bluuebunny/crossref_metadata_2025 huggingface dataset:
EFFECT OF PHYSICAL RESTRAINT ON THE LIMITS OF THERMOREGULATION IN TELEMETERED RATS
Authors: Aydin Cenk , Grace Curtis , Gordon Christopher
Published: June 2021

To understand how ambient temperature (Ta) affects the thermoregulatory response to restraint, core temperature (Tc) and heart rate (HR) were monitored by telemetry in rats subjected to one hour of physical restraint while Ta was maintained at 14 to 30 °C in 2 °C increments. Tc of unrestrained rats was unaffected by Ta. During restraint, Tc was elevated at all Ta's with exception to 14°C where the rats became mildly hypothermic. There was an inverse relationship between Ta and HR in both unrestrained and restrained rats; however, HR was significantly elevated in restrained rats at all Ta's except 22 and 24 °C. Heat loss from the tail, estimated from Tc and tail skin temperature, was markedly reduced at all but the highest Ta's in restrained rats. The data suggest that the Ta limits of normothermia (LIN) are narrowed in the restrained rat. That is, between 16 and 20 °C, the rat maintains a relatively stable Tc that is slightly elevated above that of the unrestrained rat. Overall, the ideal Ta for restrained rats appears to be 20 and no higher than 22°C for the thermoregulatory system to maintain a regulated Tc in rats well adapted to physical restraint. This is an abstract of a proposed presentation and does not reflect US EPA policy.
Prior Exposure To Sustained Hypoxia Impairs The Acute Hypoxic Ventilatory Response In Rat Pups Following Postnatal Chronic Intermittent Hypoxia
Authors: MacFarlane Peter , Ao Jingning , Mayer Catherine , Di Fiore Juliann , Wilson Christopher , Martin Richard
Published: June 2021

Chronic intermittent hypoxia (CIH) associated with apnea of prematurity (AOP) is associated with an enhanced hypoxic ventilatory response (HVR); a similar effect is observed in developing rats exposed to CIH. In the current study, we investigated whether prior exposure to sustained hypoxia (SH, 11% O2, P0–P5) disrupts the HVR following subsequent CIH (5% O2/5mins, 8hrs/day, 10d; P5–P15). Using whole‐body plethysmography, the magnitude of the HVR following 10d of CIH was enhanced (58.8±6.6% above baseline) at P16 compared to normoxia raised pups (48.9±6.4%). In contrast, the HVR was absent (10.8±5.4%) in rats pretreated with SH prior to CIH. Further, SH alone did not affect the HVR at P16. The hypercapnic ventilatory response was not different between treatment groups suggesting a selective effect on the oxygen sensing mechanisms. In summary, postnatal exposure to either CIH or SH alone has little or no effect, respectively, on the HVR, but together they have a profound effect on postnatal development of the ventilatory sensitivity to hypoxia. We propose that CIH may promote favorable adaptive respiratory responses to acute hypoxia, which are disturbed by a prior experience to sustained hypoxia. These data could provide useful insight into the importance of avoiding SH in preterm infants during their vulnerable early postnatal period. Funding: Dept. Pediatrics, CWRU, Rainbow Babies & Children's Hospital.
Opioid receptors modulate excitatory cardiovascular reflex responses to myocardial ATP stimulation through a P2 receptor mechanism
Authors: Fu Liang-Wu , Barvarz Sherwin , Ho Jesse , Longhurst John
Published: June 2021

Activation of cardiac spinal afferents evokes excitatory cardiovascular reflexes. We have demonstrated that extracellular ATP stimulates ischemia‐sensitive cardiac afferents through P2 receptor mechanisms. The present study tested the hypothesis that through activation of P2 receptors ATP provokes excitatory cardiovascular reflexes and the reflexes are facilitated by blockade of opioid receptors. Hemodynamic parameters were recorded in anesthetized rats after bilateral vagotomy. Intrapericardial injection of α,β‐meATP (31–500 nmol), a P2X receptor agonist, increased mean arterial pressure (MAP) by 4±2 to 49±4 mmHg in a dose‐dependent manner in seven rats. After blockade of cardiac neural transmission with intrapericardial procaine, the α,β‐meATPevoked pressor responses were almost eliminated (n=6). Application of α,β‐meATP‐evoked consistent reflexes in six control rats. Intrapericardial ADP, a P2Y receptor agonist, also increased MAP in five rats by 9 to 13±3 mmHg, although we observed no changes in two and a decrease in two rats. Finally, blockade of opioid receptors with naloxone enhanced the α,β‐meATP‐mediated reflexes from increase in MAP by 27±3 to 37±3 mmHg (n=6). These data suggest that extracellular ATP provokes cardiac sympathetic afferent reflexes mainly through P2X receptor mechanisms and that the magnitude of these reflexes is partially dependent on intact opioid receptors.
Changes in expression of markers of GABA transmission in nucleus of the solitary tract (NTS) of ovariectomized (OVX) female rats
Authors: Spary Emma Jane , Maqbool Azhar , Drinkhill Mark J , Batten Trevor FC
Published: June 2021

Estrogen is a modulator of autonomic function and we previously showed differences in estrogen receptor expression in NTS associated with plasma estrogen levels during the estrous cycle in female rats. As promoter sequences for several receptors, receptor associated proteins, enzymes and transporters involved in GABA transmission include potential estrogen response element consensus sites we analyzed levels of mRNA expression by real time PCR in NTS micropunches from control cycling females and 3 week OVX rats. Plasma estradiol levels were measured by ELISA and blood pressures recorded via implanted telemetry probes. For 6 of the GABA‐A subunits analyzed (alpha1; alpha3; beta1; gamma1, gamma3, delta) there were significant reductions in mRNA expression in OVX compared to rats in either the low estrogen (metestrus) or high estrogen (proestrus) phases of the cycle (p<0.05; ANOVA; n = 6). Expression of the GABA‐B receptor B1 subtype showed a highly significant reduction (p<0.01) in OVX, as did the expression levels for GABARAP and GABARAPL1/2. Differences (p<0.05) between the metestrus and OVX expression levels were found for GAD65, GAD67 and vGAT. The data suggest that circulating estrogen acts in NTS to modulate inhibitory GABAergic influences on autonomic reflexes through affecting both the GABA release mechanism and GABA receptor composition.
Acute phosphodiesterase inhibition ameliorates functional muscle ischemia in dystrophin‐deficient mdx mice
Authors: Li Liang , Victor Ronald G , Thomas Gail D
Published: June 2021

We previously have shown that the loss of sarcolemmal nitric oxide synthase (nNOS) in the dystrophin‐deficient muscles of mdx mice and boys with Duchenne muscular dystrophy (DMD) renders the diseased muscles susceptible to ischemia during exercise. We now are extending this finding to men with Becker muscular dystrophy (BMD). We therefore hypothesized that treatment with a phosphodiesterase (PDE) inhibitor to reduce cGMP breakdown and enhance the NO signal from residual nNOS would prevent functional muscle ischemia. To test this, we compared norepinephrine (NE)‐mediated vasoconstriction in resting and contracting hindlimbs of mdx mice after acute treatment with vehicle or a PDE inhibitor (tadalafil, 8 mg/kg; zaprinast, 4 mg/kg). In vehicle‐treated mice, the usual effect of muscle contraction to attenuate NE‐mediated vasoconstriction was impaired as shown by similar decreases in femoral vascular conductance in contracting vs resting hindlimbs (attenuation ratio = 0.87 ± 0.11; n = 10). NE‐induced ischemia in the contracting hindlimbs was partially reversed in mice treated with the selective PDE5A inhibitor tadalafil (0.61 ± 0.06; n = 6; P < 0.05 vs vehicle) or the nonselective PDE inhibitor zaprinast (0.46 ± 0.10; n = 7; P < 0.05 vs vehicle). The effect of PDE inhibition to ameliorate functional muscle ischemia in mdx mice suggests a novel potential use for the treatment of DMD/BMD patients. Supported by MDA, 201149.
SIRT1 acutely modulates glucose sensing of POMC neurons in the hypothalamus
Authors: Shi Xuemei , Li Depei , Chan Lawrence , Guan Xinfu
Published: June 2021

POMC neurons in the hypothalamus are glucose‐excited neurons to sense glucose via AMPK and KATP channel. NAD+‐dependent SIRT1 deacetylase in POMC neurons is essential for maintaining energy balance. However, it is unknown if SIRT1 modulates glucose sensing by POMC neurons. Our aims were to determine if SIRT1 in POMC neurons is essential for homeostatic control of blood glucose in addition to body weight; and if SIRT1 directly modulates glucose sensing by POMC neurons. [1] Using the Cre‐LoxP system, we generated POMC‐specific SIRT1 knockout (KO) mice. Using hyperinsulinemic euglycemic clamp, we demonstrated that the KO mice had increased hepatic glucose production and decreased peripheral glucose removal during the clamp. [2] Using the whole‐cell patch clamp, we showed that firing rate of POMC neurons was significantly lower (50%) in the KO brain slice at 5 mM glucose than that in the WT brain slice, indicating that POMC neurons with SIRT1 deletion require a higher level of glucose to be excited. Firing rate of POMC neurons in the WT slice decreased by 57% when glucose decreased from 5 mM to 3 mM; however, it was not altered in the KO slice. Moreover, 80% of POMC neurons in the WT slice responded to changes in glucose concentration, while only about 10% of POMC neurons in the KO slice did. These data suggest that POMC neurons with SIRT1 deletion are not sensitive to glucose levels. [3] 60% of POMC neurons in the WT slice were sensitive to genipin (UCP2 blocker, 20 μM) by increasing (+120%) firing rate at 3 mM glucose. However, POMC neurons with SIRT1 deletion were not responsive to genipin anymore. We conclude that SIRT1 in POMC neurons is essential for glucose homeostasis; and SIRT1 deletion in POMC neurons impairs glucose sensing in a UCP2‐dependent manner. We propose that SIRT1‐mediated glucose sensing in POMC neurons may play an important role in the maintenance of glucose homeostasis.
Rescue of obesity‐induced infertility in female mice due to an ovarian theca cell‐specific knockout of the insulin receptor
Authors: wu sheng , Wondisford Fredric , Wolfe Andrew
Published: June 2021

Women with Type 2DM or PCOS present with elevated LH and androgen levels, anovulation and insulin resistance in metabolic tissues. The aims of these studies were two‐fold; first, to determine whether insulin signaling in the ovary played a role in the development or function of reproductive function, and secondly, to explore whether the ovary is also a target for the pathophysiologic affects of high insulin in obesity. We disrupt the IR gene specifically in the theca cells of the ovaries (ThIRKO mouse). No changes in reproductive development or function were observed in lean ThIRKO female mice suggesting that insulin signaling in the theca cell is not essential for reproduction. To explore the effect of insulin signaling in the ovary in obesity we made mice obese (DIO). As expected, there was a significantly higher fasted glucose and body mass in WT and ThIRKO DIO mice compared to WT and ThIRKO lean mice. DIO WT mice were infertile and experienced increased circulating testosterone levels. By contrast, ThIRKO mice exhibited improved estrous cyclicity and fertility with comparable testosterone level as lean mice. These results confirm that the ovary is a target of the high levels of insulin in obesity. It highlights an important role for insulin signaling in the ovary in the development of infertility in DIO and that the reproductive axis maintains insulin sensitivity even in the setting of peripheral insulin resistance.
Activation of the novel estrogen receptor GPR30 reduces blood pressure and renal injury in the streptozotocin (STZ)‐induced diabetic rat infused with angiotensin II
Authors: Maric-Bilkan Christine , Flynn Elizabeth
Published: June 2021

Our previous studies have shown that supplementation with 17‐β estradiol is renoprotective in the STZ‐induced diabetic rat. The aim of this study was to examine if activation of the novel, membrane‐bound estrogen receptor, GPR30, could mediate some of the renoprotective effects of estrogens.

Angiotensin II (200 ng/kg/min) was infused via osmotic minipumps from the onset of diabetes (STZ, 50 mg/kg) for 6 weeks. These female diabetic Sprague‐Dawley rats were then either left untreated (D) or were treated with G1 (400 μg/kg/day via minipump), a GPR30 agonist (D+G1) for additional 3 weeks. Urine protein excretion (UPE) was measured before and after G1 treatment, while mean arterial pressure (MAP), blood glucose, glomerulosclerosis (GSI) and glomerular filtration rate (GFR) were assessed only at the end of the study.

While no difference in UPE were observed in the D+G1 between baseline and time of sacrifice, UPE was reduced by 40% compared with the untreated D at the time of sacrifice. G1 also reduced MAP by 22% and attenuated glomerulosclerosis without affecting GFR or blood glucose levels.

The study suggests that activating the GPR30 receptor is renoprotective in the STZ‐induced diabetic rat infused with Ang II, possibly via lowering MAP. Supported by RO1DK075832

D (n=4)
D+G1 (n=5)
P value

Baseline
Sacrifice
Baseline
Sacrifice

UPE (mg/day)
81.4±14.1
133±27.8*
66.2±5.78
77.9±13.7#
*0.05 vs Baseline in the same treatment group  #0.05 vs. D at sacrifice

MAP (mmHg)
‐
128±8.99
‐
100±6.30#

Blood glucose (mg/dL)
‐
348±10.9
‐
341±20.7

GFR (ml/min/kidney)
‐
1.62±0.07
‐
1.50±0.09

GSI (AU)
‐
1.60±0.05
‐
1.28±0.08#
Establishing Biomarkers of Hypertension Related Kidney Disease in a Novel Rat Model
Authors: Harmon Ashlyn Christine , Johnson Ashley , Driesbach Albert , Garrett Michael R
Published: June 2021

We recently developed a model of accelerated kidney disease through genetic modification of the Dahl salt‐sensitive (S) rat. The novel congenic model [S.SHR(11)] demonstrates significantly more renal injury including glomerulosclerosis, tubulointerstitial damage and increased renal vascular hypertrophy, culminating in a more rapid decline in kidney function compared to the already highly susceptible S rat. The model is currently being used for the identification of novel biomarkers associated with onset and progression of hypertension related kidney disease. Through the use of Multidimensional Protein Identification Technology, or MudPIT, a proteomic analysis of urine exosomes obtained under low (moderate hypertension) and/or high‐salt (severe hypertension) conditions yielded several candidate protein biomarkers (e.g. MAPK1, ODF2, APOB) which are currently being validated via western blot analyses. Validated biomarkers identified in the rat will be tested on urine exosomes obtained from a unique human chronic kidney disease (CKD) population currently being collected at the University of Mississippi Medical Center. In summary, genetic modification of the S rat generated a model of accelerated kidney disease that may provide a unique experimental model to identify novel biomarkers of onset and progression of hypertension related kidney disease in humans. Supported by NIH/NHLBI HL094446 (MRG).
Lack of specificity for AT1 angiotensin receptor antibodies in Western blot analysis
Authors: Herrera Marcela , Sparks Matthew A , Coffman Thomas M
Published: June 2021

The angiotensin II type 1 receptor (AT1R) mediates the classical actions of angiotensin II (Ang II). In rodents, there are two AT1R, AT1a and AT1b, which are highly homologous (>90% at the amino acid level) and encoded by different genes. Detection of AT1R protein is important for a broad range of experimental approaches to understand molecular mechanisms of disease. While several anti AT1R antibodies are available commercially and used by investigators for these purposes, we have had concerns about their specificity. To address this, we examined the specificity of a panel of commercial anti AT1R antibodies (Santa Cruz 1173, AbCam 18801, Alomone AAR 011) for Western blot analysis. For these studies, we used kidney tissue from wild type mice and mice with genetic deficiencies of AT1a receptors (AT1a KO) or combined deficiency of AT1a and AT1b receptors (AT1ab KO), which completely lack AT1. For all three antibodies tested, a ~42 kDa band consistent with the predicted size of the AT1 was detected in tissues from WT mice. However, identical bands were also detected in tissues from the AT1a KO and AT1ab KO mice (n=3). Along with genotyping, we verified the absence of mRNA for the AT1a and AT1b receptors in KO mice and confirmed the absence of pressor responses to Ang II. We conclude that many of the available anti‐AT1R antibodies exhibit non‐specific binding that may lead to anomalous results.
Release of iron from cellular iron stores in lysosomes: Potential involvement of divalent metal transporter 1 (DMT1) and common metabolites
Authors: Morgan Jessica , La Alice , Nguyen Trinity , Sauble Eric , Gonzalez Angelica , Nguyen Annie , Linder Maria C
Published: June 2021

Excess cellular Fe is stored in ferritin (Ft), and this is drawn upon when cells or organisms become Fe deficient. In several cell types, lysosomal protease inhibitors prevent release of Fe from Ft, indicating that degradation of the protein “shell” surrounding its Fe is required. We have begun to address several questions on the details of this process. Confocal microscopic studies with hepatoma cells showed that lysosomal protease inhibitors caused the accumulation of Ft in lysosomes, confirmed by ELISA and Fe assays of organelles separated in density gradients. The shift of Ft into lysosomes induced by incubation in a low Fe medium was accompanied by increased co‐localization of DMT1. Solubilization of the Ft ferrihydrate crystallites by common metabolites was demonstrated. Incubation of purified (insoluble) crystallites with ascorbate, glutathione (GSH) or citrate (pH 5.5 or 7) markedly increased levels of soluble Fe. Treatment of whole cells with ascorbate, GSH or other reducing agents, and manipulation of intracellular GSH concentrations, did not induce ferritin Fe release. Knockdown (90%) of DMT1 mRNA by exposure to specific siRNA for 72 h did not affect the rate of Ft Fe release. However, 72 h was not sufficient to appreciatively knock down expression of DMT1 protein. Our findings suggest that after degradation, Ft Fe is easily solubilized and returned to the cytosol via DMT1.
Transient Receptor Potential Channel 6: Role in Hemostasis and Thrombogenesis
Authors: Espinosa Enma Veronica Paez , Murad John P , Ting Harold J , Khasawneh Fadi T
Published: June 2021

Although the importance of cytosolic calcium (Ca2+) elevation in the regulation of platelet activation has been widely acknowledged, the underlying mechanism of Ca2+ entry is still unclear. Previous studies have demonstrated that the transient receptor potential channel 6 (TRPC6) is abundantly expressed in platelets, particularly in the plasma membrane. Thus, our goal was to characterize the role of TRPC6 in platelet function in vivo. To this end, we employed a genetic approach, subjecting TRPC6 knockout mice (KO) to the tail bleeding time test and a carotid artery injury thrombosis model. We found that TRPC6 KO mice displayed a prolonged bleeding time, and an increased time for occlusion of the injured carotid artery, compared to their wild‐type (WT) littermates. Regarding a possible mechanism for the observed defects, our preliminary in vitro studies revealed a defect in the thromboxane receptor‐mediated platelet aggregation in the TRPC6 KO mice, compared to the WT animals. On the other hand, TRPC6 KO mice exhibited no apparent aggregation defects in response to ADP or the thrombin receptor activating peptide 4. Taken together, our data demonstrate, for the first time, that TRPC6 deletion results in defects in hemostasis and protection against thrombogenesis, suggesting a vital role in platelet function. Furthermore, TRPC6 may define a new therapeutic target for managing multiple thrombosis‐based disorders.
The adenosine A2A receptor agonist regadenoson decreases activation of invariant NKT cells in human sickle cell subjects
Authors: Lin Gene , Field Joshua , Neuberg Donna , Nathan David , Linden Joel
Published: June 2021

Sickle cell disease (SCD) is associated with widely disseminated vaso‐occlusion caused by hyper‐adherent sickle red blood cells and leukocytes. Recurrent episodes of ischemia reperfusion‐injury in the vasculature of SCD individuals promote a pro‐inflammatory state. In a mouse model of SCD, adenosine A2A receptor (A2AR) activation significantly decreases pulmonary inflammation and dysfunction. Similar protective effects are produced by depleting invariant NKT (iNKT) cells or by blocking CD1d restricted activation of iNKT cells, indicating that A2AR agonists mediate their anti‐inflammatory effects primarily by inhibiting iNKT cell activation. We have initiated a clinical trial to investigate the safety and efficacy of a selective A2AR agonist, regadenoson, in human SCD subjects. Our findings indicate that iNKT cells in blood from normal subjects are fewer in number and less activated than from subjects with SCD. Furthermore, activated iNKT cells from SCD subjects are CD4+ and express elevated levels of phospho‐NFκB (P‐NFκB), CD69, and the A2AR. Most significantly, infused regadenoson was observed to diminish iNKT cell expression of P‐NFκB and other activation markers in a time dependent manner. These results imply that A2AR signaling and NFκB regulate iNKT cell activation and that selective A2AR agonists may be useful for treating acute vaso‐occlusion in SCD.
The Fast Skeletal Troponin Activator, CK‐2017357, Improves Resistance to Fatigue in Healthy, Conscious Rats
Authors: Kennedy Adam Richard , Heald Nathan , Pakdel Arash , Ryans Julie , Musch Tim , Poole David , Hageman Sue , Copp Steve , Malik Fady , Jasper Jeffrey
Published: June 2021

Fast skeletal muscle troponin activators, such as CK‐2017357, amplify the sub‐maximal force frequency response in ex vivo and in situ muscle studies, and decrease muscle fatigue in a rat femoral artery ligation model of vascular insufficiency. We report the effects of CK‐2017357 on running fatigue resistance in conscious, healthy rats.

Treadmill running time (aerobic ). Rats were tested at 30m/min until failure; time and distance run were recorded. Oral CK‐ 2017357 at doses of 10 and 20mg/kg, significantly increased running time on the treadmill by 20% over baseline (p<0.01) and 50% over vehicle control (p<0.01).

Rotarod running time (anaerobic). An accelerating rotarod was ramped between 12–25 revolutions per minute (RPM) over 10 minutes after which animals were allowed to continue at 25RPM for a further 5 minutes. CK‐2017357 increased rotarod running time by up to 2‐fold at doses of 1 and 3mg/kg (p<0.05 and p<0.01 respectively vs. vehicle control). Similar effects were seen with other fast skeletal muscle troponin activators but not with caffeine, creatine or phosphoserine.

Fast skeletal troponin activators, such as CK‐2017357, seem to reduce susceptibility to fatigue in healthy rats both in a long distance, endurance assay (treadmill), and in a shorter distance, high‐intensity assay (rotarod). Thus, the mechanism of action may be useful in treating disorders that increase susceptibility to muscle fatigue.
Cytokines, glucose and angiotensin II (ANG II) and the expression of Connexin (Cx) 37, 40 and 43 in cultured microvascular endothelial cells
Authors: BRAAM Branko , ZHUANG Wenqing , CUPPLES William A
Published: June 2021

Endothelial connexins emerge as molecules that strongly affect the conductive behavior of the endothelial cell layer and are involved in renin release in the kidney. Since many of the cardiovascular disease states are associated with micro‐inflammation and with increased activity of ANG II or with increased glucose, we hypothesized that these could affect expression levels of the connexins 37, 40 and 43. Immortalized microvascular endothelial cells (HMEC‐1) were exposed to Ang II (100 nM), a combination of IL6 (20 IU/ml), IFN (4 ng/ml) and TNFa (20 ng/ml) or to 25 mM Glucose for 4 hours in 50% and 100% confluent HMEC‐1 layers. Then, cells were harvested, RNA extracted and reverse transcribed and subjected to qPCR using pre‐designed primers for Cx37, Cx40 and Cx43 using GAPDH as control. Confluency by itself increased Cx37 expression, but did not affect Cx40 and Cx43 expression. The cytokine mix increased expression of 50% and 100% confluent cells 13‐ and 16‐fold (both P<0.05) for Cx40 and 2‐fold (P<0.05) and 2‐fold (NS) for Cx37. Neither ANG II or high glucose affected Cx expression. The present data indicate that inflammatory factors can affect the gene expression of Cx40 in particular. This might point toward a role for Cx40 in coupling inflammation to microvascular function and to renin release.
Mechanisms of myocardial alterations induced by chronic high‐salt intake
Authors: Katayama Isis Akemi , Dopona Ellen Priscila Brito , Heimann Joel Claudio
Published: June 2021

Objective

To evaluate the mechanisms of left (LV) and right (RV) ventricular hypertrophy induced by high salt intake.

Methods

18‐week‐old male Wistar rats were fed normal (NS: 1.3% NaCl) or high (HS 8%) salt diet since weaning. From the 7th week of age, two HS subgroups received hydralazine or losartan (HZ 15 or LO 20mg/kg/day). Tail blood pressure (BP), cardiac mass to tibial length ratio (CM/TL), cardiomyocyte transverse diameter (TD) and interstitial fibrosis (IF) were measured.

Results

(mean±SEM – p<0.05, n=8/group): At 18th week of age, BP (mmHg) was higher in HS (142±6) than in NS (112±2). BP in HS+HZ (116±6) and HS+LO (118±2) did not differ from NS. TD (μm) in the LV was higher in HS (19±0.6) and HS+HZ (17±0.8) than in NS (13±0.4) and HS+LO (13±0.8). TD in the RV was higher in HS (16±0.3) and HS+HZ (14±0.4) than in NS (12±0.2) and HS+LO (12±0.8). IF (%) was higher in HS than NS, HS+HZ and HS+LO in both ventricles (LV: HS 0.56±0.08, NS 0.22±0.04, HS+HZ 0.10±0.04 and HS+LO 0.16±0.03) (RV: HS 0.92±0.06, NS 0.32±0.02, HS+HZ 0.52±0.08 and HS+LO 0.56±0.08). CM/TL did not differ among all experimental groups.

Conclusions

In Wistar rats, salt induced cardiac hypertrophy is blood pressure independent and unrelated to changes in ventricular mass. It can be speculated that this result is an angiotensin II type 1 receptor‐dependent mechanism.

Supported by FAPESP and CNPq.
Age effects on aerobic exercise‐induced reductions in pain sensitivity in female Sprague‐Dawley rats
Authors: Dykstra-Aiello Cheryl Julia , Carlberg Karen A.
Published: June 2021

Age differences appear to exist in the pain experience. Bouts of aerobic or anaerobic exercise are known to reduce pain perception. We examined age effects on exercise‐induced reductions in pain sensitivity in aerobic exercise trained female Sprague‐Dawley rats. In a previous study, we found that running started in youth (age 11 wk) significantly reduced pain sensitivity in middle‐aged (38 wk) rats compared with sedentary rats. To examine whether starting exercise at middle‐age, rather than in youth, also affects pain sensitivity, 10 rats, starting at age 21 wk, ran 11 wk for up to 30 min every other day in motorized running wheels. Ten sedentary rats were handled every other day. A tail flick latency (TFL) test measured pain perception at 5–6 hr post‐exercise. A longer latency indicated reduced pain sensitivity. TFL was significantly longer in exercised rats compared to sedentary rats (p=0.01). After 2 wk, we examined endogenous opioid involvement with the opioid antagonist, naloxone (2 mg/kg, s.c.). Naloxone eliminated the difference, suggesting opioid mediation. No significant difference in pain sensitivity was found between rats that began exercising in youth and those that started running in middle‐age. Our results show that aerobic exercise reduces pain sensitivity in female rats independent of age at implementation of an exercise program and this effect is opioid‐mediated.
Monitoring exercise intensity during long‐term endurance exercise training in aging rats
Authors: Hoffman Marie , Akins Tiffany , Barton Gregory , McKiernan Susan , Aiken Judd , Diffee Gary
Published: June 2021

Chronic exercise is known to counter a number of deleterious effects of aging. There are a number of difficulties associated with long term exercise training in aged animals, such as matching appropriate training intensities to the declining exercise capacity as animals age. But there is little quantitative information available regarding exercise capacity in very old rats. We conducted a 1 year endurance exercise study in male FBN rats, starting with rats at age 24 mo and continuing daily until 36 mo of age. Rats were divided into 3 groups; high intensity (H), and moderate intensity treadmill exercise (M), and sedentary (S). M and H animals trained at 13 m/min for 30 min/day, with the H group at 5% incline. We monitored exercise capacity and its response to training by measurements of VO2 max and blood lactate threshold (LT) every 3 mo throughout the training. The relative training intensity for the M animals, expressed by training speed as a percent of the running speed at LT, rose significantly as the animals aged (88% at 27 mo, 104% at 30 mo, 142% at 33 mo, and 325% at 36 mo). Similar results were seen with the H animals and when using VO2 max as a measure of exercise capacity. These results indicate that even in trained animals exercise capacity declines significantly in advanced age and stress the importance of measuring exercise ability at various time points throughout a prolonged training program.

Supported by: NIH AG030423
Achilles Tendon Ablation Induces Scleraxis Expression and Neotendon Formation in the Plantaris Tendon
Authors: Flood Michael D , Phan Anthony C , Gumucio Jonathan P , Mendias Christopher L
Published: June 2021

Tendons are organized into functional cable‐like units of matrix and cells called fibrils, and surrounded by a basement membrane layer of tissue known as the epitenon. In response to mechanical stimuli tendons grow by increasing their cross‐sectional area, however it is not known whether tendons grow by adding additional fibrils (hyperplasia) or by increasing the size of existing fibrils (hypertrophy). To gain a greater understanding of the cellular mechanisms of adult tendon growth, we used a synergist ablation model whereby a tenectomy of the Achilles tendon was performed to induce growth of the synergist plantaris tendon. We hypothesized that following synergist ablation, cells in the epitenon would migrate toward the interior of the tendon and increase the size of existing tendon fibrils. Two weeks following surgery, tendons were prepared for histology and gene expression analysis. Following Achilles tenectomy, there was an outward expansion of the epitenon surrounded by eccentric layers of immature matrix containing scleraxis‐positive fibroblasts, while no change in the size or cell density of the original tendon tissue was observed. This growth was coupled with an upregulation of scleraxis and collagen I expression. Our results suggest that following mechanical loading, tendon growth occurs through the addition of new fibrils to existing tendon, rather than hypertrophy of existing fibrils.
Extensive type II muscle fiber atrophy in elderly women undergoing acute hip surgery after falling
Authors: Snijders Tim , Verdijk Lex B , Poeze Martijn , van Loon Luc J.C.
Published: June 2021

BACKGROUND

Age‐related loss of skeletal muscle mass and strength increases the risk of falls and fractures, which is the most common cause of injury‐related death in persons above 75 years. This study compares muscle fiber characteristics of elderly women undergoing acute hip fracture surgery with those of healthy young and elderly controls.

METHODS

Percutaneous needle biopsies were collected from the vastus lateralis muscle in healthy young (21±1y) and older women (79±3y), and frail elderly women admitted for acute hip fracture surgery (83±6y). Immunohistochemical analyses were performed to assess muscle fiber size and satellite cell (SC) content in a fiber type‐specific manner.

RESULTS

Type II muscle fiber size in both the healthy and frail elderly were smaller when compared with the young. Frail elderly showed a higher degree of muscle fiber atrophy and lower SC content when compared with healthy elderly, with differences being most prominent in type II muscle fibers.

CONCLUSION

Elderly at risk of falling show extensive type II muscle fiber atrophy and a decline in SC content when compared with age‐matched controls. Elderly at risk of falls and fractures may be identified by such muscle characteristics.
Contribution of group III and IV muscle afferents to ventilatory control during submaximal exercise in heart failure
Authors: Olson Thomas P , Joyner Michael J , Eisenach John H , Curry Timothy B , Johnson Bruce D
Published: June 2021

Background

Heart failure (HF) causes abnormal ventilatory responses to exercise which are inked to poor prognosis. We examined the impact of blocking fine afferents from locomotor muscles on exercise ventilatory efficiency in HF.

Methods

9 HF patients (60±6yrs, LVEF 27±6%) and 9 healthy controls (63±7yrs) underwent two 5 min cycling sessions at 65% peak workload with and without intrathecal fentanyl. VO2, VCO2, VE, breathing frequency (fb), tidal volume (VT), and chemoreceptor sensitivity (CO2 rebreathe) were measured.

Results

Fentanyl did not influence exercise VO2 in either group (HF=1.5±0.3 vs 1.5±0.3 L/min, control=1.7±0.4 vs 1.7±0.5 L/min). VE was lower with fentanyl in HF but not control (−12.3±7.1 vs 0.8±6.2%, p<0.001) due to lower fb (−10.5±4.6 vs 0.5±7.1%, p<0.01) with no change in VT (−1.2±9.4 vs 0.4±5.7%, p=0.67). VE/VCO2 improved during exercise with fentanyl in HF but not in controls (−14.8±7.1 vs ‐ 0.5±7.0, p<0.01). Fentanyl did not influence chemoreceptor sensitivity in either group.

Conclusion

Blocking group III and IV afferent feedback from the locomotor muscles during exercise improves ventilatory efficiency in HF. NIH/NCRR grant KL2‐RR024151.
The effects of aging and muscle contraction on AMPK activity
Authors: Hardman Shalene E. , Hall Derrick E. , Mitchell Alyssa J. , Black Kyler M. , Compton Richard A. , Thomson David M.
Published: June 2021

Many cell processes that are disrupted in aging skeletal muscle are also regulated by AMP‐activated protein kinase (AMPK). However, the effect of aging on skeletal muscle AMPK activation is controversial and unclear. Our purpose was to determine the effect of old‐age on endurance‐type contraction‐induced AMPK activation in skeletal muscle. Gastrocnemius muscles from YA (8 mo old) and O (30 mo old) male Fischer344 x Brown Norway F1 hybrid rats were removed after 10 minutes of electrically stimulated in situ contractions (STIM). Muscles from the resting legs served as controls (REST). AMPK phosphorylation and AMPKα2 activity were 63% and 19% lower, respectively, in O vs. YA muscles after STIM. AMPKα1 activity was unaffected by STIM in YA, but increased by 30% in O muscles. AMPK α1 protein concentration was 45% greater, while α2 content was 18% lower in O vs. YA muscles. AMPK β1, β2, and γ1 proteins were unaffected by age, but AMPK γ2 and γ3 concentrations were 75% and 85% lower in O vs. YA muscles, respectively. In conclusion, AMPKα2 activation is impaired while AMPKα1 activation is enhanced after endurance‐type in situ contractions in aged skeletal muscle. This work was funded by NIAMSD Grant AR‐51928.
Volatile organic compounds (VOCs) in exhaled breath condensate (EBC) as a biomarker of high altitude pulmonary edema (HAPE)
Authors: Figueroa Jennifer Ann , Allen Roblee , Davis Cristina , Walby William , Aksenov Alexander , Zhao Weixiang , Mansoor James , Lewis William , Schelegle Edward
Published: June 2021

HAPE is the most common cause of death at high altitude. The underlying pathophysiology of HAPE is not well understood. While certain individuals are more susceptible to developing HAPE, the ability to identify these individuals prior to ascent has yet to be determined. The present study was designed to determine if a difference exists between the profile of VOCs in EBC, hypoxic ventilatory response (HVR) and pulmonary artery systolic pressures (PASP) in healthy males with (HAPE, n=5) and without (control, n=11) a history of HAPE. On the first day of the protocol, subjects performed pulmonary function, VO2 max, and HVR tests. On the second day of the study, the subjects completed a pre and post hypoxic exposure 20 minute EBC collection, supine exercise test with Doppler Echocardiography measurement of PASP and a ninety minute hypoxic exposure (12% O2) with measurement of PASP. The main results of the study showed that the EBC profile of VOCs was significantly different between groups. In addition, this profile was strongly correlated with HVR. These results indicate that EBC analysis can be used to identify individuals who have had a previous episode of HAPE, as well as physiological responses to hypoxia. In the future, this technique could be used in the field to determine HAPE sensitivity prior to altitude exposure.
Translational Repression of Down‐Regulated in Adenoma by miR‐494 in Caco2 Cells
Authors: Anbazhagan Arivarasu N , Priyamvada Shubha , Tyagi Sangeeta , Maher Daniel B , Kumar Anoop , Borthakur Alip , Alrefai Waddah A , Kwon John , Dudeja Pradeep K
Published: June 2021

Down‐regulated in adenoma (DRA) is a Cl−/HCO3 − exchanger involved in electroneutral NaCl transport in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. In order to investigate potential modulation of DRA expression by microRNA, five different in silico algorithms were used to predict the microRNAs that target DRA. Of these microRNAs, miR‐494 was shown to have highly conserved putative binding site in the DRA 3′untranslated region (3′UTR) compared to other DRA‐targeting microRNAs in vertebrates. Transfection with pMIR‐GLO dual luciferase vector containing DRA 3′UTR resulted in a significant decrease in relative luciferase acitivity (44.13 ± 3.7% p<0.05) as compared to empty vector. Co‐transfection of the DRA 3′UTR luciferase vector with a miR‐494 mimic resulted in markedly decreased luciferase activity (69.58 ± 2.1% p<0.05), as compared to cells transfected with negative control. The transfection of a miR‐494 mimic into Caco2 cells significantly increased the expression of miR‐494 and concomitantly decreased the DRA protein expression by (50.5 ± 5.2% p<0.05). These data demonstrate a novel regulatory pathway for DRA and indicate that targeting miR‐494 using anti‐miR‐494 could be an effective therapeutic tool for diarrheal conditions. (Supported by NIDDK/VA)
Role of pregnane X receptor (PXR) in diet‐induced obesity
Authors: Gyamfi Maxwell Afari , Jones Dominique , Awumey Emmanuel
Published: June 2021

Clinical obesity is a complex metabolic disorder that affects 1 in 3 adults. While a high‐fat diet (HFD) causes obesity and insulin resistance, the specific genes that determine sensitivity to obesity are not known. Recent reports suggest that the nuclear receptor pregnane X receptor (PXR), a xenobiotic receptor that defends against toxic agents, may also play a role in obesity: PXR ligands alter patient plasma lipid levels and affect lipid homeostatic and energy metabolic gene expression. Because of differences in ligand specificity between human and mouse PXRs, we examined the role of PXR in diet‐induced obesity using male PXR‐humanized (hPXR) transgenic and PXR‐knockout (PXR‐KO) vs. wild type (WT) mice. After 16 weeks of high (45%) or normal (12%) fat diets, WT mice had a higher susceptibility to obesity compared to hPXR and PXR‐KO mice. PXR‐KO mice showed the smallest gain in white adipose tissue mass. In contrast, only HFD‐fed hPXR mice had elevated serum glucose and triglycerides and reduced hepatic adiponectin, even though all three genotypes on a HFD displayed insulin resistance, hyperleptinemia and hepatotoxicity. Taken together, our data indicate that while the mouse PXR promoted HFD‐induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and can be used to explore the role of human PXR in metabolic syndrome. [Supported by NIH grants RO1 HL064761, R25HL059868, P20 MD000175, and SC1 HL099139]
Genistein represses invasion in an in vitro colon cancer metastasis model by transcriptionally upregulating metastasis repressor NDRG1
Authors: Li Qian , Chen Hong
Published: June 2021

Cancer metastasis accounts for most cancer deaths. Genistein, a biologically active soy isoflavone, has been indicated as an inhibitor of cancer progression. In this study, we tested our hypothesis that genistein can repress colon cancer metastasis in vitro by modifying the expression of metastasis suppressor NDRG1. Cell models SW480 and SW620 which are poorly and highly metastatic human colon cancer cell lines respectively, were used. Genistein treatment reduced the proliferation and wound healing ability of both cell lines. Interestingly, genistein specifically increased the adhesion of the metastatic cell line SW620 and inhibited the migration of SW620. Moreover, genistein increased the mRNA expression of NDRG1 in both cell lines. The low NDRG1 gene expression in SW620 compared to that in SW480 is associated with altered chromatin configuration of the gene. In conclusion, genistein treatment increased expression of several genes responsible for metastasis repression in SW620. Future research will focus on illustrating the epigenetic mechanisms underlining the metastasis inhibiting effects by genistein.

Grant Funding Source : University of Illinois at Urbana‐Champaign
RNA binding protein RBM3 promotes a cancer stem cell phenotype with multidrug resistance
Authors: Venugopal Anand , Kwatra Deep , Stecklein Shane , Ramalingam Satish , Subramaniam Dharmalingam , Anant Shrikant
Published: June 2021

RNA binding protein RBM3 functions to posttranscriptionally regulate mRNA by altering its stability and translation. We recently demonstrated that RBM3 acts as a proto‐oncogene that significantly increases cell proliferation, anchorage independent growth and angiogenesis. We have also observed that RBM3 overexpression in NIH3T3 cells promotes a stem‐like phenotype with upregulation of the putative quiescent stem cell marker DCLK1. Using a doxycycline (Dox) inducible system, we overexpressed RBM3 in the colorectal cancer cell line HCT116. RBM3 induction promoted spheroid formation compared to vector control. We also observed an increase in HES‐1 mRNA and protein indicating an increase in Notch activity. In addition, there was increased resistance to the antineoplastic agents doxorubicin and paclitaxel. Induction of RBM3 upregulated expression of multidrug resistance protein 2 (MRP2) and p‐glycoprotein (P‐gp), which correlated with a decrease in doxorubicin uptake and increase in doxorubicin efflux. This decrease in uptake could be partially rescued with the MRP inhibitor MK571. We conclude that RBM3 overexpression can promote a chemotherapy resistant stem‐like phenotype in colorectal cancer cells.
Dietary flavanols maintain endothelial homeostasis
Authors: Heiss Christian
Published: June 2021

Dietary intervention studies showed that flavanols can improve endothelial function. We investigated how dietary flavanols affect the balance of endothelial homeostatic regeneration and repair processes. We show in a randomized controlled clinical study that 1 month ingestion of a high flavanol drink (750 mg total flavanols) increased endothelial function (flow‐mediated vasodilation [FMD]) and decreased systolic blood pressure in patients with coronary artery disease. This increase correlated with an increase in the number of circulating angiogenic cells (CACs; CD34+/KDR+ and CD133+/KDR+). Furthermore, flavanols led to a decrease in the numbers of endothelial microparticles (CD31+/CD41‐; CD144+), markers of endothelial injury, as measured by flow cytometry. This was inversely correlated with FMD. Platelet microparticles (CD41+) remained unaffected. In vitro, low nanomolar concentrations of (−)‐epicatechin and flavanol‐rich plasma increased the migratory capacity of CACs and endothelial cells, closure of an endothelial scratch injury, and capillary network formation. Flavanol metabolites increased eNOS activity and led to vasodilation of aortic rings. In mice, epicatechin feeding over one week improved vascular function, angiogenesis (hindlimb ischemia), and aortic GSH levels. Our data lend evidence to the concept that dietary flavanols may help maintain endothelial homeostasis.
META060 activates omega‐3 fatty acid receptor GPR120, reduces weight gain, and increases insulin sensitivity in high‐fat diet fed mice
Authors: Konda Veera R. , Desai Anuradha , Darland Gary , Vroegrijk Irene O.C.M. , van Diepen Janna A. , Voshol Peter J. , Bland Jeffrey S. , Tripp Matthew L.
Published: June 2021

Long chain ω‐3 fatty acids exert anti‐inflammatory and insulin sensitizing effects through the activation of G protein‐coupled receptor 120 (GPR120). GPR120 is expressed in macrophages, intestinal L cells and adipocytes. Activation of GPR120 has been reported to reduce TAB1/TAK1 activation and to inhibit NF‐κB signaling pathway in macrophages. GPR120, through regulation of GLP‐1, plays a major role in glucose homeostasis and insulin sensitivity and may be a novel target for the treatment of type 2 diabetes. Previously, we reported that META060, a modified hop extract, improved arthritic symptoms in a mouse model of collagen induced arthritis. To investigate the effects of META060 on inflammation associated obesity, we examined the activity of META060 at GPR120 in vitro, and the effects of META060 in endocrine cell models. In this study, we identified that META060 activated GPR120 and inhibited inflammation. Moreover, META060 stimulated secretion of GLP‐1 in human intestinal L cells (NCI‐H716) and lipogenesis in adipocytes (3T3‐L1). META060 also was evaluated in a mouse model of high fat diet (HFD) induced obesity and diabetes. Mice supplemented for 14 weeks with META060 (100 mg/kg body weight) exhibited significantly less HFD induced weight gain, reduced fat accumulation in various organs, and improved insulin sensitivity. These data indicate that META060, a novel natural agonist for GPR120, acts as an anti‐inflammatory and improves insulin sensitivity in mice, and may have clinical application for type 2 diabetes. Supported by Metagenics Inc.
Compromised intestinal barrier induces adaptive immune responses that protect from colitis
Authors: Khounlotham Manirath , Kim Wooki , Peatman Eric , Koch Stefan , Nava Porfirio , Medina-Contreras Oscar , Nusrat Asma , Denning Timothy L , Parkos Charles A
Published: June 2021

Mice lacking Junctional Adhesion Molecule A (JAM‐A) exhibit enhanced intestinal permeability, bacterial translocation, and elevated numbers of colonic T and B cells and IgA, yet do not develop colitic disease. To study whether enhanced adaptive immunity compensates for increased intestinal permeability, we examined susceptibility of Jam‐a−/−Rag−/− mice to acute, injury‐induced colitis. Data revealed negligible contribution of adaptive immunity in mice with intact epithelial barriers; however Jam‐a−/− Rag−/− mice exhibited dramatically increased microflora‐dependent colitis compared to Jam‐a−/− controls. Specific depletion of T cells and neutralization of TGFβ revealed a protective role for TGFβ‐producing CD4 T cells in Jam‐a−/− but not Jam‐a+/+ mice. A major mechanism of TGFβ‐producing CD4 T cell‐mediated protection was via increased IgA production and consistent with this observation, Jam‐a−/−Iga−/− mice displayed markedly increased colitis. These data delineate a critical protective pathway involving TGFβ‐producing CD4 T cell‐induced IgA in response to epithelial barrier defects and mucosal injury‐induced intestinal inflammation. These findings suggest that, under conditions of preexisting intestinal barrier compromise, adaptive immune mechanisms play an important compensatory role that protects from colitis. This work is supported by NIH grants DK061379, DK59888 and AA017870.