Connexin 43 Knockdown Induces Mitochondrial Dysfunction and Affects Early Developmental Competence in Porcine Embryos

2020 ◽  
Vol 26 (2) ◽  
pp. 287-296
Author(s):  
Kyung-Tae Shin ◽  
Zheng-Wen Nie ◽  
Wenjun Zhou ◽  
Dongjie Zhou ◽  
Ju-Yeon Kim ◽  
...  
Keyword(s):  
Membrane Permeability ◽  
Connexin 43 ◽  
Molecular Mechanisms ◽  
Developmental Competence ◽  
Cell Junction ◽  
Ros Generation ◽  
Blastocyst Development ◽  
Atp Production ◽  
Early Embryos

AbstractConnexin 43 (CX43) is a component of gap junctions. The lack of functional CX43 induces oxidative stress, autophagy, and apoptosis in somatic cells. However, the role of CX43 in the early development of porcine embryos is still unknown. Thus, the aim of this study was to investigate the role of CX43, and its underlying molecular mechanisms, on the developmental competence of early porcine embryos. We performed CX43 knockdown by microinjecting dsRNA into parthenogenetically activated porcine parthenotes. The blastocyst development rate and the total number of cells in the blastocysts were significantly reduced by CX43 knockdown. Results from FITC-dextran assays showed that CX43 knockdown significantly increased membrane permeability. ZO-1 protein was obliterated in CX43 knockdown blastocysts. Mitochondrial membrane potential and ATP production were significantly reduced following CX43 knockdown. Reactive oxygen species (ROS) levels were significantly increased in the CX43 knockdown group compared to those in control embryos. Moreover, CX43 knockdown induced autophagy and apoptosis. Our findings indicate that CX43 is essential for the development and preimplantation of porcine embryos and maintains mitochondrial function, cell junction structure, and cell homeostasis by regulating membrane permeability, ROS generation, autophagy, and apoptosis in early embryos.

scholarly journals Cellular and mitochondrial mechanisms of atrial fibrillation

2020 ◽  
Vol 115 (6) ◽  
Author(s):  
Fleur E. Mason ◽  
Julius Ryan D. Pronto ◽  
Khaled Alhussini ◽  
Christoph Maack ◽  
Niels Voigt
Keyword(s):  
Atrial Fibrillation ◽  
Nicotinamide Adenine Dinucleotide ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Specific Treatment ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Mitochondrial Activity ◽  
Atp Production

AbstractThe molecular mechanisms underlying atrial fibrillation (AF), the most common form of arrhythmia, are poorly understood and therefore target-specific treatment options remain an unmet clinical need. Excitation–contraction coupling in cardiac myocytes requires high amounts of adenosine triphosphate (ATP), which is replenished by oxidative phosphorylation in mitochondria. Calcium (Ca2+) is a key regulator of mitochondrial function by stimulating the Krebs cycle, which produces nicotinamide adenine dinucleotide for ATP production at the electron transport chain and nicotinamide adenine dinucleotide phosphate for the elimination of reactive oxygen species (ROS). While it is now well established that mitochondrial dysfunction plays an important role in the pathophysiology of heart failure, this has been less investigated in atrial myocytes in AF. Considering the high prevalence of AF, investigating the role of mitochondria in this disease may guide the path towards new therapeutic targets. In this review, we discuss the importance of mitochondrial Ca2+ handling in regulating ATP production and mitochondrial ROS emission and how alterations, particularly in these aspects of mitochondrial activity, may play a role in AF. In addition to describing research advances, we highlight areas in which further studies are required to elucidate the role of mitochondria in AF.

257. Addition of glycine to vitrification solutions protects oocyte and embryo physiology and health

2005 ◽  
Vol 17 (9) ◽  
pp. 104
Author(s):  
K. S. Cashman ◽  
D. A. Froiland ◽  
J. G. Thompson ◽  
M. Lane
Keyword(s):  
Gene Expression ◽  
Membrane Potential ◽  
Mitochondrial Membrane ◽  
Multiple Comparisons ◽  
Developmental Competence ◽  
Rt Pcr ◽  
Blastocyst Development ◽  
Pixel Intensity ◽  
Mitochondrial Distribution

Cryopreservation procedures for oocytes result in a significant reduction in viability. Although cryopreservation procedures cause dehydration and therefore osmotic stress, the role of osmolytes in solutions has not been considered and they have therefore not been included for routine use. The aim of this study was to assess the effects of the addition of the osmolyte glycine to vitrification solutions on the health and developmental competence of mouse oocytes. Oocytes were collected from F1 female mice and cryopreserved using cryoloop vitrification with or without glycine, with fresh oocytes examined as controls (n = 2086). Mitochondrial distribution and membrane potential as well as the morphology of the spindles and chromosomes were assessed. Oocytes were fertilised to assess their ability to develop into blastocysts, which were then assessed for their expression of Glut1, Glut3 and IGF2 by real-time RT-PCR. Statistical analysis was performed using a generalised linear model followed by multiple comparisons using an LSD test. Vitrification without glycine perturbed mitochondrial distribution (mean pixel intensity of outer region:inner region, 1.58±0.20, P<0.01) and mitochondrial membrane potential (mean pixel intensity 0.56±0.01, P<0.01) compared to control oocytes (2.34±0.24 and 0.52±0.01, respectively). The addition of glycine prevented these changes (1.97±0.16 and 0.53±0.01, respectively). Vitrification without glycine resulted in 52% of spindles and chromosomes appearing normal while this was increased to 69% with the addition of glycine, however in both treatments these abnormalities appeared to recover after culture for 2 h. Vitrification did not affect fertilisation and blastocyst development however expression of Glut3 was decreased 2.9 fold in blastocysts resulting from oocytes vitrified in the absence of glycine (P<0.01). The data presented suggests that the addition of glycine results in fewer perturbations in oocyte physiology and gene expression of the subsequent blastocysts and should therefore be considered for routine inclusion in solutions for the cryopreservation of oocytes.

scholarly journals Autophagy in unicellular eukaryotes

2010 ◽  
Vol 365 (1541) ◽  
pp. 819-830 ◽  
Author(s):  
Jan A. K. W. Kiel
Keyword(s):  
Molecular Mechanisms ◽  
Yeast Species ◽  
Developmental Change ◽  
Building Blocks ◽  
Extracellular Medium ◽  
New Host ◽  
Growth And Survival ◽  
Atp Production ◽  
Unicellular Eukaryotes

Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components will be catabolized by (macro)autophagy in order to re-use building blocks and to support ATP production. In many cases, autophagy takes care of cellular housekeeping to sustain cellular viability. Autophagy encompasses a multitude of related and often highly specific processes that are implicated in both biogenetic and catabolic processes. Recent data indicate that in some unicellular eukaryotes that undergo profound differentiation during their life cycle (e.g. kinetoplastid parasites and amoebes), autophagy is essential for the developmental change that allows the cell to adapt to a new host or form spores. This review summarizes the knowledge on the molecular mechanisms of autophagy as well as the cytoplasm-to-vacuole-targeting pathway, pexophagy, mitophagy, ER-phagy, ribophagy and piecemeal microautophagy of the nucleus, all highly selective forms of autophagy that have first been uncovered in yeast species. Additionally, a detailed analysis will be presented on the state of knowledge on autophagy in non-yeast unicellular eukaryotes with emphasis on the role of this process in differentiation.

129 Knockdown of connexin 43 effects on developmental competence in porcine parthenotes

2019 ◽  
Vol 31 (1) ◽  
pp. 190
Author(s):  
K.-T. Shin ◽  
Y.-J. Niu ◽  
Z.-W. Nie ◽  
W. Zhou ◽  
Y.-H. Kim ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Membrane Permeability ◽  
Connexin 43 ◽  
Reactive Oxygen Species Generation ◽  
Reactive Oxygen ◽  
Fluorescein Isothiocyanate ◽  
Developmental Competence ◽  
Oxygen Species ◽  
Small Molecular Weight ◽  
Western Blot Assay

Connexin 43 (Cx43) is one of the gap junction proteins that are compounds of transmembrane proteins and transports the small-molecular-weight chemicals up to 2 kDa. Lacking of Cx43 influences the junctional protein and induces autophagy and apoptosis in somatic cells. However, the function of Cx43 in porcine early embryos is still unknown. With the aim to find out the molecular mechanism of Cx43 on the developmental competence of early porcine embryos, dsRNA technology was carried out and inhibitor was used as the positive control. Additionally, it is difficult to obtain pig embryos of homogeneous quality due to the relatively high incidence of polyspermy during IVF. Therefore, diploid parthenotes have been frequently used to study early development in the pig. Connexin 43 dsRNA (1μg μL−1) was microinjected into the parthenogenetically activated porcine zygotes. Blastocyst rate [5 repeats; treatment, 8.8±1.6% (n=1356) v. control, 38.6±4.3% (n=1082)] and total cell numbers in the blastocyst [4 repeats; treatment, 20.7±3.5 (n=85) v. control, 39.8±4.1(n=97)] were significantly reduced following Cx43 knocking down. Results from fluorescein isothiocyanate-dextran and Western blot assay show that knockdown (KD) of Cx43 significantly increased membrane permeability through down-regulation of genes that are components of both adherence and tight junction in the porcine blastocyst. Reactive oxygen species were significantly increased in the Cx43 KD group compared with the control. In addition, KD of Cx43 activated Caspase 3 and significantly increased ATG8 expression and induced autophagy and apoptosis. Results suggest that KD of Cx43 influences pre-implantation porcine embryo development via increasing membrane permeability and reactive oxygen species generation and by inducing autophagy and apoptosis.

98 Effects of maternal age on oxygen consumption of oocytes and invitro-produced equine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 175 ◽  
Author(s):  
G. Catandi ◽  
Y. Obeidat ◽  
J. Stokes ◽  
A. Chicco ◽  
T. Chen ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Maternal Age ◽  
Early Stage ◽  
Calf Serum ◽  
Developmental Competence ◽  
Metabolic Function ◽  
Blastocyst Development ◽  
Follicular Maturation ◽  
Early Embryos ◽  
Student’S T

Mitochondrial replication is arrested during early cleavage stages, leaving the embryo dependent on maternally derived mitochondria for oxidative phosphorylation. Numbers of mitochondrial DNA (mtDNA) are used as indicators of functional mitochondria; however, direct comparisons for mtDNA and oxygen consumption rate (OCR) have not been performed for horses. The objectives of this study were to compare equine oocyte mtDNA copy numbers with a measure of mitochondrial function (OCR) and to determine whether maternal age of the oocyte donor impacts OCR of early-stage embryos. We hypothesised that (1) OCR in oocytes is not directly associated with mitochondrial numbers and (2) aerobic metabolism (OCR) is lower in early embryos from old than from young mares. Mares ages 6-13 years (Young, n=7) and=20 years (Old, n=8) were used as oocyte donors. Oocytes were collected from dominant follicles (=35mm) during oestrus and at 16±2h after induction of follicular maturation. Recovered oocytes were incubated in tissue culture medium 199 with 10% fetal calf serum, 25mgmL−1 of gentamicin, and 0.2mM pyruvate for 26±2h. Metaphase II oocytes (Young, n=14; Old, n=15) were fertilized by intracytoplasmic sperm injection (ICSI) using frozen-thawed sperm from one stallion. Presumptive zygotes were cultured in global medium (LifeGlobal Group). Other oocytes and early embryos were used for OCR. A microchamber containing an electrochemical sensor was used to measure OCR from individual oocytes (Young, n=9; Old, n=14) and early embryos (Young, n=8; Old, n=10). After analyses, oocytes were snap frozen, and mtDNA was later quantified by qPCR. Metabolic assays of embryos that cleaved were performed at Day 2 after ICSI. After the assay, embryos were placed back to culture until blastocyst formation at Day 7 or 8. Two-tailed Student's t-tests were used for OCR and mtDNA comparisons, and Fisher's exact tests were used to compare development rates. We found that OCR was higher (P=0.007) for oocytes from Young (mean±s.e.m.: 1.8±0.2) than from Old (1.3±0.1 fmol s−1). However, mtDNA numbers were not different (P=0.3) for Young (5.6±0.4×105) and Old (6.2±0.4×105). Cleavage rates were similar (P=0.6) between Young (11 out of 14, 79%) and Old (13 of 15, 87%). Day 2 embryos from Young had higher basal OCR compared with Old (3.8±0.1 and 3.2±0.2 fmol s−1, respectively; P=0.05). Blastocyst rates per cleaved oocytes were similar for Young (5 of 11, 45%) and Old (4 of 13, 31%; P=0.7). Lower OCR was observed in oocytes and early embryos from Old, which indicates that mitochondrial metabolic function is reduced for mitochondria originating in the oocytes of Old compared with Young. Use of mtDNA was not indicative of mitochondrial metabolic function. Although sample numbers were limited, cleavage and blastocyst development were not significantly different between Young and Old. Further developmental competence was not determined, although the compromised metabolic capacity of oocytes and embryos from old mares could ultimately contribute to lower fertility outcomes.

Abstract 372: Role of Redox-Sensitive Calcium Channel TRPM2 and Oxidative Stress in Vascular Smooth Muscle Cell Differentiation to an Osteogenic Phenotype: Implications in Hypertension.

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Augusto C Montezano ◽  
Hiba Yusuf ◽  
Glaucia E Callera ◽  
Rhian M Touyz
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
Molecular Mechanisms ◽  
Ros Generation ◽  
Phenotype Transition ◽  
And Oxidative Stress

Vascular smooth muscle cell (VSMC) transformation to an osteoblast-like phenotype is a major factor contributing to vascular calcification, often associated with chronic kidney disease and hypertension. Exact molecular mechanisms underlying VSMC transformation remain unclear but intracellular calcium and ROS have been implicated. Whether these factors are interlinked is unknown. Here, we tested the hypothesis that ROS and redox-sensitive calcium channels (TRPM2) induce an osteogenic phenotype transition in VSMCs from WKY and SHRSP rats. Cultured VSMCs from WKY and SHRSP rats were exposed to calcification medium (CaM) (Ca2+ 1.8 mmol/L, PO4 2.0 mmol/L) for 10 days in the presence/absence of tempol (superoxide dismutase mimetic), and N-(p-amylcinnamoyl)anthranilic acid (ACA, TRPM2 inhibitor). Osteocalcin (OC), BMP-2, BMP-7, TRPM2 and TRPM2-S expression, as well as p47 phox translocation (cytosol:membrane), were determined by immunoblotting. ROS generation was evaluated by chemioluminescence. ROS production (Ctl: 30 AU/ug protein; CaM: 60 AU/ug protein, p<0.05) and p47 translocation (Ctl: 0.7 AU; CaM: 1.1AU, p<0.05) were increased by the CaM in VSMCs from WKY. CaM-induced increase in OC (Ctl: 5 AU; CaM: 13 AU) and BMP-2 (Ctl: 0.60 AU; CaM: 0.75AU) (p<0.05), followed by a decrease in BMP-7 (Ctl: 1.12 AU; CaM: 0.7 AU) (p<0.05), expression in VSMCs from WKY; an effect that was inhibited by tempol and ACA. In SHRSP, the increase in OC (Ctl: 1.25 AU; CaM: 2 AU) and BMP-2 (Ctl: 0.6 AU; CaM: 0.85 AU, p<0.05) expression induced by the CaM was also blocked by tempol. TRPM2 expression was higher in SHRSP (1.40 AU) than in WKY (1.05 AU) VSMCs (p<0.05). However, TRPM2-S expression, an intracellular inhibitor of TRPM2, was decreased in SHRSP compared to WKY VSMCs (SHRSP: 0.9 AU; WKY: 1.5 AU; p<0.05), and it was further decreased by the CaM (0.6 AU). In conclusion, ROS, through TRPM-2 sensitive mechanisms, seems to play an important role in VSMC transformation to an osteogenic, a phenomenon that may be exacerbated in hypertension.

scholarly journals Molecular mechanism of mice gastric oxidative damage induced by nanoparticulate titanium dioxide

2021 ◽  
Author(s):  
Jianhui Ji ◽  
Yingjun Zhou ◽  
Fashui Hong ◽  
Yuguan Ze ◽  
Dongxue Fan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Titanium Dioxide ◽  
Antioxidant Enzymes ◽  
Molecular Mechanisms ◽  
Intragastric Administration ◽  
Nano Tio2 ◽  
Related Factors ◽  
Atp Production ◽  
Cyt C

Abstract Background Nanoparticulate titanium dioxide (Nano-TiO2) has been widely used in food industry, and it has been demonstrated to have adverse effects on mice and human stomach, but its mechanism is rarely concerned. The aim of this study is to determine the effects of nano-TiO2 on the stomach and confirm the role of oxidative stress and apoptosis in the mice gastric damage caused by nano-TiO2, as well as its molecular mechanisms. Methods Mice were continuously exposed to nano-TiO2 with 1.25, 2.5 and 5 mg/kg bw by intragastric administration for 9 months in the present study. The ultrastructure, levels of reactive oxygen species (ROS) and peroxides, activities of antioxidant enzymes and mitochondria-related enzymes, ATP contents as well as apoptosis-related factors expression in mice stomach were examined. Results Oxidative stress, apoptosis and nano-TiO2 aggregation were found in gastric mucosal smooth muscle cells after nano-TiO2 exposure. Nano-TiO2 exposure also resulted in the over-production of ROS and peroxides, decrease of ATP production and activities of antioxidant enzymes and mitochondria-related ATPases, upregulation of apoptosis-related factors including γH2AX, Cyt c, caspase 3, and p-JNK expression, and down-regulation of Bcl-2 expression in mice stomach. Conclusions The gastric toxicity of mice induced by chronic exposure to low dose nano-TiO2 may be associated with oxidative stress and mitochondria-mediated apoptosis in mice.

Melatonin as a prospective metabolic regulator in pathologically altered cardiac energy homeostasis

2021 ◽  
Vol 4 (2) ◽  
pp. 316-335
Author(s):  
Swaimanti Sarkar ◽  
Aindrila Chattopadhyay ◽  
Debasish Bandyopadhyay
Keyword(s):  
Oxidative Stress ◽  
Ischemia Reperfusion Injury ◽  
Energy Requirement ◽  
Ischemia Reperfusion ◽  
Cardiac Cells ◽  
Acid Oxidation ◽  
Ros Generation ◽  
Atp Production ◽  
Working Heart

A constant energy supply is indispensable for the relentlessly working heart. The unique metabolic flexibility of the cardiac tissue enables it to maintain its energy requirement under variable physiological conditions. However, some physiopathological statuses including aging, ischemia-reperfusion injury, diabetic cardiomyopathy, pathological cardiac hypertrophy, and heart failure frequently cause cardiac dysfunction and detrimental metabolic alteration. If the ATP supply fails to match the requirement of a working heart, the heart loses its functional capacity, resulting in slower recovery. A decrease in energy generation is often the ramifications of myocardial mitochondrial dysfunction and oxidative stress. Melatonin, a broad-spectrum antioxidant molecule has an appreciable role in the maintenance of metabolic homeostasis― from a single cell to an entire organism. Melatonin has the capacity to reduce ROS generation, preserve mitochondrial stability, and restore a robust mitochondrial function for unabated ATP production in cardiac tissues. Additionally, melatonin can promote carbohydrate and fat metabolism to further improve the ATP production in heart. In cardiac cells, melatonin upregulates GLUT4 expression either by impeding oxidative stress or by enhancing AMPK activation which accelerates fatty acid oxidation by upregulating PPAR-α and CPT-1α. Melatonin plays a pivotal role in the maintenance of calcium homeostasis in cardiomyocytes by obviating oxidative stress-mediated disruption of SERCA and NCX proteins. A possible role of melatonin to convert the Warburg effect to oxidative metabolism in pathological cardiac events has been recently contemplated. The current review will discuss the possible role of melatonin protecting against cardiac metabolic imbalances under pathological states.

scholarly journals Mitochondrial Dynamics: Functional Link with Apoptosis

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Hidenori Otera ◽  
Katsuyoshi Mihara
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Mitochondrial Dynamics ◽  
Functional Link ◽  
Iron Sulfur Cluster ◽  
Atp Production ◽  
Iron Sulfur ◽  
Cellular Apoptosis

Mitochondria participate in a variety of physiologic processes, such as ATP production, lipid metabolism, iron-sulfur cluster biogenesis, and calcium buffering. The morphology of mitochondria changes dynamically due to their frequent fusion and division in response to cellular conditions, and these dynamics are an important constituent of apoptosis. The discovery of large GTPase family proteins that regulate mitochondrial dynamics, together with novel insights into the role of mitochondrial fusion and fission in apoptosis, has provided important clues to understanding the molecular mechanisms of cellular apoptosis. In this paper, we briefly summarize current knowledge of the role of mitochondrial dynamics in apoptosis and cell pathophysiology in mammalian cells.

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