Activation Of Permeability Transition Pore Research Articles

Cell-Free Hemoglobin Induces Vascular and Mitochondrial Dysfunction in the Pulmonary Endothelium in an Oxidation-State Dependent Manner

Background: Sepsis results in significant organ damage due in part to a breakdown in endothelial vascular function. Elevated levels of cell-free hemoglobin (CFH) drive endothelial dysfunction in the lungs. CFH generates reactive oxygen species (ROS) via the iron present in the heme moiety, which can be oxidized from ferrous (CFH2+) to ferric (CFH3+). Recent studies suggest mitochondrial dysfunction may be a key pathway leading to microvascular dysfunction. We hypothesize that CFH induces mitochondrial dysfunction and microvascular endothelial barrier disruption in the lung in an oxidative-state dependent manner. Methods: CFH was converted from CFH2+ to CFH3+ by UV irradiation (validated by spectrophotometry). Primary human lung microvascular endothelial cells (HLMVECs) were treated with 1.0 mg/mL CFH2+, CFH3+, or vehicle control. Barrier dysfunction was measured via electric cell-substrate impedance sensing (ECIS) and express permeability test (XPerT) at 24h. We quantified mitochondrial superoxide (MitoSOX Red; 5 uM) by flow cytometry (1, 6, 24h) and total cellular ROS production (CellROX Deep Red; 5 uM) by fluorescence microscopy (6 and 24 h). Mitochondrial morphology (electron density, roundness, area, circularity, and aspect ratio) was assessed by transmission electron micrographs at 6h and quantified by a blinded reviewer (ImageJ). Activation of the mitochondrial permeability transition pore (mPTP) was assessed by flow cytometry using calcein-AM with cobalt chloride. DNA was purified from patient plasma and the copy number of mitochondrial genes ND4L and CYB was quantified by digital droplet PCR. Results: In HLMVECs, CFH generated excess ROS (CellROX and MitoSOX) at 6h compared to vehicle (522 vs 265 MFI, p < 0.01). Morphology analysis demonstrated increased electron density and aspect ratio and decreased circularity and roundness. mPTP activity was increased by CFH3+ (1295 vs 2197 MFI, p < 0.01) compared to vehicle or CFH2+. Barrier dysfunction caused by CFH3+ measured by ECIS peaked at 6h (-1270 vs 262 max TER drop, p < 0.0001), while XPerT confirmed dysfunction at 24h (9.41e+06 vs 6.53e+05 total fluorescent area, p < 0.01). Quantification of mtDNA copy number in patient plasma revealed that circulating mt-ND4L copies correlate with CFH (r = 0.44, p < 0.01). Conclusions: The results demonstrate significant perturbations to mitochondrial function and network dynamics caused by oxidized CFH (3+). The novel findings point towards the ferric (3+) form of hemoglobin as the primary driver of endothelial dysfunction, and the underlying mechanism is directly related to disruption of the mitochondrial network. The association of mitochondrial and vascular biomarkers demonstrates an urgent need to better understand how mitochondrial dysfunction can be prevented to limit vascular damage. This work was funded by the National Institutes of Health NHLBI 5R35HL150783 and 1F31HL167471. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.

Alpha-synuclein interaction with mitochondria is the final mechanism of ferroptotic death induced by erastin in SH-SY5Y cells

Ferroptosis has been characterized as a form of iron-dependent regulated cell death accompanied by an accumulation of reactive oxygen species and lipid oxidation products along with typical morphological alterations in mitochondria. Ferroptosis is activated by diverse triggers and inhibited by ferrostatin-1 and liproxstatin-1, apart from iron chelators and several antioxidants, and the process is implicated in multiple pathological conditions. There are, however, certain ambiguities about ferroptosis, especially regarding the final executioner of cell death subsequent to the accumulation of ROS. This study uses a typical inducer of ferroptosis such as erastin on SH-SY5Y cells, and shows clearly that ferroptotic death of cells is accompanied by the loss of mitochondrial membrane potential and intracellular ATP content along with an accumulation of oxidative stress markers. All these are prevented by ferrostatin-1 and liproxstatin-1. Additionally, cyclosporine A prevents mitochondrial alterations and cell death induced by erastin implying the crucial role of mitochondrial permeability transition pore (mPTP) activation in ferroptotic death. Furthermore, an accumulation of α-synuclein occurs during erastin induced ferroptosis which can be inhibited by ferrostatin-1 and liproxstatin-1. When the knock-down of α-synuclein expression is performed by specific siRNA treatment of SH-SY5Y cells, the mitochondrial impairment and ferroptotic death of the cells induced by erastin are markedly prevented. Thus, α-synuclein through the involvement of mPTP appears to be the key executioner protein of ferroptosis induced by erastin, but it needs to be verified if it is a generalized mechanism of ferroptosis by using other inducers and cell lines.

Mild excitotoxic effect on mitochondrial membrane potential, Ca2+ homeostasis and energy production in a mouse model of Alzheimer’s disease

AbstractBackgroundNeurons are bound to maintain a tight control over their cellular homeostasis in order to preserve their many functions, and specifically to precisely regulate ion fluxes for the generation of action potential and neurotransmission. In particular, the energetic balance has to be finely adjusted since its alteration may hamper cellular functionality with consequent cell suffering and death. Mitochondria play a central role in the homeostatic regulation within the cells due to the many vital tasks they perform in terms of energy production, participation in the regulation of Ca2+ concentration, modulation of intracellular signaling that allow the cell to respond to external solicitationMethodPrimary hippocampal neuronal cultures of 14‐17 DIV from WT and AD mouse model were challenged with μM concentration of glutamate to induce a mild excitotoxic effect; mitochondrial membrane potential, mitochondrial an cytosolic Ca2+ transients and cytosolic ATP levels were evaluated with fluorescent probes. Cells were treated also with inhibitors of the permeability transition pore to evaluate its effect on cell functionalityResultWe observed that in a model of Familial Alzheimer’s disease, hippocampal neurons are sensitive to a modest excitotoxic signal that produces a transient cytosolic and mitochondrial matrix Ca2+ with consequent partial loss of mitochondrial membrane potential and delayed recovery of mitochondrial matrix [Ca2+] which might contribute in the activation of the mitochondria permeability transition pore and consequent impairment of mitochondrial and cellular functions. This is accompanied by a reduced cytosolic ATP content that can result in an energy deficit for the cell. A brief treatment with inhibitors of the permeability transition pore is able to reduce these effects and might have protective roles against exogenous insults to the cells and help mitigate the neuronal loss characteristic of Alzheimer’s disease.ConclusionAD hippocampal neurons are more sensitive to a mild excitotoxic insult that affect mitochondrial functionality and in particular the activation of the permeability transition pore (PTP). Treatment with μM concentrations of two different inhibitors of the PTP prevent mitochondrial impairment.

Investigating the role of mitochondrial calcium uniporter beta in Alzheimer’s disease

AbstractBackgroundWe have recently shown that mitochondrial calcium (mCa2+) overload significantly contributes to the development of Alzheimer’s disease (AD). We found that either genetic inhibition of the neuronal mitochondrial calcium uniporter (MCU, the primary route for mCa2+ influx)1 or enhancement of mitochondrial Na+/Ca2+ exchanger (NCLX, the primary route for mCa2+ efflux)2 in the AD mouse model reduces AD‐associated phenotype and mitochondrial permeability transition pore (MPTP)‐induced cell death. However, blocking either the mCa2+ influx or enhancing mCa2+ efflux can also shut down the bioenergetics. Thus, it is vital to find a regulator of mCa2+ homeostasis that will positively impact Ca2+‐dependent physiological functions.MethodsTo define the new molecular regulatory mechanism to limit mCa2+ overload in AD that will maintain bioenergetics and prevent cell death, we have examined the role of Mitochondrial Calcium Uniporter Beta (MCUB), a paralog of MCU that acts as a negative regulator of mCa2+ uptake3. This study used AD patient samples, mice, and cell lines to examine MCUB expression. A neuroblastoma cell line stably expressing the human Swedish mutant amyloid precursor protein (N2a/APPswe) with adenovirus encoding MCUB was examined for mitochondrial alterations in: Ca2+ handling, ROS generation, permeability transition pore activation, oxidative phosphorylation (OxPHOS) and cell death.Results5xFAD mice and human AD brain samples displayed significant reductions in the expression of MCUB. Studies performed in N2a/APPswe neurons discovered that mCa2 handling, redox, and bioenergetics was severely impaired in AD cells. Rescue of MCUB via adenoviral expression enhanced the clearance of pathogenic mCa2+, enhanced OxPHOS, and prevented ROS and cell death.ConclusionsOur data suggest that MCUB modulation has the therapeutic potential to preserve bioenergetics and limit cell death in AD. This project will further utilize neuronal‐specific knockouts and overexpression of MCUB to increase or decrease mCa2+ uptake, respectively, in 5xFAD mouse models and will explore whether and how MCUB‐mediated changes in uptake are able to preserve metabolism, mCa2+ dynamics, and AD‐phenotype in vivo.

The ketone body β-hydroxybutyrate shifts microglial metabolism and suppresses amyloid-β oligomer-induced inflammation in human microglia.

Fatty acids are metabolized by β-oxidation within the "mitochondrial ketogenic pathway" (MKP) to generate β-hydroxybutyrate (BHB), a ketone body. BHB can be generated by most cells but largely by hepatocytes following exercise, fasting, or ketogenic diet consumption. BHB has been shown to modulate systemic and brain inflammation; however, its direct effects on microglia have been little studied. We investigated the impact of BHB on Aβ oligomer (AβO)-stimulated human iPS-derived microglia (hiMG), a model relevant to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AβO with proinflammatory activation, which was mitigated by BHB at physiological concentrations of 0.1-2 mM. AβO stimulated glycolytic transcripts, suppressed genes in the β-oxidation pathway, and induced over-expression of AD-relevant p46Shc, an endogenous inhibitor of thiolase, actions that are expected to suppress MKP. AβO also triggered mitochondrial Ca2+ increase, mitochondrial reactive oxygen species production, and activation of the mitochondrial permeability transition pore. BHB potently ameliorated all the above mitochondrial changes and rectified the MKP, resulting in reduced inflammasome activation and recovery of the phagocytotic function impaired by AβO. These results indicate that microglia MKP can be induced to modulate microglia immunometabolism, and that BHB can remedy "keto-deficiency" resulting from MKP suppression and shift microglia away from proinflammatory mitochondrial metabolism. These effects of BHB may contribute to the beneficial effects of ketogenic diet intervention in aged mice and in human subjects with mild AD.

The Reduction in the Mitochondrial Membrane Potential in Aging: The Role of the Mitochondrial Permeability Transition Pore.

It is widely reported that the mitochondrial membrane potential, ∆Ψm, is reduced in aging animals. It was recently suggested that the lower ∆Ψm in aged animals modulates mitochondrial bioenergetics and that this effect is a major cause of aging since artificially increased ∆Ψm in C. elegans increased lifespan. Here, I critically review studies that reported reduction in ∆Ψm in aged animals, including worms, and conclude that many of these observations are best interpreted as evidence that the fraction of depolarized mitochondria is increased in aged cells because of the enhanced activation of the mitochondrial permeability transition pore, mPTP. Activation of the voltage-gated mPTP depolarizes the mitochondria, inhibits oxidative phosphorylation, releases large amounts of calcium and mROS, and depletes cellular NAD+, thus accelerating degenerative diseases and aging. Since the inhibition of mPTP was shown to restore ∆Ψm and to retard aging, the reported lifespan extension by artificially generated ∆Ψm in C. elegans is best explained by inhibition of the voltage-gated mPTP. Similarly, the reported activation of the mitochondrial unfolded protein response by reduction in ∆Ψm and the reported preservation of ∆Ψm in dietary restriction treatment in C. elegans are best explained as resulting from activation or inhibition of the voltage-gated mPTP, respectively.

F-ATP Synthase Inhibitory Factor 1 in Regulation of Mitochondria Permeability Transition Pore and Metabolic Reprogramming

Mitochondrial permeability transition pore (PTP) plays an important role in mitochondrial physiology and cell fate. Emerging studies highlight PTP forms from F-ATP synthase, but whether F-ATP synthase inhibitory factor 1 (IF1) regulates the activity of PTP is basically unknown. We have recently demonstrated that IF1 interacts with p53-CyPD complex and promotes opening of the PTP, and IF1 is necessary for the formation of p53-CyPD complex. IF1, a natural inhibitor of F-ATP synthase, acts as a main driver of metabolic switch to a Warburg phenotype. In this Commentary, we intend to discuss that the PTP may act as an alternative mechanism through which IF1 regulates metabolic reprogramming. The PTP participates in physiological Ca2+/ROS homeostasis and cell fate depending on the open state. The PTP-regulatory role of IF1 provides a clue that IF1 participates in metabolic plasticity probably involving modulation of PTP activity.

In vitro study of deltamethrin-induced extracellular traps in hemocytes of Ruditapes philippinarum

Deltamethrin (DLM), a broad-spectrum pesticide, has been proven to have toxic effects on aquatic organisms. Here, we detected the formation of extracellular traps (ETosis) formation in Manila clam (Ruditapes philippinarum) hemocytes stimulated by three concentrations of DLM (0.01, 0.1 and 1 μg/mL) in vitro, and explored the underlying mechanisms induced by this pesticide. Extracellular DNA structure observation and quantitative results indicated that DLM exposure could obviously induce hemocytes ETosis, especially under high concentration of DLM induction. Moreover, DLM increased the levels of myeloperoxidase (MPO) and reactive oxygen species (ROS) in a dose-dependent manner, and enhanced the mRNA expression of several ROS-related genes. DPI (NADPH oxidase inhibitor) and ABAH (MPO inhibitor) could substantially inhibit DLM-induced extracellular traps (ETs), suggesting that the induced ETs release was caused by the induction of the ROS burst and MPO production. In addition, three concentrations of DLM-induced ETs were also accompanied by mitochondrial dysfunction, such as increasing the production of mitochondrial ROS, leading to a decrease in mitochondrial membrane potential (MMP) and activation of mitochondrial permeability transition pore (MPTP). Taken together, these results will shed new light on the immunotoxicity of DLM in clams and perhaps lays the foundation for health assessment in bivalves.

1,3,8-Triazaspiro[4.5]decane Derivatives Inhibit Permeability Transition Pores through a FO-ATP Synthase c Subunit Glu119-Independent Mechanism That Prevents Oligomycin A-Related Side Effects

Permeability transition pore (PTP) molecular composition and activity modulation have been a matter of research for several years, especially due to their importance in ischemia reperfusion injury (IRI). Notably, c subunit of ATP synthase (Csub) has been identified as one of the PTP-forming proteins and as a target for cardioprotection. Oligomycin A is a well-known Csub interactor that has been chemically modified in-depth for proposed new pharmacological approaches against cardiac reperfusion injury. Indeed, by taking advantage of its scaffold and through focused chemical improvements, innovative Csub-dependent PTP inhibitors (1,3,8-Triazaspiro[4.5]decane) have been synthetized in the past. Interestingly, four critical amino acids have been found to be involved in Oligomycin A-Csub binding in yeast. However, their position on the human sequence is unknown, as is their function in PTP inhibition. The aims of this study are to (i) identify for the first time the topologically equivalent residues in the human Csub sequence; (ii) provide their in vitro validation in Oligomycin A-mediated PTP inhibition and (iii) understand their relevance in the binding of 1,3,8-Triazaspiro[4.5]decane small molecules, as Oligomycin A derivatives, in order to provide insights into Csub interactions. Notably, in this study we demonstrated that 1,3,8-Triazaspiro[4.5]decane derivatives inhibit permeability transition pores through a FO-ATP synthase c subunit Glu119-independent mechanism that prevents Oligomycin A-related side effects.

The mitochondrial permeability transition pore in Ca2+ homeostasis

The mitochondrial Permeability Transition Pore (PTP) can be defined as a Ca2+ activated mega-channel involved in mitochondrial damage and cell death, making its inhibition a hallmark for therapeutic purposes in many PTP-related paradigms. Although long-lasting PTP openings have been widely studied, the physiological implications of transient openings (also called “flickering” behavior) are still poorly understood. The flickering activity was suggested to play a role in the regulation of Ca2+ and ROS homeostasis, and yet this hypothesis did not reach general consensus. This state of affairs might arise from the lack of unquestionable experimental evidence, due to limitations of the available techniques for capturing transient PTP activity and to a still partial understanding of its molecular identity. In this review we will focus on possible implications of the PTP in physiology, in particular its role as a Ca2+ release pathway, discussing the consequences of its forced inhibition. We will also consider the recent hypothesis of the existence of more permeability pathways and their potential involvement in mitochondrial physiology.

GLUT inhibitor WZB117 induces cytotoxicity with increased production of amyloid-beta peptide in SH-SY5Y cells preventable by beta-hydroxybutyrate: implications in Alzheimer's disease.

Inhibitors of glucose transporters are being explored as potential anti-cancer drugs. Decreased cerebral glucose utilization with reduced levels of several glucose transporters is also an important pathogenic signature of neurodegeneration of Alzheimer's disease, but its exact role in the pathogenesis of this disease is not established. We explored in an experimental model if inhibitors of glucose transporters could lead to altered amyloid-beta homeostasis, mitochondrial dysfunction, and neuronal death, which are relevant in the pathogenesis of Alzheimer's disease. SH-SY5Y cells (human neuroblastoma cell line) were exposed to an inhibitor (WZB117) of several types of glucose transporters. We examined the effects of glucose hypometabolism on SH-SY5Y cells in terms of mitochondrial functions, production of reactive oxygen species, amyloid-beta homeostasis, and neural cell death. The effect of β-hydroxybutyrate in ameliorating the effects of WZB117 on SH-SY5Y cells was also examined. We observed that exposure of SH-SY5Y cells to WZB117 caused mitochondrial dysfunction, increased production of reactive oxygen species, loss of cell viability, increased expression of BACE 1, and intracellular accumulation of amyloid β peptide (Aβ42). All the effects of WZB117 could be markedly prevented by co-treatment with β-hydroxybutyrate. Cyclosporine A, a blocker of mitochondrial permeability transition pore (mPTP) activation, could not prevent cell death caused by WZB117. Results in this neuroblastoma model have implications for the pathogenesis of Alzheimer's disease and warrant further explorations of WZB117 in primary cultures of neurons and experimental animal models.

Cardiac Myocyte-Specific Overexpression of FASTKD1 Prevents Ventricular Rupture After Myocardial Infarction.

Background The mitochondrial mRNA-binding protein FASTKD1 (Fas-activatedserine/threonine[FAST] kinase domain-containing protein 1) protects myocytes from oxidative stress in vitro. However, the role of FASTKD1 in the myocardium in vivo is unknown. Therefore, we developed cardiac-specific FASTKD1 transgenic mice to test the effects of this protein on experimental myocardial infarction (MI). Methods and Results Transgenic mouse lines with cardiac myocyte-specific overexpression of FASTKD1 to varying degrees were generated. These mice displayed normal cardiac morphological features and function at the gross and microscopic levels. Isolated cardiac mitochondria from all transgenic mouse lines showed normal mitochondrial function, ATP levels, and permeability transition pore activity. Male nontransgenic and transgenic mice from the highest-expressing line were subjected to 8 weeks of permanent coronary ligation. Of nontransgenic mice, 40% underwent left ventricular free wall rupture within 7 days of MI compared with 0% of FASTKD1-overexpressing mice. At 3 days after MI, FASTKD1 overexpression did not alter infarct size. However, increased FASTKD1 resulted in decreased neutrophil and increased macrophage infiltration, elevated levels of the extracellular matrix component periostin, and enhanced antioxidant capacity compared with control mice. In contrast, markers of mitochondrial fusion/fission and apoptosis remained unaltered. Instead, transcriptomic analyses indicated activation of the integrated stress response in the FASTKD1 transgenic hearts. Conclusions Cardiac-specific overexpression of FASTKD1 results in viable mice displaying normal cardiac morphological features and function. However, these mice are resistant to MI-induced cardiac rupture and display altered inflammatory, extracellular matrix, and antioxidant responses following MI. Moreover, these protective effects were associated with enhanced activation of the integrated stress response.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

RationaleSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. ObjectivesWe examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. MethodsWe used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. ResultsSARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. ConclusionsCoronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

PHYSIOLOGICAL ALTERATIONS OF MITOCHONDRIA UNDER DIABETES CONDITION AND ITS CORRECTION BY POLYPHENOL GOSSITAN

The state of lipid peroxidation (LPO), respiration and oxidative phosphorylation, mitochondrial permeability transition pore (mPTP) and antiradical activity of rat liver mitochondria in streptozotocin (STZ)-induced diabetes condition were studied, considered the ways of correction of detected membrane damages with the use of gossitan, isolated from cotton plant Gossypium hirsutum L. It was shown that the rate of respiration of liver mitochondria in states V3 and V4 increases during STZ-induced diabetes, which significantly reduces the respiratory control (RC) and ADP/O coefficients in comparison with the control. The findings suggest that the uncoupling of respiration and oxidative phosphorylation takes place during STZ-induced diabetes. It was shown that in the conditions of STZ-induced diabetes, the rate of swelling of rat liver mitochondria is higher than of the healthy ones; this means that mPTP of rat liver mitochondria is in the open state. Gossitan recovers mPTP to the normal condition, thereby removing the effect of STZ on mitochondria. Gossitan (per os dose is 10 mg/kg of body weight, during 8 days) eliminates the detected functional disorders of rat liver mitochondria, probably due to its antioxidant properties.

Therapeutic Targets for Regulating Oxidative Damage Induced by Ischemia-Reperfusion Injury: A Study from a Pharmacological Perspective.

Ischemia-reperfusion (I-R) injury is damage caused by restoring blood flow into ischemic tissues or organs. This complex and characteristic lesion accelerates cell death induced by signaling pathways such as apoptosis, necrosis, and even ferroptosis. In addition to the direct association between I-R and the release of reactive oxygen species and reactive nitrogen species, it is involved in developing mitochondrial oxidative damage. Thus, its mechanism plays a critical role via reactive species scavenging, calcium overload modulation, electron transport chain blocking, mitochondrial permeability transition pore activation, or noncoding RNA transcription. Other receptors and molecules reduce tissue and organ damage caused by this pathology and other related diseases. These molecular targets have been gradually discovered and have essential roles in I-R resolution. Therefore, the current study is aimed at highlighting the importance of these discoveries. In this review, we inquire about the oxidative damage receptors that are relevant to reducing the damage induced by oxidative stress associated with I-R. Several complications on surgical techniques and pathology interventions do not mitigate the damage caused by I-R. Nevertheless, these therapies developed using alternative targets could work as coadjuvants in tissue transplants or I-R-related pathologies

Urocortin I Protects against Myocardial Ischemia/Reperfusion Injury by Sustaining Respiratory Function and Cardiolipin Content via Mitochondrial ATP-Sensitive Potassium Channel Opening.

Objective Our experiments were aimed at probing whether urocortin I postconditioning was beneficial for maintaining the mitochondrial respiratory function and inhibiting the surging of reactive oxygen species. In addition, our experiments also intended to reveal the relationships between urocortin I postconditioning and mitochondrial ATP-sensitive potassium channel. Methods Langendorff and MPA perfusion systems were used to establish myocardial ischemia-reperfusion injury model and cardiomyocytes hypoxia-reoxygenation injury model in rats, respectively. Isolated hearts and cardiomyocytes were randomly divided into normal group, ischemia-reperfusion/hypoxia-reoxygenation group, urocortin I postconditioning group, and 5-hydroxysolanoic acid (5-HD)+urocortin I group. At the end of balance (T1) and reperfusion (T2), cardiac functions, mitochondrial state3 respiratory, respiratory control ratio, mitochondrial respiratory enzyme activity, and mitochondrial cardiolipin content were measured. Our experiments also observed the ultrastructure of myocardium. The changes of cardiomyocyte mitochondrial permeability transition pore, mitochondrial membrane potential, reactive oxygen species, expression of apoptosis protein, and cardiomyocytes activity were detected at the end of reoxygenation. Results The cardiac functions, mitochondrial respiratory function, and enzyme activity of the normal group were better than other three groups at T2, and urocortin I postconditioning group was better than the IR group and 5-HD+urocortin I group. LVEDP, +dp/dtmax, mitochondrial respiratory function, and enzyme activity of IR group were worse than 5-HD+urocortin I group. Cardiolipin content of the normal group was higher than the other three groups at T2, urocortin I postconditioning group was higher than the IR group and 5-HD+urocortin I group, and 5-HD+urocortin I group was still higher than the IR group. The ultrastructure of the normal group maintained the most integrated than the other groups, IR group suffered the most serious damage, and ultrastructure of the urocortin I postconditioning group was better than the IR group and 5-HD+urocortin I group. At the end of reoxygenation, activity of mitochondrial permeability transition pore and generation of reactive oxygen species of normal group were lower than the other groups, HR group and 5-HD+urocortin I group were higher than the urocortin I postconditioning group, and 5-HD+urocortin I group was still higher than the urocortin I postconditioning group. Normal group had the highest level of mitochondrial membrane potential at the end of reoxygenation, and the urocortin I postconditioning group was higher than the HR group and 5-HD+urocortin I group. The normal group had the lowest expression level of Bax and the highest expression level of Bcl-2 at the end of reoxygenation. Urocortin I postconditioning group had lower Bax expression but higher Bcl-2 expression than the HR and 5-HD+urocortin I group. Accordingly, the normal group had the highest activity of cardiomyocytes, and the urocortin I postconditioning group was higher than the HR group and 5-HD+urocortin I group. Conclusions Urocortin I postconditioning can protect the activity of cardiomyocytes after hypoxia-reoxygenation injury, improve the mitochondrial respiratory function, and enhance the contractility of isolated heart after myocardial ischemia-reperfusion injury. The alleviation of myocardial injury relates to the opening of mitochondrial ATP-sensitive potassium channel.

Holographic imaging of the mitochondrial swelling in the living cells reveals that mitochondrial depolarization occurs before activation of the high-conductance permeability transition pore

Mitochondrial permeability transition is caused by opening of the Cyclosporine A (CSA) dependent calcium-induced pore, Permeability Transition Pore (PTP). PTP activation is the central event in stress-induced cell death. Molecular details of PTP opening remain incompletely understood. PTP makes mitochondrial inner membrane permeable to the molecules up to 1.5 kDa in size and results in high-amplitude mitochondrial swelling. Swelling and solutes equilibration make mitochondria optically transparent—the phenomenon that has been widely used as a bona fide “light-scattering” PTP assay in the isolated mitochondria.

Temporal effect of melatonin posttreatment on anoxia/reoxygenation injury in H9c2 cells

Melatonin has been proven to reduce myocardial ischemia-reperfusion (MI/R) injury. However, in most studies, melatonin was administered before MI/R, thus, the results lack clinical significance in patients with acute myocardial infarction. We hypothesize that melatonin posttreatment at different times has different curative effects. Administered of Melatonin (150 μM) at different times after the onset of reoxygenation (t = -15, 0, 5, 10, 15, and 30 min). Cellular apoptosis, oxidative stress, and mitochondrial function were assessed. Mitophagy-related protein levels,mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) activity were also measured. A/R injury upregulated mitophagy, which was associated with increased cellular apoptosis, oxidative stress, and mitochondrial dysfunction. Melatonin posttreatment (t = -15, 0, 5, 10, 15, and 30 min) significantly inhibited excessive mitophagy after A/R injury, reduced cellular apoptosis and oxidative stress, restored mitochondrial function and MMP, and restrained mPTP opening. The therapeutic time window in which melatonin posttreatment protected H9c2 cells against A/R injury was large (from -15 to 30 min after the onset of reperfusion), but the earlier the melatonin administration was, the better its protective effect was. This mechanism is likely due to a reduction in mPTP activity and MMP collapse, which lead to the inhibition of mitophagy.

Ketone Ester D-β-Hydroxybutyrate-(R)-1,3 Butanediol Prevents Decline in Cardiac Function in Type 2 Diabetic Mice.

BackgroundHeart failure is responsible for approximately 65% of deaths in patients with type 2 diabetes mellitus. However, existing therapeutics for type 2 diabetes mellitus have limited success on the prevention of diabetic cardiomyopathy. The aim of this study was to determine whether moderate elevation in D‐β‐hydroxybutyrate improves cardiac function in animals with type 2 diabetes mellitus.Methods and ResultsType 2 diabetic (db/db) and their corresponding wild‐type mice were fed a control diet or a diet where carbohydrates were equicalorically replaced by D‐β‐hydroxybutyrate‐(R)‐1,3 butanediol monoester (ketone ester [KE]). After 4 weeks, echocardiography demonstrated that a KE diet improved systolic and diastolic function in db/db mice. A KE diet increased expression of mitochondrial succinyl‐CoA:3‐oxoacid‐CoA transferase and restored decreased expression of mitochondrial β‐hydroxybutyrate dehydrogenase, key enzymes in cardiac ketone metabolism. A KE diet significantly enhanced both basal and ADP‐mediated oxygen consumption in cardiac mitochondria from both wild‐type and db/db animals; however, it did not result in the increased mitochondrial respiratory control ratio. Additionally, db/db mice on a KE diet had increased resistance to oxidative and redox stress, with evidence of restoration of decreased expression of thioredoxin and glutathione peroxidase 4 and less permeability transition pore activity in mitochondria. Mitochondrial biogenesis, quality control, and elimination of dysfunctional mitochondria via mitophagy were significantly increased in cardiomyocytes from db/db mice on a KE diet. The increase in mitophagy was correlated with restoration of mitofusin 2 expression, which contributed to improved coupling between cytosolic E3 ubiquitin ligase translocation into mitochondria and microtubule‐associated protein 1 light chain 3–mediated autophagosome formation.ConclusionsModerate elevation in circulating D‐β‐hydroxybutyrate levels via KE supplementation enhances mitochondrial biogenesis, quality control, and oxygen consumption and increases resistance to oxidative/redox stress and mPTP opening, thus resulting in improvement of cardiac function in animals with type 2 diabetes mellitus.