is widely accepted that mPTP opening occurs upon reperfusion due to normalization of cytosolic pH. However, our results suggest a role for the complex I substrate pyruvate in Ca2+‐induced mPTP opening and thus, mitochondrial utilization of succinate during reperfusion may prove beneficial against mPTP opening.

nitric oxide (NO) plays an important role in intracellular signaling, and stole the limelight in 1992 by winning science's equivalent of an “Oscar”: Science magazine's “Molecule of the Year.” Subsequently, the importance and versatility of its known roles has expanded. Furthermore, this

Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening

Mitochondria-associated oxidative stress plays a crucial role in Alzheimer’s disease (AD). Grape seed proanthocyanidins (GSPs) have been reported to prevent oxidative stress. In this study, we investigated the underlying mechanisms of GSPs in protecting neurons against oxidative injury in an experimental model of sporadic AD. Primary mouse cortical neurons were subjected to streptozotocin (STZ) to mimic neuronal oxidative damage in vitro, and mice were subjected to intracerebroventricular (ICV) injection of STZ as an in vivo sporadic AD model. GSPs not only significantly ameliorated neuron loss and mitochondrial dysfunction in mouse cortical neurons pretreated of STZ, but also reduced cognitive impairments, apoptosis and mitochondrial oxidative stress in the cerebral cortex and hippocampus of sporadic AD mice. Moreover, GSPs increased phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), Akt and glycogen synthase kinase 3β (GSK-3β) at its Ser9. Notably, GSPs inhibited STZ-induced mitochondrial permeability transition pore (mPTP) opening via enhancing phosphorylated GSK-3β (p-GSK-3β) binds to adenine nucleotide translocator (ANT), thereby reducing the formation of the complex ANT-cyclophilin D (CypD). In conclusion, GSPs ameliorate neuronal oxidative damage and cognitive impairment by inhibiting GSK-3β-dependent mPTP opening in AD. Our study provides new insights into that GSPs may be a new therapeutic candidate for treatment of AD.

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.

Simple SummaryDiabetes mellitus as a systemic metabolic disease is one of the most serious threats to global health in this century. Diabetic cardiomyopathy is increasingly recognized as one of the most important complications of the disease, which is associated with impaired cell energy metabolism and damage to mitochondria in cardiomyocytes. Therefore, targeting mitochondrial dysfunction by pharmacological agents can be used as a therapeutic strategy in diabetic heart disease. The aim of the work was to study the effect of the mitochondria-targeted agent alisporivir on the development of mitochondrial dysfunction in the heart of mice with experimental diabetes mellitus. Alisporivir has been recently identified as a non-immunosuppressive analogue of cyclosporin A, a selective inhibitor of cyclophilin D and the mitochondrial permeability transition pore opening, with a potential in a wide range of therapeutic indications. Our results indicated that alisporivir alleviates diabetes-induced abnormalities in the ultrastructure and functions of mitochondria in cardiomyocytes and increases the rate of glucose utilization in diabetic mice. The data suggest that alisporivir acts as a mitochondria-targeted metabolic reprogramming agent and attenuates oxidative damage to the heart tissue of diabetic mice.Diabetes mellitus is a systemic metabolic disorder associated with mitochondrial dysfunction, with the mitochondrial permeability transition (MPT) pore opening being considered as one of its possible mechanisms. The effect of alisporivir, a non-immunosuppressive cyclosporin derivative and a selective inhibitor of the MPT pore opening, on the ultrastructure and functions of the heart mitochondria of mice with diabetes mellitus induced by a high-fat diet combined with streptozotocin injections was studied. The treatment of diabetic animals with alisporivir (2.5 mg/kg ip for 20 days) increased the rate of glucose clearance during the glucose tolerance test. The blood glucose level and the indicator of heart rate in alisporivir-treated diabetic mice tended to restore. An electron microscopy analysis showed that alisporivir prevented mitochondrial swelling and ultrastructural alterations in cardiomyocytes of diabetic mice. Alisporivir canceled the diabetes-induced increases in the susceptibility of heart mitochondria to the MPT pore opening and the level of lipid peroxidation products, but it did not affect the decline in mitochondrial oxidative phosphorylation capacity. The mRNA expression levels of Pink1 and Parkin in the heart tissue of alisporivir-treated diabetic mice were elevated, suggesting the stimulation of mitophagy. In parallel, alisporivir decreased the level of mtDNA in the heart tissue. These findings suggest that targeting the MPT pore opening by alisporivir alleviates the development of mitochondrial dysfunction in the diabetic heart. The cardioprotective effect of the drug in diabetes can be mediated by the induction of mitophagy and the inhibition of lipid peroxidation in the organelles.

it is has been over two decades since Murry and colleagues ([15][1]) published their seminal study describing ischemic preconditioning, a phenomenon whereby brief antecedent periods of ischemia-reperfusion profoundly protect the heart against a subsequent lethal period of ischemia and reperfusion.

The future of fatty livers

Inhibiting mitochondrial permeability transition pore (mPTP) opening is a key protection of the myocardium from ischemia/reperfusion (I/R) injury. Here, we investigated age-associated differences in the ability of cyclosporine A (CsA) to protect the heart and to modulate mPTP opening during I/R injury in vivo and its opening induced by reactive oxygen species (ROS) in vitro. Fischer 344 male rats were assigned from their respective age groups, young or old groups, to (1) I/R or (2) I/R+CsA. All animals were subjected to 30 min of ischemia following 120 min of reperfusion to determine myocardial infarct size in vivo. To measure mPTP opening in vivo, left ventricular tissues were collected 10 min after reperfusion and nicotinamide adenine dinucleotide (NAD(+)) levels were measured. In parallel experiments, rat ventricular myocytes were prepared from young and old hearts, loaded with tetramethylrhodamine ethylester and then subjected to oxidative stress in the presence or absence of CsA, and the mPTP opening time was measured using laser scanning confocal microscopy. CsA reduced myocardial infarct size in young I/R rats. Whereas CsA failed to significantly affect myocardial infarct size in old I/R rats, NAD(+) levels were better preserved in young CsA-treated rats, but this relative improvement was not observed in old rats. CsA also significantly prolonged the time necessary to induce mPTP opening in young cardiomyocytes, but not in cardiomyocytes isolated from the old rats. mPTP regulation is dysfunctional in the aged myocardium and this could account for loss of cardioprotection with aging.

Background: Protoporphyrin IX (PpIX) and its derivatives are widely used in photodynamic therapy (PDT) to kill cancer cells. Studies showed that the application of these drugs could cause systemic toxic effects in human. However, the molecular pathways involved in PpIX-induced cytotoxicity are not well-defined. Macrophages represent the primary system for protecting tissues from toxicants and initiating the resolution of inflammation. Thus, this study aims to investigate the toxicity of PpIX on macrophages and provide strategies to prevent the toxic effects. Methods: THP-1 macrophages were incubated with PpIX and cell death was measured by MTT assay and Annexin V-PI staining. Intracellular reactive oxygen species (ROS) were evaluated by 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and MitoSOX® Red staining and mitochondrial membrane potential (ΔΨm) was detected by tetramethylrhodamine methyl ester (TMRM) staining. Mitogen-activated protein (MAP) kinase activation was assayed by western blotting. Mitochondrial permeability transition pore (mPTP) opening was measured by calcein loading/Co²⁺ quenching technique and evaluating the release of mitochondrial content. Results: PpIX reduced cell viability in a dose- and time-dependent manner. The cell death was characterized by increasing PI-positive cells, ATP depletion, LDH releasing and rapid ΔΨm loss favoring necrotic features. In addition, PpIX successively induced ROS production, c-Jun N-terminal protein kinase (JNK) activation and mPTP opening. ROS scavengers, N-acetylcysteine (NAC) and deferoxamine (DFX), JNK inhibitor, SP600125, and mPTP inhibitor, cyclosporin A (CsA), all significantly rescued this cell death. Furthermore, mPTP opening was directly regulated by ROS/JNK pathway. Conclusion: PpIX induces a necrotic cell death in THP-1 macrophages through ROS production, JNK activation, and mPTP opening. It is tempting to speculate that blocking the pathways involved in the cytotoxic effects of PpIX will alleviate its side effects.