bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Mitochondrial Defect in the Ion Channel, VDAC3, Alters ENaC Activity Via ROS
Authors: Linck Valerie A , Zhou Li , Zai Yu‐Jia , Galarza‐Paez Laura , Li Linda Wenyi , Thai Tiffany L , Ma He‐Ping , Eaton Douglas Charles 
Published: November 2023

It has been suggested that voltage‐dependent anion channels (VDACs) control the release of superoxide from mitochondria. We have shown that reactive oxygen species like superoxide (O2−) and hydrogen peroxide (H2O2) stimulate epithelial sodium channels (ENaC) in sodium‐transporting epithelial tissue including cortical collecting duct (CCD) principal cells. Therefore, VDACs could regulate ENaC by modulating cytosolic ROS levels. Our patch‐clamp data show that there was a significant difference in ENaC single‐channel activity between wild‐type (WT) and VDAC3 knockout mice kept on high salt (NaCl) diet for two weeks, but not on normal diet. Western blots show that a high salt diet significantly increases the expression of cleaved g‐ENaC in VDAC3 knockout mice, but not in WT mice. Consistent with this observation, systolic blood pressure was significantly increased in VDAC3 knockout mice but not in WT mice on a high salt diet for two weeks. Electron microscopy shows that a significant morphological change occurrs in the CCD cells of VDAC3 knockout mice on a high salt diet compared to WT mice which may be caused by superoxide overload in the mitochondria. These data suggest that deletion of VDAC3 sensitizes ENaC to high salt challenge and promotes salt‐sensitive hypertension. This is presumably due to ROS release in close proximity to ENaC in the apical membrane since mitochondria are concentrated in a band 1 to 2 microns below the apical membrane. Mitochondrial superoxide would be trapped in the mitochondria of VDAC3 knockout mice, but it can be degraded by mitochondrial superoxide dismutase to produce H2O2 which is known to freely move across the outer membrane of mitochondria into the cytosol. Therefore, the activation of ENaC could be a direct effect of H2O2 on ENaC. Alternatively, recent studies have shown that H2O2 stimulates ENaC‐targeted protease activity, so H2O2 could also promote ENaC cleavage to increase activity. However, the specific underlying mechanism remains to be determined.Support or Funding InformationSupported by NIH R25‐DK101390 to VAL and R01‐DK100582 to HPM
Dose Dependent Nitric Oxide Production by Poloxamer 188 in Rat Isolated Hearts
Authors: Douglas Hunter F , Salzman Michele M , Hackel Benjamin J , Bartos Jason A , Yannopolous Demetris , Riess Matthias L 
Published: November 2023

BackgroundPoloxamer 188 (P188) is a tri‐block copolymer surfactant that has demonstrated post‐ischemic cardioprotective effects ex‐ and in‐vivo. The exact mechanism is unknown; however, preliminary results suggest the protective effects may be mediated by nitric oxide (NO) production. We hypothesized that P188 will result in NO production, and this is abolished by NO synthase (NOS) inhibition.MethodsLangendorff‐prepared hearts from 8 week old Brown Norway rats were perfused with Krebs solution at 37°C, saturated with 95% oxygen (O2), 5% carbon dioxide (CO2) at a constant perfusion pressure of 70mmHg and allowed to stabilize. Hearts were then loaded with 10μM of the NO‐sensitive fluorescent dye DAF‐FM diacetate (4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate). P188 was administered in increasing concentrations (0mM, 0.1mM, 0.3mM, 1.0mM, and 3.0mM) ± 100μM of the nonspecific NOS inhibitor L‐NAME (Nω‐Nitro‐L‐arginine methyl ester). After 10min of exposure to each concentration of P188, DAF‐fluorescence was measured using a bifurcated fiber optic probe in contact with the left ventricular epicardial wall (495/515nm excitation/emission). Values are ± SEM. Statistics: ANOVA followed by Student Newman Keul; p<0.05 *vs control.ResultsP188 led to a concentration‐dependent increase in DAF‐fluorescence of *3.6%±0.7 vs 1.0%±0.4, 7.3%±3.3 vs 4.9%±1.4, 10.1%±4.0 vs 2.5%±1.3, and *26.2%±5.1 vs 1.1%±0.4%. This was completely abolished by L‐NAME.ConclusionOur results support the notion that the protective effect of P188 is, at least in part, mediated by NO release.Support or Funding InformationThis work was supported by the NIH (R01 HL095122) and institutional funds. None of the authors have a conflict of interest.
The Presence of Mitochondrial Associated ER Membranes (MAM) in Cortical Collecting Duct Principal Cells
Authors: Galarza‐Paez Laura , Zou Li , Ma He‐Ping , Eaton Douglas Charles , Thai Tiffany L. 
Published: November 2023

Mitochondria have an established role as the ‘powerhouse of the cell’; additionally, they have been recently implicated in the formation of calcium signaling microdomains. In most epithelia, mitochondria can associate directly with IP3 receptors on the endoplasmic reticulum forming mitochondria‐associated ER membranes or MAMs. This interaction is facilitated by the tethering of the voltage dependent anion channel (VDAC) in the outer mitochondrial membrane and the IP3RIII on the ER. Ca2+ can move directly through the IP3RIII into the mitochondria via the VDAC1 channel. Interestingly, IP3RIII is the same isoform of IP3R known to mediate the inhibition of the epithelial sodium channel (ENaC) downstream of Gq proteins. Based on the knowledge that IP3R and mitochondrial Ca2+ both regulate ENaC activity, we hypothesized that MAMs exist in the cortical collecting duct (CCD) cells and are involved in the regulation of ENaC. MpkCCD cells were stained with mitotrackerRED and ERtrackerBlue and imaged with confocal microscopy, super resolution microscopy and we found that there is a strong association between both organelles. TEM images for mouse principal cells were obtained and further confirmed the association between ER and mitochondria. Wild type and VDAC1 kidneys were stained with fluorescently tagged VDAC1 and IP3R antibodies against these proteins. We found that the proteins co‐localize in kidney tissue and VDAC1 is present in mitochondrial membranes (as expected), but also in the plasma membrane of the CCD principal cells. Because of the ionic conditions across the outer mitochondrial membranes and across the plasma membrane, only VDAC1 in the mitochondria are likely to be active pathways for the movement of calcium. We concluded that mitochondria and ER associate in kidney cortical collecting duct principal cells through VDAC1 and IP3R complexes. In the future we will perform experiments to test whether MAMs are involved in the regulation of ENaC and their underlying mechanisms.Support or Funding InformationThis work was supported by APS Summer Undergraduate Research Programs to LG‐P and by NIH R01‐DK100582 to HPM
Disrupted Ammonia Handling, Glutamine Metabolism and Mitochondrial Dysregulation in Slc4a11 KO Corneal Endothelium
Authors: Ogando Diego G. , Zhang Wenlin , Li Shimin , Choi Moonjung , Kim Edward , Bonanno Joseph A. 
Published: November 2023

PurposeCorneal endothelial dystrophies are associated with mutations in SLC4A11. SLC4A11 shows NH3:H+ cotransport activity. NH3 is predominantly produced in mitochondria from metabolism of glutamine. We asked if glutamine metabolism, ammonia production, ROS production and mitochondrial activity were altered in the Slc4a11 KO mouse corneal endothelium and cell lines derived from WT and KO.MethodsCorneal endothelial tissue from Slc4a11−/− mice were evaluated for glutaminolysis enzyme expression, oxidative stress (MitoSox, protein nitrosylation, OHdGuanine & Hmox1) and autophagy (LC3B). Conditionally immortalized cell lines of Slc4a11−/− and WT mouse corneal endothelial cells (MCEC) expressing temperature sensitive Large T antigen were generated. In MCEC, cell death was quantified by AnV‐FITC + PI, mitochondrial membrane potential was measured by JC1, ATP was determined by luciferin‐luciferase assay, reactive oxygen species was quantified by Carboxy‐H2DCFDA and NH3 production was measured by colorimetric assay. Relative glucose and glutamine flux was measured by GC‐MS.ResultsQPCR indicated increased Heme Oxygenase1, GLS1 (glutaminase) and decreased GLS2 and GGT (g‐glutamyltranspeptidase) in Slc4a11−/− corneal endothelium (CE). Increased autophagy was observed by LC3B staining in Slc4a11−/− (CE). Resting pHi is increased and NH3‐dependent H+ transport is decreased in Slc4a11−/− CE. MitoSox staining indicates mitochondrial oxidative stress in Slc4a11−/− CE at 12 weeks of age (KO: 274,404 ± 3138 integrated intensity/cell vs WT: 80,105 ± 2147, n=2, p=0.012). OHdG staining (marker of oxidative modification of DNA) was increased in Slc4a11−/− at 40 weeks. Glutamine (0.5mM) induced increased ROS, apoptosis, & MMP depolarization with decreased ATP and NH3 production in Slc4a11−/− relative to WT MCEC. Glucose derived TCA cycle intermediates increased from 35% in WT to 50% in Slc4a11−/− MCEC. MitoQ significantly inhibited Gln‐induced cell death. Gln‐induced cell death is partially inhibited by GLS1‐specific inhibitors BPTES and CB839 and totally inhibited by Dimethyl‐α‐ketoglutarate.ConclusionLoss of Slc4a11 induces aberrant glutamine metabolism, NH3 related toxicity, mitochondrial dysfunction and oxidative stress. This suggests that the corneal edema seen in Slc4a11 KO mice is caused by a reduced ability to generate ATP, secondary to mitochondrial dysfunction.Support or Funding InformationNational Eye Institute RO1 EY08834
The Effect of SLC4A11 Mutations on Intracellular pH
Authors: Li Shimin , Ogando Diego G , Bonanno Joseph A 
Published: November 2023

SLC4A11 is a ubiquitously expressed transporter showing Na+‐OH−, NH3‐H+ cotransport, and water permeability. SLC4A11 mutations have been associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED2) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). To address the role of known human SLC4A11 mutations on cellular physiology we measured intracellular pH regulation in PS120 fibroblasts that have been stably transfected with SLC4A11 mutant variants.Mutations were made in the Wild‐Type (WT) construct phCMV2‐hSLC4A11 by site‐directed mutagenesis. Mutants with a single amino acid change were obtained and referred to as R125H, C386R, V507I and N639A respectively. PS120 cells were stably transfected with those constructs and empty vector (EV) using SuperFect, and selected by Geneticin. For intracellular pH (pHi) measurement, PS120 cells grown on coverslips were loaded with a pH‐sensitive fluorescent dye, 2′7′‐Bis‐(2‐Carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF‐AM). The coverslip was mounted in a perfusion chamber (37°C) on the stage of a fluorescence microscope. Perfusion was performed with a Bicarbonate‐free solution (BF) to obtain stable baseline pH, followed by pulsing cells with 10 mM NH4Cl for 5 minutes, and changed back to BF perfusion until pHi was stable. Fluorescence ratio (495/440 nm) was converted to pHi following calibration with high K+/Nigericin. Results are means ± SE from 3 trials.The baseline pHi was 7.211 ± 0.136, 7.877 ± 0.056, 7.391 ± 0.069, 7.226 ± 0.232, 7.504 ± 0.104 and 7.359 ± 0.221 for WT, EV, R125H, C386R, V507I and N639A, respectively. The pHi increase from NH4Cl pulse was 0.486 ± 0.014, 0.161 ± 0.018, 0.298 ± 0.05, 0.307 ± 0.07, 0.304 ± 0.011 and 0.442 ± 0.084 respectively. The rate of pHi increase (ΔpH/min) was 2.184 ± 0.33, 0.469 ± 0.037, 1.004 ± 0.124, 1.024 ± 0.362, 1.17 ± 0.142 and 1.818 ± 0.405, respectively. The subsequent rate of pHi decrease in the presence of NH4Cl was (ΔpH/min) 0.153 ± 0.038, 0.006 ± 0.012, 0.012 ± 0.005, 0.011 ± 0.003, 0.033 ± 0.002 and 0.009 ± 0.007, respectively. Removal of NH4Cl‐BF resulted in maximum acidification of 1.205 ± 0.122, 0.343 ± 0.074, 0.671 ± 0.019, 0.409 ± 0.122, 0.747 ± 0.041 and 0.748 ± 0.048 pH units. NH4Cl induced pHi changes were WT > N639A > V507I > R125H > C386R > EV.Expression of SLC4A11 WT and mutants lower resting pHi relative to EV, consistent with a H+ permeability. NH4Cl induced pHi changes were consistent with reduction of NH3:H+ linked cotransport in SLC4A11 mutants.Support or Funding InformationNIH R01 EY08834‐23 & P30EY019008
Role of transcript variants of Mitochondrial Ca2+ Uniporter
Authors: Valkov Nedyalka , Yang Donqin , Jhun Bong Sook , Zhang Peng , O‐uchi Jin 
Published: November 2023

Recent discovery in the molecular identity of the mitochondrial Ca2+ uniporter complex (mtCUC) (a pore forming subunit MCU and regulatory subunits) provides new possibilities for applying genetic approaches to study the mitochondrial Ca2+ influx mechanism and its significant impact to mitochondrial and cellular functions. Indeed, rapid progress was attained in characterizing the role of MCU in cardiomyocytes by genetically knocking down/out the components of mtCUC in situ or in vivo. However, it is not clear how mt Ca2+ are regulated under pathophysiological condition in the heart by MCU gene expression and/or its protein modifications, though the alteration of mtCUC function may play a key role in non‐cardiac diseases in human.Recently, we reported that a post‐translational modification (PTM) of MCU protein by a tyrosine (Tyr) kinase name Pyk2 is the critical regulatory mechanism for upregulating MCU function in cardiomyocytes. We found that Pyk2‐dependet Tyr phosphorylation of MCU accelerates MCU oligomerization to form more number of terameric channels at inner mitochondrial membrane (IMM), which initiates mtCa2+overload, mtCa2+‐dependent ROS overproduction and activation of apoptotic signaling under alpha1‐adrenoceptor (alpha1‐AR) stimulation. In addition to the discovery of the PTMs of MCU, we identified the existence of a novel MCU transcript variant (termed MCU‐S) in human and mouse cells/tissues including hearts in addition to the original MCU (renamed as MCU‐L) reported in 2011. We found that modulation of MCU‐S/MCU‐L ratio in situ by overexpression of MCU‐S inhibits mitochondrial Ca2+ influx in HEK293T cells. Finally, we also tested in rat neonatal cardiomyocytes that introduction of MCU‐S inhibits the mitochondrial Ca2+ uptake, mtCa2+‐dependent ROS overproduction and activation of apoptotic signaling under alpha1‐adrenoceptor stimulation. These results indicate that strategy of switching MCU transcript variants will provide strong antioxidative and anti‐apoptotic effects similar to the beneficial effect by the blockade of MCU Tyr phosphorylation. Elucidation of the role of MCU transcript variants will provide us new insights into the molecular basis of mitochondrial Ca2+ handling under the physiological and pathophysiological conditions in the heart.Support or Funding Information16SDG27260248 AHA Scientist Development Grant
Calcium Activates Ferroportin‐Mediated Cellular Iron Efflux
Authors: Ruwe T. Alex , Shawki Ali , Mackenzie Bryan 
Published: November 2023

The Ohio Physiological Society (OPS) became the first APS chapter during the Spring Meeting of Council in 1995. The OPS was founded in 1986 as a professional association of Ohio physiologists. It was established with the purpose of enhancing and advancing the field of physiology as a coordinated discipline consisting of the many subdisciplines working at the molecular, cellular, and organ system levels of organization in both basic and applied areas. The current membership is from a variety of academic institutions and industries across Ohio. Travel awards are made to students demonstrating exemplary research while presenting at the Annual OPS Meeting. These funds help defray the cost of attending the Experimental Biology Meeting in order to present their research.Support or Funding Information[Ohio Physiological Society, Peter K. Lauf Travel Award 2016]
Functional Studies of TRPM7 – a Candidate Gene for Stillbirth?
Authors: Cartwright James , Aziz Qadeer , Harmer Stephen , Tinker Andrew , Munroe Patricia 
Published: November 2023

Stillbirth is classified as a baby born dead after 24 weeks gestation and over 2.6 million stillbirths occurred in 2015. Damaging mutations in genes that cause long QT syndrome (LQTS), short QT syndrome (SQTS), and Brugada Syndrome are known to cause sudden cardiac death (SCD) in adults and children. Prenatal diagnosis of LQT has been possible for decades, creating a disease spectrum where channelopathies may fatally influence pregnancy 1.Our study aim was to identify genetic variants in cardiac ion channels that may cause stillbirth. We sequenced 35 candidate genes [12 LQTS genes and 13 candidate genes from genome‐wide association studies of QT interval and SCD] in 70 stillbirth cases. Thirty‐nine cases harboured a novel or low frequency predicted damaging missense variant. Two novel and two rare variants were detected in the transient receptor potential melastatin 7 (TRPM7) gene, these were selected for follow‐up functional studies.TRPM7 is an ion channel that conducts cations through a tetrameric pore and is required for cardiac development and conduction 2. The four stillbirth variants (p.G179V, p.R494Q, p.T860M and p.E1205G) were inserted into a TRPM7 expression plasmid using site‐directed mutagenesis. Wild‐type (WT) and mutant channels were expressed in HEK293 and CHO‐K1 cells and whole‐cell patch clamp experiments were performed to assess channel function. TRPM7 mRNA and protein levels were measured using qRT‐PCR and western blotting, respectively.TRPM7 currents were recorded following transfection of HEK293 and CHO‐K1 cells. Gene expression analysis of TRPM7 mRNA in transfected HEK293 cells found similar levels between WT and variants (P = 0.6506). HEK293 cells expressing G179V or T860M variants showed significantly reduced current at +80mV compared to WT channels (39.2 & 36.3 pA/pF respectively, vs 133.2 pA/pF, P < 0.05). Conversely, in CHO‐K1 cells expressing the R494Q variant there was a significant increase in current density compared to WT channels (273.6 pA/pF vs 110.0 pA/pF, P < 0.0001). Western blot analyses failed to detect full length TRPM7 in HEK293 cells transfected with either G179V or T860M compared to WT, R494Q and E1205G expressing cells, where a 210kDa protein was detectable.Three TRPM7 variants were found to alter channel function. G179V and T860M reduced protein expression and current density when expressed in HEK293 cells. In CHO‐K1 cells, R494Q expression results in increased current density compared to WT. These differences in channel activity were observed despite similar levels of mRNA expression. These preliminary data suggests that our recently discovered pathogenic variants in TRPM7 could be a contributory causal factor in unexplained stillbirths.Support or Funding InformationResearch support: British Heart Foundation, Stillbirth & Neonatal Death Charity, Wellness of Women, UK.
TRPV4‐Mediated Ion Transport in the Choroid Plexus
Authors: Preston Daniel , Simpson Stefanie , Danko Caleb , Schroten Horst , Schwerk Christian , Blazer‐Yost Bonnie 
Published: November 2023

The Choroid Plexus (CP), which is responsible for the production of cerebrospinal fluid (CSF), is found in the lateral, third, and fourth ventricles of the brain. The CP is composed of a high resistance epithelium which surrounds a network of capillaries. The primary function of the barrier epithelium is regulated transport of ions and water that controls the production and movement of CSF. Transient Receptor Potential Vanilloid‐4 (TRPV4) is a calcium‐permeable ion channel on the apical membrane of the CP epithelium that is activated by a wide variety of stimuli including hypotonicity, fluid sheer stress and arachidonic acid metabolites. Our laboratory has shown that treatment with a TRPV4 antagonist inhibits hydrocephalus in a rat model of the disease. When TRPV4 is activated, the channel allows Ca2+ to flow into the cells. The resulting increase in intracellular Ca2+ can activate Ca2+‐activated channels. The current study was designed to identify the channels secondarily activated in response to stimulation of TRPV4. A recently described high resistance porcine choroid plexus cell line (PCP‐R) was used to determine the effect of activation of TRPV4 with a specific channel agonist, GSK1016790A. Ussing chamber electrophysiology was used to monitor the transepithelial ion flux and transepithelial electrical resistance changes. Based on the direction of the ion flux stimulated in response to the agonist, it is likely that activation of TRPV4 mediates the secretion of a cation. One of the most likely families of cation channels are the Ca2+‐activated K+ channels. Neither the intermediate conductance potassium (IK, SK4) channel inhibitor TRAM34 nor the big potassium (BK) channel inhibitor iberiotoxin blocked the agonist‐stimulated transport. However, fluoxetine which inhibits the small conductance Ca2+‐activated K+ channels (SK1, SK2 and SK3), blocked TRPV4‐mediated transepithelial ion flux. The SK2‐specific inhibitor Tamapin, a potent scorpion toxin, was ineffective in blocking the TRPV4‐mediated transepithelial transport. These data suggest that the TRPV4 stimulated transepithelial ion flux involves either SK1 or SK3. TRPV4 activity may play a role in the homeostatic regulation of the composition of the CSF and the current data suggest that both TRPV4 and SK channels may be targets for the treatment of hydrocephalus.Support or Funding InformationHydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.
Use of Human Umbilical Cord Mesenchymal Stem Cells to Treat Corneal Opacity Acquired through Injury and Infection
Authors: Elzarka Mohamed , Call Mindy , Kao Winston W.Y. 
Published: November 2023

optical refractive power can be attributed to the cornea. An important tissue within the cornea is the corneal stroma, which accounts for 90% of corneal thickness and which is composed of interwoven collagen fibrils, proteoglycans, and sparsely distributed keratocytes. The lattice arrangement and spacing of the collagen fibrils in the stroma allows for transparency, permitting light to refract through the stroma correctly. Disruption of the collagen fibril architecture through conditions such as corneal injury, bacterial infection, and complications from surgical interventions, can lead to increased corneal opacity and a loss in vision acuity. Previous work in our lab has shown that human umbilical cord mesenchymal stem cells (hUMSCs) have been able to partially rescue the thin, cloudy corneas of collagen V‐null mice with little to no immune rejection or inflammation. This projects aims to translate this learning into insight on how to aid in corneal repair, and hypothesizes that hUMSCs would similarly improve corneal opacity brought about by corneal injury and bacterial keratitis infection.Wildtype C57BL/6 mice were used to create two study groups. The first group was used to model corneal injury and received bilateral keratectomies (2mm in diameter) of the corneal epithelium and anterior corneal stroma. The second group modeled corneal infection and was bilaterally infected with Pseudomonas aeruginosa (strain PA01). Mice in each group were subsequently treated on one eye with hUMSCs through a stem‐cell‐infused fibrin gel applied directly to the corneal surface. Corneal opacity was assessed in these eyes and in contralateral controls that did not receive hUMSC treatment using Heidelberg Retinal Tomography. Our results indicate that among eyes that underwent keratectomy, hUMSC‐treated eyes had significantly reduced corneal opacity as compared to their contralateral controls. Similarly, infected eyes treated with stem cells show a reduction in opacification as compared to non‐treated, infected eyes. These results implicate the efficacy of using hUMSCs in treating acquired corneal disease and have led to ongoing experimentation in assessing the hUMSC immunomodulatory response in bacterial infection.