bluuebunny/crossref_metadata_2025 huggingface dataset:
Minimally invasive transverse aortic banding as a model to study heart disease in SD, SS and Mas−/− rat strains
Authors: Anene‐Maidoh Ogugua T. , Greene Andrew S.
Published: June 2021

Minimally invasive transverse aortic banding (MTAB) is a model for inducing ventricular dysfunction that has been used exclusively in mice. Our objective was to test the efficacy of the MTAB model in rats by using the Sprague Dawley, Dahl salt‐sensitive hypertensive and the recently developed Mas receptor knock out rat. The Mas receptor mediates the physiological effects of the angiotensin peptide ANG1‐7 and thus modulates cardiovascular function. A rat knockout of the Mas1 receptor was generated using zinc‐finger nuclease (ZFN) mediated engineering on the SS genetic background. A 10‐bp deletion in exon1 of the Mas1 gene led to a severely truncated protein and a loss of function. Cardiac function was assessed following MTAB using cardiac ultrasound, PV loop analysis, and histology.

Weekly echocardiography showed rat MTAB resulted in significant LV hypertrophy associated with a biphasic change in fractional shortening compared to sham controls. Six weeks after banding, acute measurements of LV pressures and volumes revealed significant changes in systolic and diastolic function associated with decreases in ventricular contractility and compliance. Histology showed increases in fibrosis. Differences in severity of disease between rat strains were observed suggesting MTAB combined with ZFN engineering can reveal functional cardiac effects and supports the role of the Mas1 receptor in cardiac fibrosis.

HL082798
Deletion of protein tyrosine phosphatase 1B (PTP1B) prevents type 1 diabetes‐induced endothelial dysfunction.
Authors: Herren David , Tremblay Michel L , Marrero Mario B , Stepp David W , Belin de Chantemele Eric J
Published: June 2021

PTP1B dephosphorylates receptors tyrosine kinase and acts as a brake on various signaling pathways. Deletion of PTP1B protects against metabolic dysfunction by increasing insulin sensitivity, but little is known about its role in the control of vascular function. We hypothesized that PTP1B deletion prevents vascular dysfunction in the absence of insulin. Insulin secretion was abolished in Balb/C and PTP1B‐deficient mice with streptozotocin (STZ) injection. 24 days after treatment, aortic reactivity was assessed via myography. STZ induced similar increases in glycemia in Balb/C and PTP1B KO mice (384±24 vs 444±21 mg/dL, Balb/C vs PTP1B). Phenylephrine (PE)‐induced constriction was increased by STZ treatment in Balb/C mice only (115±13 vs 136±6% of KCl, vehicle vs STZ). Endothelial function assessed via acetylcholine (ACh)‐mediated relaxation was not affected by PTP1B deficiency but was reduced by STZ in Balb/C mice (63±5 vs 43±5% of precontraction, vehicle vs STZ). In PTP1B KO mice, STZ treatment did not affect maximum relaxation (56±7 vs 58±7% of preconstriction, vehicle vs STZ) but increased ACh sensitivity (logEC50 7.4±0.3 vs 8.2±0.2, vehicle vs STZ). Western blot analysis revealed a trend towards increased baseline Akt activation without changes in eNOS expression or activation. These data indicate that PTP1B deletion protects against the harmful vascular effects of type I diabetes.
EGCG protects against oxLDL‐induced endothelial dysfunction by inhibiting LOX‐1‐mediated signaling
Authors: Ou Hsiu‐Chung , Tsai Kun‐Ling , Wu Yun Jhen , Chiu Tsan‐Hung , Lee Shin‐Da
Published: June 2021

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), originally identified as the major receptor for oxidized low‐density lipoprotein (oxLDL) in endothelial cells, plays a major role in the pathology of vascular diseases. In this study, we hypothesize that the most abundant polyphenolic compound in tea, epigallocatechin‐3‐gallate (EGCG), can down‐regulate parameters of endothelial dysfunction by modulating LOX‐1‐regulated cell signaling. In cultured human umbilical vein endothelial cells (HUVECs), exposure to oxLDL increased the expression of LOX‐1 at the RNA and protein levels. OxLDL rapidly activated the membrane translocation of Rac1 and p47phox and the subsequent induction of reactive oxygen species generation. It also increased p38MAPK phosphorylation and decreased the phosphorylation of Akt as well as nuclear translocation of NF‐κB. In addition, oxLDL diminished the expression of endothelial NO synthase, enhanced the expression of endothelin‐1 and adhesion molecules, and increased the adherence of monocytic THP‐1 cells to HUVECs. Pretreatment with EGCG, however, exerted significant cytoprotective effects. Results from this study may provide insight into a possible molecular mechanism by which EGCG suppresses oxLDL‐mediated vascular endothelial dysfunction. (This study was supported by grants from the National Science Council; NSC98‐2320‐B‐039‐020‐MY3).
Baicalein attenuates oxLDL‐induced oxidative functional damages in endothelial cells
Authors: Lin Chun‐Chao , Ou Hsiu‐Chung , Pi I , You Ya‐Ching
Published: June 2021

Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized low‐density lipoprotien (oxLDL) pormotes inflammatory and thrombotic processes. Baicalein, a flavonoid obtained from the root of Chinese medicinal herb Scutellaria baicalensis, possesses a multitude of pharmacological activities including anti‐oxidant, anti‐inflammation and anti‐tumor. In this present study, we tested the hypothesis that baicalein could protects vascular endothelial cells against the proatherosclerotic stressor oxLDL and explored the possible mechanisms. Human umbilical vein endothelial cells (HUVECs) were incubated with baicalein (2.5–20 μM) for 2 hours and then incubated with oxLDL (150 μg/ml) for an additional 24 hours. Our results showed that baicalein ameliorated the oxLDL‐diminished eNOS protein expression and reduced the oxLDL‐induced adhesion molecules and the adherence of monocytic THP‐1 cells to HUVECs. Furthermore, several apoptotic features caused by oxLDL including ROS production, intracellular calcium accumulation, mitochondrial membrane potential collapse, disturbed the balance of bcl‐2 family proteins, and triggered subsequent cytochome c release to cytosol and activation of caspase‐3 were attenauted by pretreatment with baicalein. Findings from this study suggest that baicalein may have clinical implications in the prevention of atherosclerotic vascular disease.
The effects of a multiflavonoid supplement on blood pressure and oxidative stress
Authors: Campbell Kristen Joanne , Kappus Rebecca M. , Nieman David C. , McAnulty Steven R. , Cook Carol , Hubner Martin , Collier Scott R.
Published: June 2021

Antioxidant supplementation has been shown to reduce blood pressure. Moderate intensity aerobic exercise also elicits a reduction in blood pressure immediately following exercise, termed post exercise hypotension (PEH). The purpose of this study was to test the effects of a multi‐flavonoid supplement plus exercise on PEH following a 2‐week dosing period.

Methods

12 normotensive subjects between the ages of 18–24 were randomized into either a placebo or a multiflavonoid supplement. Each subject was studied pre, immediately before and after exercise and at 30 minutes post‐exercise both prior to and following 2 weeks supplementation. Blood was taken both pre and immediately following the exercise sessions and was assayed for ferric reducing antioxidant power (FRAP) and the oxygen radical absorbance capacity (ORAC).

Results

Significant decreases in systolic blood pressure in the treatment vs. placebo group (−3 ± 0.44 mmHg vs. −0.3 ± 0.56 mmHg, respectively, interaction p = 0.05,) and significant increases in FRAP (742 ± 41 to 956 ± 46 μmol/L vs. 658 ± 41 to 670 ± 46 μmol/L, interaction p=0.024) were found following supplementation and no differences in the control group. ORAC produced no significant differences with supplementation.

Conclusions

These data suggest that Q‐EGCG supplementation increases FRAP in young normotensives which may be a mechanism for the resting blood pressure reduction.
Membrane fatty acid composition predicts lipoxidative stress in the rat and human myocardium
Authors: Collins Nathaniel B , Mulligan Christopher M , Le Catherine H , Routh Melissa A , Sterrenberg Timothy R , Bouma Gerrit J , Medway Allen , Chicco Adam J
Published: June 2021

Peroxidation of membrane polyunsaturated fatty acids is a hallmark feature of myocardial injury associated with aging and heart failure. We examined the extent to which changes in myocardial membrane fatty acid composition predict markers of lipoxidative stress associated with aging and heart failure in the rat and human myocardium. In Fisher 344 rats, aging was associated with a marked desaturation of myocardial phospholipids, represented by increases in 22:6 and the 20:4/18:2 ratio that correlated closely with increases in malondialdehyde (MDA) and 4‐hydroxnonenal (HNE)‐protein adducts. These phenomena occurred at an earlier age and to a greater extent in SHHF rats genetically predisposed to hypertensive cardiomyopathy. No independent effect of aging on these parameters was observed in human hearts, but the presence of dilated cardiomyopathy significantly increased membrane 22:6 content, which closely paralleled increases in MDA. No robust or consistent relationships were found between glutathione peroxidase‐4 or superoxide dismutase (SOD) isozyme protein content and MDA or HNE, though both tended to correlate positively with MDA in rats and humans, and SODs correlated positively with 22:6 in the human heart. In conclusion, the desaturation of membrane phospholipids may represent an important contributor to lipoxidative stress in aged and/or failing myocardium. Supported by a grant from the AHA.
Gene expression differences in the brachial and femoral arteries of Rapacz Familial Hypercholesterloemic swine at birth are related to atherosusceptibility
Authors: Bahls Martin , Bidwell Christopher A. , Juan Hu , Granada Juan F , Krueger Christian G , Reed Jess D , VanAlstine William G , Newcomer Sean C
Published: June 2021

The mechanisms underlying the greater prevalence of atherosclerosis in the femoral compared to brachial arteries of both humans and quadrupeds are still unclear. We hypothesized that brachial and femoral differences in genes associated with atherosclerosis occur during vascular development. To test this hypothesis five femoral and brachial arteries were harvested from one week old Rapacz swine. The presence of atherosclerosis was quantified through histological staining (SudanIV, VVG, H/E). Total RNA was isolated for microarray analysis using a Macherey‐Nagel NucleoSpin RNAII kit. Gene expression was quantified by Affymetrix GeneChip Porcine Genome Arrays. Bioconductor was used to run Robust Multi‐chip Average (RMA) expression values on a paired t‐test model. No atherosclerotic disease was detected in either the brachial or femoral arteries. Of the 16526 expressed genes, RMA analysis revealed that 71 genes were differentially expressed to a significant level (FDR<0.05 + min. 2fold difference) in the brachial and femoral arteries. 29 transcripts had higher gene expression in the femoral arteries, while 42 were higher in the brachial arteries. In conclusion, inherent gene expression differences between the brachial and femoral arteries of swine exist at birth and may contribute to the unequal distribution of atherosclerosis between these vasculatures later in life.
cGMP is involved in Cutaneous Vasodilation in Local Skin Warming in Humans
Authors: Kellogg Dean L , Zhao Joan L , Wu Yubo , Johnson John M
Published: June 2021

Nitric oxide (NO) dependent systems typically involve cGMP generation via NO activation of soluble guanylyl cyclase(sGC) as a step in vasodilation. We hypothesize that this mechanism participates in the vasodilation induced by increased local skin temperature (Tloc). We examined effects of a specific sGC inhibitor, ODQ, on increases in skin blood flow (SkBF) caused by increased Tloc. 3 intradermal microdialysis probes (MD) were inserted into forearm skin in 11 subjects. Skin blood flow was monitored by Laser‐Doppler Flowmetry (LDF). Local temperatures at the MD sites were controlled by specialized LDF probe‐holder/local‐heater units. The study began with perfusion of Ringer's at all sites, followed by 2%DMSO in Ringer's or 1mM ODQ dissolved in 2%DMSO/Ringer's at 2 separate MD sites. (Preliminary studies showed that 1mMODQ antagonized NO mediated vasodilation by sodium nitroprusside.) Ringer's was continuously perfused at the 3rd control site (CTR). In the protocol, Tloc was initially held at 34°C and then was raised to 41.5°C to cause local vasodilation. Finally, 58mM nitroprusside was perfused at all MD sites to cause maximal vasodilation for data normalization. Results: no SkBF differences were found between sites with Tloc=34°C (p>0.05 between sites). SkBF rose at all sites as Tloc was increased (p<0.05 vs Tloc=34C)). SkBF increase was attenuated at the ODQ treated site with Tloc=41.5°C (p<0.01 vs Ringer's or 2%DMSO sites). These data suggest that sGC activation participates in the vasodilation induced by increased local skin temperature. (supported by NIH Grant HL065599)
Impaired ATP release during exercise in older adults: potential mechanism for impaired skeletal muscle blood flow with advancing age
Authors: Kirby Brett S , Crecelius Anne R , Voyles Wyatt F , Dinenno Frank A
Published: June 2021

Aging is associated with impaired control of skeletal muscle blood flow during exercise. In young adults, circulating adenosine triphosphate (ATP) aids in the control of muscle vascular tone during exercise. We tested the hypothesis that increases in plasma [ATP]v and ATP release during forearm exercise are impaired in aging humans. We measured forearm blood flow (FBF; Doppler ultrasound) and [ATP]v, and calculated ATP release (FBF x [ATP]v) during 5, 15, and 25% MVC rhythmic handgrip exercise in 14 young (22±1) and 12 older (63±2 yrs) adults. Deep venous blood samples were mixed in stop solution to preserve [ATP]v for plasma measurement. FBF tended to be lower during 5% MVC and was lower during 15 and 25% MVC in older adults (P<0.05). At rest, there were no age‐group differences in [ATP]v (Y=214±16; O=183±35 nmol/L; P=0.4) or ATP release. [ATP]v increased above rest during all exercise intensities in young adults (P<0.05); in contrast there was no significant increase in [ATP]v in older adults. ATP release increased in a graded fashion in both age groups, however this increase was blunted ~50–60% at all exercise intensities in older adults (P<0.05 vs young). ATP release was related to FBF during exercise in both groups (r2=0.73). We conclude that the increase in [ATP]v and ATP release during forearm exercise is impaired in healthy older adults and relates to the observed declines in exercise hyperemia.

Supported by HL‐087952
Estrogen and sympathetic modulation of hindlimb blood flow variability in rats
Authors: Mattar Louis , Noble Earl , Shoemaker J. Kevin
Published: June 2021

Recent evidence suggests that males and females rely on different physiologic mechanisms to maintain blood flow and vasculature conductance. To test the hypothesis that sympathetic inputs affect hindlimb flow variability, the coefficient of variation (CV = standard deviation/mean) of hindlimb flow and conductance were measured in male and female animals of various rat strains (Sprague Dawley, Spontaneously Hypertensive and Wistar‐Kyoto). One thousand consecutive heart beats at baseline and during sympathectomy (25mg/kg Hexamethonium; Hex) were used to assess each CV. Female rats (any strain) exhibited greater CV in hindlimb conductance at baseline compared to males (grouped gender averages: 35±19AU vs. 12±11AU, respectively; P<0.05). A subgroup of female hypertensive animals that were ovariectomized (OVX) exhibited lower CV (25±21AU) compared to their intact counterparts (41±14AU; P<0.05), and OVX animals given estrogen (51±22AU; P<0.05). The variability in female animals was driven by changes in hindlimb flow, and was blunted (i.e. not different from males) following Hex (19±13AU vs. 10±6AU, n.s. between female and male animals respectively). Therefore, blood flow variability was greater in the hindlimb of female rats of various strains and this variability was related to both estrogen and sympathetic mechanisms.

Supported by CIHR.
Acute Antioxidant Consumption Improves Vascular Function in the Elderly
Authors: Wray D. Walter , Nishiyama Steven K , Harris Ryan A , McDaniel John , Fjeldstad Anette , Hayman Melissa A , Ives Stephen J , Richardson Russell S
Published: June 2021

Oxidative stress has been implicated in the age‐related decrement in vascular health, and may contribute to the reduced vasodilatory capacity in the elderly. To examine the impact of oxidative stress on age‐related vascular dysfunction, 27 young (26 ± 1 yrs) and 20 older (71 ± 2 yrs) healthy volunteers partook in a flow‐mediated vasodilation (FMD) protocol following consumption of either placebo or an antioxidant (AO) cocktail (Vitamin C, E, and Alphalipoic acid). Ultrasound Doppler was utilized to determine blood velocity and brachial artery diameter, and reactive hyperemia was quantified to normalize FMD for the shear stimulus. An age‐related decline in brachial artery vasodilation was observed (6.7 ± 0.7, young vs. 4.9 ± 0.3 %, older), but this age difference was no longer significant after FMD was normalized for shear rate (0.31 ± 0.03, young vs. 0.27 ± 0.03 %/s‐1•s). Following AO consumption, FMD improved in the elderly (4.9 ± 0.3 to 7.7 ± 0.7 %, placebo vs AO) but tended to decline in young (6.7 ± 0.7 to 5.7 ±0.7, placebo vs. AO), effects which remained after shear rate normalization. These data demonstrate that consumption of an AO cocktail at over‐the‐counter doses acutely improves endothelial function in the elderly, restoring brachial artery vasodilation to that of their younger, healthy counterparts. These findings thus support a possible role of AOs in the maintenance of vascular health in the elderly.
Nitrite supplementation reverses vascular endothelial dysfunction in old mice via improved nitric oxide bioavailability
Authors: Sindler Amy L , Calvert John W , Fleenor Bradley S , Zigler Melanie L , Lefer David J , Seals Douglas R
Published: June 2021

Aging causes vascular endothelial dysfunction as indicated by impaired endothelium‐dependent dilation (EDD), and this is mediated by reduced nitric oxide (NO) bioavailability. Administration of nitrite (NO2−), a product of NO oxidation, is associated with enhanced NO bioavailability. We hypothesized that treatment with NO2− would enhance EDD with aging. Old (27 mo, n = 7) adult C57 male mice had a lower ex vivo carotid artery acetylcholine (ACh)‐induced EDD (70.9 ± 3.7 vs. 94.0 ± 2.9%, P < 0.05) and NO component of EDD (mean dilation to ACh + L‐name minus mean dilation to ACh alone, −26.6 ± 5.9 vs. −50.5 ± 9.5%, P < 0.05) compared with young adult controls (6 mo, n = 6). In old mice (n = 4), 3 weeks of dietary supplementation with sodium NO2− (50 mg/l in drinking water) restored EDD (96.6 ± 1.9%, P < 0.05) and its NO component (−60.2 ± 5.0%, P < 0.001). Sensitivity to ACh and endothelium‐independent dilation were not different in young, old and old NO2− supplemented mice. These preliminary results support the idea that NO2− supplementation may be an effective strategy for reversing vascular endothelial dysfunction with aging by enhancing NO bioavailability.

Work supported by NIH R37 AG013038, HL 007822
Age‐ and limb‐related differences in the vascular response to limb dependency in humans
Authors: Monahan Kevin D. , Solaiman Adil Z. , Snyder Katherine A.M. , Sielatycki John A. , Feehan Robert P. , Wilson Thad E.
Published: June 2021

To determine if age‐ and limb‐related differences exist in the vascular response to limb dependency we studied 9 young (25±1 yrs, mean±SE) and 7 older adults (65±3 yrs). We hypothesized that vasoconstriction (VC) during limb dependency would be greater in the leg than arm and attenuated with age. Supine subject's arms and legs were lowered 30 cm below heart level while measurements of skin blood flow velocity (BFV; laser Doppler flowmetry) and limb BFV (brachial and popliteal arteries) were obtained. Limb dependency induced significant VC (%Δvascular resistance; MAP/MBV) in the arm (Δ27.5±8.2%) and leg (Δ49.8±8.1%) of young adults although the magnitude of this response was greater in the leg (P<0.05 arm vs. leg). In older adults limb dependency induced significant VC in the arm (Δ23.6+6.8%) and leg (Δ22.4±4.3%; P>0.05 arm vs. leg). Leg VC during limb dependency was attenuated with age (Δ49.8±8.1% vs. Δ22.4±4.3% in young and older, respectively; P<0.05). In contrast skin VC during limb dependency was similar in the arm (Δ165±27% and Δ156±29% for young and older, respectively) and leg (Δ167±27% and Δ162±23%; all P<0.05 from baseline) of young and older adults. These findings identify important age‐, limb‐, and possible tissue‐related differences in the vasoconstrictor response to limb dependency, which may be attributable to altered myogenic and or venoarteriolar responses.

Support: NIH HL92309 and AG24420
Food insecurity is a risk factor for elevated levels of depression symptoms among low‐income pregnant Latinas
Authors: Hromi‐Fiedler Amber , Bermúdez‐Millán Angela , Chapman Donna , Segura‐Pérez Sofia , Damio Grace , Melgar‐Quiñonez Hugo , Pérez‐Escamilla Rafael
Published: June 2021

The objective of this study was to determine predictors of elevated levels of prenatal depression symptoms among low‐income Latinas (N=134). Depression symptoms were assessed using the 20‐item Center for Epidemiologic Studies Depression Scale (CES‐D). 24.1% reported elevated levels of depressive symptoms (cutoff value ≥ 24). Multivariate logistic regression was used to determine significant predictors (p<0.10) of elevated levels of prenatal depression symptoms. Participants who experienced food insecurity during pregnancy, measured by an adapted version of the US Household Food Security Survey Module, were 2.7 times more likely to experience elevated levels of prenatal depression symptoms compared to those who were food secure (95% CI: 1.01–7.09). Other risk factors for elevated levels of prenatal depression symptoms were lower levels of social support, experiencing heartburn, self‐perceived poor/fair health, being Puerto Rican, and lower maternal age. These findings suggest that food insecurity, self‐perceived health, and the presence of physical symptoms during pregnancy are important factors for clinicians to measure when evaluating depression risk among low‐income pregnant Latinas. Funded by Connecticut NIH EXPORT Center for Eliminating Health Disparities among Latinos (NIH‐NCMHD Grant # P20MD0017650).
THE CARDIAC ANTI‐HYPERTROPHIC EFFECT OF AN ORALLY ACTIVE MAS AGONIST IS ASSOCIATED TO A DECREASE IN BETA‐ARRESTIN IN DOCA‐SALT HYPERTENSIVE RATS.
Authors: Santiago Nívia Maria , Honorato‐Sampaio Kinulpe , Isoldi Mauro C , Santos Robson AS , Campagnole‐Santos Maria J
Published: June 2021

In addition to the role of β‐arrestin in AT1 receptor second messenger desensitization, more recent evidence revealed that β‐arrestins act as scaffolds for numerous signaling networks, such as the MAPKs. On the other hand, Ang‐(1‐7) anti‐trophic effect in the cardiomyocytes can be mediated by alterations in the MAPKs pathways. In this study we evaluated the effect of AVE0991 (AVE), a nonpeptide Ang‐(1‐7) mimic, on the signaling pathways involved in the heart hypertrophy induced by DOCA‐salt hypertension (DSH). After 4 weeks of induction of DSH, rats received AVE (1mg/kg) by gavage for 2 weeks. AVE attenuated the DSH‐induced hypertrophy, as quantified by myocyte diameter measurements (Normotensive rats: 12.97±0.15 μm; DSH: 14.41±0.23 μm; DSH+AVE: 13.39±0.15 μm; n=3 each), without changing the increased blood pressure (tail cuff pletismography). DSH rats showed an increased mRNA expression (real time PCR) of β‐arrestin in the left ventricle (LV) (5.96±1.44 A.U. vs 1.01±0.07 A.U. in normotensive rats; p<0.05) that was blocked after AVE (0.84±0.13 A.U.). In addition, AVE induced an increased AT1 receptor expression in the LV (1.95±0.32 vs 1.01±0.09 in normotensive rats; p<0.05). No alteration in cardiac adenylate cyclase expression was observed. These data suggest that Ang‐(1‐7) may modulate DSH‐induced cardiac hypertrophy by altering the β‐arrestin pathway. Financial Support: CNPq and INCT
Renal epithelial sodium channel (ENaC) is required for blood pressure (BP) maintenance in pregnant rat
Authors: West Crystal , Masilamani Shyama
Published: June 2021

To define the molecular mechanisms responsible for Na retention, plasma volume expansion (PVE) and BP maintenance in pregnancy, we studied virgin (V), mid pregnant (MP, day 13/14) and late pregnant (LP, day 19/20) rats. We previously found αENaC (the rate limiting subunit for channel formation) is upregulated in MP and LP rats and in vivo ENaC activity (natriuretic response to ENaC blockade with benzamil (Bz), 0–3 hour urine collection) is increased in MP and LP vs V rats. To assess the importance of ENaC activity for these adaptations in pregnancy, P rats were placed on chronic Bz (iv via perfusion pump, 0.7 or 1.05 mg/kg adjusted to account for PVE as pregnancy progresses). This prevented the increase in in vivo ENaC activity in MP (MP=361±92; time control V=388±60 μM/hr, NS) and LP (LP=585±100; time‐control V=663±194 μM/hr, NS) rats. Compared to control rats, Bz reduced PV by 0.92±0.16 ml (p<0.05) in MP rats and by 0.697±0.13 ml in LP rats. Bz reduced BP (radio‐telemetry) in LP rats by 29.9±9.4 mmHg ( p<0.05) but not in V rats. LP rats receiving mineralocorticoid receptor (MR) blockade with eplerenone (epl) overnight, net UNaV was decreased to V levels (LP + epl=571±172, V=368±64, normal LP=985±118 * μM/hr, *=p<0.05 V vs normal LP). These data suggest that ENaC activity is required for PVE and BP maintenance in pregnancy and the mechanism of ENaC activation in pregnancy is via the MR. (Support: NIH K22 award and CHRB award)
Decreased Venous Contraction and Enhanced Relaxation and Nitric Oxide Production in the Inferior Vena Cava of Pregnant Rat
Authors: Xia Yin , Cost Diana V. , Khalil Raouf A.
Published: June 2021

Normal pregnancy (Norm‐Preg) is associated with increased cardiac output and plasma volume, partly due to increased nitric oxide (NO) production by endothelial cells in various arterial beds; however, the role of venous endothelium is unclear. We tested whether Norm‐Preg is associated with increases in venous relaxation and NO production. Segments of inferior vena cava (IVC) from Norm‐Preg (day 19) and virgin Sprague‐Dawley rats were used to measure isometric contraction/relaxation. The IVC was loaded with the NO‐sensitive DAF‐FM diacetate (DAF) for simultaneous measurement of NO production. The α‐adrenergic agonist phenylephrine (PHE, 10−5 M) and membrane depolarization by 96mM KCl caused less contraction in IVC of Norm‐Preg (20.64±8.75 and 230.07±31.40) than virgin rats (70.90±15.60 and 308.32±95.65 mg/mg tissue). Acetylcholine (Ach, 10−5 M) caused greater relaxation of KCl‐induced contraction in IVC of Norm‐Preg (46.85±12.95) than virgin rats (36.05±8.07%). Basal NO was slightly greater in IVC of Norm‐Preg (0.12±0.02) than virgin rats (0.10±0.02). In IVC precontracted with PHE, Ach (10−5 M) caused greater NO production in Norm‐Preg (0.37±0.08) than virgin rats (0.22±0.03). Thus Norm‐Preg is associated with decreased venous contraction and enhanced Ach‐induced relaxation and NO production, which could lead to more distensible veins and venous insufficiency during pregnancy.
Pre‐inspiratory and inspiratory hypoglossal motor output during hypoxia induced plasticity in the rat
Authors: Lee Kun‐Ze , Fuller David D
Published: June 2021

Respiratory discharge in the hypoglossal (XII) nerve is composed of pre‐inspiratory (pre‐I) and inspiratory (I) activity. Our first purpose was to test the hypothesis that hypoxia‐induced plasticity in XII motor output is differentially expressed in pre‐I vs. I XII bursting. Short‐term potentiation (STP) of XII motor output was induced in vagotomized and ventilated rats by isocapnic hypoxia. Both pre‐I and I XII discharge abruptly increased at beginning of hypoxia, and the relative increase in amplitude was much greater for pre‐I vs. I bursting. In addition, STP was expressed in I but not pre‐I bursting following hypoxia. Specifically, I activity remained elevated following hypoxia but pre‐I bursting abruptly returned to pre‐hypoxia levels. Our second purpose was to test the hypothesis that STP of I XII activity results from recruitment of silent XII motoneurons (MNs) vs. rate coding of active MNs. Single fiber recordings were used to classify XII MNs as I, expiratory‐inspiratory (E‐I), or silent based on baseline discharge patterns. STP of I XII activity following hypoxia was associated with increased discharge frequency in active I and silent MNs, but not E‐I MNs. We conclude that the expression of respiratory plasticity is differentially regulated between pre‐I and I XII activity. Moreover, both recruitment of silent MNs and rate coding of active I MNs contribute to STP of XII motor output following hypoxia.
Impaired intracellular calcium homeostasis during acute prolonged severe hypoxia in rat carotid body cells
Authors: Kim Insook , Yang DJ , Carroll John L
Published: June 2021

CB glomus cells depolarize in hypoxia, leading to calcium influx. As a first line defense against hypoxia, their response to prolonged, acute hypoxia is important. We studied the effects of 15 min of severe, acute hypoxia. CBs were harvested from 14–17 day rats, dissociated, loaded with fura‐2, and perfused with HCO3‐buffered salt solution (BSS) equilibrated with 21% O2+5% CO2 (normoxia, N), 0% O2+5% CO2 (severe hypoxia, SH), 1–2% O2+5% CO2 (moderate hypoxia, MH) or 20 mM KCl in N. Cells were exposed to 1 min KCl, 2 min SH, 15 minute SH and final 2 min SH, with 5 min recovery in between. Glomus cells were identified by morphology and response to KCl. Results (n>50): 100% of glomus cells exposed 2 min to SH exhibited a brisk rise in intracellular calcium ([Ca2+]i) and returned to baseline upon return to N. 15 min of SH resulted in a brisk rise in [Ca2+]i and decline to a lower steady state, but [Ca2+]i homeostasis was severely impaired, with persistent elevation of [Ca2+]i. Many cells responded again to hypoxia following SH, indicating viability. 15 min exposure to KCl showed a [Ca2+]i response profile nearly identical to 15 min SH, but [Ca2+]i homeostasis remained intact. Impaired [Ca2+]i handling was also observed with MH (PO2 ~ 8–9 mmHg) but much less with mild hypoxia (~ 15–18 mmHg). Preliminary results suggest that prolonged, acute hypoxia markedly impairs [Ca2+]i homeostasis in CB glomus cells, which may have clinical relevance.
Hyperoxia decreases brain derived neurotrophic factor (BDNF) production in the carotid body and brainstem and catecholaminergic neurons in nucleus tractus solitarii (nTS).
Authors: Mason Ariel Vanessa , Chavez‐Valdez Raul , Johnson Sheree , Gauda Estelle B
Published: June 2021

BDNF in the carotid body (CB) has trophic effects on the development of primary sensory afferents. Hyperoxic exposure (HYP) during early postnatal development causes hypoplasia of the CB, reduces sensory neurons, and ablates hypoxic chemosensitivity. We hypothesized that HYP would directly affect BDNF production in the CB and brainstem (BS) and tyrosine hydroxylase (TH) expression in the nTS.

Methods

Time‐dated SD rats were exposed to HYP (60% O2) or (21% O2) for 48h prior to delivery and for 7 or 14 days after delivery and then returned to 21% O2 until postnatal day 9, 16 or 22 when the CB and BS were harvested and processed for gene (qRT‐PCR) and protein expression.

Results

HYP exposure for 7days reduced BDNF mRNA levels by 80% and 50% in the CB and BS, respectively (p=0.002) along with a 90% reduction in protein expression (p=0.04) in both tissues. HYP also up‐regulated p75 receptor gene expression in BS (1.6‐fold, p=0.002) without significant change in TrkB receptor. BS BDNF mRNA levels decreased from day 9 to 16 (p=0.04) but was not different between HYP and control animals at day 16. At day 22, there was a reduction in TH‐ir+ neurons and in TH protein levels (15%) in the nTS region of animals exposed to HYP, while no change in TH protein levels was observed in the locus coeruleus.

Conclusion

The reduction in TH+ neurons in the nTS in hyperoxic exposed animals may be linked to reduced BDNF production in the CB.

Supported by NIH R01HL080725
Neurotrophic Factors and the Growth of the Carotid Body
Authors: Kostuk Eric Wildon , Pichard Luis , Shirahata Machiko
Published: June 2021

Hypoxia is potentially a life‐threatening event for humans and animals. In the mammal, the carotid body is a major sensory organ detecting hypoxia. The carotid body function in developing animals differs from that of adult animals. The response of the carotid body to hypoxia is minimal at birth, and then gradually reaches the adult level. Environmental factors as well as genetic factors appear to play a role in the development of the carotid body function and morphology. Our previous studies have shown the vast morphological and genetic differences of the carotid body between the DBA/2J and A/J mice (4‐week old). Interestingly, the morphological differences of the carotid body are not apparent in 1‐day and 1‐week old mice. We hypothesize that neurotrophic factors contribute to the differential growth of the carotid bodies in these strains of mice. Thus, we examine the expression levels of glial cell line‐derived neurotrophic factor (Gdnf) and its family ligand (neurturin: Nrtn) and their receptors (Gdnf family receptor‐α1 and α2: GFR‐α1, GFR‐α2; Ret receptor) in the carotid body during postnatal development. Carotid bodies were harvested from deeply anesthetized mice (2‐day, 1‐week, 2‐week and 4‐week old), and real‐time PCR were performed. At 2‐day and 1‐week old, expression of all these genes was not significantly different between the strains. Gdnf expression was significantly higher in DBA/2J mice than A/J mice at 2‐week and 4‐week old. The results suggest that GDNF may be important for the growth of the carotid body and that 2‐week age may be a critical window for the growth of the carotid body in mice.

Supported by HL81345.
Femoral occlusion increases hypoxia‐inducible factor‐1α in dorsal root ganglion and hindlimb muscle
Authors: Gao Wei , Li Jianhua
Published: June 2021

Hypoxia inducible factor‐1 (HIF‐1) has an important contribution to pathophysiological changes of homeostasis under conditions of oxygen deprivation as well as ischemia. We employed western blotting methods to examine effects of femoral occlusion on protein expression of HIF‐1α in dorsal root ganglion (DRG), different fiber types of muscle i.e. oxidative and glycolytic muscle. DMOG was used to enhance HIF‐1α activity following local intramuscular injection (IM). Our data show that HIF‐1α was increased in lumbar DRG after formal artery ligation by using a rat model in different time courses as compared with sham control (3.4±0.5 vs 1.1±0.1 fold at 6 hrs; 3.1±0.4 vs 1.2±0.2 fold at 24 hrs; 2.3±0.4 vs 1.1±0.2 fold at 72 hrs; p<0.05). This effect was mimicked by IM of DMOG (5.0±0.3 vs 1.3±0.2 fold at 24 hrs, p<0.05) in lumbar DRG. In contrast, HIF‐1α outside lumbar DRG was not altered, which suggests HIF‐1α was increased due to specifically localized hindlimb. In addition, HIF‐1α was increased in both red and white muscle after femoral occlusion compared with non‐occlusion control; however, the level of HIF‐1α was greater in white muscle as compared with red muscle. In conclusion, femoral occlusion increases HIF‐1α expression in DRG, and differentially increases HIF‐1α expression in different fiber types of muscle. We anticipate that HIF‐1α is likely to contribute to autonomic responses to activation of muscle afferents.
Non‐NAD(P)H‐oxidase generated superoxide contributes to aortic relaxation in a physically inactive type II diabetic rat
Authors: Bunker Aaron , Arce‐Esquivel Arturo , Laughlin M. Harold
Published: June 2021

We tested the hypothesis that physical inactivity (SED) leads to impaired endothelium‐dependent relaxation (EDR) in Otsuka Long‐Evans Tokushima Fatty (OLETF) rat aorta and decreased superoxide scavenging contributes to impaired EDR. OLETF and control rats were divided into SED and physically active (PA, via running wheels) groups (n=5–8) at 4 weeks of age (W). At 40W acetylcholine (Ach) and sodium nitro‐prusside (SNP) were used to examine EDR in aortic rings in vitro. To evaluate the role of nitric oxide, superoxide dismutase, and NAD(P)H‐oxidase; EDR was evaluated following aortic treatment with N‐nitro‐L‐arginine methyl ester, TEMPOL (TEMP), or Apocynin (Apo), respectively. TEMP, but not Apo, reduced Ach EDR (p<0.05, 10−5–10−4M) and SNP induced relaxation (p<0.05, 10−9–10−4M) in OLETF SED. This effect was not observed in OLETF PA or control groups. 40W OLETF SED developed hyperglycemia and obesity (169±9.4mg/dl, 32.5±0.77%body fat) versus OLETF PA (104.7±4.6mg/dl, 12.4±0.87%), control PA (87.5±2.9mg/dl, 9.1±0.49%), and control SED (98.5±5.9mg/dl, 13.1 ±0.84%). OLETF SED rats did not demonstrate significantly impaired EDR compared to OLETF PA or control groups. We conclude that non‐NAD(P)H‐oxidase generated superoxide contributes to Ach and SNP induced aortic relaxation in the OLETF SED and that PA alone was not sufficient to maintain OLETF endothelial function. Grant support: HL36088 and HL52940
Physical inactivity rapidly alters glycemic control in young, lean, previously active volunteers
Authors: Mikus Catherine Renee , Thyfault John P
Published: June 2021

Postprandial hyperglycemia is a better predictor of cardiovascular disease (CVD) and all cause mortality than fasting blood glucose (FBG) or hemoglobin A1c, regardless of diabetes status. Further, physical inactivity (PIA) is strongly associated with insulin resistance (IR) and CVD. To determine whether transitioning from a high to low level of physical activity (PA) adversely affects glycemic control (GC), we equipped young (30 ± 1 y), lean (24 ± 1 kg·m−2), healthy, physically active (>10,000 steps·d−1) volunteers (N=10) with continuous BG monitors for 3 d during separate ACTIVE (habitual PA) and INACTIVE (≤ 5,000 steps·d−1) phases (diet replicated across phases). During the INACTIVE phase, PA decreased from 15,758 ± 1,103 to 4,334 ± 294 steps·d−1, FBG did not change (84 ± 3 to 87 ± 1 mg·dL−1), fasting insulin increased (4 ± 1 to 6 ± 1 μIU·mL−1; p = 0.02), and, when BG responses to all meals were pooled, the amplitude of glycemic excursions (post‐ minus pre‐meal BG; an index of GC) increased at 30 (16 ± 4 to 30 ± 4 mg·dL−1; p = 0.02) and 60 min (8 ± 2 to 22 ± 5; p = 0.04) post‐meal. We conclude that brief, modest reductions in PA rapidly diminish GC in young, lean individuals, suggesting PIA may: 1) play a key role in the development of IR by impairing GC, 2) cause aberrations in GC prior to changes in fitness and adiposity, and 3) be a useful tool to study early events in the etiology of IR.

Support: MU iCATS, T32 AR048523 (CRM), VHA CDA (JPT).
Insulin Resistance‐Independent Impairments to Arterial Endothelial Function with Depressive Symptoms in Mice
Authors: Beckett Jordan A.L. , James Milinda E. , Stapleton Phoebe A. , Goodwill Adam G. , d'Audiffret Alexandre C. , Frisbee Jefferson C.
Published: June 2021

Previous studies have associated the presence of depressive symptoms with evolution of impaired vascular function. To determine contributing mechanisms, male mice were exposed to 8 weeks of unpredictable chronic mild stress (UCMS; e.g., wet bedding, predator sound/smell, random disruption of light/dark cycles), with indices of depressive behavior (coat status, grooming and mobility) becoming exacerbated vs. controls. In these studies, we consistently demonstrated an increased vascular oxidant stress, impaired vascular dilator reactivity, a loss in nitric oxide bioavailability and the development of H2O2 as a compensatory dilator mechanism. However, we also determined that UCMS mice experience increased, but highly variable, insulin resistance and chronic inflammation. As such, while vascular impairments are consistent across animals, the predictive power of systemic markers of CVD risk, including fasting insulin, and plasma levels of TNF‐α and CRP were limited. Additionally, there was no clear correlation between insulin resistance severity and plasma levels of TNF‐α or IL‐1β in UCMS mice. As such, while there are trends between IR or inflammation and vascular dysfunction in UCMS mice, these are not robust relationships, suggesting that an unidentified mechanism outside of glycemic control or chronic inflammation may be a superior predictor of vascular outcomes in this murine model of depression.
AMP‐activated protein kinase α1 silencing provides evidence for a Metformin‐induced decrease in FA uptake and oxidation and increase in glucose uptake and SIRT1 activity in L6 skeletal muscle cells
Authors: Bogachus Lindsey D , Turcotte Lorraine P
Published: June 2021

AMP‐activated protein kinase (AMPK) and SIRT1 have been shown to be key regulators of glucose and fatty acid (FA) metabolism. L6 skeletal muscle cells were treated using RNAi technology to genetically silence AMPKα1 (α1) and AMPKα2 (α2) and incubated ± AICAR (A: 2mM, 2hr) or Metformin (M: 2mM, 3hr). After treatment, glucose uptake (GU) and FA uptake (FAU) & oxidation (FAO) were measured using [2‐3H]deoxyglucose or [1‐14C]palmitate. AMPKα1 & AMPKα2 phosphorylation (~P), PGC‐1α acetylation (~A), and SIRT1 activity were measured. The effects of genetic and pharmacological treatments on metabolic parameters were assessed with ANOVA. AMPKα1 & AMPKα2 ~P measurements verified that the RNAi transfections were efficient (P<0.05). Silencing of α1 eliminated the M‐induced decrease in FAU & FAO and blunted the M‐induced increase in GU (P<0.05). Silencing of α2 eliminated the A‐induced increase in GU & FAO and reduced A‐induced FAU (P<0.05). A & M treatments increased SIRT1 activity (P<0.05). While silencing of α1 increased M‐induced SIRT1 activity, silencing of α2 increased both A‐ and M‐induced SIRT1 activity (P<0.05). PGC‐1α ~A data confirmed the SIRT1 activity measurements. Our data show that in muscle cells the stimulatory effects of A on muscle metabolism occur via preferential ~P of AMPKα2 and that the inhibitory effects of M on FA metabolism occur via preferential ~P of AMPKα1. Funding: WiSE & IEB Research Award, USC
Oxidative stress leads to reduced coupling of oxidative phosphorylation in in vivo resting mouse skeletal muscle
Authors: Siegel Michael , Knowles Gary , Harper Mary‐Ellen , Marcinek David
Published: June 2021

The “Uncoupling to Survive” Hypothesis suggests that oxidative stress induces reduced coupling of oxidative phosphorylation (P/O), which then reduces reactive oxygen species production by the mitochondria. This study sought to test whether oxidative stress leads to mitochondrial uncoupling in skeletal muscle in vivo. We subjected mice to two different doses of Paraquat (PARA), a redox cycling agent known to stimulate production of reactive oxygen species (ROS) in mitochondria. After two weeks of treatment, we used magnetic resonance spectroscopy to measure ATP flux and optical spectroscopy to measure oxygen uptake to determine the P/O value in the distal hind limb. PARA treatment led to a significant dose dependent decrease in mitochondrial P/O values in vivo (P<0.05), supporting the hypothesis that oxidative stress leads to mitochondrial uncoupling. We are currently investigating the mechanism of this reduced coupling using a UCP3 knockout mouse. Early results indicate no change in the coupling of resting skeletal muscle in the absence of UCP3. Studies are currently underway to determine if the presence of UCP3 is necessary for oxidative stress‐induced uncoupling in vivo.

This work was supported by NIH grants AG022385 and AG028455 and the Ellison Medical Foundation.
Rats selectively bred for low aerobic capacity become promptly fatigued and have slow metabolic recovery after stimulated muscle contractions
Authors: Torvinen Sira , Silvennoinen Mika , Gröhn Olli , Närväinen Johanna , Tuunanen Pasi , Koch Lauren , Britton Steven , Kainulainen Heikki
Published: June 2021

Good aerobic capacity has a major role in health and longevity. Changes in oxidative metabolism underlie also several complex diseases, such as type 2 diabetes. Koch and Britton (Physiol Genomics, 2001) have developed two distinct rat strains by artificial selection: high‐capacity runner (HCR) and low‐capacity runner (LCR) rats. These rat strains differ in their intrinsic aerobic capacity. The aim of the study is to get more information from HCR/LCR rats as models of metabolic diseases and to find out the effects of aerobic training and aging in longevity, metabolism and muscle function. Muscle function was studied in electrically stimulated contracting gastrocnemius muscle using 1H‐magnetic resonance imaging (MRI) and 31P‐MR spectroscopy. The simultaneous force output was measured with an ergometer connected to foot pedal. Our results from 31P‐MR spectroscopy and mechanical measurement show that there was a significant difference between HCR and LCR groups in the isometric force decrease during transcutaneous stimulation, the decrease being greater in LCR rats (P<0,001). The phosphocreatine resynthesis after stimulation was also significantly slower in LCR group (P<0,02). In conclusion, 31P‐MR spectroscopy seems to be a valid non‐invasive method to investigate energy metabolism in this disease model, making also longitudinal studies feasible. Study is supported by the Ministry of Education, Finland
Low RIP140 Expression Partially Restores Metabolic Flexibility in L6 Muscle Cells Treated with High FA
Authors: Constantinescu Silvana , Turcotte Lorraine P.
Published: June 2021

To elucidate the role of RIP140 in the regulation of fatty acid uptake (FAU) and oxidation (FAO) and glucose uptake (GU) in insulin‐resistant muscle cells, L6 myotubes were transfected with RNAi to genetically silence a control sequence (CS) or RIP140 (RIP). Cells were then incubated ± palmitic acid (FA: 400μM, 12h) to induce insulin resistance followed by incubation ± insulin (I: 1000 nM) before measurement of glucose and FA kinetics. In line with the presence of insulin resistance, FA treatment raised basal GU (122.2±5.2% vs 230.0±18.8%; P<0.05) and obliterated the I‐induced increase in GU (P<0.05). FA treatment decreased basal FAU (107.1±3.7% vs 45.4±10.5%; P<0.05) and FAO (176.8±19.6 vs 82.7±13.9%; P<0.05). I‐induced FAU and FAO was reduced by 24% (P<0.05) and 44% (P<0.05) with FA treatment. Silencing of RIP140 in FA‐treated cells did not affect basal GU or FAO but it was associated with higher FAU (45.4±10.5% vs 75.0±12.1%; P<0.05). With I stimulation, silencing of RIP140 in FA‐treated cells was associated with higher GU (29%; P<0.05), lower FAU (35%; P<0.05) and no change in FAO (P<0.05). Our data show that reduced RIP140 expression in FA‐treated muscle cells rescues insulin‐mediated GU and basal FAU but does not affect basal or I‐mediated FAO. Together, our results indicate that low RIP140 expression may partially restore insulin sensitivity when insulin resistance is induced by high FA treatment.

USC Women in Science and Engineering, USC Integrative and Evolutionary Biology and USC Zumberge Research Innovation Fund