bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of combined resveratrol plus metformin therapy in db/db diabetic mice
Authors: Duarte‐Vázquez Miguel Angel , Gómez‐Solis Antonieta , Gómez‐Cansino Rocio , Reyes‐Esparza Jorge , Rosado Jorge Luis , Rodriguez‐Fragoso Lourdes
Published: November 2023

The prevalence of Type 2 Diabetes mellitus in the world is associated with other conditions that trigger metabolic syndrome. Although there are several studies on benefits of resveratrol, few have assessed in combination of metformin. The aim of this study was to investigate at the effects of combined therapy (metformin/resveratrol) on body weight gain, biochemical clinical findings, liver and renal function of db/db diabetic mice. Diabetic mice were treated whit resveratrol (20 mg/kg/day), metformin (150 mg/kg/day) and combined therapy for 5 weeks. Histopathological tissues analyses and biochemical parameters (glucose, insulin, triglycerides and cholesterol), functional liver enzymes (AP, AST and GGT) and renal parameters (urea and uric acid) were examined. Our results clearly showed that combined therapy of resveratrol and metformin therapy reduced obesity, glucose and triglyceride levels, as well as improving renal function and partially improving liver function in diabetic mice. The combined therapy may enhance remedial effects in diabetic patients and also in other metabolic disorders as such as metabolic syndrome.

Support or Funding Information

CONACYT‐PEI Mexico for its financial support (Project 231344).
Phage Display cDNA Screens Reveal a Specific Region of Plasmodium falciparum Glutamic Acid‐rich Protein that Binds to Human Red Blood Cells. A Functional Role in Malaria Pathogenesis
Authors: Almukadi Haifa S. , Hanada Toshihiko , Chishti Athar H.
Published: November 2023

Severe malaria caused by Plasmodium falciparum is a serious disease that affects a large number of people regardless of the mode of treatment with currently existing antimalarial drugs. In 2015, the Centers for Disease Control and Prevention (CDC) estimated nearly 214 million cases of malaria infection worldwide resulting in ~400,000 deaths mainly in sub‐Saharan Africa. Infection of red blood cells (RBCs) by P. falciparum, the most lethal species among the human malaria parasites, leads to several complications such as cytoadherence, which involves binding of infected red blood cells (iRBCs) to endothelial receptors, and rosetting, which is the binding of iRBCs to uninfected RBCs. These features contribute to specific organ damage and exacerbate disease pathogenesis. To elucidate the biological mechanisms of P. falciparum infection in human RBCs, we are currently investigating the virulence phenomena including parasite invasion and rosetting. Using phage display technology, we previously identified several novel ligand‐receptor interactions at the blood stage of malaria infection. Here, we used two independent phage display cDNA library screens and identified P. falciparum glutamic acid‐rich protein (Pf‐GARP) as a novel parasite ligand that binds to the surface of human RBCs. Specifically, we screened phage display cDNA libraries constructed from P. falciparum FCR‐3 and 3D7 strains and identified several overlapping Pf‐GARP clones expressing peptides that bind to intact RBCs. Two phage clones encoding Pf‐GARP392–437 and Pf‐GARP356–552 revealed a specific binding region located nearly in the middle of full length Pf‐GARP1–673. Using bioinformatics tools, we chemically synthesized a codon‐optimized segment, Pf‐GARP370–444, located within the overlapping phage clones. The phage cDNA inserts were cloned into pET‐32b vector, and recombinant peptides were expressed in E. coli. Recombinant fusion proteins encoded by all three Pf‐GARP cDNA were confirmed to bind human RBCs in vitro. The codon‐optimized Pf‐GARP370–444 was cloned into the pRE4 vector, and expressed on the surface of Chinese hamster ovary (CHO) cells. The transfected CHO cells formed rosette‐like clusters in the presence of uninfected human RBCs in vitro. Importantly, direct biochemical interaction between purified Pf‐GARP370–444 protein and human RBCs was abolished upon treatment of RBCs with chymotrypsin but not with trypsin and neuraminidase. These results indicate that a centrally‐located segment of Pf‐GARP recognizes a specific chymotrypsin‐sensitive receptor(s) on the surface of host RBCs. Our findings suggest a functional role of Pf‐GARP in the rosetting and invasion phenomena with implications in inter‐membrane transport and vaccine development against malaria.

Support or Funding Information

Funding source: NIH‐HL060961
Virtual Surgical Planning & 3D Printing in Prosthetic Orbital Reconstruction with Percutaneous Implants: A Technical Case Report
Authors: Huang Yu‐hui
Published: November 2023

Osseointegrated titanium implants to the cranial skeleton for retention of facial prostheses have proved to be a reliable replacement for adhesive systems. However, improper placement of the implants can jeopardize prosthetic outcomes, and long‐term success of an implant‐retained prosthesis.

3D computer imaging, virtual planning and 3D printing have become accepted components of the preoperative planning and design phase of treatment. Computer‐aided design (CAD) and computer‐assisted manufacture (CAM) that employ cone‐beam computed tomography (CBCT) data offer benefits to patient treatment contributing to greater predictability and improved treatment efficiencies with more reliable outcomes in surgical and prosthetic reconstruction.

3D Printing enables transfer of the virtual surgical plan to the operating room by fabrication of surgical guides. Previous studies have shown that accuracy is considerably improved with guided implantation when compared to conventional template or freehand implant placement.

This clinical case report demonstrates the use of a 3D technological pathway for preoperative virtual planning through prosthesis fabrication, utilizing 3D Printing, for a patient with an acquired orbital defect which was restored with an implant‐retained silicone orbital prosthesis.

Support or Funding Information

This work was partially funded by financial support from Face the Future Foundation.
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 Information

Supported 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

Background

Poloxamer 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.

Methods

Langendorff‐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.

Results

P188 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.

Conclusion

Our results support the notion that the protective effect of P188 is, at least in part, mediated by NO release.

Support or Funding Information

This 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 Information

This 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

Purpose

Corneal 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.

Methods

Corneal 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.

Results

QPCR 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.

Conclusion

Loss 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 Information

National 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 Information

NIH 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 Information

16SDG27260248 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 Information

Research 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 Information

Hydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.
Mechanism of hERG current inhibition by 4‐hydroxynonenal (4‐HNE), a polyunsaturated fatty acid‐derived electrophile
Authors: Kim Sung Joon , Choi Seong Woo , Jang Ji Hyun , Lee Hyang‐Ae , Zhang Yin Hua Hua
Published: November 2023

Under oxidative stress, peroxidation of ω6‐polyunsaturated fatty acids produces 4‐Hydroxynonenal (4‐HNE), a highly reactive electrophile forming 4‐HNE‐protein adducts. Here we investigate the effects of 4‐HNE on cardiac hERG current that is a major target of arrhythmogenic conditions. Acute application of 4‐HNE (30~100 μM) decreased the tail current of hERG (IhERG,tail), that was reversed by TCEP, a membrane impermeable reducing agent. The action potential duration (APD) of guinea‐pig ventricular myocytes (GPVMs) was prolonged by 4‐HNE. Chronic incubation (1–3 h) with lower concentration of 4‐HNE (10 μM) induced time‐dependent suppression of IhERG,tail and prolongation of APD in GPCMs, that was not reversed by TCEP treatment. Western blot analysis revealed that mature glycosylated hERG (155 kDa) is reduced by the chronic 4‐HNE (10 μM, 1–12 h). In total cell preparations, the decrease of mature hERG was partly rescued by proteasome‐ and lysosome‐dependent degradation inhibitors, bortezomib and bafilomycin, respectively. However, reduction of hERG proteins in the plasma membrane fraction and the inhibition of IhERG,tail by the chronic 4‐HNE treatment were not prevented by bortezomib and bafilomycin. Taken together, it is suggested that the electrophilic binding of 4‐HNE with extracellular domain acutely inhibits hERG activity. Also, the sustained exposure to 4‐HNE may accelerate the degradation of hERG proteins through proteasome‐ and lysosome‐dependent process. The suppression of hERG by 4‐HNE may participate in proarrhythmic effects of endogenous lipid peroxidants under pathological conditions.
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.
Modulation of TRPV4‐stimulated Ion Transport in Choroid Plexus Epithelia by Arachidonic Acid
Authors: Otun Ayodamola , Preston Daniel , Danko Caleb , Schroten Horst , Schwerk Christian , Blazer‐Yost Bonnie
Published: November 2023

Hydrocephalus is a condition that can be caused by overproduction of cerebrospinal fluid (CSF) in the choroid plexus (CP) seated in the brain ventricles. Our laboratory has recently discovered that an antagonist of an osmotic‐ and mechano‐sensitive calcium channel, TRPV4, is effective in treating hydrocephalus in a rat model. Hydrocephalus involves a high level of inflammation due to the fluid pressure exerted on brain tissue. It is, therefore, of interest to study the role of inflammation on ion transport mediated by TRPV4 so as to further understand endogenous modulators involved in hydrocephalic development. To determine the effect of potential endogenous mediators, we conducted Ussing style electrophysiological experiments to measure transepithelial ion transport in a high resistance porcine choroid plexus cell line (PCP‐R). This cell line has been shown to demonstrate similar characteristics as the native choroid plexus and responds to the TRPV4 agonist GSK1016790A with an increase in transepithelial ion flux and a decrease in transepithelial resistance. Arachidonic acid is an endogenous molecule produced during an inflammatory response and is also a precursor to multiple prostanoids some of which are known to activate TRPV4. A short (10 minute) pre‐incubation with arachidonic acid has no effect on basal ion flux in confluent PCP‐R cells but potentiates TRPV4‐stimulated transepithelial ion flux by approximately 150%. The transepithelial resistance of the cells was not altered in response to arachidonic acid but arachidonic acid did not alter the TRPV4‐mediated decrease in resistance. It is hypothesized that the overproduction of fluid in hydrocephalus is due, in part, to the abnormal flux of ions into the CSF and, likewise, inflammatory responses cause changes in ion and fluid balance. These data indicate that either arachidonic acid or a metabolite formed by enzymes in the choroid plexus cells modulate ion transport responses rather than initiate them. These studies are important for understanding the formation of excess CSF during hydrocephalic development and in other inflammatory brain diseases.

Support or Funding Information

Hydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.
Activation of TRPA1 and TRPV1 in vagal nociceptive sensory neurons by inhibitors of mitochondrial function
Authors: Stanford Katherine R , Barannikov Ivan R , Taylor‐Clark Tom E
Published: November 2023

Inflammatory signaling pathways such as tumor necrosis factor alpha and interleukin‐1β result in mitochondrial dysfunction and induce mitochondrial reactive oxygen species (ROS) production, depolarization, calcium release as well as morphological changes such as remodeling and fission. Mitochondrial dysfunction is linked to nociceptive disorders which involve sensory nerve hyperexcitability including asthma, and diabetic painful neuropathy. However, it is not known how mitochondrial signaling events may influence nociceptive signaling. Our lab has previously demonstrated that Antimycin A, a mitochondrial electron transport chain complex III inhibitor, activates bronchopulmonary C‐fibers via gating of transient receptor potential channels (TRP) Ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). Both TRPA1 and TRPV1 are selectively expressed in nociceptive subtypes of vagal, trigeminal and dorsal root afferents. Here we characterized the response to three inhibitors of mitochondrial function: Rotenone (complex I inhibitor), Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP, mitochondrial inner membrane proton ionophore), and Antimycin A (complex III inhibitor) using live cell imaging of dissociated vagal neurons. We used co‐imaging of mitochondrial superoxide (MitoSox Red) and calcium (FURA‐2AM) to simultaneously measure ROS production and neuronal activation. Antimycin A and CCCP caused ROS production in both nociceptive and non‐nociceptive neurons. Rotenone had little effect on ROS production. Ca2+ imaging showed that mitochondrial modulators failed to activate non‐nociceptive neurons, but selectively stimulated approximately 50% of nociceptive neurons. Interestingly, this activation was not correlated with ROS production. Antimycin A‐induced nociceptor activation was abolished in TRPA1−/− neurons pretreated with the TRPV1 inhibitor iodoresiniferatoxin. However, only a fraction of the Antimycin A‐induced nociceptor activation was prevented by the reducing agent dithiothreitol (DTT). Taken together, this suggests that modulation of mitochondrial function activates nociceptive neurons via ROS‐mediated and ROS‐independent mechanisms linked to TRPA1 and TRPV1.

Support or Funding Information

NHLBI R01 (HL119802)
Selection of High Affinity Binders Against the Thumb‐Domain of αENaC using a Bacterial Two‐Hybrid System
Authors: Svenningsen Per , Dimke Henrik
Published: November 2023

The objective of project was to develop reagents that facilitate the isolation of extracellular vesicles (EVs) containing the epithelial Na+ channel (ENaC). The ENaC subunits are highly conserved, and the traditional method of generating antibodies against the extracellular domain of ENaC does often not produce high affinity binders. We therefore sought to develop a recombinant affinity reagent using a bacterial two‐hybrid screen. The thumb domain of αENaC (Gly415‐Lys504) was used as bait to isolate high affinity binders. As binders, we generated a library of the small antibody mimetics termed affibodies. The isolated affibodies was subsequently characterized by producing a fusion protein consisting of biotin‐acceptor peptide (AP), the affibody and an intein/chitin‐binding domain. The affibody fusion protein was readily expressed in bacteria and was purified using chitin‐resin. Subsequently, the AP‐fused affibody was effectively eluted from the chitin beads using thiol‐induced cleavage. As demonstrated by streptavidin gel‐shift assay, co‐expression of the AP‐fusion protein with biotin protein ligase BirA biotinylated the affibody, thereby producing an effective tag for the subsequent isolation of the affibody. One of the affibodies was further tested in preliminary experiments for its ability to isolate ENaC positive urinary EVs. Compared to urinary EVs isolated by ultracentrifugation, affinity isolated samples were depleted in Tamm‐Horsfall protein as judged by coomassie‐stained SDS‐PAGE. Immunoblots showed that urinary EVs isolated by affibodies and ultracentrifugation were positive for αENaC, AQP2, and the exosomal marker ALIX. Thus, our results indicate that affibodies are useful affinity reagents for enrichment of specific urinary EVs.

Support or Funding Information

Lundbeck Foundation and Novo Nordisk Foundation
Intracellular and Near‐Membrane Dynamics of L‐type CaV1.2 Channel
Authors: Ghosh Debapriya , Nieves‐Cintrón Madeline , Navedo Manuel F
Published: November 2023

L‐type CaV1.2 channels (LTCCs) are involved in myriad of physiological functions ranging from excitation, contraction, transcription and proliferation. Numerous studies had been conducted to understand the electrophysiological and biochemical properties of LTCC. Yet, the intracellular and near‐membrane dynamics of LTCCs are poorly understood. Here, using 4D spinning‐disk‐confocal and near‐membrane Total Internal Reflection Microscopy (TIRFM) recordings, we found that LTCCs exhibit diverse and dynamic intracellular trafficking patterns executed via compartments in form of punctate and larger ovoid vesicles as well as elongated tubules. Total Internal Reflection Fluorescence Recovery After Photobleaching (TIR‐FRAP) showed that both actin and microtubules are equally important in active plasma membrane trafficking of these compartments, where LTCC demonstrate either ‘kiss and run’ or ‘kiss and linger’ type fusion events, while perhaps also supporting distinct patterns of intracellular LTCC movements. These LTCC carrying compartments seems to undergo constitutive homotypic fusion and fission events, near the cell membrane. In conclusion, our study reveals very diverse and unique intracellular and near‐membrane dynamics of LTCCs with profound implications for ion channel trafficking.

Support or Funding Information

This work is supported by NIH R01HL098200 and R01HL121059.
Cardioprotection by Genetic Deletion of Kvβ2 in Ischemia Reperfusion Injury
Authors: Chapalamadugu Kalyan C. , Badole Sachin L. , Tur Jared , Tipparaju Srinivas M.
Published: November 2023

Background

Ischemia reperfusion (I/R) injury increases the levels of NADH in the heart leading to increased NADH/NAD+ ratio. Voltage‐gated potassium channel (Kv) β‐subunits bind to ion channels and contractile proteins resulting in modulation of cardiac function. Previous studies demonstrated high affinity binding of voltage‐gated potassium channel beta subunit 2 (Kvβ2) to pyridine nucleotides (NAD[P]+, NAD[P]H).

Hypothesis

We hypothesized that Kvβ2 plays a major role in sensing I/R injury.

Methods and Results

Hearts from wild type (WT) and Kvβ2 knockout (KO) mice were perfused using Langendorff apparatus and subjected to 45 min no‐flow global ischemia followed by 120 min reperfusion. At the end of reperfusion, hearts were sectioned and stained with 2,3,5‐triphenyltetrazolium chloride (TTC). Cardiac perfusates were collected before ischemia and during reperfusion at 0, 30 and 120 min, and cardiac troponin I (cTnI) was measured. Intracardiac levels of NAD+ and NADH were measured in I/R injured hearts. Infarct size was measured, and normalized to total area of the heart. I/R challenge of WT mice hearts resulted in a significant myocardial damage where the infarct size was 46.6±5.4%. However, infarct size in Kvβ2 KO hearts was only 12±2.3% (n=5), which clearly demonstrates that the KO hearts show significant resistance to injury (p<0.05), despite similar changes in NADH/NAD ratio in both groups. Baseline cTnI levels (ng/ml) in the perfusate were similar between the WT and KO hearts (n=3). During reperfusion, cTnI levels in the perfusate from KO hearts were significantly (p<0.05) decreased when compared to WT.

Conclusions

This study shows that Kvβ2 deletion is cardioprotective in I/R injury. It is plausible that Kvβ2:NADH/NAD axis may serve as a metabolic sensor in I/R injury, where Kvβ2 subunit senses and relays the increased NADH/NAD levels to target proteins in WT hearts, whereas deletion of Kvβ2 abrogates relaying the information leading to protection.
ClC‐3 and LRRC8A interact physically and co‐localize with endosomal superoxide production.
Authors: Nguyen Hong‐Ngan , Choi Hyehun , Lamb Fred S
Published: November 2023

Both the ClC‐3 Cl−/H+ antiporter and LRRC8A anion channels are required for TNFα‐induced, Nox1‐dependent superoxide (O2−•) production and subsequent signaling in vascular smooth muscle cells (VSMC). Both proteins have been associated with the swelling‐activated anion conductance (IClswell) which can modulate Nox1 activity, and thereby TNFα signaling. It is now clear that LRRC8 family (LRRC8A‐E) proteins constitute IClswell channels. However, it is not known why ClC‐3 expression also affects the magnitude of this anion current. We hypothesized that a physical relationship and functional interdependence between ClC‐3 and LRRC8A explains this finding. Confocal microscopy and immunoprecipitation were used to localize expressed ClC‐3 and LRRC8A with O2−• (ROSstar 550) in HEK293 cells expressing fluorescent fusion proteins. The endogenous proteins were localized by immunostaining of cultured human aortic VSMC. As previously demonstrated, ClC‐3‐GFP localizes to the membrane of very large intracellular vesicles (1–10 μm diameter). These compartments are now revealed to be multivesicular bodies, and the smaller vesicles within (0.1–0.6 μm) are a prominent site of ROSstar staining. When expressed alone, GFP‐LRRC8A localizes to small cytoplasmic vesicles which also are strongly ROSstar positive, suggesting that this is an important site of O2−• production. Co‐expression of ClC‐3‐GFP and mCherry‐LRRC8A reveals that small vesicles, which are rich in LRRC8A, are contained within ClC‐3‐expressing multivesicular bodies. In VSMC, antibodies to ClC‐3, LRRC8A and Nox1 stain both localized regions of the plasma membrane and scattered intracellular vesicles in the 0.1–0.60 μm range. Overlapping staining for all three proteins is seen in a subset of these vesicles, and in scattered multivesicular clusters. We have previously demonstrated that LRRC8A co‐immunoprecipitates with Nox1 and its p22phox subunit (Choi, FRBM, 2016). We now observe that immunoprecipitation of endogenous ClC‐3 pulls down LRRC8A. These findings demonstrate co‐localization and physical association between ClC‐3 and LRRC8A. This association, at sites where Nox1 is producing O2−•, may explain why both proteins play a role in Nox1‐mediated O2−• production and TNFα signaling.

Support or Funding Information

HC is supported by an SDG from the American Heart Association. FL is supported by R01 HL128386.
Instantaneous Down‐Regulation of NMDA Receptor Activity by Nanomolar Concentrations of Ouabain
Authors: Westin Linda , Akkuratov Evgeny E , De Marothy Minttu , Lindskog Maria , Brismar Hjalmar , Aperia Anita
Published: November 2023

The N‐methyl‐d‐aspartate (NMDA) receptor plays an essential role for glutamatergic transmission and synaptic plasticity. Calcium influx via the NMDA receptor (NMDAR) activation modulates both long‐term potentiation (LTP) and long‐term depression (LTD). Hyperactivity of the NMDAR can trigger events leading to calcium mediated neurotoxicity, a common cause of brain damage. Recent studies have suggested that the Na+,K+‐ATPase (NKA) may interact with NMDAR and that ouabain, a highly specific NKA ligand, has a neuro‐protective effect. Here we have tested whether subsaturating concentrations of ouabain may attenuate the calcium response from an activated NMDAR. Single hippocampal neurons derived from E18.5 rats and cultured for 3 weeks were exposed to 10μM NMDA with or without 10nM ouabain, and the dendritic calcium response was recorded. The calcium influx was immediately and transiently attenuated with approximately 40% when ouabain was co‐administered. The ouabain effect was not due to NMDAR internalization, changes in membrane potential or alterations in Src family phosphorylation. Studies on HEK cells expressing NMDAR subunits, the ubiquitous NKA catalytic alpha1 subunit and the neuron specific NKA alpha3 subunit, confirmed that ouabain attenuates the calcium response from an activated NMDAR and indicated that ouabain attenuation of NMDAR calcium response was mediated via the alpha3 and not the NKA alpha1 subunit . Since ouabain locks NKA alpha subunits in one of its two major conformations, we speculated that ouabain bound NKA alpha3 can act as an allosteric modifier of NMDAR. Using dual‐color direct stochastic optical reconstruction microscopy (dSTORM) we showed a spatial correlation between the NMDARs and the NKA alpha1 and alpha3 subunits. The distance between NKA alpha3 and the NR2 subunits of the NMDAR was less than 50 nm for 50% of the NKA alpha3 and less than 70 nm for 50% of the NKA alpha1 subunits. In summary, our results demonstrate a novel mechanism for modulation of NMDAR signaling and offer an explanation for the neuro‐protective effect of ouabain reported by other investigators.