bluuebunny/crossref_metadata_2025 huggingface dataset:
rHuEpo treatment increases exercise performance exclusively by increasing Hb mass
Authors: Lundby Carsten , Robach Paul , Rasmussen Peter , Boushel Robert , Calbet Jose AL
Published: June 2021

This study was performed to test the hypothesis that administration with recombinant human erythropoietin (rHuEpo) increases exercise performance by augmenting maximal oxygen transport. For this purpose eight subjects were hemodiluted isovolumic following 13 weeks of rHuEpo treatment. Oxygen delivery was increased at maximal exercise from 1777.0 ± 102.0 before rHuEpo treatment to 2079.8 ± 120.7 ml.min−1after treatment. Following hemodilution oxygen delivery was decreased to the pre value (1710.3 ± 138.1 ml.min−1). The a‐vO2 difference was increased following rHuEpo treatment at maximal exercise, the values being 166.4 ± 8.0 ml before treatment, 182.2 ± 6.7 after rHuEpo administration, and then decreased to 161.7 ml after hemodilution. At maximal exercise intensity leg VO2 increased from 1511.0 ± 130.1 ml.min−1 before treatment to 1793.0 ± 148.7 ml.min−1 with rHuEpo, and decreased after hemodilution to 1428.0 ± 111.6 ml.min−1. VO2max increased from 3950.0 ± 160.7 before administration to 4254.5 ± 178.4 ml.min−1 with rHuEpo, and decreased to 4059.0 ± 161.1 ml.min−1with hemodilution (P=0.22 compared to pre rHuEpo). The main finding in the present study is that rHuEpo exerts its main effects on performance through an increase in red cell mass. In addition the data demonstrate that mean arterial pressure increases because of increased blood viscosity and that this does not limit maximal cardiac output.
M‐cadherin Mediates Myogenic Differentiation by Suppressing Id2 via beta‐catenin
Authors: Wang Yan , Always Stephen E.
Published: June 2021

The purpose of this study was to determine if M‐cadherin mediates myogenic differentiation by modulating β‐catenin/Canonical Wnt signaling and suppressing Id2 transcription. C2C12 myoblasts were serum starved to induce myogenic differentiation. Myotube formation was confirmed by immunofluorescent staining of myosin heavy chain and calculation of the fusion index. M‐cadherin expression was either knocked‐down via RNA interference or overexpressed via transient transfection of M‐cadherin construct. Phospho‐ and total β‐catenin and GSK‐3β and total Id2 protein expression were measured by immunoblotting. C2C12 myoblasts had greater myogenic differentiation at high cell density than at low cell density in response to serum starvation. At high cell density, GSK‐3β‐dependent inhibitory phosphorylation of β‐catenin was increased and this was accompanied by downregulation of Id2 expression (both mRNA and protein), which could be rescued by knocking down M‐cadherin expression, resulting in reduced myogenic differentiation in response to serum starvation. Conversely, overexpression of M‐cadherin had the opposite effect. These data suggest that M‐cadherin mediates myogenic differentiation of C2C12 myoblasts by suppressing Id2 transcription via modulating β‐catenin transcription activity. Supported by NIH AG021530
Phosphorylation of muscle Akt, AS160, and S6K1 are reduced following 8 weeks of increased physical activity in fasting rats
Authors: Fry Christopher S , Glynn Erin L , Lujan Heidi L , DiCarlo Stephen E , Rasmussen Blake B
Published: June 2021

Overactivation of the mTOR pathway increases S6K1 activity resulting in an enhanced serine phosphorylation of IRS‐1 and an inhibition of insulin signaling in skeletal muscle. To characterize the effect of increased energy expenditure on regulatory proteins associated with both the insulin and mTOR signaling pathway we examined these proteins while in the fasting state. Soleus muscle was collected from 4 male Sprague‐Dawley sedentary rats and from 5 rats exposed to 8 weeks of free access to running wheels. We utilized immunoblotting methods to measure phosphorylation status and total protein abundance of mTOR, 4E‐BP1, Akt/PKB, S6K1, IRS‐1, TSC2 and AS160. Rats exposed to the chronic increase in energy expenditure tended to have a lower body weight as compared to sedentary rats (336±13g vs 356±30g, P=0.09), and soleus muscle weights were similar between groups (P>0.05). The phosphorylation status of S6K1 (Thr389), AKT/PKB (Ser473) and AS160 (Ser/Thr) were significantly reduced with an increase in energy expenditure (P<0.05). Phosphorylation of TSC2, 4E‐BP1, and IRS‐1 (Ser636/639) were unchanged. The total protein abundance of mTOR, S6K1, 4E‐BP1, Akt/PKB, TSC2, AS160 and IRS‐1 were unaffected with chronic energy expenditure (P>0.05). We conclude that a chronic increase in energy expenditure downregulates Akt signaling to AS160 and S6K1 phosphorylation in fasting skeletal muscle. Funded by NIH grants R01 HL074122 and R01 AR049877
Nutritional energy in the regulation of human muscle mTOR signaling following resistance exercise
Authors: Glynn Erin L , Fry Christopher S , Drummond Micah J , Dreyer Hans C , Dhanani Shaheen , Volpi Elena , Rasmussen Blake B
Published: June 2021

Resistance exercise (Ex) stimulates the mammalian target of rapamycin (mTOR) signaling pathway. The ingestion of leucine‐enriched essential amino acids and carbohydrate (EAA+CHO) 1 hr following Ex further stimulates mTOR signaling. The purpose of this study was to determine whether the addition of nutritional energy to a EAA+CHO stimulus following Ex enhances mTOR signaling. Sixteen male subjects were randomized to two groups containing an equivalent amount of EAA (∼20g) but different amounts of CHO. Both groups ingested the nutrient solution 1 hr after a bout of Ex. mTOR signaling was assessed by immunoblotting from muscle biopsy samples at baseline, immediately following, and 1 and 2 hr post‐Ex. There were no differences between groups for any proteins measured prior to nutrient ingestion. At 2 hr post‐Ex (following nutrient ingestion) the phosphorylation status of mTOR and its downstream effectors S6K1 and 4E‐BP1 increased with no differences between groups (P>0.05). However, Akt/PKB phosphorylation was higher in the extra energy group, likely due to increased blood insulin concentration (P<0.05). We conclude that mTOR signaling is not further enhanced, despite increased Akt activation, by the availability of additional energy (carbohydrates) following Ex as compared to a stimulus containing the same amount of EAA and approximately half the caloric energy.

Funded by NIH Grant # 5R01AR049877
Metabolic effects of electrical stimulation in C2C12 myocytes
Authors: Philp Andrew , Donnelly Kenny , Baar Keith
Published: June 2021

Members of the Peroxisome proliferator‐activated receptor (PPAR)‐γ coactivator (PGC) family are believed to control cellular metabolism and mitochondrial biogenesis. PGC‐1α is the most characterized of the family members with gain of function studies suggesting a role in slow fiber transition, mitochondrial biogenesis and glucose and fatty acid metabolism. Less is known about the role, if any, of the other two family members, PGC‐1β and PRC. The aim of this study was to investigate the activation of the PGC‐1 family members following electrical stimulation. Three hours of 1Hz stimulation resulted in an 81% increase in CaMKII (Thr 286) phosphorylation (P=0.003), a 43.5% increase in AMPK (Thr 172) phosphorylation (P=0.04) and a 23.1% increase in PKA (Thr 197) phosphorylation (P=0.12). None of the PGC family members were transcriptionally activated immediately following stimulation. PGC‐1α transcription peaked (4.37‐fold increase) at 6 hours whereas PGC‐1β and PRC were not significantly increased at any time point. These data indicate that low frequency electrical stimulation induces the activation of CaMKII, AMPK, and PKA and induces the transcription of PGC‐1α. This model is well suited for rapid identification of factors that are essential in the activation of PGC‐1α and mitochondrial biogenesis.
A novel mechanism for metformin in improving insulin signaling in skeletal muscle
Authors: Bikman Benjamin Thomas , Donghai Zheng , Cortright Ron N , Neufer P Darrel , Kane Daniel A , Anderson Ethan J , Woodlief Tracey L , Price Jesse W , Dohm G Lynis
Published: June 2021

One possible link between obesity and insulin resistance may be through the NF‐κB pathway‐associated serine kinase, IKK. Metformin is a widely used insulin‐sensitizing drug that has been shown to activate AMPK in skeletal muscle though it is unknown whether metformin mediates inflammatory activity in this same tissue. The aim of this study was to 1) determine the ability of metformin to attenuate IKK, 2) determine whether changes in AMPK are associated with IKK in skeletal muscle, and 3) examine whether changes in AMPK and IKK are consistent with increased insulin sensitivity, as evidenced by reduced phosphorylation of Ser307 on IRS1. Lean and obese Zucker male rats received either placebo or metformin by gavage daily for four weeks (four groups of seven). IP and Western blot analysis were used to determine levels of all proteins in white gastrocnemius (WG). pAMPK increased overall with metformin treatment (P<0.05). Metformin increased IκBα levels (P<0.05), indicating lower activity of IKK, and significantly reduced IRS1 Ser307 in the obese rats (P<0.001). Differences in IκBα levels between groups closely followed differences in pAMPK. From these data we conclude that metformin's inhibitory influence on IKK activity, as evidenced by elevated IκBα levels, results in meaningful differences in IRS1 Ser307 in the muscle from obese rats.
Relationships between oxidative stress, HIF‐1α transcription, erythropoietin and vascular endothelial growth factor during sustained hypoxia in humans
Authors: Pialoux Vincent , Mounier Rémi , Brown Allison D , Steinback Craig D , Poulin Marc J
Published: June 2021

The aim of this study was to investigate the relationships between reactive oxygen species (ROS), hypoxia inducible factor (HIF‐1α) transcription, and HIF‐1α target genes erythropoietin (EPO) and vascular endothelium growth factor (VEGF) in humans. Five healthy men (32±7 yrs, mean±SD) were exposed to 12 h of sustained hypoxia (PetO2=60 Torr). DNA oxidation (8‐Hydroxy‐2′‐deoxyguanosine, 8‐OHdG) oxidation protein products (AOPP), EPO and VEGF were measured in plasma and HIF‐1α mRNA was assessed in leucocytes before, and after 1, 2, 4, 6, 8, 10, 12 h of exposure to hypoxia.

HIF‐1α mRNA amount increased during the first 2h of hypoxic exposure (+ 68%; p=0.03), then returned to baseline levels. VEGF increased at 4h (+121%; p=0.02) whereas EPO increased progressively from 4h to 12h (+114%; p<0.01). AOPP increased continuously from 4 h (+ 69%, p=0.04%) to 12h (+ 216 %, p=0.03) of hypoxic exposure while 8‐OHdG increased after 6 h (+ 78%, p<0.01) and remained elevated until 12h. During the “acute” increase phase of HIF‐1α (i.e., between 0 and 2h), 8‐OHdG was positively correlated with HIF‐1α (r=0.55, p=0.02).

These findings demonstrate that hypoxia induces oxidative stress via an overproduction of ROS. Finally, this in vivo study in humans corroborates the previous in vitro findings demonstrating that ROS is involved in the stabilization of HIF‐1α transcription.

Supported by Alberta Heritage Foundation for Medical Research, Heart & Stroke Foundation of Canada, Canadian Institutes of Health Research.
Intermittent Hypoxia (IH)‐induced hypertension in mice requires NFATc3
Authors: de Frutos Garcia Sergio , Alo Dominique , Berry Tammy , Walker Mary , Kanagy Nancy , Bosc Laura Gonzalez
Published: June 2021

Sleep apnea (SA) is an intermittent respiratory arrest during sleeping associated with increased incidence of hypertension and peripheral vascular disease. Exposing rodents to brief periods of IH during sleep mimics the cyclical hypoxia/normoxia of SA. We have previously shown that IH induces systemic hypertension in mice, increases NFAT transcriptional activity in aorta (Ao) and mesenteric arteries (MA) and cyclosporin A (CsA), NFAT inhibitor, prevents IH‐induced hypertension. In addition, we have established that smooth muscle (SM) α‐actin and soluble guanylyl cyclase α‐1 (sGC) are positively regulated by NFAT to potentially maintain SM contractile phenotype. The goal of this study was to determine if NFATc3 mediates IH‐induced hypertension and its target genes. Mean arterial pressure (MAP) was measured in NFATc3 knockout (KO) and wild type (WT) mice exposed to air for 5 days and then up to 7 days of IH cycling (20 exposures/h to 5% O2/5% CO2) for 7 h/day. WT showed a significant elevation in MAP (Δ 23.5±6.1 mmHg) at every time point whereas MAP failed to increase in the KO (Δ −3.9±5.7). Expression levels of SM‐α‐actin and sGC were determined in MA and Ao of air, V‐IH and CsA‐IH mice (25 mg/kg/day). SM‐α‐actin mRNA was elevated in MA (2−ddCT 3.3±0.3) and Ao (2.0±0.3) of V‐IH mice and CsA significantly attenuated that increase (MA 2.1±0.2; Ao 0.7±0.2). sGC was only elevated in MA from V‐IH, suggesting that both genes are up‐regulated by NFAT during IH. These results indicate that IH‐induced increase in MAP requires NFATc3 and that NFATc3 may contribute to the vascular changes associated with IH‐induced hypertension. Supported by SDG AHA.
Mitochondrial ROS is involved in downregulation of hERG by hypoxia
Authors: Nanduri Jayasri , Wang Ning , Bergson Pamela , Yuan Guoxiang , Prabhakar Nanduri R
Published: June 2021

We previously reported that hypoxia attenuated hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we examined the role of reactive oxygen species (ROS) in hypoxia‐induced downregulation of hERG. Hypoxia elevated ROS levels and the time course of the response correlated with hERG downregulation. ROS scavengers restored hERG expression in hypoxia exposed cells. Treatment of cells with X/XO (500μm/5milliunits/ml) under normoxic conditions mimicked the effect of hypoxia. Cellular sources of ROS include inhibition of the electron transport chain (ETC) of the mitochondria as well as NADPH oxidases. Apocyanin (1mM) and DPI (10μM), NADPH oxidase inhibitors, were ineffective in preventing hypoxia induced down‐regulation of hERG protein. In contrast, stigmatellin (1μm) and myxthiazole (1μM), mitochondrial complex III inhibitors prevented hypoxia‐induced downregulation of hERG. More importantly, effects of hypoxia on hERG protein were markedly attenuated or abolished in ρ0 cells deficient in mitochondrial DNA. Moreover, hypoxia did not increase ROS levels in ρ0 cells. These results suggest that ROS generated by mitochondrial ETC are involved in hypoxia‐evoked downregulation of hERG protein. (Supported by NIH‐HL‐25830).
Profile of E‐cadherin mobility in the endothelial junction
Authors: Quadri Sadiqa K , Sun Li , Bhattacharya Jahar
Published: June 2021

The adherens junction protein, E‐cadherin determines lung endothelial (EC) barrier properties (2003. 278:13342‐9). To determine EC cadherin dynamics, we over expressed full length of GFP‐E‐cadherin in confluent monolayers of rat lung microvascular EC (RLMEC). Viewed by confocal microscopy, E‐cadherin fluorescence (Io) was patchy, being high (>100 grey levels) in some regions, but low (<100 grey levels) in 75±5% of the cell perimeter (mean±SE; n=10 cells). To determine E‐cadherin mobility, at different sites along the EC perimeter we determined the fluorescence recovery after photobleaching (FRAP) to 50% of Io. To rule out errors due to non‐specific bleaching, we corrected FRAP against fluorescence of reference landmarks. At regions of low E‐cadherin expression, FRAP was 40±6% of Io in 2.5 min. At regions of high E‐cadherin expression, FRAP was slower, being 27±4% of Io (P<0.01). We conclude that in a substantial part of the EC perimeter, E‐cadherin expression is relatively low and that in these regions E‐cadherin mobility is higher than in regions of high E‐cadherin expression. We speculate that E‐cadherin mobility decreases as the junctions stabilize and endothelial barrier properties increase (Support: HL36024).
Roscovitine Inhibits ERK1/2 Activation Induced by Angiotensin
Authors: Wen Jin‐kun , Li Ai‐ying , Han Mei
Published: June 2021

R‐Roscovitine (CYC202) is often as a biological tool in cell cycle and apoptosis studies. Our recent study showed that roscovitine significantly inhibited VSMC proliferation stimulated by angiotensin II (Ang II) or fetal bovine serum (FBS). However, the mechanism of action and the real targets of roscovitine in vascular smooth muscle cells (VSMCs) remain nuclear. To elucidate the molecular mechanism of action of roscovitine in VSMCs, in the present study, VSMCs were isolated from the thoracic aortas of male Sprague‐Dawley rats and treated with Ang II after pretreating VSMCs with roscovitine for 15 h. We found that ERK1/2 phosphorylation induced by Ang II is abrogated by pretreating VSMCs with roscovitine for 15 h. Pretreating VSMCs with roscovitine also inhibits Ang II‐induced c‐Jun expression and phosphorylation, leading to negative regulation of downstream gene angiotensinogen gene. Our data provided a possible molecular mechanism whereby the inhibitive effect of roscovitine on VSMC proliferation is mediated. This study provided the first evidence that roscovitine could inhibit ERK1/2 phosphorylation and block the c‐Jun induction and activation, consequentially inhibiting Ang II‐induced its precursor gene expression. The results give an evidence that roscovitine may be feasible and effective in treating cardiovascular diseases such as atherosclerosis and restenosis.
Exercise training decreases the size and alters the composition of the neointima in a porcine model of coronary restenosis
Authors: Fleenor Bradley Stephen , Ivey Jan , Casati Jennifer , Bowles Douglas K.
Published: June 2021

Exercise training reduces adverse events following coronary angioplasty in humans. Therefore, our aim was to determine if exercise reduces the size or alters the composition of the neointima (NI) in a porcine balloon coronary injury (BCI) model. Miniature Yucatan swine were randomly assigned to cage confinement (SED, n=7) or exercise (EX, n=8) training on a treadmill. After 4 months the animals underwent BCI of the left anterior descending (LAD) and left circumflex (LCX) arteries. The animals recovered for 2 days and resumed the previous protocol of SED or EX. Additionally on day 12 following the BCI, each animal received an intravenous injection of BrdU to label proliferating cells. At 28 days post BCI the animals were euthanized and the LAD and LCX were excised. Injured and uninjured sections from each artery underwent standard histological procedures and were stained with VVG, pico Sirius red and antibodies for BrdU and fibronectin. Our results demonstrate that exercise decreased lesion size and NI proliferation (−48%) in the LAD (p<.05) but not the LCX. Exercise significantly increased NI collagen (5.9%) and decreased NI fibronectin (−7.9%) in both arteries (p<.05). However, in the uninjured media collagen decreased in the EX (−5.2%, p<.05) with no effect on fibronectin. In conclusion, exercise may decrease post angioplasty events by reducing lesion size and altering NI composition.

Supported by: NIH HL 52490
Evidence for NK‐1 Receptors in the Thermal Hyperemic Response in Human Skin
Authors: Wong Brett J , Minson Christopher T
Published: June 2021

We tested the hypothesis that functional NK1 receptors are required for normal expression of the thermal hyperemic response in human skin. Nine subjects were equipped with four microdialysis fibers on the forearm. Sites received: 1) Ringer's solution as a control; 2) L‐NAME to inhibit NO synthase; 3) substance P (SP); or 4) SP plus L‐NAME. Skin blood flow was measured via laser‐Doppler flowmetry (LDF) and local heaters were used to control skin temperature. Sites 2 and 4 were perfused with L‐NAME for 30 minutes. Sites 3 and 4 were then perfused with SP and SP plus L‐NAME, respectively, for 15 minutes and the SP‐induced vasodilation was allowed to return to baseline. Once a stable baseline was achieved, local heaters were increased from 33°C to 42°C (0.5°C/5s). Cutaneous vascular conductance (CVC) was calculated as LDF/MAP and scaled to maximal vasodilation via nitroprusside. The initial peak in L‐NAME (43 ± 3 %CVCmax) and L‐NAME + SP sites (53 ± 3 %CVCmax) were significantly reduced compared to control (79 ± 3 %CVCmax) and SP sites (79 ± 2 %CVCmax; p<0.001 for both). The secondary plateau in L‐NAME (36 ± 4 %CVCmax) and SP + L‐NAME sites (56 ± 6 %CVCmax) were significantly reduced compared to control (86 ± 3 %CVCmax) and SP sites (91 ± 1 %CVCmax; p<0.001 for both). L‐NAME sites were significantly reduced compared to SP + L‐NAME sites (p<0.01). These data demonstrate NK1 receptor desensitization, alone or with NO synthase inhibition, significantly alters the cutaneous thermal hyperemic response.

Supported by NIH HL‐70928
Vascular hypercontractility to endothelin 1 in mice lacking endothelial PPARG
Authors: de Lange Willem Jacobus , Beyer Andreas M. , Halabi Carmen M. , Sigmund Curt D.
Published: June 2021

Peroxisome proliferator‐activated receptor gamma (PPARG), a ligand‐activated nuclear hormone receptor, plays an important role in modulating blood pressure. This is evidenced by observations that thiazolidinediones, synthetic PPARG ligands used as antidiabetic drugs, lower blood pressure, while certain PPARG dominant negative (DN) mutations cause diabetes and early onset severe hypertension in humans. As PPARG is expressed in the vasculature, we postulated that it may exert its effect on blood pressure by modulating vascular function and reactivity. In order to test this hypothesis, we generated mice in which PPARG is knocked out in the endothelium (PPARG−/−), using the Cre‐LoxP system. Vascular function was assessed by studying the response of aortas from ePPARG−/− mice to an array of vasodilators and constrictors under baseline conditions. Vasorelaxation in response to acetylcholine and sodium nitroprusside, as well as vasoconstriction to serotonin and protaglandin F2a were normal. Endothelin 1 (ET‐1) mediated vasoconstriction was, however, significantly enhanced in ePPARG−/− mice. This finding is in step with high fat diet‐induced hypertension in ePPARG−/− mice reported previously, as well as findings that DN PPARG mutations cause ET‐1 mediated hypercontractility. Together these findings indicate that endothelial PPARG may play an essential role in modulating vascular reactivity to ET‐1.
Decreased sustained potassium current contributes to different firing patterns of celiac neurons in DOCA‐salt hypertensive and normotensive Rats
Authors: Wang Xiaohong , Kreulen David L
Published: June 2021

It is not known if changes in the electrophysiological properties of sympathetic ganglia neurons contribute to increased sympathetic activity in hypertension. Using whole‐cell patch clamp dissociated celiac neurons from hypertensive and normotensive rats were divided into three groups based on their action potential (AP) firing patterns to a constant suprathreshold depolarizing current (100pA; 400 ms). Phasic neurons fired several APs and adapted quickly; tonic‐phasic neurons fired several APs and adapted slowly; tonic neurons fired continuously. The distribution of these three groups was significantly different in neurons from hypertensive rats. There were more phasic neurons, less tonic‐phasic neurons and similar amount of tonic neurons in hypertensive rats. The firing frequency in tonic neurons was higher in hypertensive rats compared to normotensive rats. Potassium channel currents were tested to explore the reason of these neuronal firing changes in hypertension. Sustained potassium current was significantly lower in celiac neurons from hypertensive rats compared to those from normotensive rats. The M‐type and A‐type potassium currents were not different between hypertensive and normotensive rats. In normotensive rats, 23.44mM tetraethylammonium (TEA) reduced the amplitude of sustained potassium currents by 50%. TEA also decreased the firing frequency in tonic neurons and converted tonic‐phasic neurons to phasic neurons. The different celiac neuronal firing patterns contributed by decreased sustained potassium current may play a compensatory role in elevated sympathetic activity in hypertension.

Support HL70687
OXIDATIVE STRESS IN SYMPATHETIC PREMOTOR NEURONS CONTRIBUTES TO RENOVASCULAR HYPERTENSION
Authors: Oliveira‐Sales Elizabeth Barbosa , Nishi Erika Emy , Boim Mirian Aparecida , Dolnikoff Miriam Sterman , Bergamaschi Cássia Toledo , Campos Ruy Ribeiro
Published: June 2021

Based on previous data, we hypothesized that overactivity of NAD(P)H oxidase‐derived reactive oxygen species and lowered activity of CuZnSOD, an endogenous antioxidant within the rostral ventrolateral medulla (RVLM), could contribute to 2K‐1C hypertension. Using real‐time PCR, we found that mRNA expression of NAD(P)H oxidase subunits (p47phox and gp91phox) was greater in 2K‐1C compared to the control group in the RVLM and CuZnSOD expression was similar in both groups. To test the functional significance of whether oxidative stress was involved in the maintenance of sympathetic vasomotor tone and blood pressure in 2K‐1C, we injected (1 and 5 nmol/100nl) of superoxide dismutase mimetic (Tempol) into the RVLM. In 2K‐1C, Tempol ‐1nmol resulted in a higher fall in mean arterial pressure (MAP) than the other dose (15 ± 1%, P<0,05) followed by a significant reduction in renal sympathetic nerve activity (RSNA) (11± 2%). Tempol 5 nmol decreased MAP 12± 4% (P<0,05) and RSNA 20 ±7% (P<0,05). In the control group, all doses of Tempol did not change MAP, however, Tempol 1 and 5 nmol decreased RSNA (6 ± 2%, 2 ± 2%, respectively). Taken altogether, the results support the idea that an increase in oxidative stress within the RVLM plays a major role in maintaining hypertension and sympathetic drive in 2K‐1C. Supported by FAPESP.
Osteopontin Deficiency Attenuates Tubulointerstitial Injury in Angiotensin II‐Infused Mice.
Authors: Wolak Talya , Ren Yuelan , Kim Jason , Collins Alan , Hsueh Willa , Nicholas Susanne
Published: June 2021

Our aim was to examine the role of Osteopontin (OPN) in tubulointerstial injury induced by AngiotensinII (AngII). We infused OPN‐null (OPN−/−; n=24) and wild type (OPN+/+; n=22) mice with AngII (2.5μg/kg/min) or vehicle. Blood pressure (BP) and 24‐hour urine albumin‐to‐creatinine ratio (ACR) were measured. After 28 days, kidneys were sectioned for immunhistochemistry (IHC) and immunofluorescence (IF) to detect macrophage with MOMA‐2 and CD68 and epithelial cell transformation (EMT) with α‐smooth muscle actin (α‐SMA). Kidney cortex mRNA was used for MCP‐1.Kidney tubular epithelial (LLC‐PK1) cells were incubated with recombinant OPN (10nM) for 4, 8, 16 and 24 h (n=4) and then analyzed for TGFβ‐1 and α‐SMA protein. BP and ACR increased equally in AngII‐infused OPN+/+ and AngII‐infused OPN−/−. There was significantly lower tubulointerstial macrophage infiltration (by MOMA‐2; 1.6±0.2 vs. 2.8±0.2, p<0.01 and CD68; 0.58±0.15 vs. 2.18±0.25, p<0.01) and α‐SMA (4.9±0.5 vs. 9.1±0.6%, p<0.01), in the AngII‐infused OPN−/− vs. AngII‐infused OPN+/+. MCP‐1 mRNA was 4.5 fold higher in AngII‐infused OPN−/−vs. AngII‐infused OPN+/+ mice (p<0.05). Recombinant OPN increased α‐SMA and TGFβ‐1 expression in LLC‐PK1 after 8 and 24 hours of culture, respectively. The results of our study indicate that OPN recruits macrophages and enhances the expression of EMT markers. However, inflammation is modulated by down regulation of MCP‐1.
Long‐term cytokine blockade attenuates hypertensive response and protects against renal injury in spontaneously hypertensive rats
Authors: Elks Carrie M. , Haque Masudul , Mariappan Nithya , Guggilam Anuradha , Francis Joseph
Published: June 2021

Introduction: Recent findings support a role for cytokines in renal dysfunction. In this study, we examined whether cytokine blockade protects against renal injury in spontaneously hypertensive rats (SHR).

Methods: After measurement of baseline blood pressure, male SHR and WKY controls were treated with etanercept (ETN, 1 mg/kg/day), a recombinant protein which binds circulating TNF‐α; pyrrolidine dithiocarbamate (PDTC, 100 mg/kg/day), a NF‐κB blocker; or vehicle for 15 weeks. Blood pressure was measured weekly using tail plethysmography. Renal function was measured, rats were sacrificed, and renal tissue was collected for structural and functional mitochondrial assays. Oxidative stress in kidney medulla was measured using electron paramagnetic resonance (EPR). mRNA levels of cytokine and oxidative stress genes in renal tissue were measured using real time RT‐PCR.

Results: Untreated SHR had higher mean arterial pressure and demonstrated glomerular injury and decreased renal function. These animals also had increased expression of cytokine and oxidative stress genes, accompanied by mitochondrial dysfunction. Treatment with ETN or PDTC in SHR attenuated hypertension, renal damage, and mitochondrial damage.

Conclusion: Long‐term cytokine blockade protects against renal injury in SHR.
Effect of Exercise Training and Carvedilol Treatment on Cardiac Function and Structure in Sympathetic Hyperactivity‐Induced Heart Failure Mice
Authors: Medeiros Alessandra , Vanzelli Andrea Somolanji , Brum Patricia Chakur
Published: June 2021

The present investigation was undertaken to study the isolated effect of β‐blockers and exercise training (ET) on cardiac structure and function, and overall functional capacity in a genetic model of sympathetic hyperactivity induced heart failure in mice (DKO). DKO and wild type (WT) were studied from 3 to 5 mo of age, and randomly assigned into control, carvedilol‐treated and exercise‐trained groups. Eight weeks of carvedilol treatment or ET (sessions of 60 min, 5 days/wk) were performed. At 3 mo of age, no significant difference in FS or exercise capacity was observed between WT and DKO mice. At 5 mo of age, DKO presented exercise intolerance and higher HR associated with reduced FS. In addition, cardiac myocyte hypertrophy, ventricular fibrosis and capillary rarefaction were observed. ET and carvedilol similarly improved FS of DKO mice. The effect of ET was mainly associated with an improved exercise tolerance and improved skeletal muscle capillary density while β‐blocker therapy had a greater impact on cardiac reverse remodeling. Collectively, these data provide direct evidence for the beneficial effects of ET and carvedilol in DKO mice preventing cardiac dysfunction. The different mechanisms associated with beneficial effects of ET and carvedilol empowers future studies associating both therapies.
Tissue Doppler indices of cardiac contractile function during whole‐body heat stress
Authors: Brothers R Matthew , Bhella Paul S. , Shibata Shigeki , Wingo Jonathan E. , Levine Benjamin D. , Crandall Craig G.
Published: June 2021

Previously we demonstrated that heat stress increases ejection fraction, suggestive of improved contractile function. However ejection fraction is highly load dependent and may be an imprecise measure of contractile function. Therefore, we sought to use tissue Doppler mitral annular systolic velocity as a myocardial specific and less load dependent index to test the hypothesis that heat stress improves contractile function when compared to normothermic conditions. Peak systolic septal and lateral mitral annular systolic velocities were measured during normothermic and heat stress (i.e., Δ internal temperature 1.0 ± 0.05°C) conditions in six healthy male volunteers. Normothermic measurements were performed following a 45‐minute supine rest period and always preceded the heat stress measurements. Heat stress significantly increased septal mitral annular systolic velocity (normothermia: 8.8 ± 0.4 cm/s; heat stress: 11.1 ± 0.8 cm/s; P=0.01) and lateral mitral annular systolic velocity (normothermia: 10.4 ± 0.9 cm/s; heat stress: 13.9 ± 1.5 cm/s; P=0.03) relative to normothermic conditions. These data support our previous findings suggesting that the contractile state of the left ventricle is improved during heat stress conditions.

Supported by NIH Grant NIH‐HL61388 & HL84072
In vivo assessment of resting and catecholamine stimulated cardiac function in rat models of low and high aerobic running capacity
Authors: Morgan Eric E. , Ways Justin A. , Cicila George T.
Published: June 2021

Our laboratory has previously identified inbred rat strains divergent for aerobic running capacity (ARC); low performing Buffalo (BUF) and high performing DA rats. Subsequent studies of isolated DA and BUF hearts showed a strong correlation between ARC and cardiac output (CO). In the present study, we tested the hypotheses that 1) there is increased in vivo basal LV function in DA compared with BUF rats and 2) the cardiac response to catecholamine stress is increased in DA vs. BUF rats. A high‐fidelity microtip pressure‐volume catheter was used to examine cardiac function in intact male BUF (n=13) and DA (n=12) rats at rest and during dobutamine infusion. Resting DA hearts outperformed BUF hearts as evidenced by a significantly higher cardiac output (39±2 vs 63±6 mL/min, P<0.05) and contractility index (136±4 vs 154±7 mmHg2/sec, P<0.05). Following dobutamine infusion (10μg/kg/min), CO was significantly greater in DA compared to BUF rats (50±2 vs 92±9 mL/min, P<0.05). DA rats showed a greater increase in stroke volume whereas BUF rats displayed a greater increase in heart rate following dobutamine infusion (Figure). We conclude that cardiac function is greater at rest in DA compared to BUF rats, and that DA and BUF hearts differ in their response to catecholamine stress.

This work was supported by NIH grant HL67276 (GTC) and a Medical Student Research Fellowship from The University of Toledo College of Medicine (EEM).
Field trials of a new physiological data logger in active fishes
Authors: Clark Timothy Darren , Frappell Peter B , Taylor Brian , Farrell Anthony , Hinch Scott , Seymour Roger S
Published: June 2021

We have designed and constructed implantable data loggers that measure heart rate (HR) and body temperature of free‐ranging animals. We present data from the first field trials of these loggers in penned southern bluefin tuna (Thunnus maccoyii) under aquacultural conditions, and in spawning sockeye salmon (Oncorhynchus nerka) under natural conditions. Bluefin tuna (~ 15.2kg) maintained visceral temperature (VT) between 0.4 – 2.6°C above ambient water temperature (18.5°C) during non‐digesting periods, while HR ranged from 33 – 74 beats/min. VT rose to 1.4 – 6.5°C above ambient at the peak of the digestive period, and the magnitude of this increase was dependent on ration size. HR followed a similar trend during the digestive period, reaching levels of 52 – 117 beats/min and implying that the postprandial increase in oxygen consumption is assisted largely by an increase in HR. The duration of digestion ranged from 10.7 to 30.9 h depending on ration size.

Sockeye salmon were implanted with data loggers in October 2007 when they had migrated from the ocean to their natal stream to spawn and subsequently die. Data analyses are ongoing, but it has been confirmed that salmon carrying data loggers do not have a reduced lifetime on the spawning ground, implanted salmon display normal courting and mating behaviours, and implanted salmon completed their lifecycle normally by spawning before death.
The two‐pore domain K+ channel K2P1.1 (TWIK‐1) is non‐functional in the heart
Authors: Kim Donghee , Kang Dawon
Published: June 2021

K2P1.1 mRNA is expressed in many regions including human and rat heart. Recent microarray studies suggest that K2P1.1 may be involved in atrial fibrillation, and knockout mice shows deficiency in phosphate/water transport in the kidney. These studies suggest that K2P1.1 would produce a functional K+ current. In the original study, K2P1.1 expressed a functional current, but a later study showed that K2P1.1 was silent because it was normally sumoylated. Thus, desumoylation or K274E mutation to prevent sumoylation was found to open K2P1.1 in oocytes and Cos‐7 cells. A more recent study found that K2P1.1 does not undergo the sumoylation process and that K274E mutation simply increases the current due to a charge effect. In our study, transfection of Cos‐7 cells with K2P1.1 or K274E mutant failed to produce K+ currents. In Cos‐7 cells or in rat cardiac myocytes that express K2P1.1 mRNA, desumoylation also did not activate any K+ currents. Causing oxidative stress using buthionine sulfoxime or hydrogen peroxide (that may alter sumoylation) also failed to cause activation of any new K+ channels in ventricular myocytes. These results support the view that K2P1.1 does not form a functional channel under different physiological conditions. The role of K2P1.1 in cardiac and renal function remains to be determined.
MRI volumetry of the amygdala: an anatomic background
Authors: Brabec Jiri , Horinek Daniel , Krasensky Jan , Vaneckova Manuela , Seidl Zdenek , Petrovicky Pavel
Published: June 2021

MRI volumetry of amygdala is much disputed in literature. The complexity of amygdalar structure and the small differences in the intensities of the magnetic resonance signals for the temporal lobe structures makes it difficult to elaborate the methodology that would accurately reflect its true anatomic relations.

The goal of our study was to elaborate standard protocol for the MRI volumetry of the amygdala by means of classic neuroanatomic methods and MRI examinations in a sample of 70 healthy adults (18 – 99 years of age). On the basis of comparison of amygdalar cytoarchitecture and intensities of the MR signal, two methodological approaches emerged: classical approach and expanded approach.

Neither volumetric differences between two sexes nor interhemispheric differences were significant for absolute volumes of amygdala in either of the two approaches. A significant correlation between the size and age of the amygdala was not found.

In spite of the fact that it is not possible for clinicians to measure all necessary dimensions, another goal was to find a simplified measurement technique, which could serve as an indicator of the actual size of the amygdala. From a wide variety of approaches, a single measurement of the plane in the level of the most anterior tip of the hippocampus is very promising.

Supported by grants IGA MZ 2006 NR8931‐4, and No. 0021620806 from the Ministry of Education of the Czech Republic
S100A1 expression in mouse embryos
Authors: Cheung Siu Yuen , Wang Xia , Bao Li Hua , Heizmann Claus W , Chan Wood Yee
Published: June 2021

S100A1 protein is an EF‐hand type calcium‐binding protein with unique functions in adult tissues. However, its role in the embryonic development is largely unknown. To better understand its developmental role, we sought to determine the spatio‐temporal expression of S100A1 with reverse transcription‐PCR and specific immunohistochemical localization in normal and S100A1‐deficient mouse embryos which were generated previously by gene trap mutagenesis. The S100A1 mRNA started to express in normal embryos at E9.5 and was steadily increased until birth. At E15.5 and E17.5, S100A1 protein expression was observed in the heart, cartilages, bones, liver, lung and hindbrain. The expression was not detected in S100A1‐deficient embryos at all stages examined and S100A1‐deficient embryos did not exhibit any morphological anomalies. The S100B protein, which forms heterodimers with the S100A1 protein, was expressed in both normal and S100A1‐deficient embryos, indicating that the absence of the S100A1 protein did not influence S100B protein expression. Taken together, our results suggest that the widely expressed S100A1 protein in embryos may be a functionally redundant protein whose developmental functions can be supplemented by other proteins.

Acknowledgements: The work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK4421/05M).
Digital Atlas of Human Embryonic Development by High Resolution Imaging
Authors: YAMADA Shigehito , Anderson Stasia A , Samtani Rajeev R , Lee Elaine S , Lockett Elizaqbeth C , Uwabe Chigako , Shiota Kohei , Lo Cecilia W
Published: June 2021

Embryos undergo complicated morphogenetic changes during organogenesis. Detailed characterization of the remodeling will help in elucidating the etiology of the congenital anomalies. The analysis of such changes require the visualization of embryonic structures in three‐dimensions (3D). To generate high resolution 3D images through the period of organogenesis, human embryos from the Kyoto Collection at Carnegie stages 13 to 23 were imaged by magnetic resonance imaging (MRI) and with episcopic fluorescence image capture (EFIC) ‐ a novel high resolution histological technique that provides perfectly registered serial 2D images suitable for high fidelity 3D rendering. Embryos were first MRI scanned using a 7T super‐conducting magnet, followed by EFIC imaging. Obtained high‐resolution images were resectioned digitally in multiple planes, allowing detailed visualization of anatomic structures in the developing embryo. Using these data sets, we constructed a digital atlas, with each stage represented by serial 2D image stacks shown in three orthogonal planes. The Atlas also includes annotations delineating important anatomic structures and developmental milestones, as well as 3D Quicktime movies in different perspectives. This Atlas of the Human Embryo should serve as an invaluable resource for morphologic information of human development, and for studying the etiology of the congenital anomalies.
SYNTHESIS OF ESTROGENS AND ANDROGENS IN THE GONADS OF THE FETAL SHEEP
Authors: Gomez Yolanda , Infante Veronica Chavez , Ramirez Esther Bautista , Pardo Marta Romano
Published: June 2021

In this work we investigated the secretion of 17B‐estradiol (E2) and testosterone (T) in the gonads of ovine fetuses of 3.6 and 12 inches in crown‐rump (C‐R) length. The fetus were obtained immediately after the slaughtering of animals and transferred to phosphate‐buffer in cold. The gonads were carefully dissected and cells were obtained by dissociation trypsin treatment, the cells isolated were incubated in Dulbecco's medium plus 0.1 BSA during one hour to 370C. The secretion of E2 and T were measured by radioimmunoassay in the incubation medium of the cells.

The 17B‐ estradiol was the predominant estrogen produced by ovaries during the developing sheep gonad. The fetuses of 12 in showed the highest secretion of E2 58pg/mg protein whereas, the minimum secretion level of E2 were 10 pg/mg protein in the fetuses of 8 in. The amount of testosterone in these ovaries was not detectable in the previous fetuses until the 8 in fetuses an average of 15pg/mg protein and the secretion was maintain during all fetuses development.

Testosterone was the principal androgens synthesized by the testes. The maximum level of T secretion 600pg/mg protein was found in the feto 6.4 in. By the other hand, the E2 secretion was low during the process.

These results show that during the development of the ovine fetal gonads synthesizes estrogens and androgens, which strongly suggests the activity of P450‐aromatase and 17B‐HSD.

CONACYT 41423 Q
Meiotic recombination hotspot ade6‐M26: Phosphorylation‐independent regulation of Atf1‐Pcr1 heterodimer by SAP/MAP kinase Spc1
Authors: Gao Jun , Davidson Mari K , Wahls Wayne P
Published: June 2021

Homologous recombination occurs at a high frequency in meiosis, is clustered at hotspots, and aids chromosome segregation. In the fission yeast S. pombe, Atf1‐Pcr1 heterodimer (ATF/CREB proteins) binds to and activates a meiosis‐specific recombination hotspot, ade6‐M26. The stress‐activated protein kinase Spc1 (a homolog of human p38) phosphorylates Atf1 and is also essential for hotspot activity. We determined the roles of Atf1, Pcr1, and Spc1 in hotspot recombination. Deletion analyses revealed that only the bZIP (DNA binding/protein dimerization) domain of Pcr1 was required. In contrast, bZIP and non‐bZIP regions of Atf1 affected hotspot recombination. Remarkably, a mutant form of Atf1 that can not be phosphorylated by Spc1 kinase still activated the hotspot. Furthermore, artificial tethering of Atf1 to ade6 promoted recombination in the absence of its own bZIP domain, or Pcr1, or Spc1 kinase. Therefore: 1) An amino‐terminal “activation domain” of Atf1 is necessary and sufficient to promote hotspot recombination. 2) Pcr1 confers DNA binding site specificity to Atf1‐Pcr1 heterodimer in vivo, thus targeting the Atf1 activation domain to specific chromosomal sites. 3) Phosphorylation of Atf1 by Spc1 kinase is, unexpectedly, not required for hotspot activity. 4) The Spc1 kinase regulates hotspot recombination only at or prior to binding of Atf1‐Pcr1 to ade6‐M26 DNA.

Research supported by NIH R01 GM062244
Characterization of the hOGG1‐nucleosomal DNA ternary complex
Authors: Loh Ken H , Sokol Ethan S , Haushalter Karl A
Published: June 2021

Human 8‐oxoguanine DNA glycosylase (hOGG1) is a bifunctional DNA repair enzyme with both base excision and abasic site lyase activity, allowing it to repair DNA damaged by reactive oxidative species. The hOGG1 repair mechanism proceeds through a transient imine intermediate which can be reduced using sodium borohydride to a stable amine linkage. This allows for subsequent isolation and characterization of the binary repair enzyme:DNA complex. The hOGG1 mechanism is well understood for free DNA but little work has been carried out for nucleosomal DNA. Sodium borohydride trapping has been used to generate the hOGG1:nucleosomal DNA ternary complex. Characterization of the purified complex will provide insights into the possible mechanisms that DNA repair enzymes use to recognize and excise DNA lesions in nucleosomes.
A model of Chlorella DNA ligase substrate discrimination and binding analysed by surface plasmon resonance
Authors: Brooke Clare , Odell Mark
Published: June 2021

The recent structure of Paramecium bursaria Chlorella virus 1 (PBCV‐1) DNA ligase complexed with DNA highlights differences in the substrate engagement of this minimal ligase when compared with the multi‐domain human DNA ligase 1 (huLig1). HuLig1 consists of a catalytic core, equivalent to the Chlorella enzyme, with additional structural domains including a DNA‐binding domain (DBD). Chlorella ligase lacks a DBD but possesses a ‘latch’ region that stabilises DNA interaction.

We have studied binding of a number of Chlorella and huLig1 mutants with various synthetic DNA substrates; a double‐strand (ds) nick (5′PO4 juxtaposed to 3′OH), an intact ds duplex and a duplex containing a one‐nucleotide gap. Chlorella ligase has been shown by gel shift analysis to discriminate between nicked dsDNA and an intact duplex. We observe similar on rates for all DNA species however the ligase remains more tightly bound to nicked DNA. A polypeptide derived from huLig1 equivalent to the Chlorella ligase, shows poor substrate discrimination and a ∼15‐fold lower affinity for dsDNA compared to the Chlorella enzyme.

Alanine replacement of two conserved phenylalanine residues in the Chlorella enzyme significantly alters the DNA interaction profile. We surmise that following stabilisation of DNA by the latch the two phe residues interdigitate into the DNA sequentially, phe286 inserting first (closest to the 5′PO4) followed by phe75.
Identification of a sno‐like RNA in Haloferax volcanii
Authors: Williams Meghan , Nist Richard , Ginn‐Pease Margaret E. , Hemmingsen Jens , Daniels Charles J.
Published: June 2021

A putative sno‐like RNA (sRNA) has been identified and characterized in Haloferax volcanii (Hvo), a moderately halophilic archaeon. Based on its sequence characteristics and the mechanism of snoRNA‐mediated modifications, this sRNA would target 2’‐O‐ribose methylation of cytidine‐34 in Hvo methionine tRNA. Bioinformatics studies have identified the sRNA gene sequence in all known halophilic archaeal genomes. RT‐PCR and Northern blot analysis have detected expression of the sRNA with a length of 120 nucleotides. RT‐PCR results also show that the sRNA is initially transcribed as part of the 3’ untranslated region of an mRNA and is subsequently processed. A mutant strain of Hvo lacking the sRNA gene exhibits normal growth, indicating that the sRNA‐mediated modification is not essential for viability. This study advances understanding of RNA processing in archaea.