bluuebunny/crossref_metadata_2025 huggingface dataset:
Mdivi‐1 Mitigates ROS and Mitophagy, Improves Healthy Mitochondrial Pool in Hyperglycemic Cardiomyocytes
Authors: Sharawat Surender , Nandi Shyam , Kesherwani Varun , Shahshahan Hamid , Mishra Paras
Published: June 2021

Cardiac function is contingent upon mitochondrial health and turn over. To test the hypothesis that Mdivi‐1 mitigates reactive oxygen species (ROS) and mitophagy, and improves healthy mitochondrial pool in hyperglycemic cardiomyocytes, we used four treatment groups of HL‐1 cardiomyocytes: low (LG‐5mM) and high (HG‐25mM) doses of D‐glucose, HG+Mdivi‐1, and HG+DMSO (di‐methyl sulfoxide). To evaluate ROS, mt‐ΔΨ, mitophagy and mitochondrial biogenesis, we determined the levels of DHE, MitoSox, JC1 (hypopolarized green vs hyperpolarized red color), Beclin1, LC3B, PGC‐1α, Mfn‐1, and ratio of mitochondrial to nuclear DNA in the four groups by qPCR, Western blotting and immunofluorescence. Our results revealed that DHE intensity, MitoSox intensity, Beclin1 and LC3B are up regulated by 3.54±0.08, 1.6±0.03, 1.4± 0.05 and 3.6±1.9 folds in HG but down regulated by 1.18±0.3, 1.1±0.03, 0.9±0.03 and 1.96±0.07 folds in HG+Mdivi‐1, respectively. The mt‐ΔΨ decreased in HG by 0.8± 0.06 folds but increased after treatment with Mdivi‐1 by 0.9±0.1 folds. The ratio of mitochondrial DNA (D‐loop, ND‐6) vs nuclear DNA (GAPDH, PGC‐1α) increased 1.01±0.08, 1.07±0.04 folds in HG. However, it was decreased by 0.95±0.02, 1.0±0.5 folds after treatment with Mdivi‐1. Further, Mdivi‐1 treatment decreased expression of PGC‐1α, Mfn‐1 by 0.92±0.7, 0.86±0.1 folds respectively. Based on these findings, we conclude that Mdivi‐1 mitigates glucose mediated induction of ROS, and depolarization of mitochondria, and restores healthy mitochondrial pool in cardiomyocytes.
Role of brain Angiontensin‐(1‐7) in the gene expression of angiotensinergic receptors in the heart
Authors: Kangussu Lucas , Prestes Thiago , Alves Fabiana , Santos Robson , Campagnole‐Santos Maria Jose
Published: June 2021

Hypertensive rats subjected to chronic intracerebroventricular (ICV) infusion of Angiotensin‐(1‐7) presented attenuation of arterial hypertension, improvement the sensitivity of the baroreflex, restoration the cardiac autonomic tone and alteration the expression of cardiac RAS components. In this study we evaluated whether the acute increase in the brain levels of Ang‐(1‐7) modulates the gene expression of angiotensinergic receptors in the left ventricle of normotensive and hypertensive rats. Sprague‐Dawley (SD) and hypertensive rats that overexpress renin (TGR) were used. The animals were divided into four experimental groups: SD, ICV infusion of saline (3 mL/hour), n=5; SD, ICV infusion of Ang‐(1‐7) (2,5 mg/hour), n=5; TGR, ICV infusion of saline (3 mL/hour), n=4 and TGR, ICV infusion of Ang‐(1‐7) (2,5 mg/hour), n=4. Immediately after the ICV infusion, the animals were euthanized by decapitation and the left ventricle was removed and frozen on dry ice. The mRNA expression of AT1, AT2 and Mas receptor were evaluated by semi quantitative real time‐PCR using the delta delta CT method. The acute infusion of saline or Ang‐(1‐7) for 3 hours did not alter the gene expression of AT1 and Mas receptors in the left ventricle of the normotensive or hypertensive rats. In contrast, the acute infusion of Ang‐(1‐7) was capable of increasing the gene expression of AT2 receptors in SD (2.78±0.55 a.u. vs.1.17±0.35 a.u. in ICV saline group) and TGR(mRen2)27 (3.37±0.92 a.u. vs. 1.05±0.15 a.u. in ICV saline group). Short‐term ICV infusion of Ang‐(1‐7) in the brain alters gene expression of the AT2 receptor in the left ventricle of normotensive and hypertensive rats.
Estradiol replacement attenuates stress‐induced pressor response by regulating renin‐angiotensin system in ovariectomized rats
Authors: Nagatomo Yu , Tazumi Shoko , Itoh Risa , Yoshimoto Misa , Takamata Akira , Morimoto Keiko
Published: June 2021

We examined whether chronic estrogen replacement has suppressive effects on psychological stress‐induced pressor response by attenuating renin‐angiotensin system (RAS) in ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized. After 4 wk, the rats were assigned either to a placebo‐treated (Pla) group or a group treated with 17β‐estradiol (E2) subcutaneously implanted with either pellet. Two wk later, they were implanted with radiotelemetry devices for blood pressure (BP) and heart rate (HR) measurements, and the half of them were denervated renally. These rats underwent cage‐switch stress 2 wk after the operation. The stress elevated the BP and HR rapidly and continuously both in the Pla and E2 groups. However, these responses to the stress were attenuated significantly in the E2 group compared with the Pla group. An angiotensin II type 1 receptor blocker, losartan, abolished the difference in the BP response to the stress between the two groups. In addition, the stress induced elevation of plasma renin activity and angiotensin II concentration only in the Pla group, but not in the E2 group. Furthermore, the renal sympathetic nerve denervation attenuated the pressor response in the Pla group. These results suggest that estradiol replacement attenuates psychological stress‐induced pressor response by suppressing RAS activation in ovariectomized rats.
Brainstem Serotonin (5‐HT) and Arterial Blood Pressure Regulation in Neonatal Rats Exposed to Intermittent Asphyxia
Authors: Magnusson Jennifer , Cummings Kevin
Published: June 2021

In neonatal rats, a systemic loss of 5‐HT exaggerates the drop in arterial blood pressure (BP) during severe hypoxia. In adult animals, intermittent hypoxia or asphyxia (IA) leads to a long‐termt increase in ventilation (VE), sympathetic activity and BP (long‐term facilitation, LTF). The consequences of a central loss of 5‐HT on the acute BP response to asphyxia and the long‐term regulation of BP have not been studied in the neonatal period. We hypothesized that 1) a partial loss of 5‐HT neurons increases the drop in BP during asphyxia; 2) BP increases following IA; and 3) the hypertension and respiratory LTF following IA would be reduced in 5‐HT‐deficient pups. We utilized 2 week‐old Sprague Dawley rats treated i.c.v. with 5,7‐dihydroxytryptamine (n=11), lesioning 39% of brainstem 5‐HT neurons. Littermate controls were injected with saline (CTRL, n=12). We measured VE with the barometric method and BP with a femoral artery catheter. Heart rate (HR) was derived from BP. After 15 min in room air, animals were exposed to 20 1‐min challenges of moderate asphyxia (10% O2, 5% CO2) followed by 1 hr in room air. A loss of 5‐HT neurons significantly reduced systolic (p=0.016), but not diastolic BP (p>0.1) compared to CTRL. 5‐HT‐deficiency reduced HR in both normoxic and asphyxic conditions (p<0.001). Surprisingly, the drop in BP during asphyxia was attenuated in 5‐HT‐deficient pups compared to CTRL (p=0.008). Systolic and mean BP increased significantly (p =0.003 and 0.045, respectively) following IA in 5‐HT‐deficient pups, but not in CTRL. A loss of 5‐HT neurons had no effect on the degree of respiratory LTF. Our results suggest that systolic BP is reduced by a partial deficiency of central 5‐HT neurons, an effect that can be reversed by IA.
Monitoring of intracellular free Zn2+ and Ca2+ changes in cardiomyocytes from metabolic syndrome rats
Authors: Toy Aysegul , Okatan Esma , Degirmenci Sinan , Turan Belma
Published: June 2021

Metabolic syndrome (MetS) is the name for a group of risk factors that raises important risk for heart disease and other health problems. The main features of metabolic syndrome include insülin resistance and hypertension. Although evidence is emerging that changes in cardiac energy metabolism might contribute to the development of contractile dysfunction, a depressed sarcoplasmic reticulum (SR) function seems to be a good candidate to underlie MetS‐induced cardiac dysfunction. Therefore, the present study was designed to determine whether the properties of Zn2+ (with FluoZin‐3 loading) and Ca2+ (with Fluo‐3 loading) sparks might provide insight into the role of SR in the altered Zn2+ and Ca2+ signaling in MetS cardiomyocytes when they were analyzed together with Zn2+ and Ca2+ transients. MetS was induced with high‐sucrose drinking water during 16‐week period in male rats. Basal Zn2+ and Ca2+ levels as well as frequencies of Zn2+ and Ca2+ sparks in cardiomyoctes significantly increased compared to aged‐matched controls. The Ca2+ transients exhibited significantly reduced amplitude and prolonged time course while no change in amplitude and prolongation in time course of Zn2+ transients in cardiomyocytes from MetS rats. Spatio‐temporal properties of both Zn2+ and Ca2+ sparks were also significantly altered to being almost parallel to the changes of Zn2+ and Ca2+ transients. In addition, RyR2 from MetS group was hyperphosphorylated while its protein level was depressed, similar to the hyperphosphorylation of RyR2 with Zn2+ in a dose‐dependent manner. These data show that cardiomyocytes from MetS rats exhibit altered local Zn2+ and Ca2+ signaling with increased basal Zn2+ and Ca2+ levels.
Deleterious Effects of Low and Medium Doses of Clonidine on Left Ventricular Function in Diabetic Rats
Authors: Crespo M J. , Casiano H , Morales M , Torres H , Torres‐Grajales M , Hernandez C , Rivera M , Cruz N , Quidgley J
Published: June 2021

During the perioperative period, α‐2 receptor agonists have been suggested to reduce cardiovascular (CV) risk by decreasing catecholamine‐induced cardiac stress. Diabetics have a higher incidence of perioperative CV complications due to autonomic dysfunction and hemodynamic instability. The potential benefits of clonidine, an α‐2 receptor agonist, in maintaining CV homeostasis in diabetics during the perioperative period are unknown. In this study, we evaluated with echocardiography the effects of clonidine (250, 50 and 10 µg/kg, IP) on the CV status of streptozotocin (STZ)‐induced diabetic and control (CT) rats while under anesthesia. The oxidative‐stress markers MDA and 4‐HAE were also measured in cardiac and aortic homogenates after 1‐hour incubation with clonidine (0.1, 1.0 and 10 µg/ml). Heart rate was reduced by clonidine in diabetic rats and CT. In contrast, high (250 µg/kg) and medium (50 µg/kg) clonidine doses did not affect stroke volume, cardiac output, or ejection fraction in either diabetic rats or CT. In diabetic, but not in CT, low (10 µg/kg) and medium (50 µg/kg) clonidine doses increased left ventricular end‐systolic and diastolic volumes by 80% and 50 %, respectively (n= 6, P<0.05). Clonidine did not alter MDA or 4‐HAE levels in cardiac and aortic homogenates from either diabetic rats or CT, suggesting that oxidative stress is not modified by acute clonidine incubation. Our results indicate that low and medium clonidine doses have a deleterious effect on ventricular function in diabetic rats, possibly because the negative cardiac inotropic and chronotropic effects outweigh the reductions in afterload that are secondary to α‐2 receptor activation by the drug.
Even Small Changes in Body Temperature Can Affect the Cardiac Function in Anesthetized Mice
Authors: Tsuchimochi Hirotsugu , Inagaki Tadakatsu , Shirai Mikiyasu
Published: June 2021

Heart rate (HR) increases at the beginning of dynamic exercise, then remains constant or gradually increases in response to the exercise intensity. Both central command and the exercise pressor reflex (EPR) are thought to play an important role for this tachycardic response. Recently, the contribution of transient receptor potential vanilloid 1 (TRPV1) channel to the EPR has been reported (Smith SA et al, J Physiol 2010). However, the role of the TRPV1 channel on the exercise tachycardia during dynamic exercise remains largely unknown. We therefore examined the effect of systemic TRPV1 channel blockade or activation on the HR response to treadmill exercise in rats. Male Wistar rats were previously implanted with telemetry transmitters (TRM54P, Millar) to measure HR and arterial blood pressure (AP), and were trained to run on a treadmill. Both HR and mean AP (MAP) responses to treadmill running (20 or 40 m/min, 60 s) were compared before and after administration of TRPV1 channel agonists or antagonists. Sciatic nerve‐selective desensitization of TRPV1 channel by resiniferatoxin (10 µg, under anesthesia) significantly attenuated the HR response to exercise. Systemic administration of potent TRPV1 channel antagonist iodoresiniferatoxin (0.3 mg/kg ip), which has a partial agonist‐like effect, also attenuated it. On the other hand, either the TRPV1‐selective (SB366791; 3 mg/kg) or a nonselective (ruthenium red; 5 mg/kg) antagonists did not affect the HR response to exercise. In conclusion, desensitization of the TRPV1 channel‐expressing neurons attenuated the EPR‐mediated HR response during dynamic exercise in rats, suggesting that TRPV1‐expressing neurons play an important role for the HR response during dynamic exercise.
EVALUATION OF LEFT VENTRICULAR FUNCTION IN RATS TREATED WITH NANDROLONE DECANOATE SUBMITTED TO EXERCISE OF WEATHERED
Authors: NASCIMENTO Andrews , LIMA EWELYNE , BRASIL GIRLANDIA , CALIMAN IZABELA , BISSOLI NAZARÉ , ANDRADE TADEU
Published: June 2021

There are several evidences of cardiovascular damage by use anabolic steroids androgenic (AAS). However, little is known about the cardiac effects of abusive use of AAS by women. Aim of this study was to evaluate the influence of Nandrolone Decanoate (ND) treatment over cardiac function in female rats submitted to a resistance exercise training. Female Wistar rats was divided in four groups (n=8): Sedentary vehicle (S), sedentary treated (SD), exercise vehicle (E) and exercise treated (ED). The animals were treated with ND (20 mg/kg/week) or vehicle (peanut oil) for 28 days, approved by the Ethics Committee in Animal Experimentation of the UFES (031/2012).

The results showed that ND promoted cardiac hypertrophy (area of myocytes, S:11421±312, SD:13304±233, E:12262±198 and ED:13418±153, p<0.01), (number of myocytes, S:7.8±0.2, SD:4.0±0.1, E:5.7±0.2 and ED:3.8±0.1, p<0.01) without modifying, HR, MAP and DAP, but increase intraventricular pressure (PVE; S:99.23±6.36, SD:137.06±5.56, E: 101.22±10.45 and ED:117.29±7.54 mmHg, p<0.01). The evaluation of left ventricular contractile indices shows that the values +dP/dt (S:5240.00±525.56, SD:8654.55±415.73, E:5239.14±160.56 and ED:6443.97±214.50 mmHg/s, p<0.01) and ‐dP/dt (S:‐2590.43±330.09, SD:‐5263.04±319.45, E:‐2502.83±225.32 and ED:‐3492.38±282.12 mmHg/s, p<0.01) were higher in the treated groups. Treatment with ND in female rats promoted cardiac hypertrophy and was able to change the cardiac function particularly with respect to contraction and diastolic function.

Groups
S
SD
E
ED

MAP
106±3
100±4
108±4
110±3

HR
355±12
361±7
353±7
353±1

DAP
86±3
88±5
86±4
87±3

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Aging is associated with reduced vasodilation to insulin in subcutaneous adipose arteries in B6D2F1 mice
Authors: Henson Grant , Hazra Sugata , Reihl Kelly , Donato Anthony , Lesniewski Lisa
Published: June 2021

The subcutaneous adipose tissue (sqAT) phenotype differs from that of the often studied visceral adipose tissue. Compared to visceral adipose, sqAT receives relatively less blood flow associated with lower rates of lipid mobilization and the promotion of long term lipid storage. Previously, we demonstrated endothelial dysfunction in arteries from visceral adipose of aged animals to be associated with impaired function in this adipose depot per se as well as with systemic metabolic dysfunction. Although the effect of aging on sqAT adipose arterial function is unknown, endothelial dysfunction in this depot may contribute to impaired lipid storage. The volume of the subcutaneous adipose per gram body mass, assessed by computed tomography, was reduced by 71% (P < 0.05) in old (28 – 31 mo; N = 5) compared to young (4 – 6 mo; N = 7) B6D2F1 mice. Contrary to our previous observation in visceral adipose arteries, dilation to acetylcholine was preserved with aging (92 ± 2 v 88 ± 4%, P > 0.05) and the nitric oxide inhibitor, LNAME, reduced dilation by ~38% in both groups (P < 0.05), indicating preserved nitric oxide dependent dilation in these arteries. However, dilation to insulin was lower in old mice (67 ± 2 v 42 ± 10%, P < 0.05) compared to young and LNAME did not abolish this difference, indicating a nitric oxide independent mechanism of dilatory dysfunction. Dilation in response to sodium nitroprusside was similar between groups (P > 0.05) indicating intact smooth muscle function. These data suggest that aging impairs insulin‐induced dilation in sqAT arteries, an effect that may have implications for altered lipid handling in advancing age.
Nicorandil Ameliorates Myocardial Fibrosis by Inhibition of RhoA/Rho‐kinase Signaling in Infarcted Rats
Authors: Lee Tsung‐Ming , Yang Chen‐Chia
Published: June 2021

We have demonstrated that ATP‐sensitive potassium (KATP) channel agonists attenuated fibrosis. Since RhoA has been identified as an important mediator of cardiac fibrosis, we sought to determine whether the anti‐fibrotic effects of KATP channel agonists were mediated via inhibition of the RhoA/RhoA‐kinase (ROCK) cascade. Wistar rats after induction of myocardial infarctionwere randomized to either vehicle, an agonist of KATP channel nicorandil, an antagonist of KATP channel glibenclamide, an antagonist of ROCK fasudil, or a combination of nicorandil and glibenclamide or fasudil and glibenclamide starting 24 hours after infarction and for 4 weeks. Post‐infarction was associated with increased cardiac fibrosis, as measured bySirius Red staining and hydroxyproline assay. RhoA/ROCK activation was observed after infarction by increasing RhoA localization from the cytosol to the membrane and phosphorylating the ROCK substrate. Nicorandil diminished myocardial fibrosis by inhibiting RhoA/ROCK activation, connective tissue growth factor expression and α‐smooth muscle actin‐positive myofibroblast infiltration. Fasudil showed similar antifibrotic effect of nicorandil. The beneficial effects of nicorandil were blocked by adding glibenclamide. However, glibenclamide can not abolish the attenuated fibrosis of fasudil, implying that RhoA/ROCK is a downstream effector of KATP channel activation. Chronic treatment with nicorandil, initiated at 24 hours after infarction can expedite the attenuation of infarction‐induced myocardial fibrosis probably through inhibition of RhoA/ROCK‐dependent connective tissue growth factor independent of its infarct‐sparing effect.
Dynamic Regulation of MicroRNAs in Cardiac Fibroblasts upon Activation
Authors: Valkov Nedyalka , King Michelle , Moeller Jacob , Zhang Peng
Published: June 2021

Remodeling is a common feature in cardiac disease, which entails fibroblast activation and often leads to fibrosis. MicroRNAs (miRs) are novel regulators in cardiac remodeling. In contrast to myocytes, very little is known about miRs in cardiac fibroblasts. In this study, we established the miR expression profile in adult rat ventricular fibroblasts and assessed their dynamic changes upon fibroblast activation, using high throughput TaqMan miR arrays and a culture model that mimics the phenotypic and functional changes of cardiac fibroblasts in the diseased heart. Among 518 miRs included in the arrays, 180 miRs were detected and 52 miRs were more than 4‐fold up‐ or down‐regulated upon fibroblast activation. Cardiomyocyte‐restricted miR‐208 was undetectable, indicating lack of myocyte contamination. MiR‐21, a well‐studied miR in cardiac fibroblasts and fibrosis development, was increased 3.1‐fold upon fibroblast activation, which is consistent with a 5‐fold up‐regulation reported in fibroblasts from failing hearts. We identified several previously unidentified miRs in cardiac fibroblasts. One of them is miR‐1, which has been extensively studied in myocytes. Using gain‐ and loss‐of function approaches and 3′UTR luciferase assays, we detected a novel role for miR‐1 as negative regulator of fibroblast proliferation via a mechanism that involves direct targeting of key cell cycle regulators (cyclin D1, cyclin D2 and CDK6). Together, our data suggest dynamic regulation of miR‐1 and other miRs in cardiac fibroblasts that regulate fibroblast activation and function. (The work is supported by an IDeA grant P20GM103652 from the NIH/NIGMS)
Macrophage Activation by Chronic P. gingivalis Endotoxin Attenuates Fibroblast Matrix Deposition Post‐Myocardial Infarction
Authors: DeLeon‐Pennell K , Flynn E , Jin Y , Buchanan W , Lindsey M
Published: June 2021

Periodontitis is associated with an increased risk for heart failure, however the mechanisms remain unknown. Previously, we showed chronic P. gingivalis endotoxin (Pg) accelerated macrophage infiltration resulting in increased cardiac rupture post‐myocardial infarction (MI). We hypothesized that the early influx of macrophages would decrease myofibroblast activation leading to impaired scar formation post‐MI. Mice (4‐6 months old; n=6/sex) were infused with Pg (0.8 µg/g/day) or saline starting 28 days before and throughout the duration of MI. Expression of anti‐fibrotic cytokines (IL‐1β, Tnfα, and Ccl3) were 2‐fold higher in macrophages isolated from the infarct (LVI) of Pg mice compared to saline controls at D1 post‐MI (all p˂0.05). At D7 post‐MI, Pg exposure decreased collagen I, collagen III, fibronectin, and lysyl oxidase by 2‐fold in the LVI compared to controls (all p˂0.05). Post‐MI, fibroblasts differentiate into myofibroblasts to stimulate extracellular matrix (ECM) production. Alpha smooth muscle actin, a myofibroblast marker, decreased in the LVI of Pg mice compared to controls (p˂0.05). Our study uncovered a novel mechanism for periodontitis induced cardiac dysfunction and impaired healing post‐MI through down‐regulation of myofibroblast activation and reduced ECM deposition post‐MI.
Neuregulin‐1β Alters the Context‐dependent Production of Immunomodulatory Factors in Human Ventricular Fibroblasts
Authors: Galindo Cristi , Sengsayadeth Seng , Tikhomirov Oleg , Ryzhov Sergey , Sawyer Douglas
Published: June 2021

Neuregulin‐1β (NRG‐1β) is a growth and survival factor critical for cardiac development and repair. We recently showed that NRG‐1β reduces cardiac fibrosis in infarcted swine and reduces basal and tumor necrosis factor β (TGFβ)‐induced α‐smooth muscle actin (α‐SMA) expression. In normal human ventricular fibroblasts, we also found that NRG‐1β caused a reduction in TGFβ‐induced α‐SMA, but we did not observe significantly lower α‐SMA levels in response to NRG‐1β under basal conditions. We thus hypothesized that the anti‐fibrotic effects of NRG‐1β might be context dependent. Indeed, when primary rat cardiac fibroblasts were cultured on softer substrates NRG‐1β had little effect on α‐SMA expression. NRG‐1β did not alter fibroblast collagen gel contraction in the presence or absence of TGFβ, nor did NRG‐1β treatment alter proliferation or viability of rodent or human fibroblasts. Nonetheless, these cells expressed all three NRG‐1β receptors (ErbB2, 3, and 4), and NRG‐1β alone reduced transcript levels of tumor necrosis factor‐α (TNF‐α) and prominin 1 (CD133) after 2 hours of treatment in the absence of serum. NRG‐1β inhibition of CXCL10, HLA‐DQB1, and adiponectin (ADIPOQ) on the other hand required the presence of TGFβ, as did NRG‐1β‐mediated increase of the TGFβ‐signaling inhibitor SMAD7. Most interesting was the induction of IL10 transcription in response to the combination of NRG‐1β and TGFβ, neither of which had any effect on IL10 transcript levels in the absence of the other. These results suggest that NRG‐1β modulates inflammation via cardiac fibroblast signaling and moreover that these signaling events are context‐dependent.

Work was supported by the NIH (K01 HL121045; U01 HL100398).
Investigating the Effect of Inhibiting 'Reactive Oxygen Species' on Intracellular Signaling Pathways in Endothelial Cells
Authors: Ashworth Isa , Obeidat Yasin , Puentes Laura , Torina Anali , Shafique Ehtesham , Abid Ruhul
Published: June 2021

Objective

Elevated Reactive Oxygen Species (ROS) in Endothelial Cells (EC's) have been implicated in the pathogenesis of cardiovascular diseases, and much research has gone into elucidating the mechanisms through which oxidative stress leads to these diseases. Research in the last decade, however, has uncovered the central role lower levels of ROS play in physiological intracellular signaling pathways, such as those stimulated by Vascular Endothelial Growth Factor (VEGF) and Tumour Necrosis Factor‐α (TNF‐α) in EC's. Our objective in this study was to determine the effect of a lack of ROS in EC's on the intracellular signaling pathways stimulated by VEGF and TNF‐α.

Methods

The activity of these pathways can be measured by the expression of key downstream proteins that are common to these two pathways such as Akt, ERK‐1/2, IKKα/B, AMPK, eNOS, mTOR and FOXO1. To investigate these pathways in vitro, Bovine Endothelial Aortic Cells (BAEC's) were treated with ROS inhibitors (NAC, DPI) and then exposed to VEGF or TNF‐α to stimulate their respective pathways. Protein was then extracted and transferred to a membrane by Western Blotting then visualized by immunoflourescence.

Results

Western blots demonstrated that reduction of ROS levels by NAC inhibited VEGF‐induced Akt phosphorylation but not phosphorylation of ERK1/2 in BAEC. The findings of TNF‐induced signaling activation were not conclusive.

Conclusion

Whereas VEGF‐mediated activation of PI3K‐Akt activation requires ROS, ERK1/2 activation is independent of ROS in ECs.

This research was funded by National Institutes of Health (NIH) and American Heart Association (AHA) grants.
Role of Increased Serine1176 Phosphorylation in Maintaining Normal Endothelial Responses in Male and Female Heterozygous Endothelial Nitric Oxide Synthase (NOS)‐Deficient Mice
Authors: Didion Sean
Published: June 2021

Although endothelial responses in male and female heterozygous endothelial nitric oxide synthase (eNOS)‐deficient mice are normal, it is not known whether there is compensation for the loss of a single eNOS gene by vasodilator mechanisms other than NOS and nitric oxide. The goal of the present study was to test the hypothesis that endothelial responses in male and female heterozygous endothelial nitric oxide (eNOS)‐deficiency are due to NOS‐dependent signaling pathways. Responses of carotid arteries from male and female eNOS+/+ and eNOS+/‐ mice to acetylcholine (Ach) and nitroprusside (NP) were examined in the presence of vehicle (saline), L‐NNA (100 µM; a NOS inhibitor), or ODQ (1 µM; an inhibitor of soluble guanylyl cyclase, sGC). Responses to Ach and NP were similar in male and female eNOS+/+ and eNOS+/‐ mice treated with vehicle. Carotid artery responses to Ach were significantly inhibited in male and female eNOS+/+ mice in the presence of L‐NNA and ODQ. For example, acetylcholine produced 89±2, 21±8 and 3±5% relaxation in vehicle, L‐NNA, and ODQ treated vessels from male eNOS+/+ mice. L‐NNA and ODQ produced a similar degree of inhibition of Ach‐induced relaxation in male and female eNOS+/‐ mice. Although vascular eNOS expression was reduced by 蠅60%, phosphorylation at Ser1176 (a site associated with increased eNOS activity) was markedly higher in eNOS+/‐ mice. These findings demonstrate that endothelial responses in male and female eNOS+/‐ mice maintain their dependency on NOS and sGC. Increased eNOS phosphorylation appears to be a compensatory mechanism that serves to maintain endothelial responses in the absence of a single eNOS gene. Supported by NIH HL‐089884 and HL‐107632.
Assessment of Cardiac Function Using Pressure Volume Loops in Rbm20‐/‐ Rats
Authors: Sanchez Walk Andrea , De Loera Denise , Bosch Luiza , Guo Wei , Ren Jun , Chew Herbert
Published: June 2021

RNA binding motif 20 (Rbm20) is a muscle‐specific splicing factor that mainly regulates isoform transition of titin, a giant sarcomeric protein responsible for diastolic properties of the heart. Rbm20‐/‐ (KO) rats fail to splice titin from fetal to adult isoforms, resulting in less stiff ventricular walls and thus systolic dysfunction and heart failure. Although previous data have shown that ejection fraction in KO rats is significantly lower than that of Rbm20+/+ (WT) rats in 10 m old rats, little is known of cardiac function in KO rats versus age‐matched WT rats with development using pressure volume loop analysis. We hypothesized that KO rats would develop dysfunction early in life. Preliminary results of 6 m old rats (n=8 per group) show significant (p<0.05) decreases in cardiac output (KO 18.6±2.1; WT, 43.0±2.9 mL•min‐1), heart rate (KO, 215.0±12.9; WT, 274.4±18.5 b•min‐1), stroke volume (KO, 85.2±4.9; WT, 156.9±3.3 µL), ejection fraction (KO, 30.2±5.0; WT, 53.5±6.6 %), stroke work (KO, 6.17±0.60; WT, 13.0±0.80 mmHg•mL), and cardiac efficiency (KO, 0.464±0.031; WT, 0.655±0.073). End‐systolic elastance was significantly lower in KO (KO, 0.405±0.094; WT, 0.825±0.063 mmHg•µL‐1), indicating an increase in systolic compliance, while neither end‐diastolic elastance (KO, 0.095±0.030; WT, 0.040±0.003 mmHg•µL‐1) nor Tau (KO, 13.5±1.48; WT, 12.8±0.85 ms), both indexes of diastolic compliance, were different. We conclude that KO rats show significant evidence of heart failure as early as 6 m of age.

Supported by NIH P20 GM103432, the University of WY Agricultural Experimental Station, and USDA‐NIFA.
What The HIV‐Associated Gut Microbiome May Tell As about The Role of The Adaptive Immune System in Matching Our Gut Microbes to Our Diet
Authors: Lozupone Catherine , Neff Preston , Rhodes Matthew , Donnelly Jody , Campbell Thomas , Palmer Brent
Published: June 2021

Consistent with an important role for adaptive immunity in modulating interactions between intestinal bacteria and host, dramatic alteration in the composition of gut microbes occurs during chronic HIV infection. 16S rRNA sequencing of feces from HIV‐infected individuals revealed that HIV infection is associated with highly characteristic gut community changes, and antiretroviral therapy does not consistently restore the microbiota to an HIV‐negative state. Surprisingly, the microbiota of HIV‐infected individuals in the US was strikingly similar compositionally to a Prevotella‐rich community composition typically observed in healthy individuals in agrarian cultures in Malawi and Venezuela and related to that of US individuals with carbohydrate‐rich/ protein and fat poor diets. This surprising relationship with compositions previously associated with diet/culture indicates a role for adaptive immunity in shaping the microbiota in the context of its environment.
Estrogen Receptor‐Alpha Activation of AMPK in Vascular Smooth Muscle Cells and Vascular Tone
Authors: Kim Seong , Hamblin Milton
Published: June 2021

The cardioprotective effects of estrogen have long been known to result, in part, from its influence on vascular tone. The classical ERs, ERalpha (ERα) and ERbeta (ERβ), can localize within the cell and function as ligand‐activated transcription factors, modulating gene expression in response to estrogen. ERα and ERβ can also localize to the plasma membrane and participate in rapid cellular activation by interacting with cellular signaling pathways. Estrogen has been shown to activate AMPK‐activated protein kinase (AMPK), suggesting that AMPK may mediate some of the effects of estrogen, including its influence on vascular tone. In this study, the selective ERα agonist 4,4′,4′‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT), AMPK activators 5‐aminoimidazole‐4‐carboxamide (AICAR) and metformin, and the AMPK inhibitor Compound C were used to examine the importance of ERα‐AMPK signaling in the relaxation of aortic rings from male and female C57BL/6 mice. The results suggest that activation of AMPK represents a novel pathway through which ERα affects vasomotor tone. (Supported by 5K12HD043451).
Microvascular Dysfunction is Evident in the Cutaneous Circulation of Psoriatic Patients
Authors: Alba Billie , Greaney Jody , Ferguson Sara , Alexander Lacy
Published: June 2021

Vascular dysfunction in conduit and resistance vessels is evident in adults with plaque psoriasis, a chronic systemic inflammatory disease. However, potential impairments of cutaneous microvascular function in psoriatic patients remain incompletely understood. We hypothesized that microvascular function would be impaired in the cutaneous circulation (non‐lesional) of psoriatic patients. One intradermal microdialysis fiber was placed in the forearm skin of 5 healthy adults (NP: 1M, 4F; 49 ± 1 yr) and 5 adults with moderate (>5% body surface area affected) plaque psoriasis (P: 1M, 4F; 48 ± 1 yr). The site was perfused with lactated Ringer's while red blood cell flux was measured using laser‐Doppler flowmetry during standardized local heating (42°C) to elicit eNOS‐dependent vasodilation. NO‐dependent vasodilation was further quantified after the perfusion of 20 mM L‐NAME (nonspecific NO‐synthase inhibitor). Data were expressed as a percentage of maximum cutaneous vascular conductance (%CVCmax; 28 mM SNP). The local heating‐induced plateau was attenuated in psoriatic patients (NP: 99 ± 1 %CVCmax vs. P: 75 ± 9 %CVCmax; p<0.05). Quantified eNOS‐dependent vasodilation appears to be reduced in the psoriatic group (NP: 57 ± 8 %CVCmax vs. P: 41 ± 8 %CVCmax; p=0.15). These preliminary results suggest that microvascular dysfunction is evident in psoriatic patients and may be mediated by reduced NO bioavailability. Further, the assessment of cutaneous vascular function has particular relevance for this clinical population because the skin‐related symptomology represents the outward manifestation of this systemic inflammatory disease process.

FUNDING: Penn State College of HHD Grant
Muscle Metaboreflex‐Induced Vasoconstriction of Ischemic Active Skeletal Muscle
Authors: Kaur Jasdeep , Spranger Marty , Krishnan Abhinav , Machado Tiago , Alvarez Alberto , Hanna Hanna , Altamimi Yasir , O'Leary Donal
Published: June 2021

Metabolite accumulation due to ischemia of active skeletal muscle stimulates group III/IV chemosensitive afferents eliciting reflex increases in arterial blood pressure and sympathetic activity, termed the muscle metaboreflex. We and others have previously demonstrated sympathetically‐mediated vasoconstriction of coronary, renal and forelimb vasculatures with muscle metaboreflex activation (MMA). Whether MMA elicits vasoconstriction of the ischemic muscle from which it originates is unknown. We hypothesized that the vasodilation in the active skeletal muscle with imposed ischemia becomes progressively restrained by the increasing sympathetic vasoconstriction during MMA. We activated the metaboreflex during mild, dynamic exercise in chronically instrumented canines via graded reductions in hindlimb blood flow (HLBF) before and after α1‐adrenergic blockade (prazosin; 50 µg/kg). Hindlimb resistance (Rh) was calculated as hindlimb arterial pressure / HLBF. During mild exercise HLBF must be reduced below a threshold level before MMA occurs. With initial reductions in HLBF, vasodilation occurred with the imposed ischemia. Once muscle metaboreflex was elicited, Rh increased. This increase in Rh was abolished by α1‐adrenergic blockade. We conclude that metaboreflex activation during submaximal dynamic exercise causes sympathetically‐mediated α‐adrenergic vasoconstriction of the ischemic skeletal muscle. HL‐55473
Predictors of Walking impairment in Patients With Claudication Using Linear & Non‐Linear Models
Authors: Abraham Pierre , Feuilloy Mathieu , Mezdad Tininhan , Chauvet Pierre , Schang Daniel
Published: June 2021

Objective

To identify new variables associated to maximal walking time on treadmill (MWT) in patients with claudication.

Material

We retrospectively analysed data of 1120 patients referred for transcutaneous exercise oxymetry (TcpO2).

Methods

The outcome measurement was the absolute walking time (MWT) on treadmill (3.2 km/h, 10% slope). We used linear regression analyses (LRA) and a non‐linear analysis (NLA) combining Support Vector Machines and Genetic Explanatory in 800 patients with the following resting variables: Age, gender, body mass index (BMI), the presence of diabetes (Db), minimal ankle to brachial index at rest (ABI), usual walking speed over 10 m (WS10), Number of comorbid conditions (CMC), smoking behaviour (Smo), resting heart rate (HR), pre‐test glycemia (Gly) and haemoglobin (Hg), beta‐blocker use (BB), and exercise‐derived variables: minimal value of pulse oxymetry (Sat), resting chest tcpO2 (TcpO2‐rest), decrease in chest tcpO2 during exercise (DCT), presence of buttock ischemia defined as a DROP index < ‐15 mmHg (Butt‐I).

Results

Independent variables associated to MWT, by decreasing importance in the models, were : ABI, Age, BMI, WS10m, DC‐TcpO2, Smo, Butt‐I, Gly, HR for the NLA, and Age, ABI, WS10; TcpO2‐rest, BMI, Smo, Butt‐I, HR and BB for the LRA (r=0.518; p<0.01). Gender, Db, CMC, Hb, Gly and Sat were not detected as significantly associated to MWT on treadmill with the LNA and LRA models. Testing of models against MWT over 320 new patients gave r=0.509 for LRA and 0.575 for NLA (both p<0.05).

Conclusions

In addition to ABI, Age, BMI and Smo were found new variables associated to MWT : Butt‐I, HR and WS10.
Effect of Muscle Fiber Denervation on Mitochondrial Hydrogen Peroxide Generation in Adjacent Innervated Muscle Fibres: Role in Sarcopenia?
Authors: Pollock Natalie , Vasilaki Aphrodite , McArdle Anne , Jackson Malcolm
Published: June 2021

Nerve transection causes dramatic increases in mitochondrial generation of hydrogen peroxide (H2O2) in the denervated skeletal muscle of rodents. We hypothesized that denervation of a limited number of fibers, such as that observed in muscles during aging, could influence H2O2 generation by both denervated and innervated fibers throughout the muscle. Thy1‐YFP mice (expressing YFP in neurons) underwent full transection of the peroneal nerve or transection of only one of the 3 branches entering the anterior tibialis (AT) muscle. Following up to 10days recovery, generation of H2O2 by mitochondria in permeablised fibers with intact mitochondra from different portions of the AT was assessed using Amplex Red. Results demonstrated increased H2O2 generation by 3days after full transection compared with sham control, which remained elevated at 10days. In situ imaging showed loss of innervation of endplates with no evidence of post‐synaptic changes. Immuno‐fluorescent staining for markers of denervation showed neuronal cell adhesion molecule positive muscle fibers by 7days post‐surgery. Transection of the single branch of the peroneal nerve caused denervation of a portion of the AT and mitochondrial H2O2 generation in the denervated portion was elevated by 3days post‐transection in a similar manner to the fully denervated muscle. Denervation of some just fibers was associated with increased mitochondrial H2O2 generation in all areas of the muscle. Thus data support the hypothesis that the effect of partial denervation on muscle H2O2 generation is mirrored in fully innervated fibers of the same muscle, an effect which may be mirrored in sarcopenia. Supported by the UK Biotechnology and Biological Sciences Research Council.
Mitochondrial properties in Rhabdomyosarcoma: An insight into the molecular mechanisms of muscle differentiation
Authors: Joseph Rosette , Hood David
Published: June 2021

Rhabdomyosarcoma (RMS) is a type of cancer that arises in muscle tissue and primarily affects children. RMS cells remain immature and thus, their lack of differentiation into mature muscle cells is one of the hallmarks of this disease. The cause of this lack of differentiation remains unknown. In this study, we hope to gain an insight into the molecular mechanisms of RMS cell differentiation and how mitochondrial properties may be involved in this process, particularly related to mitochondrial movement, morphology and protein markers of mitochondrial biogenesis. For the purpose of this study, human skeletal muscle cells (HSKM) and rhabdomyosarcoma (RD) were induced to differentiate for a period of 7 days. While no difference was observed in mitochondrial speed, acceleration and distance travelled from the origin in HSKM cells after 7 days of differentiation, RD cells exhibited an approximate 2‐ fold increase in speed and acceleration after 7 days of differentiation. Moreover, mitochondria in RD cells had a more punctate appearance after 7 days of differentiation. p53, a regulator of cell cycle arrest, was markedly elevated in RD cells. However, BAX, a regulator of apoptosis, remained highly expressed in HSMK cells throughout the 7 day differentiation period. While these changes occurred in HSMK and RD cells, no difference in PGC1‐α, a marker of mitochondrial biogenesis, was observed. This study suggests that mitochondrial properties and motility are altered in RD cells when induced to differentiate, and this could be related to the degree of tumorigenesis. Supported by NSERC and Mitacs.
Intermittent Physical Activity Produces a Leaner but “Sedentary Obese” White Adipose Tissue Phenotype
Authors: Wainright Katherine , Flemming Nicholas , Rowles Joe , Welly Rebecca , Zidon Terese , Park Young‐Min , Scroggins Rebecca , Anderson‐Bacum Emily , Hasty Alyssa , Vieira‐Potter Victoria , Padilla Jaume
Published: June 2021

Adipose tissue (AT) dysfunction plays a pivotal role in the development of insulin resistance in obesity. Regular physical activity (PA) is effective in reducing AT mass and inflammation; however, whether intermittent PA affords beneficial effects is unknown. The purpose of this study was to evaluate the influence of intermittent PA on adiposity and retroperitoneal AT expression of genes related to inflammation and oxidative stress. Male C57BL/6 mice (age 5 weeks) were randomly assigned to four groups (n=10/group): low‐fat diet sedentary (LFD‐SED), high‐fat diet sedentary (HFD‐SED), HFD regular PA (HFD‐RPA), and HFD intermittent PA (HFD‐IPA) for 24 weeks. All animals were single‐caged. RPA mice had access to voluntary running wheels throughout the study period, whereas IPA mice had access to running wheels for three‐week intervals (i.e., 3 weeks on/3 weeks off) throughout the study period. Both HFD‐RPA and HFD‐IPA were associated with a similar reduction in retroperitoneal AT mass (~‐24%, p<0.05) relative to HFD‐SED. However, HFD‐RPA, but not HFD‐IPA, exhibited a decrease in retroperitoneal adipose mRNA markers of inflammation (F4/80, CD11c, leptin, VCAM‐1) and oxidative stress (p22phox and p47phox). Collectively, these data suggest that episodic PA over a prolonged period of time leads to a reduction in adiposity albeit with retention of a “sedentary obese” white AT phenotype.
Quantification of Impaired Cumulative Muscle Hemodynamics During Continuous Non‐Steady State Exercise and Recovery in Aging Humans
Authors: Racine Matthew , Hearon Christopher , Richards Jennifer , Dinenno Frank
Published: June 2021

Aging is associated with impaired muscle blood flow during exercise; however, much of our understanding is derived from comparisons of steady state hemodynamics. In 8 young (22 ± 3 yrs) and 6 older (66 ± 5 yrs) adults, we continuously measured forearm blood flow (FBF; Doppler ultrasound) and calculated forearm vascular conductance (FVC; mean arterial pressure from finometry) during 3 min of rhythmic handgrip exercise at 10 and 20% maximum voluntary contraction (MVC; 1.5 min each) and 2 min of post‐exercise recovery. Two randomized, continuous non‐steady state exercise trials consisted of either 1.5 min at a low workload (10% MVC) immediately followed by 1.5 min at a high workload (20% MVC) (L2H) or vice versa (H2L). FBF and FVC were quantified in 3‐sec bins and area under the curve (AUC) was calculated throughout. In both exercise trials, FBF (~30‐40%) and FVC (~40‐50%) were reduced with age at the end of the first workload, and this persisted throughout the next workload transition and into recovery. When quantified as an absolute cumulative response over time in the L2H trial, the age‐related deficit in FBF increased from ~600 mL at 1.5 min to ~3000 mL at end recovery, and similar results were obtained for FVC (~900‐4400 mL/100mmHg). Similar trends were observed for the H2L trial, but the magnitude of the age‐related deficit was greater (FBF ~1500‐3800 mL; FVC ~2200‐5500 mL/100mmHg). We conclude that during non‐steady state exercise and recovery, the magnitude of FBF and FVC deficit with age increases over time, which may have implications for understanding muscle perfusion during activities of normal daily living in older adults. Support HL095573
Influence of Prenatal Exercise on Fetal/Infant Health
Authors: May Linda , Strickland Diana , Newton Ed , Gross McMillan Amy , Steed Dennis , Biko David
Published: June 2021

Over 30% of children, as young as 6 years, are overweight or obese with increased risk factors of heart disease. Excess adipose, especially abdominal, is associated with increased risk of heart disease. Research has shown an association between obesity, cardiovascular disease, and autonomic imbalance. It has been suggested that a major focus of disease prevention should target the prenatal period, since this is a critical time of organ development. Our previous studies found prenatal exercise is associated with improved fetal, infant, and child heart health and autonomic control. Though these results are promising, exercise was self‐reported and subject to bias. Therefore we conducted a preliminary exercise intervention study with the goal to test the hypothesis that prenatal exercise intervention will improve fetal and infant measures of heart rate (HR), obesity, and nervous system development. Participants either exercised (5+45 min,3X per wk,16‐36 wks pregnant) or did not. Statistical analyses included t‐tests & ANOVA. Preliminary data show lower HR and adipose accumulation, with increased HR variability and neurodevelopmental scores in fetus and infants of exercisers compared to offspring of controls. This study is of public import regarding prevention of heart disease and obesity by targeting the critical time period before birth in order to improve child health, potentially decrease health care costs, while being a safe, low‐cost, non‐pharmacologic intervention available to the general public.
The Role of Cytokines in Muscle Fatigue in Patients with Chronic Fatigue Syndrome (CFS).
Authors: Earl Kate , Sakellariou Giorgos , Owens Daniel , Sinclair Melanie , Fenech Manuel , Close Graeme , Lawton Clare , Dye Louise , Beadsworth Micheal , McArdle Anne
Published: June 2021

CFS is characterized by profound levels of persistent/recurrent fatigue. It is proposed that chronic, low level inflammation may play a role in this fatigue. We recruited 100 untreated patients with CFS (average age 33±12) and 100 age and sex matched healthy controls (HCs). Serum levels of TNF‐α were assessed using ELISA. Subjective fatigue was determined by questionnaire and muscle function tests were undertaken in subgroups in which maximal voluntary contraction (MVC), electrically stimulated muscle force generation and rate of fatigue were assessed in the quadriceps muscle. Subjective fatigue was higher in patients with CFS compared with HCs. Preliminary analyses showed that serum TNF‐α was undetectable in 97% of HCs, whereas 15% of patients with CFS had detectable (4.4+/‐0.18pg/ml) serum TNF‐α. MVC was significantly reduced in subjects with CFS compared with HCs. No difference was seen in stimulated muscle fatigue between groups. This preliminary data suggests that a sub‐group of patients with CFS may have low level inflammation and analyses are underway to further characterise other inflammatory markers in serum and muscle of these patients and to determine whether such changes could affect indices of muscle function or central fatigue. Funded by MRC, BBSRC and the ME Association.