Mitochondrial Transition Pore Opening Research Articles

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists invitro. Invivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, invitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

Alcohol exposure during pregnancy induces cardiac mitochondrial damage in offspring mice.

Prenatal alcohol exposure (PAE) has been linked to congenital heart disease and fetal alcohol syndrome. The heart primarily relies on mitochondria to generate energy, so impaired mitochondrial function due to alcohol exposure can significantly affect cardiac development and function. Our study aimed to investigate the impact of PAE on myocardial and mitochondrial functions in offspring mice. We administered 30% alcohol (3 g/kg) to pregnant C57BL/6 mice during the second trimester. We assessed cardiac function by transthoracic echocardiography, observed myocardial structure and fibrosis through staining tests and electron transmission microscopy, and detected cardiomyocyte apoptosis with dUTP nick end labeling assay and real-time quantitative PCR. Additionally, we measured the reactive oxygen species content, ATP level, and mitochondrial DNA copy number in myocardial mitochondria. Mitochondrial damage was evaluated by assessing the level of mitochondrial membrane potential and the opening degree of mitochondrial permeability transition pores. Our findings revealed that PAE caused cardiac systolic dysfunction, ventricular enlargement, thinned ventricular wall, cardiac fibrosis in the myocardium, scattered loss of cardiomyocytes, and disordered arrangement of myocardial myotomes in the offspring. Furthermore, we observed a significant increase in mitochondrial reactive oxygen species content, a decrease in mitochondrial membrane potential, ATP level, and mitochondrial DNA copy number, and sustained opening of mitochondrial permeability transition pores in the heart tissues of the offspring. These results indicated that PAE had adverse effects on the cardiac structure and function of the newborn mice and could trigger oxidative stress in their myocardia and contribute to mitochondrial dysfunction.

Genetic enhancement of mitochondrial Ca2+ buffering capacity prevents apoptotic signaling activation in response to cytosolic Ca2+ elevation

Introduction: Over the past decade, multiple groups have used genetically engineered mitochondrial Ca2+ (mtCa2+)-sensitive biosensors to delineate the role of mtCa2+ uptake in various cell types/tissues. It has been widely shown that inhibition of mtCa2+ influx via the main mtCa2+ channel, mtCa2+ uniporter (MCU), can mitigate increases in free mitochondrial-matrix Ca2+ concentration ([Ca2+]mt) in response to cytosolic Ca2+ elevation, which prevents overproduction of reactive oxygen species, opening of mitochondrial transition pore (mPTP), and activation of apoptotic signaling. However, chronic interruption of the mtCa2+ handling system may be unfavorable for maintaining physiological cellular functions. Inhibiting mtCa2+ influx may secondarily increase cytosolic Ca2+ concentration ([Ca2+]c) in the absence of MCU activity for cytosolic Ca2+ clearance, activating signaling pathways that cause cellular damage. [Ca2+]mt is determined by the balance of mtCa2+ influx, efflux, and Ca2+ buffering capacity at the mitochondrial matrix. However, the molecular identity of mitochondrial Ca2+ buffering machinery is still unknown. Hypothesis: Enhancement of mtCa2+ buffering capacity by overexpressing mtCa2+ biosensors can prevent mtCa2+ overload by chelating excessive free Ca2+ at the mitochondrial matrix and providing anti-apoptotic effects during cytosolic Ca2+ elevation. Methods: HEK293T cells and H9c2 cardiac myoblasts transfected with mtCa2+ biosensors underwent biochemical, cell biological, and physiological assays. In this experiment, we tested Mitycam, a mitochondria-targeted inverse pericam (Kettlewell S, et al, 2009), and its mutant Mitycam-E31Q, a lower Ca2+-binding affnity version of the original Mitycam (Haviland S, et al, 2014). Results: First, we used Western blotting with an anti-GFP antibody to confirm that the original and mutant Mitycams were expressed at a similar protein size in HEK293T cells and H9c2 cells. The expression level of Mitycam-E31Q was significantly higher than the original Mitycam (55.4±12.9% increase, n=3, p<0.05) incresedespite the equal amount of plasmids transfected. Next, we observed that both Mitycams were exclusively localized in mitochondria by confocal microscopy after co-transfection with mitochondrial matrix-targeted DsRed (mt-RFP) in HEK293T cells and H9c2 cells. A decrease in Mitycam fluorescence in response to transient [Ca2+]c elevation was detected in H9c2 cells following treatment with thapsigargin, an inhibitor of sarco/endoplasmic Ca2+-ATPase (SERCA). These changes were abolished when cells were co-transfected with a dominant-negative pore-forming subunit of MCU, MCUb, confirming that Mitycam and Mitycam-E31Q are capable of sensing [Ca2+]mt elevation by mtCa2+ uptake via MCU. Lastly, H9c2 cells were transfected with Mitycams or mitochondrial matrix-targeted GFP (mt-GFP) as a control, and exposed to prolonged [Ca2+]c elevation by ionomycin, a membrane-permeable Ca2+ ionophore. Cells expressing mt-GFP showed apoptotic signaling activation after ionomycin treatment as assessed by the increased the amount of cleaved caspase-3 by Western blotting (40.1 ±9.4% increase after treatment compared to before treatment, n=4, p<0.05). Importantly, both Mitycams significantly inhibited the increase in cleaved caspase-3 after ionomycin stimulation, with the largest inhibition from Mitycam-E31Q (25.0±9.8% and 47.1±10.1% inhibition in Mitycam and Mitycam-E31Q, respectively. n=4). Conclusion: Overexpressing mitochondrial Ca2+ biosensors may serve as additional artificial buffering machinery in the mitochondrial matrix, which can reduce mtCa2+ overload and prevent apoptotic signaling activation in response to [Ca2+]c elevation. NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.). 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.

Glucose-Induced Cellular Morphological Changes Across Mammalian Species

All organisms encounter environmental fluctuations and must contend with these changes to maintain homeostasis. Some mammals tolerate a wide range of intracellular conditions while others do not. Altered glucose levels are an example of a physiological challenge that can be encountered by cells. We studied responses of dermal fibroblasts obtained from skin biopsies to altered glucose treatments in culture using a comparative approach. Specifically, we investigated responses of mammals known to tolerate a range of blood glucose levels (fruit-eating geladas and Egyptian rousettes) as well as a hibernator (ground squirrel), which exhibits a marked winter decrease in glucose metabolism. As a comparison, we investigated species that maintain fairly stable blood glucose (human and rat). Fibroblasts were cultured at baseline (8mM glucose), then subjected to hypoglycemic (2.5mM) and hyperglycemic (30mM) treatment over 24h. We assayed glycolytic rate in human and rat fibroblasts using a Seahorse XFp analyzer, and found no significance between oxygen consumption or glycolysis across glucose treatments (all p>0.05). To investigate underlying cell phenotypes, we used Cell Painting, employing fluorescent stains to mark organelles. Hoechst 34580 stained DNA, to quantify number and size of nuclei, offering insights into proliferation. MitoTracker Deep Red visualized mitochondria, providing information about energy generation. Alexa Fluor 488 Concanavalin A conjugate marked endoplasmic reticulum. F-actin was stained with Alexa Fluor 555 Phalloidin conjugate, detecting cytoskeletal organization and overall cell structure. Images were analyzed with CellProfiler software, which extracts ~1400 morphological cell features, including area, compactness, fluorescence intensity, and cell connectivity to quantify subtle changes in phenotype. Each extracted feature will be compared across treatments and within a comparative context. We predict that fibroblasts exposed to hyperglycemia exhibit mitochondrial fragmentation and redistribution (MitoTracker) and increased production of reactive oxygen species (assessed with MitoSOX), which is linked to the opening of mitochondrial permeability transition pores. Cell Profiler analysis will identify structural changes that could indicate negative outcomes, such as mitophagy. Hyperglycemia may also induce endoplasmic stress, increasing lipid droplets and altering shape and size of the endoplasmic reticulum. In contrast, hypoglycemia will cause mitochondrial degradation. In both hyper- and hypoglycemia, proliferation rates are predicted to decrease, resulting in fewer nuclei and reduced cell connectivity. Phenotypic differences across species, evaluated in the context of their organismal phenotype, will help explain the mechanisms of cell function in mammals under environmental stress. Funding by NSF #2022046. 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.

Contribution of the Type III Secretion System (T3SS2) of Vibrio parahaemolyticus in Mitochondrial Stress in Human Intestinal Cells.

Vibrio parahaemolyticus is an important human pathogen that is currently the leading cause of shellfish-borne gastroenteritis in the world. Particularly, the pandemic strain has the capacity to induce cytotoxicity and enterotoxicity through its Type 3 Secretion System (T3SS2) that leads to massive cell death. However, the specific mechanism by which the T3SS2 induces cell death remains unclear and its contribution to mitochondrial stress is not fully understood. In this work, we evaluated the contribution of the T3SS2 of V. parahaemolyticus in generating mitochondrial stress during infection in human intestinal HT-29 cells. To evaluate the contribution of the T3SS2 of V. parahaemolyticus in mitochondrial stress, infection assays were carried out to evaluate mitochondrial transition pore opening, mitochondrial fragmentation, ATP quantification, and cell viability during infection. Our results showed that the Δvscn1 (T3SS2+) mutant strain contributes to generating the sustained opening of the mitochondrial transition pore. Furthermore, it generates perturbations in the ATP production in infected cells, leading to a significant decrease in cell viability and loss of membrane integrity. Our results suggest that the T3SS2 from V. parahaemolyticus plays a role in generating mitochondrial stress that leads to cell death in human intestinal HT-29 cells. It is important to highlight that this study represents the first report indicating the possible role of the V. parahaemolyticus T3SS2 and its effector proteins involvement in generating mitochondrial stress, its impact on the mitochondrial pore, and its effect on ATP production in human cells.

Mitochondrial calcium signaling in non-neuronal cells: Implications for Alzheimer's disease pathogenesis

Mitochondrial dysregulation is pivotal in Alzheimer's disease (AD) pathogenesis. Calcium governs vital mitochondrial processes impacting energy conversion, oxidative stress, and cell death signaling. Disruptions in mitochondrial calcium (mCa2+) handling induce calcium overload and trigger the opening of mitochondrial permeability transition pore, ensuing energy deprivation and resulting in AD-related neuronal cell death. However, the role of mCa2+ in non-neuronal cells (microglia, astrocytes, oligodendrocytes, endothelial cells, and pericytes) remains elusive. This review provides a comprehensive exploration of mitochondrial heterogeneity and calcium signaling, offering insights into specific differences among various brain cell types in AD.

Current understanding of the molecular mechanisms of chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is the most common off-target adverse effects caused by various chemotherapeutic agents, such as cisplatin, oxaliplatin, paclitaxel, vincristine and bortezomib. CIPN is characterized by a substantial loss of primary afferent sensory axonal fibers leading to sensory disturbances in patients. An estimated of 19-85% of patients developed CIPN during the course of chemotherapy. The lack of preventive measures and limited treatment options often require a dose reduction or even early termination of life-saving chemotherapy, impacting treatment efficacy and patient survival. In this Review, we summarized the current understanding on the pathogenesis of CIPN. One prominent change induced by chemotherapeutic agents involves the disruption of neuronal cytoskeletal architecture and axonal transport dynamics largely influenced by the interference of microtubule stability in peripheral neurons. Due to an ineffective blood-nerve barrier in our peripheral nervous system, exposure to some chemotherapeutic agents causes mitochondrial swelling in peripheral nerves, which lead to the opening of mitochondrial permeability transition pore and cytochrome c release resulting in degeneration of primary afferent sensory fibers. The exacerbated nociceptive signaling and pain transmission in CIPN patients is often linked the increased neuronal excitability largely due to the elevated expression of various ion channels in the dorsal root ganglion neurons. Another important contributing factor of CIPN is the neuroinflammation caused by an increased infiltration of immune cells and production of inflammatory cytokines. In the central nervous system, chemotherapeutic agents also induce neuronal hyperexcitability in the spinal dorsal horn and anterior cingulate cortex leading to the development of central sensitization that causes CIPN. Emerging evidence suggests that the change in the composition and diversity of gut microbiota (dysbiosis) could have direct impact on the development and progression of CIPN. Collectively, all these aspects contribute to the pathogenesis of CIPN. Recent advances in RNA-sequencing offer solid platform for in silico drug screening which enable the identification of novel therapeutic agents or repurpose existing drugs to alleviate CIPN, holding immense promises for enhancing the quality of life for cancer patients who undergo chemotherapy and improve their overall treatment outcomes.

Mitochondria-tau association promotes cognitive decline and hippocampal bioenergetic deficits during the aging

Current studies indicate that pathological modifications of tau are associated with mitochondrial dysfunction, synaptic failure, and cognitive decline in neurological disorders and aging. We previously showed that caspase-3 cleaved tau, a relevant tau form in Alzheimer's disease (AD), affects mitochondrial bioenergetics, dynamics and synaptic plasticity by the opening of mitochondrial permeability transition pore (mPTP). Also, genetic ablation of tau promotes mitochondrial function boost and increased cognitive capacities in aging mice. However, the mechanisms and relevance of these alterations for the cognitive and mitochondrial abnormalities during aging, which is the primary risk factor for AD, has not been explored. Therefore, in this study we used aging C57BL/6 mice (2–15 and 28-month-old) to evaluate hippocampus-dependent cognitive performance and mitochondrial function. Behavioral tests revealed that aged mice (15 and 28-month-old) showed a reduced cognitive performance compared to young mice (2 month). Concomitantly, isolated hippocampal mitochondria of aged mice showed a significant decrease in bioenergetic-related functions including increases in reactive oxygen species (ROS), mitochondrial depolarization, ATP decreases, and calcium handling defects. Importantly, full-length and caspase-3 cleaved tau were preferentially present in mitochondrial fractions of 15 and 28-month-old mice. Also, aged mice (15 and 28-month-old) showed an increase in cyclophilin D (CypD), the principal regulator of mPTP opening, and a decrease in Opa-1 mitochondrial localization, indicating a possible defect in mitochondrial dynamics.Importantly, we corroborated these findings in immortalized cortical neurons expressing mitochondrial targeted full-length (GFP-T4-OMP25) and caspase-3 cleaved tau (GFP-T4C3-OMP25) which resulted in increased ROS levels and mitochondrial fragmentation, along with a decrease in Opa-1 protein expression. These results suggest that tau associates with mitochondria and this binding increases during aging. This connection may contribute to defects in mitochondrial bioenergetics and dynamics which later may conduce to cognitive decline present during aging.

Dibutyl phthalate exposure induced mitochondria-dependent ferroptosis by enhancing VDAC2 in zebrafish ZF4 cells

Dibutyl phthalate (DBP) contamination has raised global concern for decades, while its health risk with toxic mechanisms requires further elaboration. This study used zebrafish ZF4 cells to investigate the toxicity of ferroptosis with underlying mechanisms in response to DBP exposure. Results showed that DBP induced ferroptosis, characterized by accumulation of ferrous iron, lipid peroxidation, and decrease of glutathione peroxidase 4 levels in a time-dependent manner, subsequently reduced cell viability. Transcriptome analysis revealed that voltage-dependent anion-selective channel (VDAC) in mitochondrial outer membrane was upregulated in ferroptosis signaling pathways. Protecting mitochondria with a VDAC2 inhibitor or siRNAs attenuated the accumulation of mitochondrial superoxide and lipid peroxides, the opening of mitochondrial permeability transition pore (mPTP), and the overload of iron levels, suggesting VDAC2 oligomerization mediated the influx of iron into mitochondria that is predominant and responsible for mitochondria-dependent ferroptosis under DBP exposure. Furthermore, the pivotal role of activating transcription factor 4 (ATF4) was identified in the transcriptional regulation of vdac2 by ChIP assay. And the intervention of atf4b inhibited DBP-induced VDAC2 upregulation and oligomerization. Taken together, this study reveals that ATF4-VDAC2 signaling pathway is involved in the DBP-induced ferroptosis in zebrafish ZF4 cells, contributing to the in-depth understanding of biotoxicity and the ecological risk assessment of phthalates.

Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP-Activated Exchange Protein-1.

Mitochondrial dysfunction contributes to the development of secondary brain injury (SBI) following intracerebral hemorrhage (ICH) and represents a promising therapeutic target. Celastrol, the primary active component of Tripterygium wilfordii, is a natural product that exhibits mitochondrial and neuronal protection in various cell types. This study aims to investigate the neuroprotective effects of celastrol against ICH-induced SBI and explore its underlying mechanisms. Celastrol improves neurobehavioral and cognitive abilities in mice with autologous blood-induced ICH, reduces neuronal death in vivo and in vitro, and promotes mitochondrial function recovery in neurons. Single-cell nuclear sequencing reveals that the cyclic adenosine monophosphate (cAMP)/cAMP-activated exchange protein-1 (EPAC-1) signaling pathways are impacted by celastrol. Celastrol binds to cNMP (a domain of EPAC-1) to inhibit its interaction with voltage-dependent anion-selective channel protein 1 (VDAC1) and blocks the opening of mitochondrial permeability transition pores. After neuron-specific knockout of EPAC1, the neuroprotective effects of celastrol are diminished. In summary, this study demonstrates that celastrol, through its interaction with EPAC-1, ameliorates mitochondrial dysfunction in neurons, thus potentially improving SBI induced by ICH. These findings suggest that targeting EPAC-1 with celastrol can be a promising therapeutic approach for treating ICH-induced SBI.

Bioinsecticide PA1b alleviates lipopolysaccharide-induced inflammation and impairment of bovine mammary epithelial cells

Pea Albumin 1, subunit b (PA1b) is a 37 amino acid peptide. It was extracted from pea seeds and showed significant insecticidal activity against certain insects, such as the mosquitoes Culex pipiens and Aedes aegyptii, cereal weevils (genus Sitophilus), and certain species of aphids. Considering that pea seeds are regularly consumed by humans and mammals, PA1b is assumed to be a promising bioinsecticide with no allergenicity or toxicity to hosts. To clarify this aspect, PA1b was applied to bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). The results revealed that LPS induced inflammatory cytokine tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL6) and monocyte chemoattractant protein 1 (MCP-1) secretion, while PA1b depressed these cytokines release via inhibiting NF-κB signaling activation. In addition, PA1b protected mammary epithelial cells from impairment caused by LPS, because it reduced cell membrane permeability and subsequently reconstructed mammary epithelial cell viability. Moreover, it inhibited cell apoptosis accompanied with alleviated oxidative stress. Furthermore, PA1b prevented opening of mitochondrial permeability transition pores, in turn up-regulated mitochondrial membrane potential and ATP production. Therefore, PA1b improved mitochondrial function, which contributed to re-construction of mammary epithelial cell viability. In conclusion, PA1b alleviates LPS-induced inflammation of bovine mammary epithelial cells via inhibiting NF-κB signaling activation and protects bovine mammary epithelial cells by improving mitochondrial function. PA1b is a good therapeutic survival factor for mammary epithelial cells.

Direct toxicity of cigarette smoke extract on cardiac function mediated by mitochondrial dysfunction in Sprague-Dawley rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes.

Cigarette smoke has been recognized as a major risk factor for cardiovascular disease. However, its direct effects on rodent and human cardiomyocytes and its cellular mechanisms are not fully understood. In this study, we examined the direct effects of cigarette smoke extract (CSE) on contractile functions, intracellular Ca2+ dynamics, and mitochondrial function using cultured or freshly isolated rat ventricular myocytes and human induced pluripotent stem cell (iPS)-derived cardiomyocytes. In rat cardiomyocytes, CSE (≥0.1%) resulted in a time- and concentration-dependent cessation of spontaneous beating of cultured cardiomyocytes, eventually leading to cell death, which indicates direct toxicity. In addition, 1% CSE reduced contractile function of freshly isolated ventricular myocytes. Similar contractile dysfunction (declined spontaneous beating rate and contractility) was also observed in human iPS-derived cardiomyocytes. Regarding intracellular Ca2+ dynamics, 1% CSE increased the Ca2+ transient amplitude by greatly increasing systolic Ca2+ levels and slightly increasing diastolic Ca2+ levels. CSE also accelerated the decay of Ca2+ transients, and triggered spike-shaped Ca2+ transients in some cells. These results indicate that CSE causes abnormal Ca2+ dynamics in cardiomyocytes. Furthermore, CSE induced a cascade of mitochondrial dysfunctions, including increased mitochondrial reactive oxygen species, opening of mitochondrial permeability transition pore, reduction of mitochondrial membrane potential, and release of cytochrome c from mitochondria. These results suggest that CSE-induced contractile dysfunction and myocardial cell death is caused by abnormal Ca2+ dynamics and subsequent mitochondrial dysregulation, which would result in reduced bioenergetics and activation of cell death pathways.

Розвиток мітохондріальної дисфункції при гострій кардіотоксичній дії доксорубіцину у дорослих щурів

Doxorubicin is a potent cytotoxic antibiotic that is the most widely prescribed in the world and is effective against a wide range of cancers. At the same time, the cardiotoxic effects of this drug often require discontinuation of treatment before the effect is achieved. Mitochondria are important mediators of cellular life, and cardiomyocyte death due to mitochondrial mechanisms of internal killing is the basis of many heart diseases. The aim of the study was to investigate the effects of short-term doxorubicin administration on Ca2+-induced opening of the nonspecific mitochondrial permeability transition pore (mPTP) in the heart of adult rats. To reproduce and evaluate acute cardiotoxicity in rats, which is the main complication in patients taking doxorubicin, a short-term doxorubicin cardiomyopathy model was used. A comparative ultrastructural study of myocardial tissues was performed at total cumulative doses of doxorubicin of 8, 13 and 15 mg/kg administered intraperitoneally and spread over two days. It was shown that the drug caused damage and death of the myofibrillar apparatus, mitochondria and cardiomyocytes and exhibited a dose-dependent effect. Therefore, further experiments were carried out at the most indicative dose, namely 15 mg/kg. We have shown that the content of reactive oxygen species in the heart mitochondria, namely, •O2-, Н2О2, •ОН, increased after doxorubicin administration by 10.5, 5.3 and 3.4 times, respectively, indicating a significant increase in free radical processes. It is important that at the same time, the content of endogenous H2S decreased by 2.6 times. This activated mPTP opening in the rat heart: the amplitude of spontaneous swelling doubled, Ca2+-induced swelling increased by 53% compared to the control, and an increase in mPTP sensitivity to Ca2+ was observed at all applied concentrations. Thus, the acute cardiotoxic effect of doxorubicin resulted in the induction of mPTP opening, which led to mitochondrial and cardiomyocyte death.

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.

Predicting molecular mechanism of silymarin-potentiated diclofenac toxicity: Insight from in silico molecular docking

Silymarin was shown to enhance diclofenac toxicity by inducing the loss of mitochondrial membrane permeability (MMP) in Caco-2 cells, independent of endoplasmic reticulum stress. This study employed in silico molecular docking to further investigate the potential interaction between silymarin and specific mitochondrial proteins involved in the loss of mitochondria integrity, aiming to elucidate the underlying mechanism of potentiation. The target proteins for our docking analysis included mitochondrial complex I and III, voltage-dependent anion-selective channel (VDAC), and cyclophilin D (CypD). Our results indicated that diclofenac could bind to both mitochondrial complex I and III. In contrast, silymarin exhibited a strong interaction with mitochondrial complex I with the binding energy (ΔG) −7.74 kcal/mol and the inhibition constant (Ki) 2.12 µM, while not showing significant interaction with mitochondrial complex III. Additionally, silymarin had the potential to induce the opening of mitochondrial permeability transition pore by binding with VDAC in the outer mitochondrial membrane with ΔG −6.08 kcal/mol and Ki 34.94 µM. However, silymarin did not exhibit significant interaction with CypD in the inner mitochondrial membrane. Therefore, mitochondrial complex I and VDAC could be the potentiation targets of silymarin, resulting in the disruption of mitochondria integrity and enhancing the toxicity of diclofenac.

Mitochondrial Volume Regulation and Swelling Mechanisms in Cardiomyocytes.

Mitochondrion, known as the "powerhouse" of the cell, regulates ion homeostasis, redox state, cell proliferation and differentiation, and lipid synthesis. The inner mitochondrial membrane (IMM) controls mitochondrial metabolism and function. It possesses high levels of proteins that account for ~70% of the membrane mass and are involved in the electron transport chain, oxidative phosphorylation, energy transfer, and ion transport, among others. The mitochondrial matrix volume plays a crucial role in IMM remodeling. Several ion transport mechanisms, particularly K+ and Ca2+, regulate matrix volume. Small increases in matrix volume through IMM alterations can activate mitochondrial respiration, whereas excessive swelling can impair the IMM topology and initiates mitochondria-mediated cell death. The opening of mitochondrial permeability transition pores, the well-characterized phenomenon with unknown molecular identity, in low- and high-conductance modes are involved in physiological and pathological increases of matrix volume. Despite extensive studies, the precise mechanisms underlying changes in matrix volume and IMM structural remodeling in response to energy and oxidative stressors remain unknown. This review summarizes and discusses previous studies on the mechanisms involved in regulating mitochondrial matrix volume, IMM remodeling, and the crosstalk between these processes.

Isolation, characterization and modulatory potentials of β-stigmasterol, ergosterol and xylopic acid from Anchomanes difformis on mitochondrial permeability transition pore in vitro

ObjectiveSecondary metabolites and polyphenolic compounds from medicinal plants have been demonstrated to have multiple biological functions with promising research and development prospects. This study examined the effect of β-stigmasterol (with ergosterol) and xylopic acid isolated from Anchomanes difformis on liver mitochondrial permeability transition pore (mPTP). MethodsThe compounds were isolated by vacuum liquid chromatography. Mitochondrial swelling was assessed as changes in absorbance under succinate-energized conditions. Results1H and 13C NMR spectroscopic elucidation of the isolates affirmed the presence of β-stigmasterol with ergosterol (1:0.3) and xylopic acid. The isolates reversed the increase in lipid peroxidation and inhibited the opening of mitochondrial permeability transition pores caused by calcium and glucose. Pharmacological inhibition of mPTP offers a promising therapeutic target for the treatment of mitochondrial-associated disorders. ConclusionReduction in the activity of calcium ATPase and the expression of Caspase-3 and −9 were observed, suggesting that they could play a role in protecting physicochemical properties of membrane bilayers from free radical-induced severe cellular damage and be useful in the management of diseases where much apoptosis occurs.

Expanded bioinformatic analysis of Oximouse dataset reveals key putative processes involved in brain aging and cognitive decline

The theory that aging is driven by the damage produced by reactive oxygen species (ROS) derived from oxidative metabolism dominated geroscience studies during the second half of the 20th century. However, increasing evidence that ROS also plays a key role in the physiological regulation of numerous processes through the reversible oxidation of cysteine residues in proteins, has challenged this notion. Currently, the scope of redox signaling has reached proteomic dimensions through mass spectrometry techniques. Here, we perform a comprehensive bioinformatics analysis of cysteine oxidation changes during mouse brain aging, using the quantitative data provided in the Oximouse dataset. Interestingly, our unbiased analysis identified hundreds of putative cysteine redox switches covering several pathways previously associated with aging. These include the ubiquitin-proteasome pathway and one-carbon metabolism (folate cycle, methionine cycle, transsulfuration and polyamine pathways). Surprisingly, cysteine oxidation changes are enriched in synaptic proteins in a highly asymmetric distribution: while postsynaptic proteins tend to increase cysteine oxidation with age, the opposite occurs for presynaptic proteins. Additionally, cysteine oxidation changes during aging are associated with proteins involved in the regulation of the mitochondrial transition pore opening and synaptic calcium homeostasis. Our analysis reinforces the concept that brain aging is associated with selective changes in the oxidation state of key proteins, rather than an overall trend toward increased oxidation. Also, we provide a prioritized list of specific cysteine residues with putative impact in aging processes for future experimental validation.

Maresin1 alleviates liver ischemia/reperfusion injury by reducing liver macrophage pyroptosis

BackgroundCell pyroptosis has a strong proinflammatory effect, but it is unclear whether pyroptosis of liver macrophages exacerbates liver tissue damage during liver ischemia‒reperfusion (I/R) injury. Maresin1 (MaR1) has a strong anti-inflammatory effect, and whether it can suppress liver macrophage pyroptosis needs further study.MethodsThis study aimed to investigate whether MaR1 can alleviate liver I/R injury by inhibiting macrophage pyroptosis. The effects of MaR1 on cell pyroptosis and mitochondrial damage were studied by dividing cells into control, hypoxia/reoxygenation, and hypoxia/reoxygenation + MaR1 groups. Knocking out RORa was used to study the mechanism by which MaR1 exert its protective effects. Transcriptome analysis, qRT‒PCR and Western blotting were used to analyze gene expression. Untargeted metabolomics techniques were used to analyze metabolite profiles in mice. Flow cytometry was used to assess cell death and mitochondrial damage.ResultsWe first found that MaR1 significantly reduced liver I/R injury. We observed that MaR1 decreased liver I/R injury by inhibiting liver macrophage pyroptosis. Then, we discovered that MaR1 promotes mitochondrial oxidative phosphorylation, increases the synthesis of ATP, reduces the generation of ROS, decreases the impairment of mitochondrial membrane potential and inhibits the opening of mitochondrial membrane permeability transition pores. MaR1 inhibits liver macrophage pyroptosis by protecting mitochondria. Finally, we found that MaR1 exerts mitochondrial protective effects through activation of its nuclear receptor RORa and the PI3K/AKT signaling pathway.ConclusionsDuring liver I/R injury, MaR1 can reduce liver macrophage pyroptosis by reducing mitochondrial damage, thereby reducing liver damage.

Synthesis, characterization, molecular docking, RNA-sequence and anticancer efficacy evaluation in vitro of ruthenium(II) complexes on B16 cells

In recent years, the studies of the ruthenium(II) complexes on anticancer activity have been paid great attention, many Ru(II) complexes possess high anticancer efficiency. In this paper, three ligands CPIP (2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline), DCPIP (2-(3,4-dichlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline), TCPIP (2-(2,3,5-trichlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) and their three ruthenium (II) complexes [Ru(dip)2(CPIP)](PF6)2 (1, dip = 4,7-diphenyl-1,10-phenanthroline), [Ru(dip)2(DCPIP)](PF6)2 (2) and [Ru(dip)2(TCPIP)](PF6)2 (3) were synthesized and characterized. 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assay was used to investigate in vitro cytotoxicity of complexes against various cancer cells. The results showed that complexes 1–3 exhibited pronounced cytotoxic effect on B16 cells with low IC50 values of 7.2 ± 0.1, 11.7 ± 0.6 and 1.2 ± 0.2 μM, respectively. The 3D model demonstrated that the complexes can validly prevent the cell proliferation. Apoptosis determined using Annexin V-FITC/PI double staining revealed that complexes 1–3 can effectively induce apoptosis in B16 cells. The intracellular localization of 1–3 in the mitochondria, the levels of intracellular reactive oxygen species (ROS), the opening of mitochondrial permeability transition pore as well as the decline of mitochondrial membrane potential were investigated, which demonstrated that the complexes 1–3 led to apoptosis via a ROS-mediated mitochondrial dysfunction pathway. The RNA-sequence indicated that the complexes upregulate the expression of 74 genes and downregulate the expression of 81 genes. The molecular docking showed that the complexes interact with the proteins through hydrogen bond, π-cation and π-π interaction. The results show that ruthenium(II) complexes 1, 2 and 3 can block tumor cell growth and induce cell death through autophagy and ROS-mediated mitochondrial dysfunction pathways.