bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of CaMKII activity on cardiac and skeletal muscle pathology in a mouse model of Duchenne muscular dystrophy (1102.3)
Authors: Johnson Nicolette , Levy Jennifer , Grumbach Isabella , Anderson Mark , Campbell Kevin
Published: June 2021

Patients with Duchenne Muscular Dystrophy (DMD) lack functional dystrophin, an essential member of the dystrophin‐glycoprotein complex. Without dystrophin, the actin cytoskeleton is not anchored to the extracellular matrix, which leads to membrane damage. Patients with DMD have dystrophic and weakened skeletal muscle and often develop dilated cardiomyopathy. The aim of this project is to study the effects of Ca2+ and Calmodulin‐dependent protein kinase II (CaMKII) activity on the development of the dystrophic phenotype. CaMKII is an intracellular signaling protein involved in many pathways, including excitation‐contraction coupling and Ca2+ homeostasis. Hyperactivity of CaMKII is linked with cardiac hypertrophy, arrhythmias, and heart failure. We hypothesized that increased CaMKII activity would cause an increase in skeletal muscle and cardiac pathology in mdx mice, which lack dystrophin and serve as a model of DMD. A mouse model of inhibited CaMKII activity expresses a transgenic peptide inhibitor of CaMKII. A mouse model of CaMKII hyperactivity lacks the CaMKII regulatory protein, Methionine Sulfoxide Reductase A. We crossed the mouse models of inhibited and hyperactive CaMKII activity onto a dystrophic mdx background. Several pathological parameters were used to quantitatively measure disease progression in the different models.

Grant Funding Source: Supported by a Wellstone Muscular Dystrophy Cooperative Research Center grant (KPC), HHMI, APS (NMJ)
Collective effects of low cell pH and inorganic phosphate on force, velocity, stiffness, and power in rat slow and fast muscle fibers (1102.7)
Authors: Nelson Cassandra , Fitts Robert
Published: June 2021

Muscle fatigue is a loss of power resulting from a decline in both force and velocity. Intense exercise induces high rates of ATP hydrolysis and glycolysis, increasing inorganic phosphate (Pi) and H+. These factors are thought to induce fatigue by interfering with key steps in the cross bridge cycle, including ATP hydrolysis, ADP release, and rate and extent of cross‐bridge transition from the low‐ to high‐force state. Skinned fibers from rat soleus (slow) and gastrocnemius (fast) were isolated, and the combined effects of elevated H+ (pH 6.2) and Pi (30 mM) on force, velocity, stiffness, and power at low (15°C) and high (30°C) temperatures studied. At 30°C, pH 6.2 plus 30 mM Pi depressed force by 39% (158 to 97 kN/m2) in slow fibers and 42% (189 to 111 kN/m2) in fast fibers. Velocity was depressed by 17% (Vmax 4.23 to 3.56 Fl/s) in slow fibers and 44% (6.91 to 3.88 Fl/s) in fast fibers. The a/Po ratio was depressed in slow fibers but increased in fast fibers in pH 6.2, 30 mM Pi conditions, resulting in a greater power loss in slow (67%) versus fast (58%) fibers. Force‐stiffness plots normalized to rigor tension and stiffness show a higher number of low‐force cross bridges in fast versus slow fibers in pH 6.2, 30 mM Pi conditions. Our results show that the effects of elevated H+ and Pi are fiber‐type specific, and the larger decrease in force and velocity in type II fibers may in part be explained by a higher population of low‐force cross bridges.

Grant Funding Source: Supported by a Marquette University Way Klinger Fellowship to RH Fitts
Hyperbaric oxygen therapy alters vascular reactivity independent of ATP (1103.6)
Authors: Hake Benjamin , Eovaldi Benjamin , Sylvester Francis
Published: June 2021

The purpose of this study was to record the acute effects of hyperbaric oxygen (HBO) on vascular reactivity and determine the potential role of ATP in mediating these effects. Porcine pulmonary arteries were dissected and mounted in isolated organ baths to record changes in tension in response to potassium chloride (KCl, 15‐60 mM), phenylephrine (Phe, 10‐7‐10‐4 M), and sodium nitroprusside (SNP, 10‐7‐10‐4 M) following a 2‐hour exposure to HBO (1.75 atm). HBO augmented responses to KCl and Phe compared to control arteries exposed to room air or nitrogen at 1.75 atm as well as to room air at 1 atm. Earlier studies in our lab demonstrated that HBO similarly altered vascular reactivity in mesenteric arteries. We hypothesized that HBO increased ATP production in vascular smooth muscle leading to enhanced vascular reactivity. Consequently, ATP levels were measured in mesenteric arteries but no significant differences in ATP levels were observed regardless of hyperbaric treatment. These results suggest that HBO alters vascular reactivity independent of ATP.
Time course of heat acclimation in burn survivors with well‐healed grafted skin (1104.11)
Authors: Rivas Eric , Schlader Zachary , Ganio Matthew , Pearson James , Lucas Rebekah , Gagnon Daniel , Crandall Craig
Published: June 2021

Repeated bouts of exercise in a hot environment improves the physiological capacity for heat loss, i.e., heat acclimation. This study tested the hypothesis that the time course of this adaptive response is reduced in burn survivors with well healed skin grafts. Twenty‐nine burn survivors were stratified into two groups: <40% body surface area (BSA) of grafted skin (N=13) and >40% BSA grafted (N=16). Nine similarly aged, non‐injured subjects served as controls. Subjects underwent a 7 day heat acclimation regimen wherein they exercised daily for 90 min in 40°C, 30% relative humidity. On each day, after 90 min of exercise, heart rate (HR) and the change in core (intestinal) temperature (∆TC) were measured. Despite an increased HR and ∆TC (both P<0.05) in the >40% BSA group compared to the control group, HR and ∆TC decreased (P<0.01) across the 7‐day heat acclimation regimen similarly between groups (day x group interaction: HR: P=0.76, ∆TC: P=0.11). Independent of group, in comparison to day 1, HR was reduced by day 2 (‐8 ± 14 bpm, P=0.01) through day 7 (‐17 ± 14 bpm, P<0.01) and ∆TC was attenuated by day 6 (‐0.3°C ± 0.5, P=0.02) and day 7 (‐0.3°C ± 0.4, P<0.01). These data indicate that the time course of heat acclimation in well‐healed burned survivors with grafted skin is similar to non‐burned individuals.

Grant Funding Source: Supported by National Institute of General Medical Sciences GM068865
Large differences in body fat do not independently alter changes in core temperature during exercise (1104.2)
Authors: Dervis Sheila , Smoljanic Jovana , Jay Ollie
Published: June 2021

No previous study has isolated the independent influence of body fat (BF) on thermoregulatory responses from the biophysical factors of body mass and metabolic heat production (Hprod). Therefore, six lean (L, BF:11.1±4.2%) and six non‐lean (NL, BF:32.1±7.1%) males matched for total body mass (TBM, L: 89.6 ±7.9 kg, NL: 91.0±7.0 kg; P= 0.74), cycled for 60 min in a 28.2 ±0.2°C and 30 ±9% RH room at i) a Hprod of 490 W; and ii) a Hprod of 7.5 W/kg lean body mass (LBM). Rectal (Tre) and esophageal (Tes) temperatures, and local sweat rate (LSR) were measured continuously; while whole body sweat loss (WBSL) was measured from 0‐60 mins. At 490 W, changes in Tre (L: 0.75 ±0.16°C, NL: 0.78 ±0.15°C), Tes (L: 0.49 ±0.15°C, NL: 0.51 ±0.14°C), MLSR (L: 0.61±0.18, NL: 0.51±0.12 mgcm‐2min‐1) and WBSL (L: 568±85 mL, NL: 565±80 mL) were similar (P>0.80). At 7.5 W/kg LBM, the L group had greater changes in Tre (L: 0.92 ±0.18°C, NL: 0.53 ±0.12°C), Tes (L: 0.62 ±0.17°C, NL: 0.41 ±0.12°C), MLSR (L: 0.72±0.31, NL: 0.40±0.12 mgcm‐2min‐1) and WBSL (L: 556±104 mL, NL: 409±94 mL) (P<0.05). In conclusion, i) body fat does not independently alter thermoregulatory responses during exercise; ii) core temperature comparisons between groups differing in BF should be performed using a Hprod normalized for TBM, not LBM.

Grant Funding Source: Supported by, NSERC Discovery Grant, Grant holder: Ollie Jay
Steady‐state local sweat rate is determined by the evaporation required for heat balance relative to body surface area (1104.9)
Authors: Cramer Matthew , Jay Ollie
Published: June 2021

Recent evidence suggests whole‐body sweat rate (WBSR, g·min–1) is determined by the absolute evaporation required for heat balance (Ereq, W). However, since differences in body surface area (BSA) should theoretically modify local sweat rate (LSR, mg·cm–2·min–1) for a given absolute Ereq, we hypothesized that LSR is determined by Ereq relative to BSA (W·m–2) rather than absolute Ereq (W). Sixteen males of large (L: 2.12±0.09 m2, n=8) and small (S: 1.80±0.09 m2, n=8) BSA cycled in 25°C at workloads eliciting Ereq of 340 and 400 W, and 165 and 190 W·m–2. Heat production was estimated via indirect calorimetry. WBSR was estimated from changes in body mass between 45 and 60 min, and LSR was taken as the mean of back and forearm ventilated capsule measurements from 45‐60 min. WBSR was similar between groups at 340 and 400 W (p蠅0.54), but LSR was greater at 400 W (p=0.03), and 340 W (p=0.07), in S. At 165 and 190 W·m–2, no differences in LSR were evident (p蠅0.86), while WBSR was greater in L at 165 W·m–2 (p=0.01) but not at 190 W·m–2 (p=0.13). Differences in BSA modify LSR for a given absolute Ereq, while the prescription of Ereq in W·m–2 results in similar LSR irrespective of BSA.

Grant Funding Source: Supported by a NSERC Discovery Grant (O. Jay)
HF‐feeding‐induced endoplasmic reticulum stress links metabolic syndrome (1107.11)
Authors: Sakar Yassine , Langelier Benedicte , Devime Fabienne , Covasa Mihai
Published: June 2021

Obesity has been associated with activation of cellular stress signaling and inflammatory pathways. A key player in this cellular stress response is the endoplasmic reticulum (ER). Thus, this study examined the effect of high‐energy/high‐fat (HE/HF)‐induced obesity on ER stress activation. Using male Sprague Dawley rats, we showed that HF feeding activated ER stress response (UPR‐Unfolded Protein Response) in the small intestine, adipose tissue and liver, which was associated with altered gut peptides expression and release including glucagon‐like peptide (GLP‐1), polypeptide YY (PYY) and cholecystokinin (CCK). This effect paralleled increases in weight gain, increased gut permeability, elevated circulating inflammatory cytokines and adipokines levels and impaired calcium homeostasis through disruption of both Sarco(endo)plamsic Ca2+‐ATPase 2b (SERCA), Homocysteine‐inductible endoplasmic reticulum stress protein (HERP) and Der1‐Like Domain Family, Member 1 (DERL2). Further, HF‐diet‐induced ER stress increased systemic inflammation through proinflammatory IKKβ/NF‐κB signaling activation and diminishing insulin sensitivity in the liver tissue. Increased lipogenic and adipogenic enzyme expression were also observed in the liver and adipose tissue of HF‐fed animals. Our findings suggest that HF feeding results in profound dysfunctions of ER homeostasis promoting weight gain and increased adiposity, all of which may contribute to perpetuation of obesity.
Dopamine D2 receptors regulate leptin and IL‐6 in 3T3 L1 adipocytes (1107.5)
Authors: Cuevas Santiago , Yang Yu , Upadhyay Kiran , Armando Ines , Jose Pedro
Published: June 2021

Leptin, secreted by adipose tissue and regulates energy homeostasis, is a pro‐inflammatory cytokine that also regulates the production of other pro‐inflammatory factors. Mouse and human adipocytes express dopamine D2 receptors (D2R). We tested the hypothesis that the D2R regulates the expression of leptin and other adipokines/cytokines in adipocytes. Stimulation of the D2R with quinpirole (1μM, 24h) increased leptin (+23±8%, P<0.05,n=4 western blot) in adipocytes and in the media (+78±19%,n=4,P<0.05, ELISA) and IL‐6 (+49±7%,n=4,P<0.05, western blot). D2R stimulation also increased, leptin (1.50±0.03‐fold,n=5,P<0.05) and IL‐6 (2.44±0.08‐fold,n=5,P<0.05) gene expression (qRT‐PCR). These effects were prevented by a D2R selective antagonist (L741,626, 1μM, 24h). Conversely silencing D2R expression with siRNA decreased the D2R protein (‐51.7±9.6%,n=4, P<0.05; western blot) and leptin protein (‐51.7±9.6%,n=4 P<0.05) and mRNA (‐0.38±0.01‐fold,n=5 P<0.05), decreased leptin in the media (‐26.2±3.9% pg/mg protein, P<0.05, ELISA), and decreased IL‐6 protein (‐24.7±5.2%, n=4, P<0.05) and mRNA (0.58±0.01‐fold,n=5,P<0.05) in adipocytes. The D2R‐mediated increase in leptin was prevented by a PI3K inhibitor (LY294002, 10μM, 24h), suggesting that the D2R regulates leptin through the PI3K‐Akt pathway. The effect of the D2R agonist was blunted in adipocytes cultured in insulin‐free media but was restored by the addition of insulin (1μg/ml, 24h). We suggest that the effect of D2R on cytokines and chemokines is tissue‐specific but the mechanism is known. We also studied human adipocytes primary cells culture from subcutaneous and visceral adipocytes tissue; D2R protein was 10‐fold greater in adipocytes from visceral than subcutaneous tissue. Our results suggest that the D2R may play a significant role in the regulation of leptin and IL‐6 expression, especially in visceral adipocytes in the presence of insulin, and may modulate the role adipose tissue in energy homeostasis.
High‐fat diet alters neuromuscular transmission and smooth muscle excitability in the mouse distal colon (1110.10)
Authors: Bhattarai Yogesh , Fried David , Galligan James
Published: June 2021

We tested the hypothesis that a high‐fat diet disrupts neuromuscular transmission and smooth muscle electrical activity in the distal colon of mice. Methods: Distal colon segments from mice placed on a control (10 Kcal%) or high‐fat (HF) diet (60 Kcal%) were used in vitro. Circular muscle reactivity to electrical field stimulation (EFS) and bethanechol were measured in organ baths. Circular smooth muscle action potentials were recorded using intracellular microelectrodes in vitro. Colonic migrating motor complexes (CMMCs) were measured in vitro. Results: Bethanechol caused larger contractions in colon rings from control vs. HF mice. Colon rings from HF had reduced neurogenic contractions compared to rings from control mice. Colon segments from HF mice exhibited continuous action potential firing while control segments had bursting action potential firing. The membrane potential of the smooth muscle cells from HF mice was depolarized compared to control mice. Retrogradely propagated CMMCs occurred more often in the distal colon of HF mice compared to control mice. HF mice had reduced CMMC propagation speed compared to control mice. Conclusion: We conclude that HF diet impairs neuromuscular transmission and smooth muscle electrical activity in the colon. This would cause altered colon motility and propulsion.

Grant Funding Source: NIH (DK094932)
Ostrich oil failed to improve intestinal barrier function following 5‐fluorouracil‐induced mucositis in rats (1111.5)
Authors: Cheah Ker , Mashtoub Suzanne , Howarth Gordon
Published: June 2021

INTRODUCTION. Mucositis is characterized by intestinal barrier disruption following chemotherapy. Emu Oil promoted small intestinal repair in a rat model of mucositis (Mashtoub, 2013). However, the effects of Ostrich Oil (OO) remain unexplored. We investigated the effects of OO on mucin‐secreting goblet cells (GC) during the recovery phase of mucositis. METHODS. Female Dark Agouti rats were allocated to groups (n=8/group): Saline+Water; Saline+OO; 5‐Fluorouracil (5‐FU)+Water; 5‐FU+OO. Mucositis was induced on day 5 by 5‐FU injection and rats were euthanized on day 10. Intestinal villus height (VH), crypt depth (CD) and GC were quantified. p<0.05 was considered significant. RESULTS. Compared to water controls, VH was increased 15% by OO (p<0.05), however, VH was unaffected by 5‐FU. 5‐FU tended to decrease GC count in the villi, relative to water controls (p=0.069). GC count was unaffected by OO in 5‐FU‐treated rats compared to 5‐FU controls. CD was increased 24% by 5‐FU (p<0.01); an effect that was not altered by OO. 5‐FU reduced GC count in crypts (33%; p<0.01) compared to water controls. However, OO did not attenuate the decrease in GC count in 5‐FU‐treated rats. CONCLUSIONS. Ostrich Oil treatment failed to improve intestinal barrier function indicated by mucin‐secreting goblet cells. As this is the first report of Ostrich Oil in bowel disease, further studies are required to explore dosage and timing regimens.

Grant Funding Source: Supported by The Rural Industries Research and Development Corporation Australia
miR‐93 downregulation of PTK6 attenuates TNFα/IFNγ mediated of colonic epithelial barrier dysfunction (1113.2)
Authors: Haines Ricci , Beard Richard , Yuan Sarah , Wu Mack
Published: June 2021

The role of protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been mainly studied in mechanisms underlying metastasis and cancer. However, it has also been shown that PTK6 null mice demonstrate particularly strong (greater than 10 fold increase in basal TER resistance measurement relative to PTK6+/+) colonic epithelial monolayers. To this end, we investigated the role of PTK6 in TNFα/IFNγ mediated disruption of colonic epithelial barrier function. Consistent with previous findings, PTK6 knockdown increased basal resistance in YAMC monolayers. In addition, we showed that PTK6 knockdown significantly attenuated TNFα/IFNγ mediated decrease in resistance of young adult mouse colonic epithelial cell (YAMC) monolayers. In order to determine mechanisms of endogenous regulation of PTK6, we performed microRNA prediction analysis on the 3’UTR of PTK6 and identified miR‐93 as being highly likely to target PTK6 in epithelial cells. We investigated the role of miR‐93 in regulating PTK6 in YAMCs and determined that transfection of miR‐93 similarly attenuated TNFα/IFNγ mediated decrease in TER. We also show by Western blot that miR‐93 significantly decreased PTK6 expression following stimulant challenge. Using immunocytochemistry and confocal microscopy, we further characterized the process by which PTK6 modulates epithelial barrier function in YAMCs. Our results indicate that miR‐93 targeting of PTK6 may serve as a novel therapeutic strategy in treating diseases involving inflammation driven hyperpermeability of the colonic epithelium.

Grant Funding Source: Supported by VA Merit Review 5101BX000799, the NIH grants HL96640, HL61507 and HL70752
Hepatic stellate cells orchestrate clearance of necrotic cells from the liver by modulating macrophage phenotype (1115.6)
Authors: Copple Bryan , Rockwell Cheryl , Luyendyk James
Published: June 2021

Hypoxia‐inducible factor‐1α (HIF‐1α) is activated in hepatic stellate cells (HSCs) by hypoxia, and regulates genes important for tissue repair. Whether HIF‐1α is activated in HSCs after acute injury and contributes to liver regeneration, however, is not known. To investigate this, mice were generated with reduced levels of HIF‐1α in HSCs by crossing HIF‐1α floxed mice with mice that express Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (i.e., HIF‐1α‐GFAP Cre+ mice). These mice and control mice (i.e., HIF‐1α‐GFAP Cre‐ mice) were treated with a single dose of carbon tetrachloride, and liver injury and repair were assessed. Although liver injury, as measured by serum ALT activity, was not different between the two strains of mice, during resolution of injury, clearance of necrotic cells was decreased in HIF‐1α‐GFAP Cre+ mice. In these mice, the persistence of necrotic cells stimulated extensive collagen deposition. Hepatic accumulation of macrophages, which clear necrotic cells from the liver after carbon tetrachloride, was not affected by HIF‐1α deletion in HSCs. Conversion of macrophages to M1‐like, pro‐inflammatory macrophages, which have increased phagocytic activity, however, was reduced in HIF‐1α‐GFAP Cre+ mice as indicated by a decrease in pro‐inflammatory cytokines, and a decrease in the percentage of Gr1hi macrophages. Collectively, these studies have identified a novel function for HSCs and HIF‐1α in orchestrating the clearance of necrotic cells from the liver, and demonstrated a key role for HSCs in modulating macrophage phenotype during acute liver injury.
Low copper and dietary sucrose drive fibrosis pathways in a rat model of non‐alcoholic fatty‐liver disease (1116.11)
Authors: Tallino Savannah , Burkhead Jason
Published: June 2021

Nonalcoholic fatty‐liver disease (NAFLD) is increasing in prevalence worldwide with the affected US population estimated near 30%. However, the molecular changes that drive NAFLD and its progression to non‐alcoholic steatohepatitis (NASH) and fibrosis are not well understood. Recently, a negative correlation has been observed between copper (Cu) status and NAFLD/NASH severity in patients, while rodent models fed low‐Cu diets also develop NAFLD pathology. The relationship between Cu status and liver disease is further complicated by consumption of simple sugars such as sucrose. We investigated effects of marginal Cu deficiency independently and in combination with 30% w/v sucrose in a mature rat model of NAFLD. Significant reductions of hepatic and serum Cu (p < 0.05) associated with significantly increased oxidative stress (p < 0.05) and up‐regulated expression of transcripts involved in inflammation and hepatic stellate cell activation (FC >= 2, p < 0.02), while rats fed low‐Cu/high sucrose also developed insulin resistance. The severity of fibrosis in particular has been implicated as a critical diagnostic tool for chronic liver disease and is likely prognostic for long‐term outcomes of liver complications and mortality. This study reveals low dietary Cu and sucrose consumption as potential drivers for NAFLD and NASH progression and transcriptional activation of fibrosis.

Grant Funding Source: Supported by UAA Innovate Award and Alaska INBRE
Iron‐mediated upregulation of M1 and downregulation of M2 programs in macrophages suggests a role for iron in the pathogenesis of non‐alcoholic steatohepatitis (1116.13)
Authors: Handa Priya , Morgan‐Stevenson Vicki , Kowdley Kris
Published: June 2021

Background and Aim: Hepatic reticuloendothelial (RES) iron deposition is associated with nonalcoholic steatohepatitis (NASH) and advanced fibrosis in nonalcoholic fatty liver diseases. We hypothesized that iron loading of macrophages could influence their activation status and contribute to necroinflammation. Methods: Mouse bone marrow derived macrophages (BMDM) were treated with either ferric ammonium citrate (FAC) alone, or cotreated with IL‐4 to determine the effect of iron on proinflammatory or alternative (M2) activation programs. Post treatment, gene expression analysis was performed to assess the status of M1 and M2 markers by qRT‐PCR. Results: BMDMs treated with ferric ammonium citrate (a physiological form of iron) showed increased M1 activation (INOS, TNF‐α, IL‐6, CD14 gene expression) and reduced gene expression of M2 markers (Arginase1, TGF, Mgl1). Further, while cells treated with IL‐4 showed enhanced expression of M2 markers such as Arginase1, Mgl1, Ym1, IL10 and PGC1β, cotreatment with iron abrogated the expression of these M2 markers. Also interestingly, while IL‐4 treatment upregulated the gene expression level of IL‐4 receptor, FAC treatment reduced its expression. Desferrioxamine, an iron chelator, blocked the downregulatory effect of FAC on M2 gene expression. Furthermore, treatment of macrophages with iron led to an elevated expression of hepcidin (p<0.05), the anti‐inflammatory peptide regulating iron homeostasis. Addition of hepcidin to LPS‐treated macrophages resulted in downregulation of TNF‐α (p<0.01), and upregulation of SOCS3 (p=0.05), an anti‐inflammatory mediator. Conclusions: Iron administration causes upregulation of M1 and downregulation of M2 gene expression. We speculate that iron‐induced inflammatory activation may contribute to the necroinflammation, while iron‐mediated downregulation M2 pathway would impact fibrogenesis accompanying NASH. These data provide support for a mechanistic role for iron in NASH.
Reversible transgene expression in mice: now you see it, now you don’t (1121.1)
Authors: Matthaei Klaus , Morton Susan , Chaston Daniel , Baillie Brett , Hill Caryl
Published: June 2021

Transgenic mice can suffer from the “Position effect” whereby the transgene integration site, per se, influences the observed phenotype. Multiple ‘Founder’ animals must therefore be studied to determine the actual phenotype of each transgene. We describe a method whereby a transgene can be switched off and on, allowing reliable validation of phenotype.

We modified the endothelial specific Tie2 promoter with LacO sequences derived from the E. coli LacO/LacI system and generated transgenic mice expressing defective Connexin(Cx)40 molecules, with EGFP as reporter. Transgene expression was down‐regulated by breeding with mice expressing the inhibitor protein LacI and up‐regulated by Isopropyl β‐D‐1‐thiogalacto‐pyranoside (IPTG) in the drinking water. EGFP was quantified using immunohistochemistry and Western blotting. Blood pressure was measured by tail cuff plethysmography and telemetry.

EGFP expression was exclusively endothelial and correlated with transgene copy number. Defective Cx40 expression increased blood pressure. Breeding with LacI mice significantly reduced EGFP expression and blood pressure by 70‐100%. EGFP expression could be reinstated by IPTG in some, but not all, founder lines in a time dependent manner.

We conclude that the LacO/LacI system allows temporal transgene regulation and can reliably control for artifactual phenotypes arising from the “Position effect”.
In vivo microelectrode assessment of NBCe2 activity in the distal nephron during an acid load (1122.2)
Authors: Yuan yang , Li huaqing , Wen Donghai , Boettger Thomas , Sansom Steven
Published: June 2021

The electrogenic Na bicarbonate co‐transporter (NBCe2) reabsorbs one Na and two or three bicarbonates in the distal nephron but its electrogenic function has not been demonstrated in‐vivo. Micropuncture was used to determine the transepithelial potential (Vte) in the distal convoluted tubule 2 (DCT2) of wild type mice (WT) and mice with a knock‐out of NBCe2 (NBCe2‐/‐). The DCT2 is the segment where expression of ENaC‐mediated Na reabsorption and ROMK‐mediated K secretion begins. Fine tip microelectrodes were placed in the lumen of the DCT2. A ground reference electrode was placed in the surface saline solution near the luminal microelectrode. Each group: n 蠅 3. We validated the microelectrode measurements by determining the effects on Vte of proximal tubule in control (‐5.6 ± 0.3 mV) and after giving 2% IP phloridzin (Vte = ‐0.2 ± 0.2 mV), an inhibitor of Na‐glucose cotransport. The Vte of the DCT2 was ‐11.0 ± 0.6 mV in WT control diet mice. When treated with acidic (280 mM NH4Cl) drinking water, the Vte of WT decreased to 0.4 ± 1.1 mV, a value significantly greater than WT, consistent with H+ secretion and/or bicarbonate reabsorption in this segment. When treated with acid water, the Vte of NBCe2‐/‐ was ‐3.4 ± 0.3 mV, a value significantly less than that of WT on acid water. We conclude that the NBCe2 is located in the DCT2 where it contributes to a more positive luminal potential when mice consume an acid load.

Grant Funding Source: NIDDK
Prostaglandin E2 receptor EP4 signaling in vascular smooth muscle cells decreases aortic elasticity (1123.2)
Authors: Ichikawa Yasuhiro , Yokoyama Utako , Ishikawa Yoshihiro
Published: June 2021

Arterial elasticity was impaired in advancing aortic aneurysm (AA). Since we have reported that the PGE2 receptor EP4 signaling was involved in progression of AA, we further examined whether EP4 signaling in smooth muscle cells (SMCs) decreases aortic elasticity. We generated mice (EP4TG) with vascular smooth muscle‐specific overexpression of human EP4 (hEP4). hEP4 mRNA was over‐expressed in the aorta of EP4TG (n=8). Using a wire myograph, we found that vasodilative effect of EP4 agonist (10 µM) was greater in the aortae of EP4TG than those of non‐transgenic mice (non‐TG) (25.0±2.9 vs. 9.6±2.0% of phenylephrine‐induced contraction, p<0.01, n=4‐6). Under basal conditions, aortic diameter and elasticity were not different. However, pressure‐induced dilation (20 to 220mmHg) was decreased in EP4TG than in non‐TG (0.84±0.04‐fold, n=5‐6, p<0.05). After angiotensin II (ATII) infusion (3.0µg/kg/min for 28 days), the aortae of EP4TG were enlarged to a greater degree than those of non‐TG (1.1±0.02‐fold, n=5‐6, p<0.05). A zelatin zymography revealed that MMP‐2 activity is higher in the aortae of EP4TG than in that of non‐TG (1.7±0.07‐fold, n=5, p<0.05). After ATII treatment, enhancement of MMP‐9 activation was greater in EP4TG than in non‐TG (1.6±0.19‐fold, n=5, p<0.05). These data suggest that EP4 signaling in SMCs decreases aortic elasticity by increasing MMP activation.
Brain ACE2 overexpression stimulates food intake and promotes glucose intolerance in mice (1126.15)
Authors: Xia Huijing , Chhabra Kavaljit , Littlejohn Nicole , Pearson Nicole , Grobe Justin , Lazartigues Eric
Published: June 2021

The brain Renin‐Angiotensin System (RAS) plays an important role in the regulation of metabolic function. While ACE2 has been reported to modulate brain RAS activity, its role in the central regulation of metabolism is unknown. To determine the role of central ACE2 in metabolism, Body weight and intake behaviors, assessed using metabolic cages, were measured in 24 wk old non‐transgenic (NT, n=8) and transgenic mice overexpressing ACE2 in the brain (SA, n=8). Fasting blood glucose was measured after a 14 h overnight fasting. Glucose tolerance test was performed by measuring blood glucose at 15, 30, 60 and 120 min after IP injection of glucose (2 g/kg). Plasma insulin level was measured by ELISA. Interestingly, body weight was significantly higher in SA than NT mice (39±1.5 vs. 31±0.8 g, P<0.05). In addition, SA mice exhibited increased daily food intake (0.12±0.01 vs. 0.09±0.01 g/g body weight), water intake (0.14±0.01 vs. 0.08±0.01 ml/g body weight), fasting blood glucose (147.8±13 vs.106.5±7 mg/dl), impaired glucose tolerance (AUC: 51.6±2 vs.41.5±1.7) and increased plasma insulin level (5.5±1.2 vs.1.7±0.3 ng/ml), compared to NT mice (P<0.05). These data suggest that ACE2 is involved in the central regulation of energy and glucose homeostasis. Brain ACE2 over‐expression leads to increased body weight, fasting glycemia and glucose intolerance, possibly through stimulating energy intake and insulin resistance.

Grant Funding Source: Supported by NIH (NIGMS)
Synaptic and neurochemical characteristics of the nucleus tractus solitarius pars centralis neurons (1129.7)
Authors: Babic Tanja , Browning Kirsteen , Travagli R. Alberto
Published: June 2021

The nucleus tractus solitarius (NTS) receives gastrointestinal (GI), cardiovascular and respiratory visceral sensory information via A or C‐fibers. We have shown recently that, contrary to cardiovascular NTSmedialis neurons, the majority of esophageal NTScentralis (cNTS) neurons respond to vanilloid agonists while only a small subset responds to both vanilloid and purinerigic agonists.

The aim of this study was to investigate further the neurochemical and synaptic characteristics of cNTS neurons using whole cell patch clamp and single cell RT‐PCR.

Excitatory postsynaptic currents (EPSCs) were evoked in cNTS by tractus solitarius stimulation. Perfusion of the slices with αβ‐methylene ATP (αMeATP) increased the amplitude of the evoked EPSC in 9 of 23 neurons tested. Variance‐mean (V‐M) analysis was performed to determine synaptic characteristics of αMeATP‐responsive and non‐responsive neurons. αMeATP ‐responsive neurons had 40±16 release sites, quantal size was 21±9pA and 78±5% probability of release during perfusion with 2.4 mM [Ca2+]e. Conversely, non‐responsive neurons had 18±6 release sites, quantal size was 23±3pA and 75±4% probability of release at 2.4 mM [Ca2+]e. Single‐cell RT‐PCR revealed that 8/13 responsive neurons expressed mGluR8 mRNA, whereas only 3 expressed glutamic acid dehydroxylase (GAD). A significantly lower proportion of non‐responsive neurons expressed mGluR8 (2/30), whereas a greater proportion expressed GAD (12/30).

These data demonstrate that cNTS comprises distinct sub‐populations of neurons that can be distinguished based on their responses to purinergic agonists and have distinct neurochemical and synaptic characteristics, suggesting that integration of sensory inputs from the esophagus relies on a discrete organization of synapses between vagal afferent fibers and cNTS neurons.

Grant Funding Source: DK55530
The hypoxia‐sensitivity of RVLM sympathetic premotor neurons is mediated by glial ATP release (1130.1)
Authors: Bou Farah Lama , McMullan Simon
Published: June 2021

Sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM) are excited by hypoxia, but the molecular mechanisms of oxygen sensing are unknown. Here we consider two mediators of hypoxia sensing: expression of the oxygen sensing molecule heme oxygenase 2 (HO‐2) and glia ATP release.

We examined the distribution of HO‐2 immunoreactivity in bulbospinal and catecholaminergic brainstem neurons in adult (n = 3) and neonatal (n = 2) rats. Whole cell responses of bulbospinal neurons to acute hypoxia (sodium cyanide, NaCN, 5, 10 or 20 mM) were recorded in acute brainstem slices before and after blockade of HO‐2 (SnPP‐IX, 10 μM) or ATP receptors (PPADS, 30 μM) and in a specific glial toxin (Fluoroacetate, 5 mM).

NaCN caused dose dependent inward currents that were greater in bulbospinal (n = 27) compared to non bulbospinal neurons (1029 ± 316 vs. 169 ± 43 pA at 20 mM NaCN, P < 0.01, n = 12). Responses were unaffected by synaptic (TTX 10 μM, I = ‐13 ± 12%, P > 0.05, n = 5) or HO‐2 blockade (ΔI = 16 ± 26%, P > 0.05, n = 5). HO‐2 immunoreactivity was absent in catecholaminergic and bulbospinal neurons and in 12 hypoxia sensitive biocytin filled bulbospinal neurons. Responses to NaCN were reduced by ATP receptor blockade (ΔI = ‐51± 13%, P < 0.01, n = 6) and abolished by gliocide (20.6 ± 7.7 pA at 20 mM NaCN, n = 12, P < 0.001).

These data suggest a crucial role for RVLM glia, not HO‐2, in driving sympathetic responses to hypoxia.
Modulation of arousal state and temperature is unaltered but activity is increased in tryptophan hydroxylase 2 knockout mice (1130.5)
Authors: Solarewicz Julia , Richardson Chelsea , Angoa‐Pérez Mariana , Kuhn Donald , Mateika Jason
Published: June 2021

Purpose: There is widespread belief that serotonin in the central nervous system (CNS) has a role in modulating various physiological functions including temperature, activity, and arousal state. Thus, we hypothesized that the absence of serotonin would be accompanied by hypersomnia, a reduction in activity and decreased temperature modulation.

Methods: Telemetry transmitters were surgically implanted in 12 mice with a null mutation for tryptophan hydroxylase 2 (Tph2‐/‐) and 12 wild type mice (Tph2+/+). Thereafter, electroencephalography, core body temperature and activity were recorded for 24 hours.

Results: Temperature during wake and sleep was modulated in a similar manner in both groups over the 24 hr period. Likewise, the change in temperature between the sleep and wake state was similar between the Tph2‐/‐ and Tph2+/+ mice (0.34 ± 0.09 vs. 0.37 ± 0.16 °C). The percentage of time spent in a given state of arousal was similar between the Tph2‐/‐ and Tph2+/+ mice (e.g. 40.6 ± 9.6 vs. 41.3 ± 7.2, % time sleeping). However, Tph2‐/‐ mice were more active (3.3 ± 1.5 vs. 1.7 ± 0.9 counts/s, p = 0.02) for a greater percentage of wakefulness (17.8 ± 6.8 vs. 14.2 ± 5.0 %, p = 0.05).

Conclusion: The modulation of temperature within or across arousal states, as well as, the duration of sleep and wake is not impacted by the absence of serotonin in the CNS. In contrast, the level of activity during wakefulness is increased in Tph2‐/‐ mice.

Grant Funding Source: Supported by: VA Award ‐ 1IO1 RX000458 (DMK) & 1I21RX001412 (JHM)
High salt diet increases glomerular ROS formation through an ET‐1/ETA dependent mechanism (1134.9)
Authors: Heimlich J. Brett , Speed Joshua , O'Connor Paul , Pollock David
Published: June 2021

Endothelin‐1 (ET‐1) stimulates the production of reactive oxygen species (ROS) in pulmonary artery smooth muscle cells and also activates NADPH oxidase in isolated aortic rings. The connection between ROS production provides mechanistic insight into possible deleterious effects of ET‐1 dysregulation during pathological processes such as hypoxia. ET‐1 production is increased by a high salt diet (HSD), so we sought to test the effects of HSD on the capacity of glomeruli to produce ROS. C57BL6/J mice were placed on a normal or HSD and then treated with or without selective ETA antagonist ABT‐627 for one week (5 mg/kg/d p.o.). Untreated animals on a HSD had significantly increased levels of glomerular ROS production when stimulated by phorbol (PMA) mediated PKC activation compared to glomeruli from normal salt controls (77.5±7.8 v. 23.7±7.3 thousand ALU/mg protein, p<0.01, n=5‐6). However, HSD animals treated with ABT‐627 had a blunted increase in glomerular ROS production (44.3±11.5 thousand ALU/mg protein, n=6). To further test the etiology of ROS production, glomeruli were incubated with 100 µM apocynin, a NADPH oxidase inhibitor, which completely prevented PMA stimulation of ROS to sub‐control levels in normal and HSD treated animals (10.5±2.75 and 13.02±4.3 thousand ALU/mg protein, n=5‐6). These data provide evidence for ET‐1/ETA mediated glomerular injury via ROS production during increased dietary salt intake.

Grant Funding Source: Supported by the National Institutes of Health
Chronic infusion of angiotensin II changed renal hemodynamic and function (1136.11)
Authors: Casare Fernando , Oliveira‐Souza Maria
Published: June 2021

This study investigated the effect of chronic treatment with angiotensin II (Ang II) on renal injury.

Methods: 35 days old male Wistar rats were divided into groups (n = 9/group): Sham, Ang II (s.c. infusion 200 ng/kg/min) for 42 days via mini‐osmotic pump),Losartan‐AT1 receptor antagonist (s.c. injection‐ 10 mg/kg/day) for 14 days and Ang II (42 days)/Losartan (14 days). Systolic blood pressure (SBP) was evaluated by tail‐cuff method. Renal plasmatic flow (RPF) and glomerular filtration rate (GFR) were determined by para‐aminohippuric acid and inulin clearance, respectively. Renal tissue was histologically analyzed and the results are expressed as mean ± SE and p 蠄 0.05 was considered significant (*vs Sham).

Results: Ang II/AT1 increased the SBP [(mmHg) Sham: 108 ± 2, Ang II: 193 ± 7*]; renal hemodynamic [(mL.min‐1.Kg‐1) RPF ‐ Sham: 23 ± 1, Ang II: 34 ± 2* and GFR ‐ Sham: 7 ± 0.3, Ang II: 13 ± 1*], filtration fraction (Sham: 32 ± 1, Ang II: 41 ± 2%*) and urinary flow rate [(µL.min‐1) Sham: 49 ± 4, Ang II: 71 ± 3*]. Ang II/AT1 decrease renal vascular resistance, increased interlobular artery thickness and its luminal diameter; induced glomerular injury, proteinuria and podocyturia.

Conclusion: Ang II infusion for 42 days induced via AT1 receptor, severe changes on renal hemodynamic and morphology leading to outward hypertrophic remodeling and chronic kidney disease.
The effect of attenuating age‐related increases in blood pressure in male and female spontaneously hypertensive rats on the kidney (1136.17)
Authors: Li Beverly , Tipton Ashlee , Sullivan Jennifer
Published: June 2021

The kidney is critical in the long‐term control of blood pressure, and renal inflammation has been linked to the progression of hypertension. Previous experiments have shown a sex difference in protein excretion and the renal immune cell profile: male SHRs have greater indices of renal injury and more pro‐hypertensive renal T helper17 cells (Th17) while females had more BP‐lowering T regulatory cells (Tregs). Male and female SHR were randomized to receive tap water (vehicle) or the BP‐lowering drugs, hydrochlorothiazide and reserpine, in drinking water from 5 to 12 weeks of age. BP was measured by tail cuff, and kidneys were isolated for flow cytometric analysis of T cells. Treatment attenuated age‐related increases in BP in males (174±4 vs. 137±4 mmHg, p<0.05) and females (132±4 vs 161±1 mmHg, p<0.05). However, treatment did not significantly change metabolic parameters, urinary electrolyte excretion (Na+, K+, Cl‐) or urinary protein excretion in either sex. In contrast, although attenuation of the development of hypertension did not significantly change the renal T cell profile in male SHR, females exhibited a decrease in renal Tregs (p=0.0091) and Th17 (p=0.0203) counts which abolished the sex differences observed in the renal T cell profile of vehicle‐treated rats.
Overexpression of miR‐20b inhibits the expression of HIF‐1α and STAT‐3 in invasive ductal carcinoma of the breast (1138.2)
Authors: Zanetti Juliana , Darin Patrícia , Lucca Fernando , Ribeiro‐Silva Alfredo
Published: June 2021

It is known that the hypoxia inducible factor‐1α (HIF‐1α) and the signal transducer and activator of transcription 3 (STAT‐3) are targets of miR‐20b. The role played by microRNAs (miRNAs) in breast cancer has been mainly studied using tumor cells. In this study, we investigated the expression of miR‐20b in formalin‐fixed paraffin embedded (FFPE) samples, as also as HIF‐1α and STAT‐3, in samples of invasive ductal carcinoma (IDC). The expression of miR‐20b and the proteins HIF‐1α and STAT‐3 was evaluated by real time PCR and immunohistochemistry, respectively. We found that the overexpression of miR‐20b (3‐fold; p<0.05) is accompanied by the downregulation of STAT‐3 (70%; p<0.001) in FFPE samples of IDC. We also observed that HIF‐1α expression in IDC and control samples was not statistically significant. Our results suggest that miR‐20b regulates the expression of HIF‐1α and its transcription factor STAT‐3 in IDC. These findings are relevant since HIF‐1α expression has been correlated with increased intratumoral angiogenesis, cancer progression and poor prognosis in breast cancer.

Grant Funding Source: Supported by FAPESP 2012/05235‐3
The potent inhibition of human SULT1A1 by 17α‐ethinylestradiol (EE2) is due to interactions with ILE89 in loop 1 (1141.12)
Authors: Rohn‐Glowacki Katie Jo , Falany Charles
Published: June 2021

Drug‐drug interactions (DDI) are a concern for the 17% of women in the United States taking birth control pills. Competition for metabolism with phase I and II drug metabolizing enzymes is one cause of these DDI. The object of this study was to determine the mechanism of SULT1A1 inhibition by EE2. SULT1A1 is the major drug/xenobiotic conjugating isoform found mainly in liver and the GI tract, but is also widely expressed at varying levels in other tissues including breast and brain. SULT1A1 conjugates a broad range of xenobiotics and endogenous compounds such as small phenols, estrogens, and acetaminophen. The Km of EE2 for SULT1A1 is 3.5‐fold lower than the Km of 17β‐estradiol (E2). In silico docking of EE2 to SULT1A1 crystal structures and homology models suggested that EE2 may inhibit SULT1A1. The inhibition constant range for E2, 2‐naphthol, and p‐nitrophenol was 9‐19nM, a physiological range for potent inhibition of SULT1A1. We were interested in the mechanism by which EE2 could inhibit SULT1A1 at low nanomolar concentrations while EE2 is not sulfated until assay concentrations are 30‐fold higher. Therefore, further analysis of the docking studies identified two amino acids that may contribute to EE2 inhibition, TYR169 and ILE89. Mutants of each residue were made, TYR169PHE (Y169F) and ILE89ALA (I89A), and tested for sulfation by and inhibition of EE2. Both mutants displayed the same Km for E2; however, the Km of EE2 for SULT1A1‐Y169F increased 3‐fold while the Km for SULT1A1‐I89A increased 10‐fold. The SULT1A1‐Y169F mutant retained the ability of EE2 to inhibit substrate sulfation with a slightly higher Ki although still in the nanomolar range, whereas SULT1A1‐I89A inhibition by EE2 was almost completely abolished at the concentrations tested. These data provide a better understanding of the SULT1A1 active site and indicate that the potent inhibition of SULT1A1 by low nanomolar EE2 concentrations may be a source of DDI in women using EE2 containing contraceptives.

Grant Funding Source: Supported by NIH GM38953
Pharmacovigilance‐pharmacological investigations on sulfonylureas and glinides‐induced atrophy in skeletal muscle (1142.5)
Authors: Cetrone Michela , Mele Antonietta , Calzolaro Sara , Conte Diana , Tricarico Domenico
Published: June 2021

The atrophy of the skeletal muscle apparatus is a comorbidity condition in the diabetes type II representing a negative prognostic factor. The effects of the anti‐diabetic drugs on the diabetes type II‐related atrophy have never been investigated. The “in vitro” effects of the sulfonylureas (tolbutamide, glibenclamide and glimepiride) and glinides (repaglinide and nateglinide), well known KATP channel blockers, were evaluated on the protein content/muscle weight, fibers viability, mitochondrial SDH activity and channel currents in oxidative soleus(SOL), glycolitic/oxidative flexor digitorum brevis(FDB) and glycolitic extensor digitorum longus(EDL) muscle fibers of mice using biochemical and cell‐counting Kit‐8 assay, imagine analysis and patch‐clamp techniques. FDA‐adverse effects reporting system evaluation of atrophy‐related signals associated with the use of these drugs in humans has been performed. In animal experiments, all drugs after 24 h of incubation time reduced the protein content and fibers viability blocking the KATP channel and enhancing the SDH activity. Repaglinide and glibenclamide were the most potent drugs. In an eight month period, muscle atrophy was found in 0.27% of the glibenclamide reports in humans and in 0.022% of the other not sulfonylureas/glinides drugs. Glibenclamide induces atrophy in animals and in humans. Glimepiride shows a less potential of inducing atrophy.

Grant Funding Source: none
Inhibition of FAAH enzyme by pterostilbene: potential mechanism of anxiolytic action (1144.10)
Authors: El‐Alfy Abir , Patel Christina , Mazhari Nunmoula , Rimando Agnes
Published: June 2021

The primary objective of this study was to evaluate the effect of the natural product pterostilbene on enzymes responsible for the catabolism of endocannabinoids. Previous studies in our laboratory have established an anxiolytic effect of pterostilbene. The current study evaluated the compound for binding to CNS target receptors involved in anxiety. The study revealed no significant interaction of ptersotilbene with GABA, serotonin, or cannabinoid receptors. In an attempt to clarify the mechanism underlying the in vivo anxiolytic action of pterostilbene, the compound was evaluated for potential inhibition of FAAH and MAGL, enzymes primarily responsible for degradation of endocannabinoids. Results revealed that pterostilbene exerted a concentration dependent inhibition of the human recombinant FAAH enzyme, with an IC50 value of 5.42 ± 0.26 μM, with no significant inhibition of the MAGL enzyme. These results suggest that pterostilbene has the potential to become a candidate compound for therapeutic drug development for anxiety disorders.