bluuebunny/crossref_metadata_2025 huggingface dataset:
Dermatological effects of weathered petroleum exposure (844.18)
Authors: Gallucci Randle , Kemp Jesse , Luckett‐Chastain Lerin , McShan William
Published: June 2021

Nearly 5 million barrels of oil were released into the Gulf of Mexico following the Deepwater Horizon oil well failure, making it undoubtedly the largest environmental disaster response effort in U.S. history. Spill remediation workers were continuously exposed to weathered oil and appeared to suffer a myriad of health effects, including contact dermatitis. However, the mechanism by which crude oil exposure induces skin inflammation has not been examined. To investigate this pathology, a mouse model of seven day exposure to weathered crude oil or acetone (control) was utilized, employing C57BL/6 and Balb/c mice. Immunohistochemistry, multiplex protein analysis, and real‐time PCR were then conducted on skin samples isolated from each strain. Inflammatory cytokine expression showed that oil exposure increased IL‐6, CXCL10, and CCL2 levels, the extent of which differed based on mouse strain. Histopathology showed obvious inflammation in the oil exposed skin, primarily characterized by epidermal thickening and neutrophil infiltration, and this was most pronounced in Balb/c mice. Oil exposure increased M1 macrophage skin infiltration but not M2 as indicated by CD86 versus CD206 expression respectively. These results suggest that weathered oil is indeed a skin irritant, and genetic predisposition may influence the severity of the skin inflammation.

Grant Funding Source: NIH/NIEHS R03 ES020546‐01A1
Analysis of persistent organic compounds and metals in urine samples of young adults (844.5)
Authors: Ihemadu Chioma , Naidu Naga , Thomas Renard , Yakubu Momoh
Published: June 2021

Persistent organic compounds are known to cause chronic toxic effects in humans such as reproductive toxicity, neurotoxicity, and carcinogenesis. Epidemiological studies have reported correlation between these compounds and stress indicators and diseases. These toxicants include pesticides: aldrin, dieldrin, endrin, endosulfan, DDT and BPA; some of which are banned in the USA and some are of major concern to public health especially to young adults. We have investigated the presence of these toxicants along with heavy metals in the urine of young adults and correlated their levels to bilirubin. The protocol for this study was approved by the TSU Committee for the use of human subjects, consent of 21 participants (18‐35 years) was obtained and urine samples collected. HPLC was used to determine the levels of the toxicants, heavy metal levels were analyzed by ICP‐MS and bilirubin was by caffeine assay. HPLC analysis for pesticide shows a range of concentration for: aldrin 0.85 to 296 ppm, endoulfan 1 0.37 to 120 ppm, endrin 0 to 67.1 ppm, dieldrin 0 to 630 ppm and DDT 0 to 4.9 ppm; whle BPA 0 to 102.4 ppb. Metal analysis shows concentration for: Cr 0 to 1.415 ug/L, Ni 257 to 422 ng/L, AS 0 to 624 ng/L, Cd 0.02 to 1.995ug/L, Al 107 to 243.5 ng/L, and Pb 257 to 384 ng/L. Bilirubin level ranged from 157 to 1,387 ng/L. Correlation analysis was done for the levels of these organic compound with the levels of bilirubin, although some of the young adults high concentrations of these persistent organic compounds, we found only positive correlation between dieldrin and bilirubin. However, these concentrations are within the acceptable limit set by CDC and EPA.

Grant Funding Source: NHLBI: HL03674 and HL070669.
Thymoquinone protects against cigarette smoke extract‐induced vascular dysfunction through inhibition of the RhoA/Rho‐kinase signaling pathway (849.1)
Authors: Ismail Raed , El‐Sayed El‐Sayed , Mansour Ahmed , Hemann Craig , El‐Mahdy Mohamed , Zweier Jay
Published: June 2021

There is an ever‐growing focus on the role of natural products in modulating reactive oxygen species (ROS) and RhoA/Rho kinase‐mediated vascular disease. Thymoquinone (TQ), a constituent of the volatile oil derived from Nigella sativa seeds, possesses promising antioxidant and vasodilating properties via its effect on multiple signaling pathways; however, the effect of TQ on the RhoA/Rho‐Kinase pathway remains to be investigated. Cigarette smoke extract (CSE)‐exposed rat aortic rings were mounted in a wire myograph and subjected to contraction and relaxation. TQ protected against CSE‐induced impairment of acetylcholine‐induced endothelium‐dependent relaxation, and decreased CSE‐induced ROS generation and glutathione depletion. Preincubation of aortic rings for 20 min with TQ attenuated the CSE‐enhanced phenylephrine‐induced vascular tension in endothelium‐denuded rings. TQ‐pretreated rings showed a decrease in CSE‐induced RhoA activation and phosphorylation of myosin light chain‐20, myosin phosphatase‐targeting subunit 1 and protein kinase CPI‐17. These data indicate that TQ inhibited ROS generation‐induced RhoA/Rho kinase pathway activation, protecting against CSE‐induced vascular dysfunction. This study provides mechanistic insight for understanding the molecular basis and efficacy of TQ on vascular disease management.

Grant Funding Source: Supported by NIH #00000784 to JLZ; Egyptian government
Role of PPAR‐γ in A2AAR‐mediated vascular relaxation in high salt‐fed mice (849.4)
Authors: Pradhan Isha , Zeldin Darryl , Ledent Catherine , Falck John , Mustafa Jamal , Nayeem Mohammed
Published: June 2021

High salt (HS)‐induced enhanced vascular response to adenosine is known to be mediated through adenosine A2A receptor (A2AAR) and cyp‐epoxygenase‐derived EETs (epoxyeicosatrienoic acid). The aim of this study is to investigate the mechanism involved in A2AAR‐mediated vascular reactivity in HS‐fed mice using A2AAR‐/‐ and A2AAR+/+ mice. We hypothesized that HS‐induced enhanced A2AAR‐EETs‐mediated vascular relaxation is dependent on PPAR‐ɣ (Peroxisome proliferator‐activated receptor gamma) in A2AAR+/+ mice and vascular contraction is dependent on less EETs in A2AAR‐/‐ mice. Organ bath and western blot studies were conducted with 4% (HS) and 0.18% NaCl (NS) diet fed A2AAR‐/‐ and A2AAR+/+ mice. A2AAR agonist, CGS‐21680, enhanced relaxation in HS‐A2AAR+/+ (+27.59 ± 4.7 vs. NS‐A2AAR+/+ +9.6 ± 7.6%), which was blocked by EET antagonist 14,15‐EEZE (1µM; ‐1.08 ± 2.95%) and PPAR‐ɣ antagonist T0070907 (0.1µM; 10.6 ± 1.84%, p<0.05). HS‐A2AAR+/+ had increased expression of cyp2j5 and PPAR‐ɣ compared to NS‐A2AAR+/+. Interestingly, PPAR‐ɣ was downregulated in HS‐A2AAR‐/‐ compared to HS‐A2AAR+/+ mice. Adenosine analog NECA (1µM) produced relaxation (+12.09 ±4.4 %, p<0.05) in HS‐A2AAR+/+ vs. contraction in HS‐A2AAR‐/‐ (‐57.29 ± 2.84%). NECA‐induced contraction in HS‐A2AAR‐/‐ was not affected by T0070907, but attenuated by the inhibitor of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs (AUDA; 10 µM; NS: ‐15.23 ± 3.41 and HS: ‐6.62 ± 1.24%, p<0.05). Our data suggest that in A2AAR+/+ mice, HS enhances relaxation through A2AAR‐EETs via PPAR‐ɣ whereas, in A2AAR‐/‐ mice, HS promotes contraction through less availability of EETs and PPAR‐ɣ.

Grant Funding Source: Supported by HL114559 to MAN, HL027339, HL094447 to SJM, GM31278 to JRF and z01‐ES025034 to DCZ
The effects of TNFα and ceramide in insulin signaling in C2C12 myocytes (854.5)
Authors: Leung Lana , Frischknecht Jonathan , Vallejo Johana
Published: June 2021

Both tumor necrosis factor alpha (TNFα) and non‐esterified fatty acids (e.g. ceramide) have been proposed to be crucial factors in the development of the insulin‐resistant state. Skeletal muscle is a key metabolic tissue and defects in insulin signaling in this tissue are central to the pathogenesis of diabetes. Here we determined the effect of TNFα and ceramide on the total protein expression and protein phosphorylation of key signaling molecules within the insulin signaling cascade in C2C12 myocytes after treatment with TNFα or with ceramide for 24 hours, 48 hours, and 72 hours, (+/‐ 1 hour Insulin) respectively. Treatment with TNFα resulted in a significant enhancement in total protein expression of Insulin Receptor β subunit (IRβ) both after 48 hours (p=0.0099) and 72 hours (p=0.0055). However, phosphorylation of IRβ (p‐IRβ) was significantly enhanced (p=0.0002) after treatment with TNFα for 48 hours (+ Insulin), but not observed after 72 hours of treatment. Interestingly, total expression of Protein Kinase B (Akt) and phosphorylated Akt (p‐Akt) was not significantly altered in any of the TNFα treatment groups. Treatment with ceramide resulted in no significant changes in total protein expression of IRβ. However, total p‐IRβ was significantly enhanced (p=0.0251) after treatment with ceramide for 48 hours (+ Insulin). This significant enhancement in p‐IRβ was not observed after 72 hours of treatment. Total protein expression of Akt was not significantly altered by ceramide treatment. Interestingly, p‐Akt was significantly increased (p=0.0251) after treatment with ceramide for 48 hours (+ Insulin). This significant enhancement in p‐Akt was not observed after 72 hours of treatment. These findings provide evidence of a differential potential for TNFα and ceramide to independently interfere with insulin signaling and to even alter protein expression.
Reactive oxygen species and acute kidney injury after trauma in obese rats (859.2)
Authors: Mittwede Peter , Xiang Lusha , Lu Silu , Clemmer John , Hester Robert
Published: June 2021

Acute kidney injury (AKI) occurs more often in obese than in lean patients after blunt trauma, with similar results observed in obese rats. While studies have suggested that reactive oxygen species (ROS) are involved in the pathogenesis of organ failure after trauma, their role in the context of obesity is not known. We hypothesized that antioxidant treatment would prevent the development of AKI in obese rats. Trauma was induced in obese (OZ) and lean (LZ) Zucker rats (12‐13 wks old) by soft tissue injury, fibula fracture, and bone component injection in both hindlimbs. In OZ and LZ without trauma, glomerular filtration rate (GFR)(1.7±0.1 vs 1.5±0.1 mL/min/g), renal plasma flow (RPF)(5.8±0.1 vs 5.5±0.2 mL/min/g), and urine albumin excretion (0.63±0.3 vs 0.27±0.1 mg/24hrs) were not different (p=ns; n=6). One day after trauma, OZ had increased urine albumin excretion as compared to LZ (6.6±1.2 vs .22±0.1 mg/24hrs; p<.05; n=6), and decreased GFR (0.9±0.1 vs 1.4±0.2 mL/min/g; p<.05; n=6) and RPF (2.5±0.2 vs 5.2±0.3 mL/min/g; p<.05; n=6). Treatment directly after trauma with a NADPH oxidase inhibitor, Apocynin (50mg/kg i.p.), led to an average GFR of 1.6±0.3 mL/min/g, RPF of 5.1±1.1 mL/min/g, and urine albumin excretion of 2.5±1.0 mg/24hrs in OZ (n=3). These data suggest that ROS may be responsible for the increased incidence of AKI after trauma in obesity, and thus may be a potential therapeutic target in this demographic.

Grant Funding Source: Supported by NIH HL‐51971, HL‐89581, AHA‐12SDG12050525
Potassium exaggerates vasopressin response in Sus scrofa models of hemorrhagic shock but not endotoxic shock (859.4)
Authors: Uyehara Catherine , Rowland Michael , Sarkar Joy , Murata Lee‐Ann , Sato Aileen , Ichimura Wayne , Hernandez Claudia , Kajiura Lauren
Published: June 2021

We recently reported a novel role of vasopressin (VP) in regulating renal potassium (K+) excretion in severe hemorrhage where hyperkalemia develops within minutes of blood loss. The VP response to hemorrhagic shock is higher than the VP response in endotoxic shock. We hypothesized that the higher VP responsiveness in hemorrhage is due to K+ stimulation of VP release. Thus, in anesthetized Yorkshire cross piglets (8.2 + 0.2 kg) we compared the VP responses to comparable hypotension induced by rapid hemorrhage (30 ml/kg over 20 minutes, n=5) or i.v. injection of E. coli endotoxin (n=5). Despite similar mean arterial pressures (45 + 5 and 47+ 4 mm Hg, respectively), plasma VP (pVP) in endotoxic shock was 148 + 23 pg/ml vs 373 + 81 pg/ml in hemorrhage. Multiple regression analysis examining the influence of hemodynamic, endocrine, and electrolyte factors on pVP showed that in endotoxic shock, pVP was mainly related to central venous pressure (r=0.92, p<0.01) whereas in hemorrhagic shock, pVP was overwhelmingly influenced by plasma K+ (r=0.97, p<0.01). Elevated plasma K+ seen during hemorrhage (7.0 + 0.8 mEq/L) was not seen with endotoxic shock. This appears to be responsible for the exaggerated VP release observed in hemorrhagic shock. Results support an important role of VP in the regulation of K+ homeostasis in severe hemorrhage.

Grant Funding Source: Supported by US Army Medical Research and Material Command DMRDP D10_I_AR_J6_925
Pelagic sea snakes dehydrate at sea (860.19)
Authors: Lillywhite Harvey , Sheehy Coleman , Brischoux François , Grech Alana
Published: June 2021

Secondarily marine vertebrates are thought to live independently of fresh water. However, some species of sea snakes require sources of fresh water even though they possess extrarenal salt glands. Here we tested the hypothesis, and demonstrate, that the only pelagic sea snake, Pelamis platurus, dehydrates at sea and spends a significant part of its life in a dehydrated state corresponding to seasonal drought. Snakes that are captured following prolonged periods without rainfall have lower body water content, lower body condition, and increased tendencies to drink fresh water than do snakes captured following seasonal periods of high rainfall. These snakes do not drink seawater. Snakes at sea presumably rehydrate by drinking from a freshwater lens that forms on the ocean surface during large rainfall events. These new data for spontaneous and voluntary drinking of fresh water, as well as the status of hydration and body condition, indicate unequivocally that this exclusively marine vertebrate dehydrates at sea and potentially remains in negative water balance for 6‐7 months at a time.

Grant Funding Source: Supported by the U.S. National Science Foundation grant IOS‐0926802 to HBL
Endothelin‐1 promotes fetal cardiomyocyte terminal differentiation via heightened DNA methylation (862.2)
Authors: Paradis Alexandra , Xiao Daliao , Zhou Jianjun , Zhang Lubo
Published: June 2021

Hypoxia is a major stress on fetal development and leads to induction of endothelin‐1 (ET‐1) expression. We tested the hypothesis that ET‐1 stimulates the terminal differentiation of cardiomyocytes from mononucleate to binucleate in the developing heart. In fetal hearts, prepro‐ET‐1 mRNA expression was significantly increased by hypoxia (10.5% O2) treatment of pregnant rats from day 15 to day 21. Under both control and hypoxic conditions, the ET‐1 ex vivo treatment of fetal rat cardiomyocytes increased percent binucleate cells and decreased Ki‐67 expression, a proliferation marker. Hypoxia alone was able to decrease Ki‐67 expression and cardiomyocyte size. A non‐selective ET‐receptor antagonist, PD145065, blocked the ET‐1 induced changes in binucleation and proliferation. ET‐1 treatment significantly increased DNA methylation in fetal cardiomyocytes, which was further blocked by 5‐aza‐2’‐deoxycytidine, a DNA methylation inhibitor. In addition, 5‐aza‐2’‐deoxycytidine treatment abrogated the increase in binucleation and decrease in proliferation induced by ET‐1. Hypoxic stress and synthesis of ET‐1 increases DNA methylation and promotes terminal differentiation of cardiomyocytes in the developing heart. This premature exit of the cell cycle may lead to a reduced cardiomyocyte endowment in the heart and have a negative impact on cardiac function.
Quantitation of oxidative stress and base excision repair in skeletal muscle of high‐ and low‐functioning elderly individuals (863.3)
Authors: Joseph Anna‐Maria , Tornaletti Silvia , Adhihetty Peter , Buford Thomas , Wohlgemuth Stephanie , Sandesara Bhanuprasad , Marzetti Emanuele , Leeuwenburgh Christiaan
Published: June 2021

Skeletal muscle aging is partly attributed to the oxidation of macromolecules caused by increased reactive oxygen species. DNA base excision repair (BER) pathways offer protection against oxidative damage through the removal and repair of DNA lesions. While the activity of these BER enzymes is proposed to be reduced with aging, the molecular details remain elusive. The purpose of this study was to investigate oxidative stress/damage, BER enzyme activities, and antioxidant capacity in skeletal muscle obtained from two sedentary but functionally‐distinct groups of elderly (> 65 yr) individuals classified as high‐ and low‐functioning (HF and LF). Results showed increased oxidative damage in both HF and LF individuals compared to young, as indicated by higher levels of 3‐Nitrotyrosine (3‐NT) and 4‐Hydroxynonenal (4‐HNE) (P<0.05). This increase corresponded with lower levels of 8‐oxoguanine DNA glycosylase (OGG1; P<0.05) protein, the main mitochondrial oxidative DNA repair enzyme, in elderly subjects compared to young. No changes in protein content were observed for apurinic/apyrimidinic (AP) endonuclease, APE1, corresponding to similar rates of AP sites incision when protein extracts from young and elderly individuals were utilized to measure APE1 enzymatic activity. These data suggest that BER capacity may be impaired in aging muscle leading to greater susceptibility to oxidative damage.

Grant Funding Source: Supported by Claude D. Pepper OAIC
Impaired phenylephrine‐induced contraction is due to increased matrix metalloproteinases 2 and 9 gelatinolytic activities in aortas isolated from rats submitted to sepsis (865.15)
Authors: Pernomian Laena , Prado Alejandro , Gerlach Raquel , Bendhack Lusiane
Published: June 2021

Sepsis leads to systemic hypotension and reduced vasoconstriction to several agonists and matrix‐metalloproteinases (MMPs) are involved in sepsis development. This study aimed to evaluate the contractile response induced by phenylephrine (PE) and MMP‐2 and MMP‐9 gelatinolytic activities in aortas isolated from rats submitted to sepsis by ligation and puncture model (CLP). Male rats (University of São Paulo Ethics Committee #144/2011) were submitted to femoral artery catheterization 24h before the surgery to induce sepsis or sham‐surgery (CO rats). The mean arterial pressure (MAP) was measured 4h after the surgeries. Aortic rings were isolated from CO and CLP rats for vascular reactivity and zymography. Concentration‐effect curves to PE (0.1nM‐10µM) were constructed in intact endothelium (E+) or denuded aortas (E‐). The MAP was lower in CLP than in CO rats. PE‐induced contraction was lower in CLP E+ than in CLP E‐ and CO E+ groups. Gelatinolytic activity of the MMP‐2 active form in CLP was greater than in CO aortas. Gelatinolytic activities of lipocalin‐associated‐MMP‐9 (135kDa) and neutrophil‐dimeric MMP‐9 (230kDa) were enhanced in the CLP compared to CO aortas. Our results indicate that PE‐induced vasoconstriction is decreased probably due to increased gelatinolytic activities of MMP‐2 active form MMP‐9 in aortas isolated from rats submitted to sepsis.

Grant Funding Source: Supported by FAPESP and CNPq
The persistence of active smooth muscle in the female rat cervix through pregnancy (865.2)
Authors: Ferland David , Watts Stephanie
Published: June 2021

A controversy exists as to whether functional smooth muscle exists in the cervix before and during pregnancy, potentially continuous with the uterus. We hypothesized that smooth muscle persists through pregnancy, remaining active. Uteri and cervices were taken from female virgin, 11 day‐ and 20 day (near labor)‐pregnant rats. Three different smooth muscle proteins (smooth muscle α actin, SM‐22α, and Calponin‐1) allowed immunohistochemical detection of the continuous nature of the smooth muscle from vagina, cervix and uterus. Uterus and cervix from virgin and 20‐day pregnant rats were hung in isolated tissue baths for measurement of isometric contraction. The virgin and day 20 cervices contracted 2.52g±0.27g and 3.88g±0.49g (respectively) to 100 mM KCl (general smooth muscle depolarization). Cervices also contracted to carbamylcholine in a concentration‐dependent fashion (maximum=133%±18.2% KCl contraction, n=4). Uterine and cervical homogenates were also used in Western analyses. Smooth muscle α actin was detected in virgin tissue (291.3±32.2 arbitrary densitometry units/β actin; n=5), in day 11 [416.8±19.5 (n=5)] and day 20 pregnant tissue [293.0±34.4 (n=5)]. These findings strongly support that smooth muscle is: 1) present in the cervix through pregnancy, 2) continuous with the uterus and 3) supports the overarching hypothesis that the smooth muscle of the cervix plays a role in pregnancy and labor.

Grant Funding Source: Supported by a CTSI grant to Stephanie W. Watts
Failure of autophagy maturation in CD38 deficient coronary arterial myocytes due to inhibition of dynein‐mediated autophagosome trafficking (865.3)
Authors: Zhang Yang , Xu Ming , Xia Min , Li Xiang , Boini Krishna , Li Pin‐Lan
Published: June 2021

Autophagic flux refers to the process that lysosome traffics and fuses with autophagosomes (APs) leading to their maturation to form autophagolysosomes (APLs) and consequent breakdown of autophagic contents by lysosomal proteases. However, the mechanisms controlling autophagic flux, particularly autophagy maturation in vascular cells are poorly understood. Here, we revealed a role of CD38, an enzyme that metabolizes NADP+ into NAADP, in autophagy maturation in coronary arterial myocytes (CAMs). In vivo, CD38‐/‐ fed the Western diet showed increased accumulation of APs in coronary arterial media compared to CD38+/+ mice, suggesting that CD38 gene deletion results in a deranged autophagic process. In primary cultured CAMs, a novel flow cytometric CytoID analysis showed that 7‐ketocholesterol (7‐Ket, an atherogenic stimulus and autophagy inducer)‐induced APs formation was similar between CD38+/+ and CD38‐/‐ CAMs when their autophagic flux was blocked by choloquine, which indicates that CD38 is not involved in APs formation. Tandem RFP‐GFP‐LC3B assay demonstrated that CD38 gene deletion markedly enhanced 7‐Ket‐induced accumulation of APs but decreased the formation of APLs in CAMs, indicating a role of CD38 in autophagy flux. Furthermore, loss of CD38 function was implicated in impaired autophagy maturation rather than in lysosomal alkalization or protease inhibition‐associated blockade of autophagic flux because CD38‐/‐ and CD38+/+ CAMs had similar lysosomal pH and lysosomal protease activities. Importantly, CD38 gene deletion markedly inhibited 7‐Ket‐induced dynein activation and APs trafficking, which were associated with attenuated lysosomal Ca2+ release. Taken together, these results suggest that CD38 plays a critical role in the control of dynein‐mediated APs trafficking and fusion with lysosomes thus determining autophagy maturation in CAMs under atherogenic stimulation.

Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316
Expression of Ski induces apoptosis and represses autophagy in cardiac myofibroblasts (868.8)
Authors: Zeglinski Matthew , Davies Jared , Ghavami Saeid , Bathe Krista , Rattan Sunil , Dixon Ian
Published: June 2021

Following myocardial infarction (MI), relatively quiestent cardiac fibroblasts undergo phenoconversion to active, hyper‐synthetic myofibroblasts. During the late stages of wound healing, myofibroblasts are removed from the infracted region through apoptosis thus decreasing scar cellularity. Apoptosis and autophagy regulate cell fate. The role of apoptosis within the remodeling heart has been well described however the role that autophagy plays following an MI is relatively unknown. Ski is a negative regulator of TGF‐β signaling and has been implicated to play a role in the post‐MI heart. Herein we investigated the regulatory role that Ski has on apoptosis and autophagy using first passage primary rat cardiac myofibroblasts. Using an adenoviral approach for gene delivery, both MTT and Live/Dead viability/cytoxicity assays demonstrated a reduction in the number of viable cells and an increase in the number of dying cells due to the over‐expression of Ski. Additionally, over‐expression of Ski led to distinct morphological changes characteristic of apoptosis. These findings were confirmed via Western blot analysis, and caspase GLO analysis showing induction of executioner caspases‐3 and ‐7, as well as the mitochondrial regulator Bax. Furthermore, evidence of caspase‐9 cleavage, but not caspase‐8, indicated that this was primarily an intrinsic apoptotic response. Markers for autophagosome formation including LC3‐B and ATG‐7 were found to be significantly reduced following Ski over‐expression. In summary, we found that over‐expression of Ski in primary cardiac myofibroblasts leads to an induction of the intrinsic apoptotic pathway and reduction in autophagy leading to cell death.

Grant Funding Source: Canadian Institutes of Health Research
Regulation of ENaC and CFTR biogenesis by ERp29 (869.9)
Authors: Bikard Yann , Grumbach Yael , Suaud Laurence , Chanoux Rebecca , Rubenstein Ronald
Published: June 2021

The Epithelial Na+ Channel (ENaC) plays an important role in blood pressure homeostasis and, in conjunction with the Cystic Fibrosis Transmembrane Conductance regulator (CFTR), regulates airway surface liquid volume. ERp29 is a 29 kD thioredoxin‐homologous protein which primarily resides in the lumen of the endoplasmic reticulum (ER). But unlike the thioredoxins, ERp29 has only a single Cysteine instead of the usual C‐X‐X‐C motif. Our group has previously demonstrated that ERp29 promotes biogenesis of CFTR. As the biogenesis of CFTR and ENaC have a number of similar features, the present studies tested the hypothesis that ERp29 would also regulate ENaC biogenesis and functional expression, as well as further probed the mechanism by which ERp29 promotes CFTR biogenesis. In MDCK epithelial cells expressing epitope tagged αβγ‐ENaC, overexpression of wt ERp29 increased the abundance of the cleaved form of γ‐ENaC, as well as ENaC functional expression (as defined by amiloride‐sensitive Isc in Ussing chamber experiments.) In contrast, siRNA‐mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single Cysteine (C157S ERp29) decreased ENaC functional expression. Interestingly, these observations were not associated with altered expression of β‐ENaC at the apical surface, suggesting that ERp29 may modulate the cleavage state, and therefore open probability (Po) of ENaC at the apical surface. Finally, ERp29 overexpression promotes the interaction of both ENaC and CFTR with the Sec24D cargo recognition component of coat complex II ER exit machinery. Together these findings suggest a key role for ERp29 in the biogenesis of ENaC and CFTR; for ENaC this may include directing channel for cleavage by furin in the Golgi and delivery to the apical membrane in a higher Po form.

Grant Funding Source: Supported by R01 DK58046
The role of Pin1 isomerase on neuronal cell death after focal cerebral ischemic in rats (877.14)
Authors: Lee Seunghoon , Min Sang‐Hyun , Hong Yonggeun
Published: June 2021

Pin1 contains two functional domains, an N‐terminal WW domain and a C‐terminal peptidyl‐prolyl cis/trans isomerase (PPIase) domain. The WW domain is a phosphorylation specific protein interaction module that directs Pin1 to its substrates‐proteins phosphorylated at a certain serine or threonine residue followed by proline (pSer/Thr‐Pro motif). Upon this binding, the PPIase domain catalyzes conformational change of the Pin1 substrates by isomerizing specific pSer/Thr‐Pro bonds. Thus, we hypothesize that Pin1 can regulate neuronal cell death and neurogenesis through Pin1 inhibitor treatment after ischemic brain injury. To test the hypothesis, we elucidated at sub‐acute phases after focal cerebral ischemia to determine the efficacy of Pin1 inhibitor, juglone. Juglone (1mg/kg) was administered once a day during 7 days after focal cerebral ischemia. Histology, protein, and gene analyses were used to determine apoptosis and neurodegeneration at day 7. MCAo+Jug group was significantly led to downregulation of Bcl‐2 and Bax expression on protein level compared to MCAo+Veh group (p<.01). Likewise, juglone treatment was reduced to DAPK1 and Tau5 level on both western blot and immunostaining (p<.01). These results suggest that Pin1 isomerase acts to pro‐apoptotic molecule on ischemic‐induced neuronal cell death after focal cerebral ischemia, as well as Pin1 inhibition might be alleviate to acceleration of apoptotic cell death from acute to subacute after focal cerebral ischemic in rats.

Kew words: Focal cerebral ischemia, Pin1, Neuronal cell death.
Oxygen consumption in a hot hypoxic world, the Devils Hole pupfish (879.24)
Authors: Hillyard Stanley , Burg George , McKenna Ken , Urbina Natalia , van Breukelen Frank
Published: June 2021

The Devils Hole pupfish, Cyprinodon diabolis, exists in an isolated cavern administered by Death Valley National Park. Only 65 adults were counted in a recent census, down from over 500 fish in 1996. Limiting factors for population recovery are high temperatures that average 33°‐34° C in the main pool and low levels of dissolved oxygen during much of the year. A refuge population of C. diabolis propagates well in our laboratory at 28° C but less so at 33° C. The critical PO2 of 33° C acclimated fish (8.39 ± 0.16 kPa) is significantly higher than that of the 28° C acclimated fish (6.49 ± 0.14 kPa). Further, the 33° C acclimated fish produce 7.3x more ethanol suggesting a large anaerobic component of total metabolic rate. We further investigated the effect of limiting oxygen levels on developing eggs. Oxygen consumption (VO2) in air saturated water prior to hatching increased as the heartbeat appeared but remained stable thereafter, even when the larva was obviously active in the egg capsule. VO2 increased markedly when the larva emerged suggesting the egg capsule is a barrier for oxygen diffusion. Surprisingly, critical PO2 of eggs was even higher than that of adults indicating an even greater reliance on anaerobic metabolism. We suggest that the reliance on anaerobic metabolism in the presence of available oxygen is a larval adaptation that persists in the adults as paradoxical anaerobism.
Voluntary wheel running improves cardiac function in a rodent model of chronic kidney disease (884.15)
Authors: Kuczmarski James , Martens Christopher , Kim Jahyun , Lennon‐Edwards Shannon , Edwards David
Published: June 2021

The purpose of this study was to determine the effect of voluntary wheel running on cardiac function in a rodent model of chronic kidney disease (CKD). CKD (5/6 ablation‐infarction) or SHAM surgery was conducted on male Sprague Dawley rats. After 4wks of disease development, subsets of CKD animals remained sedentary (CKD‐SED) or underwent a 4wk voluntary wheel running intervention (CKD‐WR). Cardiac output (CO) was measured in all animals at 8wks post‐surgery using an isolated working heart preparation. CKD‐WR rats ran an average of 13.4±1.5km total which resulted in increased soleus muscle citrate synthase activity (p<0.05). Baseline CO was not different from SHAM in CKD‐WR (p>0.05), in contrast to CKD‐SED (p<0.05). CO remained not different from SHAM in CKD‐WR during preload & afterload manipulation (all p>0.05), whereas CKD‐SED CO was impaired (all p<0.05). Left ventricular (LV) tissue analysis revealed that LV hydrogen peroxide (H2O2) was increased and associated with decreased total nitrite + nitrate (NOx) and reduced nitric oxide synthase (NOS)‐3 in CKD‐SED (p<0.05). LV H2O2, NOx, & NOS‐3 were not different from SHAM in CKD‐WR (p>0.05). In conclusion, cardiac function is improved in a rodent model of CKD in response to a modest 4‐week voluntary wheel running intervention. This may be partly mediated by reduced H2O2 and increased NO production and/or bioavailability.

Grant Funding Source: Supported by P20‐RR016472‐12 and P20‐GM103446‐12.
Temporal changes in left ventricular mechanics: impact of bed rest and exercise (884.23)
Authors: Scott Jessica , Downs Meghan , Ploutz‐Snyder Lori
Published: June 2021

BACKGROUND: The use of more sensitive and specific echocardiographic techniques, such as speckle‐tracking imaging, may address the current limitations of conventional cardiac imaging techniques to provide insight into the magnitude and time course of cardiac deconditioning following spaceflight or head‐down tilt bed rest (HDTBR). METHODS: Speckle‐tracking assessment of longitudinal, radial, and circumferential strain and twist was used to evaluate the impact of 70 days of HDTBR (n=7) and HDTBR + exercise (n=11) on temporal changes in left ventricular (LV) mechanics. Echocardiograms were performed before (BR‐2), during (BR31, 70), and following (BR+4h) HDTBR. Repeated measures ANOVA was used to evaluate the effect of HDTBR on cardiac variables in control and exercise subjects. RESULTS: After sedentary HDTBR, longitudinal (‐19.0 ± 1.8% vs. ‐14.9 ± 2.4%) and radial (15.0 ± 1.9% vs. 11.3 ± 2.2%) strain and twist (18.0 ± 4.0° vs. 17.0 ± 3.6°) were significantly impaired. In contrast, exercise preserved LV mechanics, and there were non‐significant improvements from BR‐2 to BR70 in longitudinal strain (‐18.7 ± 1.5% vs. ‐20.4 ± 2.7%), radial strain (13.2 ± 2.4% vs. 14.2 ± 1.6%), and twist (16.3 ± 3.6° vs. 18.6 ± 5.9°). CONCLUSIONS: Speckle‐tracking echocardiography provides important new insight into temporal changes in LV mechanics during disuse and exercise training.

Grant Funding Source: NASA Johnson Space Center’s Human Research Program
The effects of hypoxia in determining larval size in Drosophila melanogaster (888.1)
Authors: Wong Daniel , Martinez‐Agosto Julian
Published: June 2021

In Drosophila, hypoxia systemically restricts larval growth, but the molecular mechanism through which it does so remains poorly characterized. We show that Sima, the alpha subunit of the Drosophila hypoxia‐inducible factor, accumulates in nuclei of larval fat body cells during hypoxia and inhibits systemic growth when overexpressed in this same tissue. Oxygen deprivation and overexpression of Sima in the fat body also cause the retention of Drosophila insulin‐like peptides (Dilps) in the larval brain, suggesting that an endocrine signal emanates from the fat body and is relayed to the brain to regulate Dilp secretion during an oxygen‐deprived state. Additionally, hypoxia and overexpression of Sima in the fat body cause lipid droplets to aggregate in this metabolic tissue but not in the larval oenocytes, a liver‐like cell type, unlike what is observed in starvation. Furthermore, hypoxia reduces total cholesterol, triglyceride, and free fatty acid levels in wild‐type larvae. Finally, ubiquitous overexpression of the wild‐type insulin receptor rescues hypoxic growth restriction, suggesting that hypoxia‐induced insulin resistance in peripheral tissues serves to inhibit systemic growth. Interestingly, one tissue that mediates hypoxic growth restriction is the larval trachea, as both overexpression of the wild‐type insulin receptor and activation of TOR (Target of rapamycin) signaling in this tissue rescue inhibition of larval growth under hypoxic conditions. Rearing wild‐type larvae in hypoxia also decreases the number of lipid droplets that stud the larval trachea, which is suggestive of a novel finding that lipid droplet mobilization to the larval trachea functions to promote growth. Collectively, our results suggest that coordination of larval size is dependent on the communication between many organs through the use of insulin signaling and alterations to lipid metabolism. These findings may be applicable to other instances in which an adaptive response to hypoxia is required.
Effect of hypoxia on nuclear high affinity Ca2+ ‐ATPase activity and nuclear Ca2+ ‐influx in the cerebral cortex of newborn piglets (888.2)
Authors: Delivoria‐Papadopoulos Maria , Wang Andy , Malaeb Shadi , Ashraf Qazi
Published: June 2021

Nuclear calcium signals control a number of critical nuclear functions including regulation of transcription factors, cell cycle regulation, gene transcription and DNA replication. Previous studies have shown that during hypoxia, there is increase in intracellular Ca2+‐and in nuclear high affinity Ca2+‐ATPase activity in the cerebral cortex of newborn piglets.We test the hypothesis that hypoxia results in increased activity of high affinity Ca2+‐ATPase and increased concentration of Ca2+‐influx in cortical neuronal nuclei of newborn piglets, and this hypoxia‐induced increase is Src kinase mediated.Piglets were divided into normoxic (Nx, n=4), hypoxic (Hx, n=4), and hypoxic pretreated with Src kinase inhibitor (Hx+Srci, n=4). Hypoxia was induced by decreasing FiO2 to 0.07 for 1 hr. Src kinase inhibitor (PP2, 1.0 mg/kg i.v.) was administered 30 min prior to hypoxia. Hypoxia was documented by levels of ATP and phosphocreatine (PCr). Nuclear 45Ca2+ concentration and activity of high affinity Ca2+‐ATPase was determined at 37°C for 30 min.Nuclear 45Ca2+ ‐influx (pmoles/mg protein) was 3.95±0.57 in Nx , 13.36±1.56 in Hx (p<0.05) and 9.61±2.76 in the Hx+Srci group. Ca2+‐ATPase activity increased following hypoxia: from 244.66±73.55 nmol Pi/mg protein/h in the normoxic group to 426.33±94.99 nmol Pi/mg protein/h in the hypoxic group (p<0.05 vs. normoxic group). In the Hx+Srci group, the high affinity Ca2+‐ATPase activity increased to 354.25±62.88 nmol Pi/mg protein/h (p<0.05 vs. hypoxic group). The data show that administration of PP2, a selective inhibitor of Src kinase, prevents the hypoxia‐induced increase in nuclear 45Ca2+ ‐influx concentration and Ca2+‐ATPase activity.We conclude that the hypoxia‐induced increase in nuclear membrane high affinity Ca2+‐ATPase activity is Src kinase‐mediated activation of the high affinity Ca2+‐ATPase, a mechanism of ATP‐dependent Ca2+uptake may lead to increase in intranuclear Ca2+during hypoxia.

Grant Funding Source: NIH HD200337
Structural organization of the renal medulla has a significant impact on oxygen distribution (890.11)
Authors: Fry Brendan , Layton Anita
Published: June 2021

A theoretical model is presented to analyze the impact on oxygen distribution of the heterogeneous organization of the rat outer and inner medulla revealed in anatomical studies.

In the inner stripe, tubules and vessels are organized around vascular bundles; in the upper inner medulla, clusters of collecting ducts form the organizing motif. The present study extends a detailed mathematical model of the urine concentrating mechanism of the rat renal medulla developed (Layton, AJP Renal 300: F356‐F371, 2011), which was formulated to represent NaCl and urea, to include transport of red blood cells (RBCs), deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), and free oxygen (O2). Both basal cellular oxygen consumption throughout the whole medulla and active oxygen consumption via active NaCl transport in proximal tubules, thick ascending limbs, and collecting ducts are considered. Model equations are based on conservation of water and solutes, as well as transmural transport, and are solved to steady state. Results from the model suggest that the structural organization of the renal medulla produces significant axial and radial PO2 gradients, and impacts the effectiveness of the medullary urine concentrating mechanism. This research was supported in part by NIH grant DK‐89066.

Grant Funding Source: NIH DK089066
Renal potassium handling in the pregnant rat (892.13)
Authors: West Crystal , Baylis Chris , Gumz Michelle
Published: June 2021

The fetus requires a large amount of potassium for normal development. To accommodate this need the normal pregnant rat accumulates ~6.3 mEq of potassium over the course of gestation, over 70% of which is retained during late pregnancy. However, the mechanism of potassium retention during pregnancy is largely unknown. The purpose of this study was to examine the mechanism permitting increased potassium reabsorption in the setting of high circulating aldosterone and enhanced distal nephron sodium reabsorption in the pregnant rat. Recent work on the WNK kinases has provided interesting targets for divergent modulation of sodium and potassium handling in the distal nephron. We measured the mRNA expression of WNK1, WNK3, WNK4, and ROMK in the renal cortex and outer medulla of virgin (V, n=5), mid pregnant (MP, n=5), and late pregnant (LP, n=7) rats using quantitative real‐time PCR. We found increased WNK3 expression in the outer medulla of LP rats compared to V (156 ±15 vs 100±10 % Control). The expression level of the other genes tested did not appear to differ with pregnancy state in either the cortex or the outer medulla. ROMK is the major potassium secretory channel and WNK3 is an inhibitor of ROMK activity. Therefore, the potassium retention of late pregnancy could be due in part to increased outer medullary WNK 3 expression as well as a failure of the expected aldosterone mediated transcriptional increase in ROMK.
CaMKII‐dependent filamin phosphorylation attenuates the MARCKS regulation of ENaC (892.24)
Authors: Alli Abdel , Bao Hui‐Fang , Montgomery Darrice , Ghant Marcus , Al‐Khalili Otor , Eaton Douglas
Published: June 2021

Filamin A is an actin crosslinking protein regulated by phosphorylation. Several kinases including protein kinase A (PKA), protein kinase B (Akt), protein kinase C (PKC), and Ca(2+)/calmodulin‐dependent protein kinase II (CaMKII) phosphorylate filamin A. Xenopus laevis distal nephron epithelial (2F3) cells pretreated with the specific CaMKII inhibitor, KN‐93, showed an increase in amiloride‐sensitive transepithelial current calculated from transepithelial voltage and resistance measurements obtained with a volt‐ohmmeter, and an increase in ENaC open probability measured by single‐channel patch clamp studies. Whole cell lysates from 2F3 cells pretreated with KN‐93 or vehicle control were used to immunoprecipitate filamin, phosphofilamin, MARCKS, and ENaC before immunoblotting for each protein using specific antibodies. Immunoblot and densitometric analysis showed the level of filamin phosphorylation decreased with KN‐93 treatment compared to vehicle control. The association between ENaC and MARCKS was augmented in cells treated with KN‐93 compared to cells treated with vehicle only. ENaC‐MARCKS interaction is necessary for normal ENaC activity. Sucrose density gradient fractions from KN‐93 or vehicle treated cells were analyzed by immunoblotting. Similar to various actin cytoskeleton proteins, filamin protein expression was observed in non‐lipid raft cytoplasmic fractions. The expression of phosphofilamin between these fractions was different between the KN‐93 treated group compared to the vehicle treated control group. Taken together, these studies indicate filamin is an integral component of the actin cytoskeleton and phosphorylation of filamin by CaMKII disrupts the interaction between MARCKS and ENaC.

Grant Funding Source: DK093255‐01
Selective serotonin reuptake inhibitors potentiate anoctamin 6 activation (893.22)
Authors: Nam Joohyun , Kim Yung Kyu , Lee Min Goo
Published: June 2021

Selective serotonin reuptake inhibitors (SSRIs) are a class of widely used antidepressants. The primary mechanism of action of these drugs involves the inhibition of the serotonin transporter (SERT or SLC6A4). In addition to the inhibition of the serotonin transporter, SSRIs also modulate a variety of ion channels including Ca2+‐activated Cl− channels (CaCCs). Recently, anoctamins (ANOs) have been identified as a family of putative Cl− channels. Several research groups have reported that ANO6 generates outward‐rectifying Cl− currents by increasing intracellular Ca2+ levels, which are activated after a delay of several minutes. However, the reason for the very slow activation of the Cl− current remains unexplained. Using the whole‐cell patch‐clamp technique, we examined the effects of SSRIs, including fluoxetine, paroxetine, and sertraline, on cloned human ANO6 expressed in HEK293 cells. In contrast to their inhibitory effect on ANO1, the SSRIs reduced the latency time for the activation of ANO6 currents in a concentration‐dependent manner and slightly enhanced the magnitude of the peak current resulting from ANO6 Ca2+‐dependent activation. However, when we excised the membrane of the cells by using an inside‐out patch‐clamp technique we found that the ANO6 currents were inhibited by SSRIs. Although the exact mechanism by which the latency time of ANO6 activation is reduced remains unexplained, SSRIs might be involved in this ANO6 activation via an indirect pathway. ANO6 channels are involved in many physiological effects. Therefore, our study might contribute to a better understanding of the therapeutic action of SSRIs and their side effects.

Grant Funding Source: This work was supported by a National Research Foundation of Korea (NRF, NO 2011‐0014404)
Anoctamin‐1 channel localization in excitable domains of the heart (893.23)
Authors: Britton Fiona , O'Driscoll Kate , Hatton William
Published: June 2021

Calcium‐activated chloride (ClCa) currents are widely distributed in cardiac tissues and have been implicated to play a role in cardiac physiology and pathophysiology. Anoctamin‐1 (Ano1) is a membrane protein that functions as a ClCa channel and has been linked to essential physiological roles in a variety of excitable cells. We hypothesized that Ano1 channels in their functional role as ClCa channels are localized in distinct regions of the heart. Our objective was to define the localization and cellular expression of Ano1 channels in heart. Confocal immunofluorescence microscopy of mouse heart indicated that Ano1 channels are clustered at the intercalated discs of cardiac myocytes. We demonstrated strong co‐localization of Ano1 with connexin‐43 (Cx43) gap junction protein in isolated ventricular and atrial myocytes. A direct interaction between Ano1 channels and Cx43 was verified by co‐immunoprecipitation using mouse heart lysates. Our findings reveal that Ano1 channels compartmentalize in the intercalated disc region and suggest that Ano1 channels may play an important role in cell‐cell communication in this excitable membrane domain.

Grant Funding Source: Supported by RO1HL091238
Metabolic status changes the interregulation of AMPK and CFTR (893.26)
Authors: Hallows Kenneth , King J Darwin , Rush Elizabeth , Xiong Sheng , Li Hui
Published: June 2021

A better understanding of CFTR Cl‐ channel regulation could suggest new therapies for cystic fibrosis (CF). We previously found that the α1 catalytic subunit of AMP‐activated kinase (AMPK) binds to the C‐terminal tail of CFTR, and AMPK inhibits PKA‐stimulated CFTR gating via direct phosphorylation of CFTR. Both binding and phosphorylation are required for CFTR regulation by AMPK, which may tune CFTR activity to metabolic and other cellular stresses. Moreover, under basal conditions tonic AMPK activity may limit CFTR activation in the absence of PKA stimulation. Conversely, the loss of normal CFTR apical expression in primary CF human bronchial epithelial cells enhances AMPK expression and activity and disrupts AMPK apical targeting in these cells, suggesting that CFTR reciprocally regulates AMPK function. Here we more closely examined the nature of the AMPK‐CFTR interaction and its potential modulation by cellular AMPK activity and metabolic status. AMPK‐α binding to CFTR was reduced with AMPK activation or chemical ischemia in co‐immunoprecipitation studies performed on HEK‐293 and colonic epithelial T84 cells. Moreover, we found that activated AMPK (AMPK‐α‐pThr172) does not appear to bind to CFTR. Changes in cellular energy status inversely regulated AMPK activity and binding to CFTR in T84 cells. Of note, the CFTR‐binding region in AMPK‐α1 overlaps with the AMPK‐β‐binding region in α1, and AMPK‐α1 subunits bound to CFTR do not appear to be in a heterotrimeric AMPK αβγ complex, suggesting that CFTR may sequester AMPK‐α monomers. In summary, our results suggest that not only does AMPK regulate CFTR function, but that CFTR, via metabolic‐dependent interactions with AMPK, regulates AMPK function and subcellular localization in epithelia. This interregulation may represent a new target for CF therapy, as disruption of AMPK binding to CFTR may prevent untoward CFTR inhibition during treatment with AMPK activators to inhibit excessive inflammation and Na+ reabsorption in the CF airway.

Grant Funding Source: Supported by NIH/NIDDK