Mitochondrial Calcium Ion Research Articles

Tilianin extracted from Xiangqinglan () inhibits apoptosis induced by mitochondrial pathway and endoplasmic reticulum stress in H9c2 cells after oxygen-glucose deprivation/reoxygenation.

To investigate the efficacy of tilianin extracted from Xiangqinglan () on apoptosis of H9c2 cell after oxygen-glucose deprivation/reoxygenation (OGD/R) and the mechanism. Tilianin was obtained from Beijing Inluck Science and Technology Development Co. Ltd., with purity ≥ 98%. The OGD/R model was established in H9c2 cells. Flow cytometry detected the mitochondrial membrane potential, apoptosis rates, mitochondrial reactive oxygen species (ROS) and calcium ion concentration. Succinate dehydrogenase (SDH) activity, succinate content and levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1 β (IL-1β) were detected with enzyme-linked immunosorbent assay. Western blot measured protein levels. Tilianin significantly reduced the apoptotic rates, ROS levels, calcium ion concentration, succinate content, and, levels of TNF-α, IL-6 and IL-1β of OGD/R cells, while significantly increased the membrane potential and SDH activity in mitochondria. Western blot analysis showed that tilianin significantly up-regulated p-Calmodulin-dependent protein kinase Ⅱ and voltage-dependent anion selective channel levels in OGD/R cells, while significantly down-regulated p-protein kinase B, Bcl-2-associated X, and dynamin-related protein 1 levels related to apoptosis in the mitochondrial pathway. Moreover, tilianin significantly up-regulated B-cell lymphoma-2 and mitochondrial protein 2 related to the inhibition of apoptosis. Furthermore, tilianin down-regulated phosphorylated-apoptosis signal-regulated kinase 1, phosphorylated-p38 and C/EBP homologous protein related to endoplasmic reticulum stress. Tilianin may inhibit OGD/R-induced H9c2 cell apoptosis mediated by mitochondrial pathway and endoplasmic reticulum stress, thus protecting cardiomyocytes.

Nano-Brake Halts Mitochondrial Dysfunction Cascade to Alleviate Neuropathology and Rescue Alzheimer's Cognitive Deficits.

Mitochondrial dysfunction has been recognized as the key pathogenesis of most neurodegenerative diseases including Alzheimer's disease (AD). The dysregulation of mitochondrial calcium ion (Ca2+ ) homeostasis and the mitochondrial permeability transition pore (mPTP), is a critical upstream signaling pathway that contributes to the mitochondrial dysfunction cascade in AD pathogenesis. Herein, a "two-hit braking" therapeutic strategy to synergistically halt mitochondrial Ca2+ overload and mPTP opening to put the mitochondrial dysfunction cascade on a brake is proposed. To achieve this goal, magnesium ion (Mg2+ ), a natural Ca2+ antagonist, and siRNA to the central mPTP regulator cyclophilin D (CypD), are co-encapsulated into the designed nano-brake; A matrix metalloproteinase 9 (MMP9) activatable cell-penetrating peptide (MAP) is anchored on the surface of nano-brake to overcome the blood-brain barrier (BBB) and realize targeted delivery to the mitochondrial dysfunction cells of the brain. Nano-brake treatment efficiently halts the mitochondrial dysfunction cascade in the cerebrovascular endothelial cells, neurons, and microglia and powerfully alleviates AD neuropathology and rescues cognitive deficits. These findings collectively demonstrate the potential of advanced design of nanotherapeutics to halt the key upstream signaling pathways of mitochondrial dysfunction to provide a powerful strategy for AD modifying therapy.

Global analyses and potential effects of extracellular vesicles on the establishment of conceptus implantation during the peri-implantation period.

Intrauterine extracellular vesicles (EVs) are involved in establishing proper conceptus-endometrial communication, which is essential for conceptus implantation and subsequent successful placentation. Despite several studies on intrauterine EVs, the composition and quantitative changes in conceptus and endometrial EVs, as well as the effects of intrauterine EVs on endometrial epithelial cells (EECs) during the peri-implantation period, have not been well characterized. To elucidate global changes in proteins in EVs extracted from uterine flushings (UFs) during the pre-implantation (P17), just-implantation (P20), and post-implantation (P22) periods, the datasets of the proteome iTRAQ analysis were compared among P17, P20, and P22 EVs. These analyses revealed that the composition and function of proteins in the EVs changed dramatically during peri-implantation in cattle. Notably, intrauterine P17 EVs affected the high expression of "Developmental Biology" and "morphogenesis of an endothelium" compared with those in P20 and P22 EVs. Furthermore, P20 EVs had the functions of the high expression of "mitochondrial calcium ion homeostasis" and "Viral mRNA Translation" compared with those in P17 EVs. Transcripts extracted from EECs treated with P17, P20, or P22 EVs were subjected to RNA-seq analysis. These analyses identified 60 transcripts in EECs commonly induced by intrauterine EVs recovered from P17, P20, and P22, a large number of which were associated with "type I interferon signaling pathway". Collectively, these findings reveal the presence and multiple functions of EVs that are potentially implicated in facilitating conceptus implantation into the uterine epithelium during the peri-implantation period.

GRP75 Modulates Endoplasmic Reticulum-Mitochondria Coupling and Accelerates Ca2+-Dependent Endothelial Cell Apoptosis in Diabetic Retinopathy.

Endoplasmic reticulum (ER) and mitochondrial dysfunction play fundamental roles in the pathogenesis of diabetic retinopathy (DR). However, the interrelationship between the ER and mitochondria are poorly understood in DR. Here, we established high glucose (HG) or advanced glycosylation end products (AGE)-induced human retinal vascular endothelial cell (RMEC) models in vitro, as well as a streptozotocin (STZ)-induced DR rat model in vivo. Our data demonstrated that there was increased ER-mitochondria coupling in the RMECs, which was accompanied by elevated mitochondrial calcium ions (Ca2+) and mitochondrial dysfunction under HG or AGE incubation. Mechanistically, ER-mitochondria coupling was increased through activation of the IP3R1-GRP75-VDAC1 axis, which transferred Ca2+ from the ER to the mitochondria. Elevated mitochondrial Ca2+ led to an increase in mitochondrial ROS and a decline in mitochondrial membrane potential. These events resulted in the elevation of mitochondrial permeability and induced the release of cytochrome c from the mitochondria into the cytoplasm, which further activated caspase-3 and promoted apoptosis. The above phenomenon was also observed in tunicamycin (TUN, ER stress inducer)-treated cells. Meanwhile, BAPTA-AM (calcium chelator) rescued mitochondrial dysfunction and apoptosis in DR, which further confirmed of our suspicions. In addition, 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was shown to reverse retinal dysfunction in STZ-induced DR rats in vivo. Taken together, our findings demonstrated that DR fueled the formation of ER-mitochondria coupling via the IP3R1-GRP75-VDAC1 axis and accelerated Ca2+-dependent cell apoptosis. Our results demonstrated that inhibition of ER-mitochondrial coupling, including inhibition of GRP75 or Ca2+ overload, may be a potential therapeutic target in DR.

Abnormal levels of mitochondrial Ca2+ channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia.

Structural alterations or quantitative abnormalities of some mitochondrial ion channels and exchangers are associated with altered neuronal functions and increased susceptibility to mental illness. Here we have assessed levels of functionally prominent mitochondrial calcium ion channel proteins in plasma neuron-derived extracellular vesicles (NDEVs) of living patients with first episodes of psychosis (FP) and matched controls (Cs). NDEVs were enriched with an established method of precipitation and immunoabsorption by anti-human CD171 neural adhesion protein (L1CAM) antibody and extracted proteins quantified with ELISAs. CD81 exosome marker-normalized NDEV levels of leucine zipper EF-hand containing transmembrane 1 protein (LETM1), transient receptor potential cation channel subfamily M, member 4 (TRPM4), and solute carrier family 8 member B1 (SLC24A6) or mitochondrial Na+ /Ca2+ exchanger (NCLX) were significantly lower for FP patients (n=10) than Cs (n=10), whereas NDEV levels of voltage-dependent L-type calcium channel subunit α-1C (CACNA-1C) were significantly higher for FP patients than Cs. Abnormal structures or mitochondrial levels of LETM1, NCLX, and CACNA-1C have been linked through analyses of individual proteins, genome-wide association studies, and whole exome protein-coding sequence studies to neurodevelopmental disorders, mental retardation, schizophrenia, and major depressive diseases. A greater understanding of the altered calcium homeostasis in schizophrenia, that is attributable to underlying mitochondrial calcium channel abnormalities, will lead to improved diagnosis and treatment.

Arachidonic Acid in Follicular Fluid of PCOS Induces Oxidative Stress in a Human Ovarian Granulosa Tumor Cell Line (KGN) and Upregulates GDF15 Expression as a Response.

Polycystic ovarian ovary syndrome (PCOS) is the main cause of ovulatory infertility and a common reproductive endocrine disease of women in reproductive age. In addition, nearly half of PCOS patients are associated with obesity, and their total free fatty acids tend to increase. Arachidonic acid (AA) is a polyunsaturated fatty acid. Oxidation products of AA reacting with various enzymes[cyclooxygenases (COX), lipoxygenases (LOX), cytochrome P450s (CYP)] can change cellular mitochondrial distribution and calcium ion concentration, and increase reactive oxygen species (ROS) production. In this study, we analyzed the follicular fluid fatty acids and found higher levels of C20:4n6 (AA) in PCOS patients than in normal control subjects. Also, to determine whether AA induces oxidative stress (OS) in the human ovarian granulosa tumor cell line (KGN) and affects its function, we treated KGN cells with or without reduced glutathione (GSH) and then stimulated them with AA. The results showed that AA significantly reduced the total antioxidant capacity (TAC) and activity of antioxidant enzymes and increased the malondialdehyde (MDA), ROS and superoxide anion(O2-)levels in KGN cells. In addition, AA was also found to impair the secretory and mitochondrial functions of KGN cells and induce their apoptosis. We further investigated the downstream genes affected by AA in KGN cells and its mechanism of action. We found that AA upregulated the expression of growth differentiation factor 15 (GDF15), which had a protective effect on inflammation and tissue damage. Therefore, we investigated whether AA-induced OS in KGN cells upregulates GDF15 expression as an OS response.Through silencing of GDF15 and supplementation with recombinant GDF15 (rGDF15), we found that GDF15, expressed as an OS response, protected KGN cells against AA-induced OS effects, such as impairment of secretory and mitochondrial functions and apoptosis. Therefore, this study suggested that AA might induce OS in KGN cells and upregulate the expression of GDF15 as a response to OS.

TME-responded Full-biodegradable nanocatalyst for mitochondrial calcium Overload-induced hydroxyl radical bursting cancer treatment

Mitochondrial calcium ions (Ca2+) overload, an abnormal accumulation of free Ca2+ in mitochondrial, has become a potential strategy for effective cancer treatment. Inspired by mitochondrial Ca2+ overload-induced ROS generation, a tumor microenvironment (TME)-responded full-degradable ACC@Cu2O-TPP NCs are successfully fabricated for effective chemodynamic therapy (CDT) via toxic hydroxyl radical (∙OH) bursting. The as-synthesized nanocatalysts are stable and safe in normal tissues but will quickly degrade into Ca2+ and cuprous ions (Cu+) under the acidic TME. The mitochondrial Ca2+ overload effectively simulates extra H2O2 generation. Then Cu+ catalyzes the produced H2O2 into toxic ∙OH to kill the tumor cells via Fenton-like reaction. Meanwhile, abundant GSH in tumor cells can further reduce Cu2+ into Cu+ to enhance the CDT effect. Both in vitro and in vivo evaluations demonstrate the efficient anti-tumor effect of ACC@Cu2O-TPP NCs via mitochondrial Ca2+ overload-induced ∙OH bursting. We believe that the development of ACC@Cu2O-TPP NCs will be an amazing inspiration for designing safe and green chemodynamic nanocatalysts for highly efficient cancer treatment.

Sanguinarine induces apoptosis in Eimeria tenella sporozoites via the generation of reactive oxygen species

Eimeria tenella (E. tenella) is the most pathogenic genus in Eimeria and can lead to a huge number of deaths of chickens, causing significant economic losses in the poultry industry worldwide. As a natural alkaloid, sanguinarine has many medicinal effects; to a certain extent, it can replace antibiotics and has good application prospects in veterinary medicine. To evaluate the effect of sanguinarine on sporozoites of E.tenella, we used flow cytometry and immunofluorescence staining to detect reactive oxygen species (ROS), mitochondrial membrane potential (MMP), calcium ion (Ca2+), and caspase-3 activation in E.tenella sporozoites treated with different concentrations of sanguinarine. The results of flow cytometry showed that sanguinarine could inhibit the invasion of sporozoites of E.tenella in vitro (P < 0.05) and increase the reactive oxygen species and calcium ions in the sporozoites (P < 0.05). The results of immunofluorescence staining showed that sanguinarine could decrease the mitochondrial membrane potential of sporozoites. Our analysis suggests that sanguinarine can induce apoptosis of E. tenella sporozoites through reactive oxygen species-mediated reduction of the mitochondrial membrane potential and an increase in calcium ion concentration. It follows that sanguinarine is likely to be a novel type of anticoccidiosis drug with good research and clinical application prospects.

RNA editing regulates lncRNA splicing in human early embryo development.

RNA editing is a co- or post-transcriptional modification through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule after transcription. Previous studies found that RNA editing may be critically involved in cancer and aging. However, the function of RNA editing in human early embryo development is still unclear. In this study, through analyzing single cell RNA sequencing data, 36.7% RNA editing sites were found to have a have differential editing ratio among early embryo developmental stages, and there was a great reprogramming of RNA editing rates at the 8-cell stage, at which most of the differentially edited RNA editing sites (99.2%) had a decreased RNA editing rate. In addition, RNA editing was more likely to occur on RNA splicing sites during human early embryo development. Furthermore, long non-coding RNA (lncRNA) editing sites were found more likely to be on RNA splicing sites (odds ratio = 2.19, P = 1.37×10−8), while mRNA editing sites were less likely (odds ratio = 0.22, P = 8.38×10−46). Besides, we found that the RNA editing rate on lncRNA had a significantly higher correlation coefficient with the percentage spliced index (PSI) of lncRNA exons (R = 0.75, P = 4.90×10−16), which indicated that RNA editing may regulate lncRNA splicing during human early embryo development. Finally, functional analysis revealed that those RNA editing-regulated lncRNAs were enriched in signal transduction, the regulation of transcript expression, and the transmembrane transport of mitochondrial calcium ion. Overall, our study might provide a new insight into the mechanism of RNA editing on lncRNAs in human developmental biology and common birth defects.

Mitochondrial Ca2+ and Reactive Oxygen Species in Trypanosomatids.

Significance: Millions of people are infected with trypanosomatids and new therapeutic approaches are needed. Trypanosomatids possess one mitochondrion per cell and its study has led to discoveries of general biological interest. These mitochondria, as in their animal counterparts, generate reactive oxygen species (ROS) and have evolved enzymatic and nonenzymatic defenses against them. Mitochondrial calcium ion (Ca2+) overload leads to generation of ROS and its study could lead to relevant information on the biology of trypanosomatids and to novel drug targets. Recent Advances: Mitochondrial Ca2+ is normally involved in maintaining the bioenergetics of trypanosomes, but when Ca2+ overload occurs, it is associated with cell death. Trypanosomes lack key players in the mechanism of cell death described in mammalian cells, although mitochondrial Ca2+ overload results in collapse of their membrane potential, production of ROS, and cytochrome c release. They are also very resistant to mitochondrial permeability transition, and cell death after mitochondrial Ca2+ overload depends on generation of ROS. Critical Issues: In this review, we consider the mechanisms of mitochondrial oxidant generation and removal and the involvement of Ca2+ in trypanosome cell death. Future Directions: More studies are required to determine the reactions involved in generation of ROS by the mitochondria of trypanosomatids, their enzymatic and nonenzymatic defenses against ROS, and the occurrence and composition of a mitochondrial permeability transition pore. Antioxid. Redox Signal. 36, 969-983.

Paeoniflorin and Hydroxysafflor Yellow A in Xuebijing Injection Attenuate Sepsis-Induced Cardiac Dysfunction and Inhibit Proinflammatory Cytokine Production.

Sepsis-induced myocardial dysfunction is a major contributor to the poor outcomes of septic shock. As an add-on with conventional sepsis management for over 15 years, the effect of Xuebijing injection (XBJ) on the sepsis-induced myocardial dysfunction was not well understood. The material basis of Xuebijing injection (XBJ) in managing infections and infection-related complications remains to be defined. A murine cecal ligation and puncture (CLP) model and cardiomyocytes in vitro culture were adopted to study the influence of XBJ on infection-induced cardiac dysfunction. XBJ significantly improved the survival of septic-mice and rescued cardiac dysfunction in vivo. RNA-seq revealed XBJ attenuated the expression of proinflammatory cytokines and related signalings in the heart which was further confirmed on the mRNA and protein levels. Xuebijing also protected cardiomyocytes from LPS-induced mitochondrial calcium ion overload and reduced the LPS-induced ROS production in cardiomyocytes. The therapeutic effect of XBJ was mediated by the combination of paeoniflorin and hydroxysafflor yellow A (HSYA) (C0127-2). C0127-2 improved the survival of septic mice, protected their cardiac function and cardiomyocytes while balancing gene expression in cytokine-storm-related signalings, such as TNF-α and NF-κB. In summary, Paeoniflorin and HSYA are key active compounds in XBJ for managing sepsis, protecting cardiac function, and controlling inflammation in the cardiac tissue partially by limiting the production of IL-6, IL-1β, and CXCL2.

Sport Performance and Manual Therapies: A Review on the Effects on Mitochondrial, Sarcoplasmatic and Ca2+ Flux Response.

The present narrative review aims to highlight the possible effects manual therapies could have on cells and mitochondria, as these effects could improve athletic performance management. To this aim, this review summarizes the relationship between mechanical stimulation, with a special focus on physical activity, and cell response based on the most recent mechanobiology findings. Mechanobiology analyzes how cells respond to mechanical stressors coming from the environment. Indeed, endogenous (e.g., blood pressure, heartbeat and gastrointestinal motility) and exogenous (e.g., physical activity and manual therapies) stimuli can induce biochemical and epigenetic modifications that alter protein synthesis with heavy consequences on cell behavior. Mechanical stress can also influence mitochondrial behavior (i.e., biogenesis, autophagy, fusion, fission and energy production), sarcoplasmic response and calcium ion (Ca2+) flux. Since manual therapies have been shown to affect the extracellular matrix, which represents a primary source of mechanical stress that may alter both the cytoskeleton and mitochondrial metabolism, it is conceivable manual therapies could also affect cellular and mitochondrial behavior. Lastly, by suggesting possible directions for future laboratory and clinical studies, the authors expect this review to inspire further research on how manual therapies could affect bioenergetic metabolism and, thus, athletic performance.

Mitochondrial Ca2+ homeostasis in trypanosomes.

Mitochondrial calcium ion (Ca2+) uptake is important for buffering cytosolic Ca2+ levels, for regulating cell bioenergetics, and for cell death and autophagy. Ca2+ uptake is mediated by a mitochondrial Ca2+ uniporter (MCU) and the discovery of this channel in trypanosomes has been critical for the identification of the molecular nature of the channel in all eukaryotes. However, the trypanosome uniporter, which has been studied in detail in Trypanosoma cruzi, the agent of Chagas disease, and T. brucei, the agent of human and animal African trypanosomiasis, has lineage-specific adaptations which include the lack of some homologues to mammalian subunits, and the presence of unique subunits. Here, we review newly emerging insights into the role of mitochondrial Ca2+ homeostasis in trypanosomes, the composition of the uniporter, its functional characterization, and its role in general physiology.

The Molecular Mechanisms of Mitochondrial Calcium Ion Channel, Calcium Uniporter

Mitochondria play a role as intracellular calcium stores as well as energy conversion functions. Mitochondrial calcium uptake is mediated by mitochondrial calcium ion channel, “calcium uniporter”. In this decade, the molecular composition of the calcium uniporter complex was discovered; the calcium uniporter consists of the 7 subunits. Using the yeast reconstitution technique, we showed that MCU and EMRE are core subunits of the complex. The yeast reconstitution technique has also enabled us to perform detailed structure-function analysis of the MCU and EMRE. In this review, we will discuss the molecular mechanism of mitochondrial Ca2+ uptake.

The Role Played by Mitochondria in FcεRI-Dependent Mast Cell Activation.

Mast cells play a key role in the regulation of innate and adaptive immunity and are involved in pathogenesis of many inflammatory and allergic diseases. The most studied mechanism of mast cell activation is mediated by the interaction of antigens with immunoglobulin E (IgE) and a subsequent binding with the high-affinity receptor Fc epsilon RI (FcεRI). Increasing evidences indicated that mitochondria are actively involved in the FcεRI-dependent activation of this type of cells. Here, we discuss changes in energy metabolism and mitochondrial dynamics during IgE-antigen stimulation of mast cells. We reviewed the recent data with regards to the role played by mitochondrial membrane potential, mitochondrial calcium ions (Ca2+) influx and reactive oxygen species (ROS) in mast cell FcεRI-dependent activation. Additionally, in the present review we have discussed the crucial role played by the pyruvate dehydrogenase (PDH) complex, transcription factors signal transducer and activator of transcription 3 (STAT3) and microphthalmia-associated transcription factor (MITF) in the development and function of mast cells. These two transcription factors besides their nuclear localization were also found to translocate in to the mitochondria and functions as direct modulators of mitochondrial activity. Studying the role played by mast cell mitochondria following their activation is essential for expanding our basic knowledge about mast cell physiological functions and would help to design mitochondria-targeted anti-allergic and anti-inflammatory drugs.

Effects of a novel polyamine metabolic enzyme inhibitor SI-4650 on proliferation of colon cancer CT-26 cells and its mechanism

To investigate the effects of a novel polyamine metabolism enzyme inhibitor SI-4650 on autophagy and apoptosis of colon cancer CT-26 cells as well as their correlation. CT-26 cells treated with 40, 80 μmol·L-1 SI-4650 alone or in combination with 3-MA were used as experimental group. CT-26 cells treated with 0 μmol·L-1 SI-4650 alone or in combination with 3-MA were used as control group. Chemiluminescence was used to analyze the effect of SI-4650 on spermine oxidase (SMO) and acetylpolyamine oxidase(APAO) activity. High performance liquid chromatography (HPLC) was performed to detect cellular polyamine content.The CCK8 method was used to detect the inhibitory effect of SI-4650 on proliferation of CT-26 cells. PI single-staining/flow cytometry (FCM) were used to analyze cell cycle. Western blot were used to analyze autophagy. Apoptosis was analyzed by PI/FITC-Annexin V double staining, JC-1 fluorescent probe and Fluo-3 AM calcium ion fluorescent probe combined with flow cytometry and Western blot. CCK8 assay showed that 24-,48-,72-hours treated with SI-4650 all could inhibit the proliferative activity of CT-26 cells in a dose- and time-dependent manner (P＜0.01) . The inhibition rate was 36.98% and 46.91% in 40 μmol·L-1 SI-4650 group and 80 μmol·L-1 SI-4650 group respectively. SI-4650 could significantly inhibit the activities of SMO and APAO interfere with polyamine metabolism and reduce the content of total polyamine in CT-26 cells (P＜0.01). SI-4650 could block CT-26 cells in G0/G1 phase, significantly reduce the number of cells in S phase(P＜0.01), and lead to a significant increase in the contents of autophagy-related Beclin-1, LC3-II in CT-26 cells(P＜0.01); At the same time, the concentration of calcium in CT-26 cells was increased, the mitochondrial membrane potential was decreased, the expressions of c-PARP and Bax were increased, the content of Bcl-2 was decreased, and the number of apoptotic cells was increased. After SI-4650 combined with autophagy inhibitor 3-MA treatment of CT-26 cells, the level of autophagy, the apoptosis-related protein, mitochondrial membrane potential and calcium ion concentration were decreased, and the number of apoptotic cells was decreased. SI-4650 has the pharmacological activity of killing colon cancer CT-26 cells, and its mechanism may be related to the interference of polyamine metabolism and induction of cell apoptosis and autophagy. In this process, autophagy is inhibited to block apoptosis, autophagy and apoptosis combined to kill tumor cells.

Sex differences in mitochondrial Ca2+ handling in mouse fast-twitch skeletal muscle in vivo.

We investigated sex differences in mitochondrial Ca2+ handling properties in mouse fast-twitch skeletal muscle. Changes in cytoplasmic Ca2+ concentration ([Ca2+]cyto) were measured in vivo using tibialis anterior muscles from male and female mice. The muscles were exposed to increasing concentrations of cyclopiazonic acid [CPA; sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor] (from 10 to 30 to 50 μM at 10 min intervals). Thirty minutes after treatment, [Ca2+]cyto was increased by 31.6 ± 2.0% and 13.5 ± 4.5% of initial [Ca2+]cyto in male and female muscles, respectively, and there was a significant difference between sexes. However, muscle preincubation for 5 min with 10 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (an inhibitor of mitochondria Ca2+ uptake) eradicated this difference between sexes with respect to the CPA-induced [Ca2+]cyto increase. Both intermyofibrillar mitochondrial number and volume, assessed in longitudinal fiber sections, were higher in females compared with males (mitochondria number: 13.1 ± 1.0 in males vs. 19.9 ± 2.3 in females; mitochondrial volume: 0.034 ± 0.004 μm3/μm3 fiber volume in males vs. 0.066 ± 0.008 μm3/μm3 fiber volume in females, both P < 0.05). There were no sex differences in the content of SR Ca2+-ATPase, mitochondrial Ca2+ uniporter, mitofusin (Mfn) 1, or Mfn2. These results suggest that 1) mitochondrial Ca2+ uptake ability is greater in female than male myocytes, and 2) this superior Ca2+ uptake ability of female myocytes is due, partly, to the higher intermyofibrillar mitochondrial content but not to the expression of mitochondrial proteins related to mitochondrial Ca2+ uptake.NEW & NOTEWORTHY This investigation presents evidence that female versus male fast-twitch muscle exhibits a greater mitochondrial calcium ion uptake capability that is partly conferred by the higher intermyofibrillar mitochondrial volume density.

Ivermectin induces apoptosis of porcine trophectoderm and uterine luminal epithelial cells through loss of mitochondrial membrane potential, mitochondrial calcium ion overload, and reactive oxygen species generation

Ivermectin is a pesticide that has been used for over 30 years in livestock. Although there are a number of studies on the therapeutic potential of ivermectin, little is known about the effects of the drug during the early stage of pregnancy. In this study, we investigated the detrimental effects of ivermectin on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. Ivermectin not only inhibited the proliferation of both cells via the regulation of cell cycle-associated genes, but also induced apoptosis in pTr and pLE cells. We also verified its effect on mitochondrial dysfunction as shown by loss of mitochondrial membrane potential, mitochondrial Ca2+ overload, and reactive oxygen species (ROS) generation in pTr and pLE cells. As a mechanistic approach, we evaluated ivermectin-mediated cell signaling interactions including PI3K, AKT and MAPK pathways. Overall, our results suggest that constant exposure to and accumulation of ivermectin may cause abnormal fetal morphogenesis and placentation during the early stages of pregnancy. Our results may further provide a comprehensive understanding of the detrimental effects of ivermectin during pregnancy and will contribute to the establishment of a complete safety profile for ivermectin and its association with environmental pollution and public health in humans and livestock.

Oxibendazole induces apoptotic cell death in proliferating porcine trophectoderm and uterine luminal epithelial cells via mitochondria-mediated calcium disruption and breakdown of mitochondrial membrane potential

The well-known and effective anthelmintic oxibendazole was recently shown to have a broad spectrum of biological abilities, such as anti-cancer and anti-inflammation activities. In contrast, the mechanism of oxibendazole's anti-proliferative effect via cell signaling pathways and its role in pre-implantation has not been studied. Therefore, in this study we demonstrated the effects of oxibendazole on the proliferation of porcine trophectoderm (pTr) cells and porcine luminal epithelial (pLE) cells, a well-known in vitro model system of the fetal-maternal interface. Cell proliferation decreased in both pTr and pLE cells in response to oxibendazole, and we determined that this was modulated through intracellular cell signal transduction. Phosphorylation of ERK1/2, P90RSK, and S6 were downregulated by exposure to a 200 nM dose of oxibendazole in both types of cells, while the expression of phosphorylated JNK, AKT, and P70S6K was upregulated. Pre-treatment with a PI3K/AKT inhibitor (Wortmannin), ERK1/2 inhibitor (U0126), and JNK inhibitor (SP600125) induced the signaling interactions of these molecules, and oxibendazole co-treatment with each inhibitor resulted in even greater decreases in cell proliferation. Furthermore, intracellular and mitochondrial calcium ion accumulation was observed, which would mean that calcium ion homeostasis was disrupted, causing damage to the mitochondrial membrane potential. These deteriorated conditions ultimately led to apoptotic cell death. Taken together, the results of the present study identified that the apoptotic effect of oxibendazole on pTr and pLE cells is regulated by cell signaling pathways, and thus oxibendazole could influence the connection between the conceptus and the maternal uterus.

Sphingosine-1-phosphate attenuates hypoxia/reoxygenation-induced cardiomyocyte injury via a mitochondrial pathway

Our previous study showed that Sphingosine-1-phosphate (S1P) could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury via the JAK-STAT pathway and maintain normal myocardial mitochondria integrity in vivo. However, it is not known yet whether S1P can relieve mitochondrial dysfunction via the mitochondrial apoptotic pathway and its detailed mechanism remains to be investigated. The aim of this study was to demonstrate the mitochondrial protective effects of S1P in a cardiomyocyte H/R injury model. In the present study, we established a H/R model in H9c2 cells. Cell viability was determined by the MTT assay, and apoptosis was evaluated by annexin V-FITC/PI staining. Mitochondrial calcium ion concentration, mitochondrial membrane potential (ΔΨm), opening of the mitochondrial permeability transition pore (mPTP), and release of cytochrome C were detected by laser confocal microscopy. The results showed that S1P inhibited the decrease in cell viability induced by H/R injury and reduced apoptosis. Confocal microscopy showed that S1P prevented loss of ΔΨm, relieved mitochondrial calcium overload, and inhibited opening of the mPTP and release of cytochrome C. The STAT3 inhibitor STATTIC can reverse the antiapoptotic effects of S1P and block the effect of S1P on mitochondria. Taken together, our results indicate that S1P protects cardiomyocytes against H/R injury by relieving mitochondrial dysfunction via the STAT3 pathway.